WO2023092768A1 - Application of serpinb3/b4 as target in drugs for treating inflammatory skin diseases such as rosacea - Google Patents

Application of serpinb3/b4 as target in drugs for treating inflammatory skin diseases such as rosacea Download PDF

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WO2023092768A1
WO2023092768A1 PCT/CN2021/139541 CN2021139541W WO2023092768A1 WO 2023092768 A1 WO2023092768 A1 WO 2023092768A1 CN 2021139541 W CN2021139541 W CN 2021139541W WO 2023092768 A1 WO2023092768 A1 WO 2023092768A1
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serpinb3
rosacea
expression
significantly
skin lesions
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Chinese (zh)
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李吉
邓智利
沙珂
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中南大学湘雅医院
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Definitions

  • the invention relates to the technical field of medicines for treating inflammatory skin diseases, in particular to the application of SERPINB3/B4 as a target in medicines for treating inflammatory skin diseases such as rosacea.
  • Rosacea and psoriasis are relatively common chronic inflammatory skin diseases with a high incidence rate, and they are prone to recurrent clinically, causing great pain to patients.
  • the pathogenesis of the two has not been fully elucidated, and most studies It is believed that immune system dysfunction plays an important role in the pathogenesis of both.
  • the treatment plan for rosacea and psoriasis still needs to be perfected. Therefore, it is of great significance to further clarify the pathogenesis of the two diseases and find a new treatment plan.
  • the function of protease plays a role in maintaining skin structure and regulating skin response to pathogens and allergens.
  • the purpose of the present invention is to provide the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as rosacea, so as to solve the problems raised in the above background technology.
  • the present invention provides the following technical solutions: the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as rosacea, first of all, the SERPINB3/B4 Expression of /B4 in serum and skin lesions of patients with rosacea and psoriasis and expression of Serpinb3a in LL37-induced rosacea-like mouse model and imiquimod-induced psoriasis-like mouse model ;
  • SC75741 (a potent NF- ⁇ B signaling pathway inhibitor) was selected to inhibit the NF- ⁇ B signaling pathway in the mouse epidermis to clarify the role of NF- ⁇ B signaling pathway in the pathogenesis of rosacea; and use CCK8 and flow cytometry analysis to select The appropriate concentration of SC75741 was studied in vitro to further clarify the interaction between SERPINB3/B4 and NF- ⁇ B signaling pathway in the pathogenesis of rosacea and psoriasis.
  • the present invention collects skin lesions of normal controls and patients with rosacea, extracts total RNA, and detects the expression of SERPINB3/B4 mRNA levels between the two groups. It is found that the two are almost not expressed in normal control skin lesions, while rose The mRNA expression level of SERPINB3/B4 in the patient's facial skin lesions is significantly higher than that of healthy controls (as shown in Figure 2A, B). Similarly, the present invention performs IGA scoring on the number of papules or pustules and skin erythema in rosacea patients' skin lesions, Correlation analysis was carried out with the expression level of SERPINB3/B4 mRNA in the skin lesions of patients with rosacea.
  • the present invention collects facial skin lesions of patients with rosacea and performs immunohistochemical staining.
  • the results show that compared with normal healthy controls, the full layer of epidermal keratinocytes in patients with rosacea can detect high-abundance SERPINB3/B4 expression , and it is mainly expressed in the cytoplasm, while healthy controls have almost no expression in the whole skin layer (as shown in Figure 2E-F).
  • the above results show that the expression of SERPINB3/B4 in skin lesions of patients with rosacea is significantly higher than that of healthy controls.
  • the present invention first collects the skin lesions of healthy controls and psoriasis patients, extracts the total RNA of skin lesions, and detects SERPINB3/B4 in the skin by qRT-PCR.
  • the present invention established a rosacea-like mouse model induced by the antimicrobial peptide LL37 according to the modeling method used in previous studies. Highly similar to human rosacea, the present invention selects 7-8 week old BALB/c female mice, removes the hair on the back of the mice, and injects 50 ⁇ l (320 ⁇ M) dose of LL37 intradermally on the back of each mouse one day later, and injects once every 12 hours , mice in the control group were given equal doses of sterile 1 ⁇ PBS solution, and injected continuously for 2 days, a total of 4 times.
  • the results showed that the expression of Serpinb3a in rosacea-like mouse skin lesions was significantly up-regulated compared with the experimental control group (as shown in Figure 2K), and the difference was statistically significant.
  • Serpinb3a is an analogue of SERPINB3/B4
  • the present invention further detects the Serpinb3a protein level expression in the rosacea-like mouse model skin lesions by immunofluorescence, and the results show that Serpinb3a expression in the rosacea-like mouse model skin lesions is closely related to that of human rosacea.
  • the present invention is based on the modeling method used in the previous research, that is, continuous external application of imiquimod for 6 days to build a psoriasis-like mouse model, collect mouse skin lesions for sampling, and collect mouse skin lesions to extract total RNA , the mRNA expression of Serpinb3a in the skin lesions of psoriasis-like mice was detected by qRT-PCR, and the results showed that the expression of Serpinb3a in the skin lesions of psoriasis-like mice was significantly up-regulated compared with the experimental control group (as shown in Figure 2M). Mouse skin lesions were immunofluorescently stained, and Serpinb3a was obviously highly expressed in psoriasis-like mouse skin lesions (as shown in Figure 2N).
  • the present invention constructs a mouse model of specifically knocking down mouse epidermis Serpinb3a by siRNA, and injects LL37 intradermally and mites Topical administration of quimod was used to establish rosacea-like and psoriasis-like mouse models, and the effects of epidermal knockdown of Serpinb3a on the phenotypes of rosacea-like mice and psoriasis-like mice were observed.
  • the present invention names the negative control sequence and the two siRNA sequences as ScrRNA, siRNA#1 and siRNA#2 respectively for the next research, and first explores the effect of knocking down Serpinb3a in the epidermis on the phenotype of rosacea-like mice , as shown in Figure 3A, 7-8 weeks old BALB/c wild-type female mice had their back hair shaved, and siRNA was injected intradermally one day before, and the experimental control group was intradermally injected with the corresponding negative control sequence , followed by intraepidermal injection of LL37 to induce a rosacea-like mouse model according to the aforementioned method, and the second siRNA injection was performed before the second injection of LL37 to strengthen the knockdown effect.
  • FIG. 2B shows the performance of rosacea-like dermatitis in the Control group and LL37 group of Scr siRNA and other 2 siRNAs two days after LL37 modeling, it can be seen After LL37 modeling, the mouse skin lesions in the Scr siRNA group showed obvious inflammatory reactions, including erythema and telangiectasia, while the inflammatory reactions in the mouse skin lesions in the two siRNA knockdown groups were significantly reduced.
  • the present invention uses Image J software to analyze The area of skin lesions in each group of rosacea-like mice was measured, and the results showed that the area of erythema in the Scr siRNA group LL37 was significantly increased after modeling, and the area of erythema in the LL37 group with two siRNAs was significantly smaller than that in the Scr LL37 group (as shown in Figure 3C ), followed by scoring the erythema severity of rosacea-like skin lesions, the results showed that the erythema score of the Scr siRNA LL37 group increased significantly after modeling, and the erythema score of the 2 siRNA LL37 group was significantly reduced after modeling (as shown in Figure 3D).
  • the present invention compares the histological situation of the mouse skin lesions in each group after LL37 modeling.
  • the skin lesions of the modeling parts are taken, paraffin-embedded and sectioned, and then HE stained.
  • the number of inflammatory cell infiltration in the dermis of the mice is observed under a microscope and counted. , as shown in Figure 3E, the number of inflammatory cells infiltrating the dermis in the Scr siRNA LL37 group was significantly higher than that in the Control group, and the number of inflammatory cells infiltrating the dermis in the 2 siRNA LL37 group was significantly lower than that in the Scr siRNA LL37 group.
  • the number of infiltrating inflammatory cells was statistically analyzed by Prism9 software. The results showed that after knocking down Serpinb3a, the number of infiltrating inflammatory cells in the LL37 group with two siRNAs was significantly less than that in the Scr LL37 group (as shown in Figure 3F), and the difference was statistically significant.
  • the present invention collects mouse skin lesions, extracts total RNA, and compares the differences in mRNA expression levels of rosacea-related genes in the skin lesions of the above-mentioned groups of mice by qRT-PCR, as shown in Figure 3G-H, Scr siRNA Compared with the Control group, the expressions of Il6 and Tnf- ⁇ genes in the LL37 group were increased, and after knocking down Serpinb3a, the expression levels of the above two genes in the LL37 group with two siRNAs were significantly lower than those in the LL37 group with Scr siRNA, and the difference was statistically significant.
  • the present invention selects the aforementioned two siRNAs, injects them into the ears of mice, and knocks down Serpinb3a in the epidermis according to the existing methods.
  • Research methods The psoriasis-like mouse model was constructed with topical imiquimod, as shown in Figure 4A schematic diagram, siRNA was injected intradermally 1 day before topical imiquimod (denoted as day-1), and the experimental control group was injected with siRNA.
  • the corresponding negative control sequence was injected internally, followed by external application of imiquimod cream once a day, and the second siRNA injection was performed on the second day to strengthen the knockdown effect.
  • the effect of Serpinb3a on the phenotype of psoriasis-like mice skin lesions, the present invention observed and compared the differences between the phenotypes of the three groups of mice (Scr siRNA, siRNA#1 and siRNA#2) on the 6th day of modeling and carried out According to the method of previous research [77], the PASI score and ear thickness of the skin lesions of the mice were measured. It can be seen that the erythema and scales of the mice in the siRNA imiquimod group were compared with those in the ScrRNA imiquimod group.
  • the cells were statistically analyzed, and it was found that the two siRNA imiquimod groups were significantly lower than the Scr siRNA imiquimod group (as shown in Figure 4F-G).
  • Influence the present invention collects mouse skin lesions, extracts total RNA, compares the difference on the mRNA expression level of psoriasis-related genes in the mouse skin lesions of the above-mentioned groups by qRT-PCR, as shown in Figure 4H-I, The Il6 and Il-1 ⁇ gene expressions of the Scr siRNA imiquimod group increased significantly compared with the Control group.
  • knocking down serpinb3a in the epidermis of mice can significantly improve the inflammatory response in skin lesions of rosacea-like and psoriasis-like mice.
  • the present invention constructs a SERPINB3/B4 overexpression plasmid in vitro, and transfects the overexpression plasmid into human HaCaT cells with a transfection reagent. After 72 hours, Collect cellular RNA, perform whole transcriptome sequencing, and verify the sequencing results through in vivo and in vitro experiments,
  • the present invention performs clustering analysis on the differential gene expression values in human HaCaT cells overexpressing SERPINB3/B4, and then normalizes the gene expression values to obtain as shown in Figure 5A, where each small square in the figure represents a gene , blue indicates down-regulation of gene expression, red indicates up-regulation of gene expression, the darker the color, the more obvious the difference in expression, each column indicates the expression of different genes in each sample, and each row indicates the expression of each gene in different samples, As shown in the figure, it can be seen that the gene expression of human HaCaT cells overexpressing SERPINB3/B4 is significantly different from that of the normal control.
  • the present invention conducts gene enrichment analysis (Gene Set Enrichment Analysis, Gene Set Enrichment Analysis, GSEA), based on rosacea is a skin inflammatory disease and previous research, inflammation-related signaling pathways play a key role in the development of rosacea, so this study focuses on signaling pathways associated with inflammation, NF- ⁇ B signaling pathway almost exists In the animal cells used, it is involved in various responses of cells to external stimuli, including stress, cytokines, free radicals, microbial infection and ultraviolet radiation. Previous studies have found that this signaling pathway regulates the expression of a large number of genes related to inflammation.
  • the NF- ⁇ B signaling pathway was significantly enriched in the human HaCaT cells overexpressing SERPINB3/B4 compared with the control cell line (as shown in Figure 5B-E), which suggests that the signaling pathway may Involved in SERPINB3/B4 regulating the pathogenesis of rosacea,
  • the present invention transfected the overexpression plasmid into the human HaCaT cell line with a transfection reagent for 72 hours, and harvested the cell protein, immunoblotting
  • the phosphorylation level of p65/NF- ⁇ B (p-p65) after SERPINB3/B4 expression in human HaCaT cell line was detected by using the method, and the gray scale of p-p65 was scanned and quantified by Image J software, as shown in Figure 5F,
  • the present invention found that after expressing SERPINB3/B4 in the human HaCaT cell line, the expression of p-p65 was significantly up-regulated, the expression of p65 was not significantly different, and p-p65/p65 was up-regulated.
  • the present invention constructed SERPINB3/B4 shRNA in vitro to establish a stable knockdown of SERPINB3 /B4 human HaCaT cell line, after incubation with TNF- ⁇ for 30 minutes, the effect of knocking down SERPINB3/B4 on the activation of NF- ⁇ B signal induced by TNF- ⁇ was detected by Western blotting.
  • the human HaCaT cell line has a significant knockdown effect on SERPINB3/B4.
  • overexpression of SERPINB3/B4 in vitro induces the upregulation of downstream molecules of NF- ⁇ B signaling pathway, and knockdown of SERPINB3/B4 in vitro can prevent the activation of TNF- ⁇ -induced NF- ⁇ B signaling.
  • inflammation-related chemokines and cytokines play an extremely important role in the occurrence and progression of inflammation.
  • a series of previous results show that SERPINB3/B4 can regulate the NF- ⁇ B signaling pathway, in order to further clarify the chemokines and cellular
  • the specific role of factors in regulating the pathogenesis of rosacea by SERPINB3/B4, the present invention further analyzes the results of RNA-seq after overexpressing SERPINB3/B4 in human HaCaT cells, as shown in Figure 6A-B, chemokines and cells Factor signaling pathways are highly enriched in human HaCaT cells with overexpression of SERPINB3/B4.
  • the present invention performs cluster analysis on the expression values of chemokine and cytokine-related differences in the samples, and normalizes the gene expression values.
  • inflammation-related chemokines CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL5, CXCL8, CXCL10, CXCL11
  • cytokines IL1A, IL1B, IL6, TNFA
  • the present invention uses qRT-PCR to detect the expression of the above-mentioned genes in human HaCaT cells after overexpressing SERPINB3/B4, and the results (as shown in Figure 6D ) shows that after overexpressing SERPINB3/B4 in human HaCaT cells, the expression of chemokines CCL2, CCL5, CCL20, CXCL2, CXCL9, CXCL10, CXCL11 and cytokines IL1B, IL6, TNF- ⁇ was significantly up-regulated, the results showed that in human HaCaT cells Cells overexpressing SERPINB3/B4 can significantly induce the upregulation of inflammation-related chemokines and cytokines,
  • Intradermal knockdown of Serpinb3a in mice can reduce the expression level of rosacea-related cytokines (Il6, Tnf- ⁇ ) induced by LL37.
  • the present invention used the same method to detect the changes of the above chemokines in the skin lesions of psoriasis-like mice after knocking down serpinb3a, and the results are shown in Figure 6F.
  • Cxcl10, Cxcl11 expression levels were significantly inhibited
  • SERPINB3/B4 induced chemokines CCL2, CCL5, CXCL9, CXCL10, CXCL11 and cytokines IL-1 ⁇ , IL-6, TNF - ⁇ was significantly up-regulated, and the expression levels of the above chemokines and cytokines were significantly decreased after co-incubation with SC75741.
  • SC75741 could down-regulate related chemokines by inhibiting the activation of SERPINB3/B4 on the NF- ⁇ B signaling pathway and expression levels of cytokines.
  • the present invention reports for the first time the expression of SERPINB3/B4 in skin lesions of patients with rosacea and psoriasis, and its correlation with disease severity;
  • the research object clarifies that SERPINB3/B4 promotes the occurrence of inflammatory response through the NF- ⁇ B signaling pathway.
  • the present invention found that the application of SC75741 (a powerful inhibitor of NF- ⁇ B signaling pathway) can significantly improve the rosacea-like mouse skin lesions induced by LL37 At the same time, it significantly inhibited the activation of NF- ⁇ B signaling pathway induced by SERPINB3/B4. Therefore, the present invention provides a new explanation for the pathogenesis of rosacea and psoriasis, and provides a new idea for the treatment of the two chronic inflammatory skin diseases.
  • Fig. 1 is a work flow chart of the present invention
  • Fig. 2 is for the SERPINB3mRNA expression level in A healthy control of the present invention and rosacea patient's skin lesion;
  • IHC Immunohistochemical staining
  • G-H The mRNA expression levels of SERPINB3 and SERPINB4 in the skin of healthy controls and psoriasis patients; HS is the healthy control group, and Psoriasis is the psoriasis group;
  • Fig. 3 is the model diagram of this mouse in vivo experiment of A of the present invention.
  • the upper picture is a representative general picture of the LL37-induced rosacea-like mouse model skin lesions; the lower picture is the result of stereoscopic magnification;
  • E is the HE staining result of the skin lesion corresponding to the picture in D;
  • G-HqRT-PCR detected the mRNA expression of Il6 and Tnf- ⁇ in skin lesions of mice in each group;
  • Figure 4 A schematic diagram of the mouse in vivo experiment
  • IMQ is the imiquimod group
  • E is the HE staining result of the skin lesion corresponding to Figure C, scale bar: 50 ⁇ m;
  • the thickness of the epidermis was measured and statistically analyzed for the HE staining results of the mice in each group, and the length unit: micron;
  • Fig. 5 is the cluster analysis of AHaCaT cells of the present invention overexpressing SERPINB3/B4 compared with blank control group cell transcriptome data difference genes, each group of cells transfected 3 times; wherein, red represents high gene expression, and blue represents low gene expression Express;
  • B-EGSEA analysis showed: comparing SERPINB3/B4 with the VECTOR group in human HaCaT cells, the ranking of KEGG pathways enriched by overlapping differentially expressed genes, among which, the NF- ⁇ B signaling pathway is represented by a red box;
  • F immunoblotting detected the change of p-p65 protein level after human HaCaT cells were transfected with SERPINB3/B4 overexpression plasmid for 72 hours; the protein level was quantitatively analyzed by ImageJ software;
  • the left picture shows the expression and localization of p65 detected by immunofluorescence after human HaCaT cells were transfected with SERPINB3/B4 overexpression plasmid for 72 hours; the arrow out indicates that p65 enters the nucleus, and DAPI staining (blue) indicates the nuclear localization, scale bar: 50 ⁇ m; the right picture : Statistical analysis was performed on the positive cells of p65 into the nucleus, and the difference was statistically significant;
  • the left picture of H-I shows the establishment of human HaCaT cells knocking down SERPINB3/B4 in vitro, and the cell line was incubated with 100ng/ml TNF- ⁇ for 30 minutes.
  • the p-p65 gray level was statistically analyzed by ImageJ software, and the difference was statistically significant;
  • J-K established human HaCaT cells knocking down SERPINB3/B4 in vitro incubated the cell line with 100ng/ml TNF- ⁇ for 30 minutes, detected the protein level and localization of p65 in the knockdown group compared with the control group by immunofluorescence, and analyzed the positive cell data of p65 nuclear entry Statistical analysis shows that the difference is statistically significant; p65 (red) is expressed on the membrane, and enters the nucleus when activated, which is nuclear expression.
  • DAPI staining indicates the location of the nucleus, and the scale bar is 50 ⁇ m;
  • Figure 6 shows the results of the A-BGSEA analysis of the present invention: in human HaCaT cells overexpressing SERPINB3/B4 compared with the VECTOR blank control group, chemokine and cytokine signaling pathways are highly expressed in SERPINB3/B4 overexpressing people's HaCaT cells enrichment;
  • GqRT-PCR was used to detect the mRNA levels of inflammatory factors.
  • FIG. 1 Figure 6 an embodiment provided by the present invention: the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as rosacea;
  • Example 1 the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as rosacea, first of all, by ELISA, immunohistochemistry, qRT-PCR, immunofluorescence, etc.
  • inflammatory skin diseases such as rosacea
  • ELISA immunohistochemistry
  • qRT-PCR immunofluorescence
  • Serpinb3a The expression of serum and skin lesions of patients with psoriasis and the expression of Serpinb3a in the rosacea-like mouse model induced by LL37 and the psoriasis-like mouse model induced by imiquimod;
  • SC75741 (a potent NF- ⁇ B signaling pathway inhibitor) was selected to inhibit the NF- ⁇ B signaling pathway in the mouse epidermis to clarify the role of NF- ⁇ B signaling pathway in the pathogenesis of rosacea; and use CCK8 and flow cytometry analysis to select The appropriate concentration of SC75741 was studied in vitro to further clarify the interaction between SERPINB3/B4 and NF- ⁇ B signaling pathway in the pathogenesis of rosacea and psoriasis.
  • Example 2 the present invention collects skin lesions of normal controls and patients with rosacea, extracts total RNA, and detects the expression of SERPINB3/B4 mRNA levels between the two groups.
  • the mRNA expression level of SERPINB3/B4 in facial skin lesions of patients with rose was significantly higher than that of healthy controls (as shown in Figure 2A, B).
  • the present invention performs IGA scoring on the number of papules or pustules and skin erythema in patients with rosacea , and correlated with the expression level of SERPINB3/B4 mRNA in skin lesions of patients with rosacea, the results showed that with the increase of SERPINB3 mRNA expression level, the IGA score of skin lesions was significantly increased, and the results showed that the expression level of SERPINB3 mRNA was significantly positively correlated with IGA score (as shown in Figure 2C), in addition, the results of the present invention showed that with the increase of SERPINB4 mRNA expression level, the IGA score of skin lesions was significantly up-regulated, and the results showed that the SERPINB4 mRNA expression level was positively correlated with the severity of skin lesions (as shown in Figure 2D),
  • the present invention collects facial skin lesions of patients with rosacea and performs immunohistochemical staining.
  • the results show that compared with normal healthy controls, the full layer of epidermal keratinocytes in patients with rosacea can detect high-abundance SERPINB3/B4 expression , and it is mainly expressed in the cytoplasm, while healthy controls have almost no expression in the whole skin layer (as shown in Figure 2E-F).
  • the above results show that the expression of SERPINB3/B4 in skin lesions of patients with rosacea is significantly higher than that of healthy controls.
  • the present invention first collects the skin lesions of healthy controls and psoriasis patients, extracts the total RNA of skin lesions, and detects SERPINB3/B4 in the skin by qRT-PCR.
  • the present invention established a rosacea-like mouse model induced by the antimicrobial peptide LL37 according to the modeling method used in previous studies. Highly similar to human rosacea, the present invention selects 7-8 week old BALB/c female mice, removes the hair on the back of the mice, and injects 50 ⁇ l (320 ⁇ M) dose of LL37 intradermally on the back of each mouse one day later, and injects once every 12 hours , mice in the control group were given equal doses of sterile 1 ⁇ PBS solution, and injected continuously for 2 days, a total of 4 times.
  • the results showed that the expression of Serpinb3a in rosacea-like mouse skin lesions was significantly up-regulated compared with the experimental control group (as shown in Figure 2K), and the difference was statistically significant.
  • Serpinb3a is an analogue of SERPINB3/B4
  • the present invention further detects the Serpinb3a protein level expression in the rosacea-like mouse model skin lesions by immunofluorescence, and the results show that Serpinb3a expression in the rosacea-like mouse model skin lesions is closely related to that of human rosacea.
  • the present invention is based on the modeling method used in the previous research, that is, continuous external application of imiquimod for 6 days to build a psoriasis-like mouse model, collect mouse skin lesions for sampling, and collect mouse skin lesions to extract total RNA , the mRNA expression of Serpinb3a in the skin lesions of psoriasis-like mice was detected by qRT-PCR, and the results showed that the expression of Serpinb3a in the skin lesions of psoriasis-like mice was significantly up-regulated compared with the experimental control group (as shown in Figure 2M). Mouse skin lesions were immunofluorescently stained, and Serpinb3a was obviously highly expressed in psoriasis-like mouse skin lesions (as shown in Figure 2N).
  • the present invention constructs a mouse model of specifically knocking down Serpinb3a in the epidermis of mice by siRNA, and injects LL37 intradermally and mites Topical administration of quimod was used to establish rosacea-like and psoriasis-like mouse models, and the effects of epidermal knockdown of Serpinb3a on the phenotypes of rosacea-like mice and psoriasis-like mice were observed.
  • Example 3 the present invention named the negative control sequence and 2 siRNA sequences as ScrRNA, siRNA#1 and siRNA#2 respectively for the next research, and first explored the effect of knocking down Serpinb3a in the epidermis on the phenotype of rosacea-like mice Effect, as shown in Figure 3A schematic diagram, 7-8 weeks old BALB/c wild-type female mice, shaved their back hair, injected siRNA intradermally one day before, and injected the corresponding negative control group intradermally Sequence, followed by intraepidermal LL37 injection to induce a rosacea-like mouse model according to the aforementioned method, and the second siRNA injection was performed before the second LL37 injection to strengthen the knockdown effect.
  • FIG. 2B shows the performance of rosacea-like dermatitis in the Control group and LL37 group of Scr siRNA and other 2 siRNAs two days after LL37 modeling. It can be seen that after LL37 modeling, the skin lesions of the mice in the Scr siRNA group showed obvious inflammatory reactions, including erythema and telangiectasia, while the inflammatory reactions of the mouse skin lesions in the two siRNA knockdown groups were significantly reduced.
  • the present invention uses Image J software The area of skin lesions in each group of rosacea-like mice was measured, and the results showed that the area of erythema in the Scr siRNA group LL37 was significantly increased after modeling, and the area of erythema in the LL37 group with two siRNAs was significantly smaller than that in the Scr LL37 group (as shown in Fig. 3C), followed by scoring the erythema severity of rosacea-like skin lesions, the results showed that the erythema score of the Scr siRNA LL37 group increased significantly after modeling, and the erythema score of the 2 siRNA LL37 group was significantly reduced after modeling (as shown in Figure 3D).
  • the present invention compares the histological situation of the mouse skin lesions in each group after LL37 modeling.
  • the skin lesions of the modeling parts are taken, paraffin-embedded and sectioned, and then HE stained.
  • the number of inflammatory cell infiltration in the dermis of the mice is observed under a microscope and counted. , as shown in Figure 3E, the number of inflammatory cells infiltrating the dermis in the Scr siRNA LL37 group was significantly higher than that in the Control group, and the number of inflammatory cells infiltrating the dermis in the 2 siRNA LL37 group was significantly lower than that in the Scr siRNA LL37 group.
  • the number of infiltrating inflammatory cells was statistically analyzed by Prism9 software. The results showed that after knocking down Serpinb3a, the number of infiltrating inflammatory cells in the LL37 group with two siRNAs was significantly less than that in the Scr LL37 group (as shown in Figure 3F), and the difference was statistically significant.
  • the present invention collects mouse skin lesions, extracts total RNA, and compares the differences in mRNA expression levels of rosacea-related genes in the skin lesions of the above-mentioned groups of mice by qRT-PCR, as shown in Figure 3G-H, Scr siRNA Compared with the Control group, the expressions of Il6 and Tnf- ⁇ genes in the LL37 group were increased, and after knocking down Serpinb3a, the expression levels of the above two genes in the LL37 group with two siRNAs were significantly lower than those in the LL37 group with Scr siRNA, and the difference was statistically significant.
  • Example 4 in order to clarify the effect of knocking down Serpinb3a in the epidermis on the phenotype of psoriasis-like mice, the present invention selects the aforementioned two siRNAs and injects them into the ears of mice, knocking down Serpinb3a in the epidermis, according to previous methods
  • siRNA was injected intradermally 1 day before topical imiquimod (denoted as day-1), and the experimental control group
  • the corresponding negative control sequence was injected intradermally, followed by external application of imiquimod cream once a day, and the second siRNA injection was performed on the second day to strengthen the knockdown effect.
  • the present invention collects mouse skin lesions, extracts total RNA, and compares the differences in mRNA expression levels of psoriasis-related genes in the mouse skin lesions of the above-mentioned groups by qRT-PCR, as shown in Figure 4H-I
  • the expression levels of Il6 and Il-1 ⁇ genes in the Scr siRNA imiquimod group increased significantly compared with the Control group. The difference is statistically significant.
  • the above results show that the deletion of Serpinb3a in the ear epidermis can significantly improve the imiquimod-induced psoriasis-like mouse model in terms of inflammatory response, histopathology and mRNA expression. development of inflammatory diseases,
  • knocking down serpinb3a in the epidermis of mice can significantly improve the inflammatory response in skin lesions of rosacea-like and psoriasis-like mice.
  • Example 5 in order to further clarify the specific molecular mechanism of SERPINB3/B4 on rosacea in vitro, the present invention constructs SERPINB3/B4 overexpression plasmid in vitro, and transfects the overexpression plasmid into human HaCaT cells with a transfection reagent for 72 hours Afterwards, the cellular RNA was collected, the whole transcriptome was sequenced, and the sequencing results were verified through in vivo and in vitro experiments.
  • the present invention performs clustering analysis on the differential gene expression values in human HaCaT cells overexpressing SERPINB3/B4, and then normalizes the gene expression values to obtain as shown in Figure 5A, where each small square in the figure represents a gene , blue indicates down-regulation of gene expression, red indicates up-regulation of gene expression, the darker the color, the more obvious the difference in expression, each column indicates the expression of different genes in each sample, and each row indicates the expression of each gene in different samples, As shown in the figure, it can be seen that the gene expression of human HaCaT cells overexpressing SERPINB3/B4 is significantly different from that of the normal control.
  • the present invention carried out gene enrichment analysis (Gene Set Enrichment Analysis, Gene Set Enrichment Analysis, GSEA), based on rosacea is a skin inflammatory disease and previous research, inflammation-related signaling pathways play a key role in the development of rosacea, so this study focuses on signaling pathways associated with inflammation, NF- ⁇ B signaling pathway almost exists In the animal cells used, it is involved in various responses of cells to external stimuli, including stress, cytokines, free radicals, microbial infection and ultraviolet radiation. Previous studies have found that this signaling pathway regulates the expression of a large number of genes related to inflammation.
  • the NF- ⁇ B signaling pathway was significantly enriched in the human HaCaT cells overexpressing SERPINB3/B4 compared with the control cell line (as shown in Figure 5B-E), which suggests that the signaling pathway may Involved in SERPINB3/B4 regulating the pathogenesis of rosacea,
  • the present invention transfected the overexpression plasmid into the human HaCaT cell line with a transfection reagent for 72 hours, and harvested the cell protein, immunoblotting
  • the phosphorylation level of p65/NF- ⁇ B (p-p65) after SERPINB3/B4 expression in human HaCaT cell line was detected by using the method, and the gray scale of p-p65 was scanned and quantified by Image J software, as shown in Figure 5F,
  • the present invention found that after expressing SERPINB3/B4 in the human HaCaT cell line, the expression of p-p65 was significantly up-regulated, the expression of p65 was not significantly different, and p-p65/p65 was up-regulated.
  • the present invention constructed SERPINB3/B4 shRNA in vitro to establish a stable knockdown of SERPINB3 /B4 human HaCaT cell line, after TNF- ⁇ incubation for 30 minutes, Western blot was used to detect the effect of knocking down SERPINB3/B4 on the activation of NF- ⁇ B signal induced by TNF- ⁇ . The results showed that the stable knockdown of SERPINB3/B4 The human HaCaT cell line has a significant knockdown effect on SERPINB3/B4.
  • overexpression of SERPINB3/B4 in vitro induces the upregulation of downstream molecules of NF- ⁇ B signaling pathway, and knockdown of SERPINB3/B4 in vitro can prevent the activation of TNF- ⁇ -induced NF- ⁇ B signaling.
  • Example 6 Inflammation-related chemokines and cytokines play an extremely important role in the occurrence and progression of inflammation.
  • a series of previous results show that SERPINB3/B4 can regulate the NF- ⁇ B signaling pathway.
  • the present invention further analyzed the results of RNA-seq after overexpressing SERPINB3/B4 in human HaCaT cells, as shown in Figure 6A-B, chemokines and Cytokine signaling pathways are highly enriched in human HaCaT cells with SERPINB3/B4 overexpression.
  • the present invention performs cluster analysis on the expression values of chemokine and cytokine-related differences in samples, and normalizes the gene expression values.
  • the figure shown in Figure 6C was obtained after treatment, compared with the normal control, inflammation-related chemokines (CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL5, CXCL8, CXCL10, CXCL11) and cytokines (IL1A, IL1B, IL6, TNFA) were significantly up-regulated,
  • the present invention uses qRT-PCR to detect the expression of the above-mentioned genes in human HaCaT cells after overexpressing SERPINB3/B4, and the results (as shown in Figure 6D ) shows that after overexpressing SERPINB3/B4 in human HaCaT cells, the expression of chemokines CCL2, CCL5, CCL20, CXCL2, CXCL9, CXCL10, CXCL11 and cytokines IL1B, IL6, TNF- ⁇ was significantly up-regulated, the results showed that in human HaCaT cells Cells overexpressing SERPINB3/B4 can significantly induce the upregulation of inflammation-related chemokines and cytokines,
  • Intradermal knockdown of Serpinb3a in mice can reduce the expression level of rosacea-related cytokines (Il6, Tnf- ⁇ ) induced by LL37.
  • the present invention used the same method to detect the changes of the above chemokines in the skin lesions of psoriasis-like mice after knocking down serpinb3a, and the results are shown in Figure 6F.
  • Cxcl10, Cxcl11 expression levels were significantly inhibited
  • SERPINB3/B4 induced chemokines CCL2, CCL5, CXCL9, CXCL10, CXCL11 and cytokines IL-1 ⁇ , IL-6, TNF - ⁇ was significantly up-regulated, and the expression levels of the above chemokines and cytokines were significantly decreased after co-incubation with SC75741.
  • SC75741 could down-regulate related chemokines by inhibiting the activation of SERPINB3/B4 on the NF- ⁇ B signaling pathway and expression levels of cytokines.

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Abstract

The present invention relates to an application of SERPINB3/B4 as a target in drugs for treating inflammatory skin diseases such as rosacea. Firstly, the expression of SERPINB3/B4 in skin lesions of a rosacea patient and a psoriasis patient and the expression of Serpinb3a in skin lesions of an LL37-induced rosacea-like mouse model and an imiquimod-induced psoriasis-like mouse model are determined by means of ELISA, immunohistochemistry, qRT-PCR, immunofluorescence and the like. The expression of Serpinb3a in mouse epidermal cells is locally knocked down by means of siRNA intradermal injection, and the influence thereof on skin lesion phenotypes and histopathology of an inflammation model is observed. In-vitro research is mainly carried out in human HaCaT cells, overexpression or knock-down of the SERPINB3/B4 in the human HaCaT cells is carried out by constructing plasmids, and a specific molecular action mechanism of the SERPINB3/B4 in vitro in inflammatory skin diseases is further determined by combining methods such as RNA-seq, immunoblotting, and immunofluorescence.

Description

SERPINB3/B4作为靶点在玫瑰痤疮等炎症性皮肤病治疗药物中的应用Application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as rosacea 技术领域technical field
本发明涉及炎症性皮肤病治疗药技术领域，具体为SERPINB3/B4作为靶点在玫瑰痤疮等炎症性皮肤病治疗药物中的应用。The invention relates to the technical field of medicines for treating inflammatory skin diseases, in particular to the application of SERPINB3/B4 as a target in medicines for treating inflammatory skin diseases such as rosacea.
背景技术Background technique
玫瑰痤疮和银屑病是较常见的慢性炎症性皮肤疾病，发病率较高，且临床上易反复发作，给患者带来极大的痛苦，关于二者的发病机制尚未完全阐明，大多数研究认为免疫系统功能紊乱在二者的发病中占重要地位。目前针对玫瑰痤疮和银屑病的治疗方案仍需完善，因此进一步明确两种疾病发病机制及寻找新的治疗方案具有重大意义，蛋白酶功能在维持皮肤结构和调节皮肤对病原体及过敏源反应中起到重要作用，在前期对玫瑰痤疮发病机制的研究过程中，对玫瑰痤疮患者皮损总量RNA进行全转录组测序，发现在玫瑰痤疮患者皮损中SERPINB3/B4明显上调，因此探讨SERPINB3/B4在炎症性皮肤疾病发病过程中的具体作用机制具有重要意义。Rosacea and psoriasis are relatively common chronic inflammatory skin diseases with a high incidence rate, and they are prone to recurrent clinically, causing great pain to patients. The pathogenesis of the two has not been fully elucidated, and most studies It is believed that immune system dysfunction plays an important role in the pathogenesis of both. At present, the treatment plan for rosacea and psoriasis still needs to be perfected. Therefore, it is of great significance to further clarify the pathogenesis of the two diseases and find a new treatment plan. The function of protease plays a role in maintaining skin structure and regulating skin response to pathogens and allergens. In the previous study on the pathogenesis of rosacea, whole transcriptome sequencing was performed on the total RNA of skin lesions of patients with rosacea, and it was found that SERPINB3/B4 was significantly up-regulated in the skin lesions of patients with rosacea. The specific mechanism of action in the pathogenesis of inflammatory skin diseases is of great significance.
现有技术中炎症性皮肤病治疗药中的应用存在的缺陷是：The defect in the application of the inflammatory skin disease therapeutic drug in the prior art is:
1、传统的药物的应用不具备抑制质量过程中出现的并发性沿着，不方便使用者判断炎症发作的反应。1. The application of traditional drugs does not have the concurrency that occurs in the process of suppressing quality, and it is inconvenient for users to judge the reaction of inflammatory attacks.
发明内容Contents of the invention
本发明的目的在于提供SERPINB3/B4作为靶点在玫瑰痤疮等炎症性皮肤病治疗药物中的应用，以解决上述背景技术中提出的问题。The purpose of the present invention is to provide the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as rosacea, so as to solve the problems raised in the above background technology.
为实现上述目的，本发明提供如下技术方案：SERPINB3/B4作为靶点在玫瑰痤疮等炎症性皮肤病治疗药物中的应用，首先通过ELISA、免疫组化、qRT-PCR、免疫荧光等手段明确SERPINB3/B4在玫瑰痤疮患者和银屑病患者血清、皮损中的表达及Serpinb3a在LL37诱导的玫瑰痤疮样小鼠模型及咪喹莫特诱导的银屑病样小鼠模型皮损中的表达情况；In order to achieve the above objectives, the present invention provides the following technical solutions: the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as rosacea, first of all, the SERPINB3/B4 Expression of /B4 in serum and skin lesions of patients with rosacea and psoriasis and expression of Serpinb3a in LL37-induced rosacea-like mouse model and imiquimod-induced psoriasis-like mouse model ;
通过siRNA皮内注射，局部敲降小鼠表皮细胞内Serpinb3a的表达，观察其对炎症模型皮损表型及组织病理学的影响，体外研究主要在人HaCaT细胞中进行，通过构建质粒在人HaCaT细胞中过表达或敲降SERPINB3/B4，并联合RNA-seq和免疫印迹、免疫荧光等方法明确其与NF-κB信号通路的关系；Through intradermal injection of siRNA, the expression of Serpinb3a in mouse epidermal cells was locally knocked down, and its effect on the phenotype and histopathology of inflammatory model skin lesions was observed. In vitro studies were mainly carried out in human HaCaT cells. Overexpress or knock down SERPINB3/B4 in cells, and combine RNA-seq, western blotting, immunofluorescence and other methods to clarify its relationship with NF-κB signaling pathway;
最后，选择SC75741(一种强效NF-κB信号通路抑制剂)抑制小鼠表皮内NF-κB信号通路明确NF-κB信号通路在玫瑰痤疮发病过程中的作用；且运用CCK8和流式分析选择SC75741的合适浓度进行体外研究，进一步明确SERPINB3/B4与NF-κB信号通路在玫瑰痤疮及银屑病发病机制中的相互作用。Finally, SC75741 (a potent NF-κB signaling pathway inhibitor) was selected to inhibit the NF-κB signaling pathway in the mouse epidermis to clarify the role of NF-κB signaling pathway in the pathogenesis of rosacea; and use CCK8 and flow cytometry analysis to select The appropriate concentration of SC75741 was studied in vitro to further clarify the interaction between SERPINB3/B4 and NF-κB signaling pathway in the pathogenesis of rosacea and psoriasis.
优选的，本发明收集正常对照和玫瑰痤疮患者皮损，提取总RNA，检测SERPINB3/B4 mRNA水平在两组之间的表达情况，结果发现在正常对照皮损内二者几乎不表达，而玫瑰患者面部皮损中SERPINB3/B4在mRNA表达水平明显高于健康对照者(如图2A,B)，同样，本发明对玫瑰痤疮患者皮损的丘疹或脓疱个数及皮肤红斑进行IGA评分，并与玫瑰痤疮患者皮损中SERPINB3/B4mRNA表达水平进行相关性分析，结果显示随着SERPINB3 mRNA表达水平的增加，皮损IGA评分明显上调，结果表明SERPINB3 mRNA表达水平与IGA评分呈明显正相关(如图2C)，此外，本发明结果显示随着SERPINB4 mRNA表达水平的增加，皮损IGA评分均明显上调，结果表明SERPINB4 mRNA表达水平与皮损严重程度呈正相关关系(如图2D)，Preferably, the present invention collects skin lesions of normal controls and patients with rosacea, extracts total RNA, and detects the expression of SERPINB3/B4 mRNA levels between the two groups. It is found that the two are almost not expressed in normal control skin lesions, while rose The mRNA expression level of SERPINB3/B4 in the patient's facial skin lesions is significantly higher than that of healthy controls (as shown in Figure 2A, B). Similarly, the present invention performs IGA scoring on the number of papules or pustules and skin erythema in rosacea patients' skin lesions, Correlation analysis was carried out with the expression level of SERPINB3/B4 mRNA in the skin lesions of patients with rosacea. The results showed that with the increase of the expression level of SERPINB3 mRNA, the IGA score of skin lesions was significantly increased. The results showed that the expression level of SERPINB3 mRNA was significantly positively correlated with the IGA score ( As shown in Figure 2C), in addition, the results of the present invention show that with the increase of SERPINB4 mRNA expression level, the IGA score of skin lesions is significantly up-regulated, and the results show that SERPINB4 mRNA expression level is positively correlated with the severity of skin lesions (as shown in Figure 2D),
同时本发明收集玫瑰痤疮患者面部皮损皮肤组织，进行免疫组织化学染色，结果显示与正常健康对照者比较，玫瑰痤疮患者皮损表皮角质形成细胞全层均可检测到高丰度SERPINB3/B4表达，且其主要表达于细胞浆，而健康对照者皮肤全层几乎无表达(如图2E-F)，综上结果表明玫瑰痤疮患者皮损中SERPINB3/B4较健康对照明显高表达，At the same time, the present invention collects facial skin lesions of patients with rosacea and performs immunohistochemical staining. The results show that compared with normal healthy controls, the full layer of epidermal keratinocytes in patients with rosacea can detect high-abundance SERPINB3/B4 expression , and it is mainly expressed in the cytoplasm, while healthy controls have almost no expression in the whole skin layer (as shown in Figure 2E-F). The above results show that the expression of SERPINB3/B4 in skin lesions of patients with rosacea is significantly higher than that of healthy controls.
为进一步明确SERPINB3/B4在银屑病皮损中表达情况，相似的，本发明首先收取健康对照和银屑病患者的皮损，提取皮损总量RNA，通过qRT-PCR检 测皮肤中SERPINB3/B4 mRNA水平在两组之间的差异，如图2G-H可见相较健康对照者皮肤银屑病患者皮损的SERPINB3/B4在mRNA水平表达明显升高，同时对皮损进行SERPINB3/B4免疫组化染色，结果如图2I-J可见银屑病皮损较正常皮肤的表皮层明显增厚，且表达更高丰度的SERPINB3/B4，上述结果均表明，SERPINB3/B4在银屑病患者皮损中被激活，In order to further clarify the expression of SERPINB3/B4 in psoriatic skin lesions, similarly, the present invention first collects the skin lesions of healthy controls and psoriasis patients, extracts the total RNA of skin lesions, and detects SERPINB3/B4 in the skin by qRT-PCR. Differences in B4 mRNA levels between the two groups, as shown in Figure 2G-H, compared with healthy controls, the expression of SERPINB3/B4 in the skin lesions of psoriasis patients was significantly increased, and the SERPINB3/B4 immunization was performed on the skin lesions at the same time Histochemical staining, the results shown in Figure 2I-J show that the epidermis of psoriatic lesions is significantly thicker than that of normal skin, and expresses a higher abundance of SERPINB3/B4. activated in skin lesions,
为进一步研究SERPINB3/B4在玫瑰痤疮发病过程中发挥的作用，本发明根据先前研究所用的造模方式，建立了抗菌肽LL37诱导的玫瑰痤疮样小鼠模型，该模型皮损表现与炎症反应模式与人类玫瑰痤疮高度相似，本发明选取7-8周BALB/c雌性小鼠，剔除小鼠背部毛发，1天后每只小鼠背部皮内注射50μl(320μM)剂量LL37，每间隔12小时注射一次，对照组小鼠给与等剂量无菌1×PBS溶液，连续注射2日，共4次，模型建立成功后观察皮肤大体表型并进行取材，其次本发明收取小鼠皮损组织并提取总量RNA，检测玫瑰痤疮样小鼠皮损中Serpinb3a的mRNA表达情况，结果可见玫瑰痤疮样小鼠皮损较实验对照组Serpinb3a在mRNA水平表达明显上调(如图2K)，差异具有统计学意义,其中Serpinb3a为SERPINB3/B4的类似物，本发明进一步通过免疫荧光检测了玫瑰痤疮样小鼠模型皮损中Serpinb3a蛋白水平表达情况，结果显示玫瑰痤疮样小鼠模型皮损中Serpinb3a表达与人类玫瑰痤疮皮损SERPINB3/B4表达类似，玫瑰痤疮样小鼠模型皮损中毛囊周围和表皮全层Serpinb3a表达明显增强，为细胞浆表达(如图2L)，综上结果表明玫瑰痤疮样小鼠皮损Serpinb3a表达水平明显上调，In order to further study the role of SERPINB3/B4 in the pathogenesis of rosacea, the present invention established a rosacea-like mouse model induced by the antimicrobial peptide LL37 according to the modeling method used in previous studies. Highly similar to human rosacea, the present invention selects 7-8 week old BALB/c female mice, removes the hair on the back of the mice, and injects 50 μl (320 μM) dose of LL37 intradermally on the back of each mouse one day later, and injects once every 12 hours , mice in the control group were given equal doses of sterile 1×PBS solution, and injected continuously for 2 days, a total of 4 times. Quantitative RNA was used to detect the mRNA expression of Serpinb3a in rosacea-like mouse skin lesions. The results showed that the expression of Serpinb3a in rosacea-like mouse skin lesions was significantly up-regulated compared with the experimental control group (as shown in Figure 2K), and the difference was statistically significant. Wherein Serpinb3a is an analogue of SERPINB3/B4, the present invention further detects the Serpinb3a protein level expression in the rosacea-like mouse model skin lesions by immunofluorescence, and the results show that Serpinb3a expression in the rosacea-like mouse model skin lesions is closely related to that of human rosacea. The expression of SERPINB3/B4 in skin lesions was similar, and the expression of Serpinb3a around the hair follicles and in the whole epidermis of the rosacea-like mouse model was significantly enhanced, and it was expressed in the cytoplasm (as shown in Figure 2L). The expression level was significantly increased,
同样，本发明根据先前研究所用的造模方式，即连续外用咪喹莫特6天，建造银屑病样小鼠模型，收取小鼠皮损进行取材，收取小鼠皮损组织提取总量RNA，通过qRT-PCR检测银屑病样小鼠皮损中Serpinb3a的mRNA表达情况，结果可见银屑病样小鼠皮损较实验对照组Serpinb3a在mRNA水平表达明显上调(如图2M)，进一步收取小鼠皮损，进行免疫荧光染色，可见银屑病样小 鼠皮损中明显高表达Serpinb3a(如图2N)，Similarly, the present invention is based on the modeling method used in the previous research, that is, continuous external application of imiquimod for 6 days to build a psoriasis-like mouse model, collect mouse skin lesions for sampling, and collect mouse skin lesions to extract total RNA , the mRNA expression of Serpinb3a in the skin lesions of psoriasis-like mice was detected by qRT-PCR, and the results showed that the expression of Serpinb3a in the skin lesions of psoriasis-like mice was significantly up-regulated compared with the experimental control group (as shown in Figure 2M). Mouse skin lesions were immunofluorescently stained, and Serpinb3a was obviously highly expressed in psoriasis-like mouse skin lesions (as shown in Figure 2N).
为进一步探讨表皮中SERPINB3/B4在玫瑰痤疮及银屑病发病过程中的重要机制，本发明通过siRNA构建特异性敲降小鼠表皮Serpinb3a的小鼠模型，并对其进行LL37皮内注射及咪喹莫特外用，建立玫瑰痤疮样及银屑病样小鼠模型，观察表皮敲降Serpinb3a后对玫瑰痤疮样小鼠及银屑病样小鼠表型的影响。In order to further explore the important mechanism of SERPINB3/B4 in the epidermis in the pathogenesis of rosacea and psoriasis, the present invention constructs a mouse model of specifically knocking down mouse epidermis Serpinb3a by siRNA, and injects LL37 intradermally and mites Topical administration of quimod was used to establish rosacea-like and psoriasis-like mouse models, and the effects of epidermal knockdown of Serpinb3a on the phenotypes of rosacea-like mice and psoriasis-like mice were observed.
优选的，本发明将阴性对照序列及2条siRNA序列分别命名为Scr RNA、siRNA#1和siRNA#2进行接下来的研究，首先探讨表皮内敲降Serpinb3a对玫瑰痤疮样小鼠表型的影响，如图3A模式图所示，7-8周龄BALB/c野生型雌性小鼠，将其背部毛发剃去，前1天进行siRNA皮内注射，实验对照组皮内注射对应的阴性对照序列，随后按照前述方法进行表皮内LL37注射诱导玫瑰痤疮样小鼠模型，在注射第2次LL37前进行第2次siRNA注射，加强敲降效果，造模完成后，观察并比较三组小鼠(Scr siRNA，siRNA#1和siRNA#2)表型之间的差异并进行取材，图2B为LL37造模两日后Scr siRNA和另外2条siRNA的Control组和LL37组玫瑰痤疮样皮炎的表现，可见LL37造模后Scr siRNA组小鼠皮损部位出现明显的炎症反应，包括红斑和毛细血管扩张，而在2条siRNA敲降组小鼠皮损的炎症反应明显减轻，本发明利用Image J软件对每组玫瑰痤疮样小鼠皮损处的面积大小进行测量，结果显示Scr siRNA组LL37造模后红斑面积明显增加，2条siRNA的LL37组红斑面积较Scr的LL37组明显减小(如图3C)，其次对玫瑰痤疮样皮损红斑严重程度进行评分，结果显示Scr siRNA的LL37组造模后红斑评分明显增加，造模后2条siRNA的LL37组红斑评分明显减轻(如图3D)，Preferably, the present invention names the negative control sequence and the two siRNA sequences as ScrRNA, siRNA#1 and siRNA#2 respectively for the next research, and first explores the effect of knocking down Serpinb3a in the epidermis on the phenotype of rosacea-like mice , as shown in Figure 3A, 7-8 weeks old BALB/c wild-type female mice had their back hair shaved, and siRNA was injected intradermally one day before, and the experimental control group was intradermally injected with the corresponding negative control sequence , followed by intraepidermal injection of LL37 to induce a rosacea-like mouse model according to the aforementioned method, and the second siRNA injection was performed before the second injection of LL37 to strengthen the knockdown effect. After the modeling was completed, three groups of mice were observed and compared ( Scr siRNA, siRNA#1 and siRNA#2) phenotype differences and collected materials, Figure 2B shows the performance of rosacea-like dermatitis in the Control group and LL37 group of Scr siRNA and other 2 siRNAs two days after LL37 modeling, it can be seen After LL37 modeling, the mouse skin lesions in the Scr siRNA group showed obvious inflammatory reactions, including erythema and telangiectasia, while the inflammatory reactions in the mouse skin lesions in the two siRNA knockdown groups were significantly reduced. The present invention uses Image J software to analyze The area of skin lesions in each group of rosacea-like mice was measured, and the results showed that the area of erythema in the Scr siRNA group LL37 was significantly increased after modeling, and the area of erythema in the LL37 group with two siRNAs was significantly smaller than that in the Scr LL37 group (as shown in Figure 3C ), followed by scoring the erythema severity of rosacea-like skin lesions, the results showed that the erythema score of the Scr siRNA LL37 group increased significantly after modeling, and the erythema score of the 2 siRNA LL37 group was significantly reduced after modeling (as shown in Figure 3D).
此外本发明比较了LL37造模后各组小鼠皮损部位的组织学情况，取造模部位皮损，石蜡包埋切片后进行HE染色，显微镜下观察并统计小鼠真皮层炎症细胞浸润数量，如图3E可见，Scr siRNA的LL37组真皮部位浸润的炎症细 胞数量较Control组明显增加，2条siRNA的LL37组造模后真皮层浸润的炎症细胞数量较Scr siRNA的LL37组明显减少，同时本发明通过Prism9软件对浸润的炎症细胞数量进行统计分析，结果可见敲降Serpinb3a后2条siRNA的LL37组浸润的炎症细胞较Scr的LL37组明显减少(如图3F)，差异具有统计学意义，最后本发明收取小鼠皮损，提取总量RNA，通过qRT-PCR比较了上述各组小鼠皮损中玫瑰痤疮相关基因在mRNA表达水平上的差异，如图3G-H所示，Scr siRNA的LL37组较Control组的Il6和Tnf-α基因表达增加，而在敲降Serpinb3a后，2条siRNA的LL37组较Scr siRNA的LL37组上述两个基因表达水平明显回降，且差异具有统计学意义，以上结果表明，表皮内Serpinb3a的缺失从皮损炎症反应表现、组织病理学情况以及mRNA表达层面均能够明显改善LL37诱导的玫瑰痤疮样小鼠模型中玫瑰痤疮的发展。In addition, the present invention compares the histological situation of the mouse skin lesions in each group after LL37 modeling. The skin lesions of the modeling parts are taken, paraffin-embedded and sectioned, and then HE stained. The number of inflammatory cell infiltration in the dermis of the mice is observed under a microscope and counted. , as shown in Figure 3E, the number of inflammatory cells infiltrating the dermis in the Scr siRNA LL37 group was significantly higher than that in the Control group, and the number of inflammatory cells infiltrating the dermis in the 2 siRNA LL37 group was significantly lower than that in the Scr siRNA LL37 group. In the present invention, the number of infiltrating inflammatory cells was statistically analyzed by Prism9 software. The results showed that after knocking down Serpinb3a, the number of infiltrating inflammatory cells in the LL37 group with two siRNAs was significantly less than that in the Scr LL37 group (as shown in Figure 3F), and the difference was statistically significant. Finally, the present invention collects mouse skin lesions, extracts total RNA, and compares the differences in mRNA expression levels of rosacea-related genes in the skin lesions of the above-mentioned groups of mice by qRT-PCR, as shown in Figure 3G-H, Scr siRNA Compared with the Control group, the expressions of Il6 and Tnf-α genes in the LL37 group were increased, and after knocking down Serpinb3a, the expression levels of the above two genes in the LL37 group with two siRNAs were significantly lower than those in the LL37 group with Scr siRNA, and the difference was statistically significant. Significance, the above results show that the deletion of Serpinb3a in the epidermis can significantly improve the development of rosacea in the LL37-induced rosacea-like mouse model from the levels of inflammatory response, histopathology and mRNA expression.
优选的，本发明为明确表皮内敲降Serpinb3a对银屑病样小鼠表型的影响，选择前述的两条siRNA，进行小鼠耳部皮内注射，表皮内敲降Serpinb3a，按照既往已有研究方法外用咪喹莫特进行银屑病样小鼠模型建造，如图4A模式图所示，外用咪喹莫特前1天(记作day-1)进行siRNA皮内注射，实验对照组皮内注射对应的阴性对照序列，其后每日一次咪喹莫特乳膏外用，第2天进行第2次siRNA注射，加强敲降效果，第6天造模完成后，为明确表皮内敲降Serpinb3a后对银屑病样小鼠皮损表型的影响，本发明造模第6天观察并比较三组小鼠(Scr siRNA，siRNA#1和siRNA#2)表型之间的差异并进行取材，根据既往研究[77]方法对小鼠皮损进行PASI评分及耳部厚度进行测量，可见siRNA的咪喹莫特组小鼠较Scr RNA咪喹莫特组小鼠皮损的红斑、鳞屑明显改善(如图4B)，PASI评分明显下降(如图4C)，耳部皮肤厚度亦明显下降(如图4D)，其次对各组小鼠皮损进行HE染色，结果可见，Scr siRNA咪喹莫特组表皮厚度明显增加，真皮层炎症细胞浸润明显，而两条siRNA的咪喹模特组表皮厚度及真皮层炎症细胞浸润程度明显改善(如图4E)，并对 表皮层厚度、浸润的炎症细胞进行统计分析，发现二者在两条siRNA的咪喹莫特组较Scr siRNA的咪喹莫特组明显下降(如图4F-G)，为进一步明确Serpinb3a对银屑病相关基因分子水平的影响，本发明收取小鼠皮损，提取总量RNA，通过qRT-PCR比较了上述各组小鼠皮损中银屑病相关基因在mRNA表达水平上的差异，如图4H-I所示，Scr siRNA的咪喹莫特组较Control组Il6和Il-1β基因表达增加2条siRNA的咪喹莫特组中上述两个基因表达水平较Scr siRNA的咪喹莫特组有明显回降，差异具有统计学意义，以上结果表明，耳部表皮内Serpinb3a的缺失从皮损炎症反应表现、组织病理学情况以及mRNA表达层面均能够明显改善咪喹莫特诱导的银屑病样小鼠模型中炎症疾病的发展，Preferably, in order to clarify the effect of knocking down Serpinb3a in the epidermis on the phenotype of psoriasis-like mice, the present invention selects the aforementioned two siRNAs, injects them into the ears of mice, and knocks down Serpinb3a in the epidermis according to the existing methods. Research methods The psoriasis-like mouse model was constructed with topical imiquimod, as shown in Figure 4A schematic diagram, siRNA was injected intradermally 1 day before topical imiquimod (denoted as day-1), and the experimental control group was injected with siRNA. The corresponding negative control sequence was injected internally, followed by external application of imiquimod cream once a day, and the second siRNA injection was performed on the second day to strengthen the knockdown effect. The effect of Serpinb3a on the phenotype of psoriasis-like mice skin lesions, the present invention observed and compared the differences between the phenotypes of the three groups of mice (Scr siRNA, siRNA#1 and siRNA#2) on the 6th day of modeling and carried out According to the method of previous research [77], the PASI score and ear thickness of the skin lesions of the mice were measured. It can be seen that the erythema and scales of the mice in the siRNA imiquimod group were compared with those in the ScrRNA imiquimod group. Significantly improved (as shown in Figure 4B), the PASI score decreased significantly (as shown in Figure 4C), and the thickness of the ear skin also decreased significantly (as shown in Figure 4D). Secondly, HE staining was performed on the skin lesions of mice in each group. The thickness of the epidermis in the Mott group was significantly increased, and the infiltration of inflammatory cells in the dermis was obvious, while the thickness of the epidermis and the infiltration of inflammatory cells in the dermis were significantly improved in the imoquine model group with two siRNAs (as shown in Figure 4E). The cells were statistically analyzed, and it was found that the two siRNA imiquimod groups were significantly lower than the Scr siRNA imiquimod group (as shown in Figure 4F-G). Influence, the present invention collects mouse skin lesions, extracts total RNA, compares the difference on the mRNA expression level of psoriasis-related genes in the mouse skin lesions of the above-mentioned groups by qRT-PCR, as shown in Figure 4H-I, The Il6 and Il-1β gene expressions of the Scr siRNA imiquimod group increased significantly compared with the Control group. With statistical significance, the above results show that the deletion of Serpinb3a in the ear epidermis can significantly improve the inflammation in the imiquimod-induced psoriasis-like mouse model from the aspects of skin lesion inflammatory response, histopathology and mRNA expression disease development,
综上所述，在小鼠表皮内敲降serpinb3a后，能够明显改善玫瑰痤疮样及银屑病样小鼠皮损处炎症反应。In summary, knocking down serpinb3a in the epidermis of mice can significantly improve the inflammatory response in skin lesions of rosacea-like and psoriasis-like mice.
优选的，为了更进一步明确SERPINB3/B4在体外对玫瑰痤疮中具体分子作用机制，本发明体外构建SERPINB3/B4过表达质粒，并通过转染试剂将过表达质粒转染人HaCaT细胞，72小时后收取细胞RNA，进行全转录组测序，并通过体内外实验对测序结果进行验证，Preferably, in order to further clarify the specific molecular mechanism of SERPINB3/B4 on rosacea in vitro, the present invention constructs a SERPINB3/B4 overexpression plasmid in vitro, and transfects the overexpression plasmid into human HaCaT cells with a transfection reagent. After 72 hours, Collect cellular RNA, perform whole transcriptome sequencing, and verify the sequencing results through in vivo and in vitro experiments,
本发明对过表达SERPINB3/B4的人HaCaT细胞中的差异基因表达值进行聚类分析，进而对基因表达值进行均一化处理从而得到如图5A，如图中的每一个小方格代表一个基因，蓝色表示基因表达下调，红色表示基因表达上调，颜色越深，表达的差异越明显，每一列表示每个样本不同基因的表达情况，每一行表示每个基因在不同样本中的表达情况，如图结果可见过表达SERPINB3/B4的人HaCaT细胞较正常对照之间基因表达差异显著，结果表明，在人HaCaT细胞中过表达SERPINB3/B4后基因表达较正常对照组模式发生明显变化，The present invention performs clustering analysis on the differential gene expression values in human HaCaT cells overexpressing SERPINB3/B4, and then normalizes the gene expression values to obtain as shown in Figure 5A, where each small square in the figure represents a gene , blue indicates down-regulation of gene expression, red indicates up-regulation of gene expression, the darker the color, the more obvious the difference in expression, each column indicates the expression of different genes in each sample, and each row indicates the expression of each gene in different samples, As shown in the figure, it can be seen that the gene expression of human HaCaT cells overexpressing SERPINB3/B4 is significantly different from that of the normal control.
为进一步明确过表达SERPINB3/B4的人HaCaT细胞中信号通路的变化， 本发明对过表达SERPINB3/B4的人HaCaT细胞和对照组细胞所有的基因表达量进行基因富集分析(Gene Set Enrichment Analysis，GSEA)，基于玫瑰痤疮为皮肤炎症性疾病及先前研究，炎症相关信号通路在玫瑰痤疮发生发展中发挥关键作用，所以本次研究着重关注与炎症相关联的信号通路，NF-κB信号通路几乎存在于所用的动物细胞中，参与细胞对外界刺激的各种反应，包括应激、细胞因子、自由基、微生物感染和紫外线照射等，既往研究发现此信号通路调控大量与炎症相关的基因表达，在本次研究中富集的炎症相关信号通路中NF-κB信号通路在过表达SERPINB3/B4的人HaCaT细胞中较对照组细胞系明显富集(如图5B-E)，这提示该信号通路可能参与了SERPINB3/B4调控玫瑰痤疮的发病，In order to further clarify the changes in the signaling pathways in human HaCaT cells overexpressing SERPINB3/B4, the present invention conducts gene enrichment analysis (Gene Set Enrichment Analysis, Gene Set Enrichment Analysis, GSEA), based on rosacea is a skin inflammatory disease and previous research, inflammation-related signaling pathways play a key role in the development of rosacea, so this study focuses on signaling pathways associated with inflammation, NF-κB signaling pathway almost exists In the animal cells used, it is involved in various responses of cells to external stimuli, including stress, cytokines, free radicals, microbial infection and ultraviolet radiation. Previous studies have found that this signaling pathway regulates the expression of a large number of genes related to inflammation. Among the inflammation-related signaling pathways enriched in this study, the NF-κB signaling pathway was significantly enriched in the human HaCaT cells overexpressing SERPINB3/B4 compared with the control cell line (as shown in Figure 5B-E), which suggests that the signaling pathway may Involved in SERPINB3/B4 regulating the pathogenesis of rosacea,
为明确在人HaCaT细胞中过表达SERPINB3/B4后在蛋白水平对NF-κB信号通路的影响，本发明通过转染试剂将过表达质粒转染人HaCaT细胞系72小时后收取细胞蛋白，免疫印迹法检测在人HaCaT细胞系中表达SERPINB3/B4后对p65/NF-κB的磷酸化水平(p-p65)，同时应用Image J软件对p-p65灰度进行扫描量化，如图5F所示，本发明发现在人HaCaT细胞系中表达SERPINB3/B4后p-p65表达明显上调，p65表达无明显差异，p-p65/p65上调，结果表明NF-κB信号通路激活，同时，为了更加直观地了解表达SERPINB3/B4后对NF-κB信号通路的影响，本发明通过细胞爬片实验及免疫荧光检测过表达质粒转染人HaCaT细胞系72小时后对p65表达的影响，结果如图5G所示，在人HaCaT细胞中表达SERPINB3/B4后p65入核的细胞数目较对照组明显增多上述结果表明在在人HaCaT细胞中表达SERPINB3/B4在蛋白水平激活NF-κB信号通路，In order to clarify the effect of overexpressing SERPINB3/B4 on the NF-κB signaling pathway at the protein level in human HaCaT cells, the present invention transfected the overexpression plasmid into the human HaCaT cell line with a transfection reagent for 72 hours, and harvested the cell protein, immunoblotting The phosphorylation level of p65/NF-κB (p-p65) after SERPINB3/B4 expression in human HaCaT cell line was detected by using the method, and the gray scale of p-p65 was scanned and quantified by Image J software, as shown in Figure 5F, The present invention found that after expressing SERPINB3/B4 in the human HaCaT cell line, the expression of p-p65 was significantly up-regulated, the expression of p65 was not significantly different, and p-p65/p65 was up-regulated. The results indicated that the NF-κB signaling pathway was activated. At the same time, in order to understand The effect of expressing SERPINB3/B4 on the NF-κB signaling pathway, the present invention detected the effect on the expression of p65 after 72 hours of transfection of the overexpression plasmid into the human HaCaT cell line by cell climbing experiments and immunofluorescence, the results are shown in Figure 5G, After expressing SERPINB3/B4 in human HaCaT cells, the number of p65 nuclear cells was significantly increased compared with the control group. The above results indicate that the expression of SERPINB3/B4 in human HaCaT cells activates the NF-κB signaling pathway at the protein level,
既往研究发现，TNF-α可强烈诱导NF-κB信号的活化，为明确SERPINB3/B4对TNF-α诱导NF-κB信号活化的影响，本发明体外构建SERPINB3/B4的shRNA，建立稳定敲降SERPINB3/B4的人HaCaT细胞系，TNF- α孵育30分钟后，免疫印迹法检测敲降SERPINB3/B4后对TNF-α诱导NF-κB信号活化的影响，结果可见，构建稳定敲降SERPINB3/B4的人HaCaT细胞系对SERPINB3/B4的敲降效果明显，在正常对照组细胞中，TNF-α孵育后p-p65表达明显上调，而敲降SERPINB3/B4后，TNF-α诱导p-p65上调的情况明显改善，同时本发明对其条带灰度进行扫描，结果统计发现差异具有明显统计学意义(如图5H-I)，结果提示，人HaCaT细胞系中SERPINB3/B4的缺失阻止了TNF-α诱导的NF-κB信号的活化，同样，本发明通过细胞爬片实验及免疫荧光检测稳定敲降SERPINB3/B4对TNF-α诱导的p65入核情况的影响，如图5J-K可见在正常对照的人HaCaT细胞系中，TNF-α孵育后30分钟后p65几乎完全入核，而敲降SERPINB3/B4后p65入核的细胞数目较对照组明显减少，同时本发明对入核细胞数目进行统计，差异明显，具有统计学意义，Previous studies have found that TNF-α can strongly induce the activation of NF-κB signaling. In order to clarify the effect of SERPINB3/B4 on the activation of NF-κB signaling induced by TNF-α, the present invention constructed SERPINB3/B4 shRNA in vitro to establish a stable knockdown of SERPINB3 /B4 human HaCaT cell line, after incubation with TNF-α for 30 minutes, the effect of knocking down SERPINB3/B4 on the activation of NF-κB signal induced by TNF-α was detected by Western blotting. The human HaCaT cell line has a significant knockdown effect on SERPINB3/B4. In the normal control cells, the expression of p-p65 was significantly upregulated after TNF-α incubation, and after knockdown of SERPINB3/B4, TNF-α induced the upregulation of p-p65. The situation has been significantly improved. At the same time, the present invention scans the gray scale of its bands, and the statistical results show that the difference has obvious statistical significance (as shown in Figure 5H-I). The results suggest that the deletion of SERPINB3/B4 in the human HaCaT cell line prevents TNF- The activation of α-induced NF-κB signal, similarly, the present invention detects the effect of stably knocking down SERPINB3/B4 on TNF-α-induced p65 nuclear entry through cell slide experiments and immunofluorescence, as shown in Figure 5J-K. In the control human HaCaT cell line, p65 was almost completely incorporated into the nucleus after 30 minutes of TNF-α incubation, and the number of cells with p65 incorporated into the nucleus after knocking down SERPINB3/B4 was significantly reduced compared with the control group. Statistically, the difference is obvious and statistically significant,
综上结果表明，体外过表达SERPINB3/B4后诱导NF-κB信号通路下游分子上调，体外敲降SERPINB3/B4后能够阻止TNF-α诱导的NF-κB信号的活化。In conclusion, overexpression of SERPINB3/B4 in vitro induces the upregulation of downstream molecules of NF-κB signaling pathway, and knockdown of SERPINB3/B4 in vitro can prevent the activation of TNF-α-induced NF-κB signaling.
优选的，炎症相关的趋化因子及细胞因子在炎症发生及进展中起到极其重要的作用，前期一系列的结果表明SERPINB3/B4能够调控NF-κB信号通路，为进一步明确趋化因子及细胞因子在SERPINB3/B4调控玫瑰痤疮发病中的具体作用，本发明进一步对在人HaCaT细胞中过表达SERPINB3/B4后RNA-seq的结果进行分析，如图6A-B所示，趋化因子及细胞因子信号通路在SERPINB3/B4过表达的人HaCaT细胞中高度富集，同时本发明对样本的趋化因子及细胞因子相关的差异的表达值进行聚类分析，并对基因表达值进行均一化处理得到如图6C所示的如图，与正常对照相比，SERPINB3/B4过表达的人HaCaT细胞中炎症相关的趋化因子(CCL2，CCL5，CCL20，CXCL1，CXCL2，CXCL5，CXCL8，CXCL10，CXCL11)及细胞因子(IL1A，IL1B，IL6，TNFA)表达明显上调，Preferably, inflammation-related chemokines and cytokines play an extremely important role in the occurrence and progression of inflammation. A series of previous results show that SERPINB3/B4 can regulate the NF-κB signaling pathway, in order to further clarify the chemokines and cellular The specific role of factors in regulating the pathogenesis of rosacea by SERPINB3/B4, the present invention further analyzes the results of RNA-seq after overexpressing SERPINB3/B4 in human HaCaT cells, as shown in Figure 6A-B, chemokines and cells Factor signaling pathways are highly enriched in human HaCaT cells with overexpression of SERPINB3/B4. At the same time, the present invention performs cluster analysis on the expression values of chemokine and cytokine-related differences in the samples, and normalizes the gene expression values. As shown in Figure 6C, compared with the normal control, inflammation-related chemokines (CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL5, CXCL8, CXCL10, CXCL11) in SERPINB3/B4 overexpressed human HaCaT cells ) and cytokines (IL1A, IL1B, IL6, TNFA) were significantly up-regulated,
为进一步明确上述细胞因子在SERPINB3/B4过表达的人HaCaT细胞中的 表达水平，本发明利用qRT-PCR的方法检测人HaCaT细胞过表达SERPINB3/B4后上述基因的表达情况，结果(如图6D)可见，在人HaCaT细胞中过表达SERPINB3/B4后，趋化因子CCL2，CCL5，CCL20，CXCL2，CXCL9，CXCL10，CXCL11及细胞因子IL1B，IL6，TNF-α表达明显上调，结果表明在人HaCaT细胞过表达SERPINB3/B4后能够明显诱导炎症相关的趋化因子及细胞因子的表达上调，In order to further clarify the expression levels of the above-mentioned cytokines in human HaCaT cells overexpressing SERPINB3/B4, the present invention uses qRT-PCR to detect the expression of the above-mentioned genes in human HaCaT cells after overexpressing SERPINB3/B4, and the results (as shown in Figure 6D ) shows that after overexpressing SERPINB3/B4 in human HaCaT cells, the expression of chemokines CCL2, CCL5, CCL20, CXCL2, CXCL9, CXCL10, CXCL11 and cytokines IL1B, IL6, TNF-α was significantly up-regulated, the results showed that in human HaCaT cells Cells overexpressing SERPINB3/B4 can significantly induce the upregulation of inflammation-related chemokines and cytokines,
通过小鼠皮内敲降Serpinb3a能够降低LL37诱导的玫瑰痤疮相关细胞因子(Il6，Tnf-α)的表达水平，为进一步明确玫瑰痤疮样小鼠表皮内敲降Serpinb3a对炎症相关的趋化因子及细胞因子的影响，本发明应用qRT-PCR的方法检测了上述相关因子的表达水平，结果如图6E可见，敲降Serpinb3a后，LL37诱导的Ccl5，Cxcl9，Cxcl10，Cxcl11及Il1β表达水平受到明显抑制，本发明应用同样的方法检测了敲降serpinb3a后银屑病样小鼠皮损中上述趋化因子变化情况，结果如图6F所示，可见敲降Serpinb3a后，IMQ诱导的Ccl5，Ccl20，Cxcl9，Cxcl10，Cxcl11表达水平受到明显抑制，Intradermal knockdown of Serpinb3a in mice can reduce the expression level of rosacea-related cytokines (Il6, Tnf-α) induced by LL37. The influence of cytokines, the present invention used qRT-PCR to detect the expression levels of the above-mentioned related factors, the results can be seen in Figure 6E, after knocking down Serpinb3a, the expression levels of Ccl5, Cxcl9, Cxcl10, Cxcl11 and Il1β induced by LL37 were significantly inhibited , the present invention used the same method to detect the changes of the above chemokines in the skin lesions of psoriasis-like mice after knocking down serpinb3a, and the results are shown in Figure 6F. , Cxcl10, Cxcl11 expression levels were significantly inhibited,
同样的方法检测相关趋化因子及细胞因子的表达水平，结果5G如图所示，SERPINB3/B4诱导趋化因子CCL2，CCL5，CXCL9，CXCL10，CXCL11及细胞因子IL-1β，IL-6，TNF-α明显上调，而加用SC75741共同孵育后上述趋化因子及细胞因子的表达水平明显回降，上述结果表明SC75741能够通过抑制SERPINB3/B4对NF-κB信号通路的诱导激活下调相关趋化因子及细胞因子的表达水平。The same method was used to detect the expression levels of related chemokines and cytokines. As shown in the figure, SERPINB3/B4 induced chemokines CCL2, CCL5, CXCL9, CXCL10, CXCL11 and cytokines IL-1β, IL-6, TNF -α was significantly up-regulated, and the expression levels of the above chemokines and cytokines were significantly decreased after co-incubation with SC75741. The above results indicated that SC75741 could down-regulate related chemokines by inhibiting the activation of SERPINB3/B4 on the NF-κB signaling pathway and expression levels of cytokines.
与现有技术相比，本发明的有益效果是：Compared with prior art, the beneficial effect of the present invention is:
1、本发明首次报道了SERPINB3/B4在玫瑰痤疮和银屑病患者皮损中的表达情况，及其与疾病严重程度的相关性；本发明以玫瑰痤疮和银屑病两种慢性炎症性皮肤疾病为研究对象阐明了SERPINB3/B4通过NF-κB信号通路促进炎症反应的发生，本发明发现应用SC75741(NF-κB信号通路强效抑制剂)能够明显改善LL37诱导的玫瑰痤疮样小鼠皮损处炎症反应，同时明显抑制 SERPINB3/B4诱导的NF-κB信号通路的激活。因此本发明为玫瑰痤疮和银屑病的发病提供了新的解释，且为两种慢性炎症性皮肤疾病的治疗提供了新的思路。1. The present invention reports for the first time the expression of SERPINB3/B4 in skin lesions of patients with rosacea and psoriasis, and its correlation with disease severity; The research object clarifies that SERPINB3/B4 promotes the occurrence of inflammatory response through the NF-κB signaling pathway. The present invention found that the application of SC75741 (a powerful inhibitor of NF-κB signaling pathway) can significantly improve the rosacea-like mouse skin lesions induced by LL37 At the same time, it significantly inhibited the activation of NF-κB signaling pathway induced by SERPINB3/B4. Therefore, the present invention provides a new explanation for the pathogenesis of rosacea and psoriasis, and provides a new idea for the treatment of the two chronic inflammatory skin diseases.
附图说明Description of drawings
图1为本发明的工作流程图；Fig. 1 is a work flow chart of the present invention;
图2为为本发明的A健康对照和玫瑰痤疮患者皮损中SERPINB3mRNA表达水平；Fig. 2 is for the SERPINB3mRNA expression level in A healthy control of the present invention and rosacea patient's skin lesion;
B玫瑰痤疮患者IGA评分与皮损中SERPINB3mRNA表达水平的相关性分析；B Correlation analysis between IGA score and SERPINB3 mRNA expression level in skin lesions in patients with rosacea;
C健康对照和玫瑰痤疮患者皮损中SERPINB4mRNA表达水平；C The expression level of SERPINB4mRNA in skin lesions of healthy controls and patients with rosacea;
D玫瑰痤疮患者IGA评分与皮损中SERPINB4mRNA表达水平的相关性分析；D Correlation analysis between IGA score and SERPINB4 mRNA expression level in skin lesions in patients with rosacea;
E-F免疫组化染色(IHC)检测玫瑰痤疮患者皮损皮肤相对健康对照者正常皮肤切片中SERPINB3/B4的表达情况；Epi表示表皮；Der表示真皮；E-F Immunohistochemical staining (IHC) to detect the expression of SERPINB3/B4 in the lesional skin of rosacea patients compared with the normal skin sections of healthy controls; Epi means epidermis; Der means dermis;
G-H健康对照者皮肤和银屑病患者皮肤中SERPINB3，SERPINB4的mRNA表达水平；HS为健康对照组，Psoriasis为银屑病组；G-H The mRNA expression levels of SERPINB3 and SERPINB4 in the skin of healthy controls and psoriasis patients; HS is the healthy control group, and Psoriasis is the psoriasis group;
I-J免疫组化检测银屑病患者皮损和健康对照者正常皮肤中SERPINB3的表达情况；D免疫组化检测银屑病患者皮损和健康对照者正常皮肤中SERPINB4的表达情况；I-J Immunohistochemical detection of the expression of SERPINB3 in the skin lesions of psoriasis patients and normal skin of healthy controls; D Immunohistochemical detection of the expression of SERPINB4 in the skin lesions of psoriasis patients and normal skin of healthy controls;
K对照组和LL37组小鼠皮损中Serpinb3a的mRNA表达结果；The mRNA expression results of Serpinb3a in the mouse skin lesions of K control group and LL37 group;
L对照组小鼠和LL37组小鼠皮损中Serpinb3a的免疫荧光染色结果，Serpinb3a(红色)定位于胞浆，DAPI染色(蓝色)为细胞核定位；Immunofluorescent staining results of Serpinb3a in the skin lesions of mice in L control group and LL37 group, Serpinb3a (red) is localized in the cytoplasm, and DAPI staining (blue) is localized in the nucleus;
M对照组和IMQ组小鼠皮损中Serpinb3a的mRNA表达结果；The mRNA expression results of Serpinb3a in the mouse skin lesions of M control group and IMQ group;
N对照组小鼠和IMQ组小鼠皮损中Serpinb3a的免疫荧光染色结果，Serpinb3a(红色)定位于胞浆，DAPI染色(蓝色)为细胞核定位；标尺：50μm；Immunofluorescence staining results of Serpinb3a in skin lesions of mice in N control group and IMQ group, Serpinb3a (red) is localized in cytoplasm, DAPI staining (blue) is localized in nucleus; scale bar: 50 μm;
图3为本发明的A本次小鼠体内实验模式图；Fig. 3 is the model diagram of this mouse in vivo experiment of A of the present invention;
B上图为LL37诱导的玫瑰痤疮样小鼠模型皮损的代表性大体图；下图为体视镜放大结果图；B The upper picture is a representative general picture of the LL37-induced rosacea-like mouse model skin lesions; the lower picture is the result of stereoscopic magnification;
C各组小鼠红斑面积统计结果；Statistical results of erythema area of mice in each group of C;
D各组小鼠红斑严重程度评分后统计分析结果；Statistical analysis results after scoring the severity of erythema in each group of mice;
E为D图对应的皮损组织HE染色结果；E is the HE staining result of the skin lesion corresponding to the picture in D;
F每组小鼠皮损中浸润的炎症细胞进行统计分析结果；The results of statistical analysis of the inflammatory cells infiltrated in the skin lesions of each group of mice;
G-HqRT-PCR检测各组小鼠皮损中Il6，Tnf-α的mRNA表达；G-HqRT-PCR detected the mRNA expression of Il6 and Tnf-α in skin lesions of mice in each group;
图4：A本次小鼠体内实验模式图；Figure 4: A schematic diagram of the mouse in vivo experiment;
B各组小鼠模型皮损的体视镜放大结果图，IMQ为咪喹莫特组；Stereoscopic magnification results of mouse model skin lesions in each group B, IMQ is the imiquimod group;
C各组小鼠皮损处PASI评分；The PASI score of the mouse skin lesions in each group of C;
D各组小鼠耳朵厚度统计结果，长度单位：毫米；D statistical results of mouse ear thickness in each group, length unit: mm;
E为C图对应的皮损组织HE染色结果，标尺：50μm；E is the HE staining result of the skin lesion corresponding to Figure C, scale bar: 50 μm;
F各组小鼠皮损中浸润的炎症细胞统计分析的结果；The results of the statistical analysis of the inflammatory cells infiltrated in the skin lesions of each group of mice;
G显微镜下对各组小鼠HE染色结果进行表皮层厚度测量，并统计分析，长度单位：微米；Under the G microscope, the thickness of the epidermis was measured and statistically analyzed for the HE staining results of the mice in each group, and the length unit: micron;
H-I各组小鼠皮损中Il6，Il-1β的mRNA表达水平的qRT-PCR结果图；The qRT-PCR results of the mRNA expression levels of Il6 and Il-1β in the skin lesions of mice in each group of H-I;
图5为本发明的AHaCaT细胞过表达SERPINB3/B4较空白对照组细胞转录组数据差异基因的聚类分析，每组3次转染的细胞；其中，红色代表基因高表达，蓝色代表基因低表达；Fig. 5 is the cluster analysis of AHaCaT cells of the present invention overexpressing SERPINB3/B4 compared with blank control group cell transcriptome data difference genes, each group of cells transfected 3 times; wherein, red represents high gene expression, and blue represents low gene expression Express;
B-EGSEA分析结果显示：在人HaCaT细胞中过表SERPINB3/B4与VECTOR组比较，重叠的差异表达基因所富集的KEGG通路的排名，其中，NF-κB信号通路以红色框表示；The results of B-EGSEA analysis showed: comparing SERPINB3/B4 with the VECTOR group in human HaCaT cells, the ranking of KEGG pathways enriched by overlapping differentially expressed genes, among which, the NF-κB signaling pathway is represented by a red box;
F免疫印迹检测过人HaCaT细胞转染SERPINB3/B4过表达质粒72小时后p-p65蛋白水平变化情况；通过ImageJ软件对蛋白水平进行定量分析；F immunoblotting detected the change of p-p65 protein level after human HaCaT cells were transfected with SERPINB3/B4 overexpression plasmid for 72 hours; the protein level was quantitatively analyzed by ImageJ software;
G左图为免疫荧光检测人HaCaT细胞转染SERPINB3/B4过表达质粒72小 时后p65的表达和定位；箭头出表示p65入核，DAPI染色(蓝色)表示细胞核定位，标尺：50μm；右图：对p65入核的阳性细胞进行统计分析，差异具有统计学意义；G The left picture shows the expression and localization of p65 detected by immunofluorescence after human HaCaT cells were transfected with SERPINB3/B4 overexpression plasmid for 72 hours; the arrow out indicates that p65 enters the nucleus, and DAPI staining (blue) indicates the nuclear localization, scale bar: 50 μm; the right picture : Statistical analysis was performed on the positive cells of p65 into the nucleus, and the difference was statistically significant;
H-I左图为体外建立敲降SERPINB3/B4的人HaCaT细胞，100ng/mlTNF-α孵育细胞系30分钟，免疫印迹检测敲降组较对照组蛋白水平p-p65表达量的变化；右图为：通过ImageJ软件对p-p65灰度进行统计分析，差异具有统计学意义；The left picture of H-I shows the establishment of human HaCaT cells knocking down SERPINB3/B4 in vitro, and the cell line was incubated with 100ng/ml TNF-α for 30 minutes. The p-p65 gray level was statistically analyzed by ImageJ software, and the difference was statistically significant;
J-K体外建立敲降SERPINB3/B4的人HaCaT细胞，100ng/mlTNF-α孵育细胞系30分钟，免疫荧光检测敲降组较对照组蛋白水平p65的表达和定位，并对p65入核的阳性细胞数据进行统计分析，差异具有统计学意义；p65(红色)膜表达，激活时入核，为核表达，DAPI染色(蓝色)表示细胞核定位，标尺：50μm；J-K established human HaCaT cells knocking down SERPINB3/B4 in vitro, incubated the cell line with 100ng/ml TNF-α for 30 minutes, detected the protein level and localization of p65 in the knockdown group compared with the control group by immunofluorescence, and analyzed the positive cell data of p65 nuclear entry Statistical analysis shows that the difference is statistically significant; p65 (red) is expressed on the membrane, and enters the nucleus when activated, which is nuclear expression. DAPI staining (blue) indicates the location of the nucleus, and the scale bar is 50 μm;
图6为本发明的A-BGSEA分析结果显示：在人HaCaT细胞中过表达SERPINB3/B4与VECTOR空白对照组比较，趋化因子及细胞因子信号通路在SERPINB3/B4过表达的人HaCaT细胞中高度富集；Figure 6 shows the results of the A-BGSEA analysis of the present invention: in human HaCaT cells overexpressing SERPINB3/B4 compared with the VECTOR blank control group, chemokine and cytokine signaling pathways are highly expressed in SERPINB3/B4 overexpressing people's HaCaT cells enrichment;
CHaCaT细胞过表达SERPINB3/B4较空白对照组细胞转录组数据差异基因的聚类分析，每组3次转染的细胞；其中，红色代表基因高表达，蓝色代表基因低表达；Cluster analysis of differential gene transcriptome data of CHaCaT cells overexpressing SERPINB3/B4 compared with blank control cells, cells transfected 3 times in each group; where red represents high gene expression, blue represents low gene expression;
DqRT-PCR检测趋化因子CCL2，CCL5，CCL20，CXCL2，CXCL9，CXCL10，CXCL11及细胞因子IL1B，IL6，TNF-α表达水平；DqRT-PCR detection of chemokines CCL2, CCL5, CCL20, CXCL2, CXCL9, CXCL10, CXCL11 and cytokines IL1B, IL6, TNF-α expression levels;
E-FqRT-PCR检测敲降Serpinb3a后玫瑰痤疮样小鼠模型及银屑病样小鼠模型皮损中趋化因子变化；E-FqRT-PCR detection of chemokine changes in skin lesions of rosacea-like mouse model and psoriasis-like mouse model after knocking down Serpinb3a;
GqRT-PCR检测炎症因子mRNA水平。GqRT-PCR was used to detect the mRNA levels of inflammatory factors.
具体实施方式Detailed ways
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行 清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
在本发明的描述中，需要说明的是，术语“上”、“下”、“内”、“外”“前端”、“后端”、“两端”、“一端”、“另一端”等指示的方位或位置关系为基于附图所述的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制。此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性。In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "front end", "rear end", "both ends", "one end", "another end" The orientation or positional relationship indicated by etc. is based on the orientation or positional relationship described in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the referred device or element must have a specific orientation, use a specific Azimuth configuration and operation, therefore, should not be construed as limiting the invention. In addition, the terms "first" and "second" are used for descriptive purposes only, and should not be understood as indicating or implying relative importance.
在本发明的描述中，需要说明的是，除非另有明确的规定和限定，术语“安装”、“设置有”、“连接”等，应做广义理解，例如“连接”，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通。对于本领域的普通技术人员而言，可以具体情况理解上述术语在本发明中的具体含义。In the description of the present invention, it should be noted that, unless otherwise specified and limited, the terms "installed", "set with", "connected", etc. should be understood in a broad sense, such as "connected", which may be a fixed connection , can also be detachably connected, or integrally connected; can be mechanically connected, can also be electrically connected; can be directly connected, can also be indirectly connected through an intermediary, and can be internal communication between two components. Those of ordinary skill in the art can understand the specific meanings of the above terms in the present invention in specific situations.
请参阅图1-图6，本发明提供的一种实施例：SERPINB3/B4作为靶点在玫瑰痤疮等炎症性皮肤病治疗药物中的应用；Please refer to Figure 1-Figure 6, an embodiment provided by the present invention: the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as rosacea;
实施例一，SERPINB3/B4作为靶点在玫瑰痤疮等炎症性皮肤病治疗药物中的应用，首先通过ELISA、免疫组化、qRT-PCR、免疫荧光等手段明确SERPINB3/B4在玫瑰痤疮患者和银屑病患者血清、皮损中的表达及Serpinb3a在LL37诱导的玫瑰痤疮样小鼠模型及咪喹莫特诱导的银屑病样小鼠模型皮损中的表达情况；Example 1, the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as rosacea, first of all, by ELISA, immunohistochemistry, qRT-PCR, immunofluorescence, etc. The expression of serum and skin lesions of patients with psoriasis and the expression of Serpinb3a in the rosacea-like mouse model induced by LL37 and the psoriasis-like mouse model induced by imiquimod;
通过siRNA皮内注射，局部敲降小鼠表皮细胞内Serpinb3a的表达，观察其对炎症模型皮损表型及组织病理学的影响，体外研究主要在人HaCaT细胞中进行，通过构建质粒在人HaCaT细胞中过表达或敲降SERPINB3/B4，并联 合RNA-seq和免疫印迹、免疫荧光等方法明确其与NF-κB信号通路的关系；Through intradermal injection of siRNA, the expression of Serpinb3a in mouse epidermal cells was locally knocked down, and its effect on the phenotype and histopathology of inflammatory model skin lesions was observed. In vitro studies were mainly carried out in human HaCaT cells. Overexpress or knock down SERPINB3/B4 in cells, and combine RNA-seq, western blotting, immunofluorescence and other methods to clarify its relationship with NF-κB signaling pathway;
最后，选择SC75741(一种强效NF-κB信号通路抑制剂)抑制小鼠表皮内NF-κB信号通路明确NF-κB信号通路在玫瑰痤疮发病过程中的作用；且运用CCK8和流式分析选择SC75741的合适浓度进行体外研究，进一步明确SERPINB3/B4与NF-κB信号通路在玫瑰痤疮及银屑病发病机制中的相互作用。Finally, SC75741 (a potent NF-κB signaling pathway inhibitor) was selected to inhibit the NF-κB signaling pathway in the mouse epidermis to clarify the role of NF-κB signaling pathway in the pathogenesis of rosacea; and use CCK8 and flow cytometry analysis to select The appropriate concentration of SC75741 was studied in vitro to further clarify the interaction between SERPINB3/B4 and NF-κB signaling pathway in the pathogenesis of rosacea and psoriasis.
实施例2，本发明收集正常对照和玫瑰痤疮患者皮损，提取总RNA，检测SERPINB3/B4 mRNA水平在两组之间的表达情况，结果发现在正常对照皮损内二者几乎不表达，而玫瑰患者面部皮损中SERPINB3/B4在mRNA表达水平明显高于健康对照者(如图2A,B)，同样，本发明对玫瑰痤疮患者皮损的丘疹或脓疱个数及皮肤红斑进行IGA评分，并与玫瑰痤疮患者皮损中SERPINB3/B4mRNA表达水平进行相关性分析，结果显示随着SERPINB3 mRNA表达水平的增加，皮损IGA评分明显上调，结果表明SERPINB3 mRNA表达水平与IGA评分呈明显正相关(如图2C)，此外，本发明结果显示随着SERPINB4 mRNA表达水平的增加，皮损IGA评分均明显上调，结果表明SERPINB4 mRNA表达水平与皮损严重程度呈正相关关系(如图2D)，Example 2, the present invention collects skin lesions of normal controls and patients with rosacea, extracts total RNA, and detects the expression of SERPINB3/B4 mRNA levels between the two groups. The mRNA expression level of SERPINB3/B4 in facial skin lesions of patients with rose was significantly higher than that of healthy controls (as shown in Figure 2A, B). Similarly, the present invention performs IGA scoring on the number of papules or pustules and skin erythema in patients with rosacea , and correlated with the expression level of SERPINB3/B4 mRNA in skin lesions of patients with rosacea, the results showed that with the increase of SERPINB3 mRNA expression level, the IGA score of skin lesions was significantly increased, and the results showed that the expression level of SERPINB3 mRNA was significantly positively correlated with IGA score (as shown in Figure 2C), in addition, the results of the present invention showed that with the increase of SERPINB4 mRNA expression level, the IGA score of skin lesions was significantly up-regulated, and the results showed that the SERPINB4 mRNA expression level was positively correlated with the severity of skin lesions (as shown in Figure 2D),
同时本发明收集玫瑰痤疮患者面部皮损皮肤组织，进行免疫组织化学染色，结果显示与正常健康对照者比较，玫瑰痤疮患者皮损表皮角质形成细胞全层均可检测到高丰度SERPINB3/B4表达，且其主要表达于细胞浆，而健康对照者皮肤全层几乎无表达(如图2E-F)，综上结果表明玫瑰痤疮患者皮损中SERPINB3/B4较健康对照明显高表达，At the same time, the present invention collects facial skin lesions of patients with rosacea and performs immunohistochemical staining. The results show that compared with normal healthy controls, the full layer of epidermal keratinocytes in patients with rosacea can detect high-abundance SERPINB3/B4 expression , and it is mainly expressed in the cytoplasm, while healthy controls have almost no expression in the whole skin layer (as shown in Figure 2E-F). The above results show that the expression of SERPINB3/B4 in skin lesions of patients with rosacea is significantly higher than that of healthy controls.
为进一步明确SERPINB3/B4在银屑病皮损中表达情况，相似的，本发明首先收取健康对照和银屑病患者的皮损，提取皮损总量RNA，通过qRT-PCR检测皮肤中SERPINB3/B4 mRNA水平在两组之间的差异，如图2G-H可见相较健康对照者皮肤银屑病患者皮损的SERPINB3/B4在mRNA水平表达明显升高，同时对皮损进行SERPINB3/B4免疫组化染色，结果如图2I-J可见银屑病皮损较 正常皮肤的表皮层明显增厚，且表达更高丰度的SERPINB3/B4，上述结果均表明，SERPINB3/B4在银屑病患者皮损中被激活，In order to further clarify the expression of SERPINB3/B4 in psoriatic skin lesions, similarly, the present invention first collects the skin lesions of healthy controls and psoriasis patients, extracts the total RNA of skin lesions, and detects SERPINB3/B4 in the skin by qRT-PCR. Differences in B4 mRNA levels between the two groups, as shown in Figure 2G-H, compared with healthy controls, the expression of SERPINB3/B4 in the skin lesions of psoriasis patients was significantly increased, and the SERPINB3/B4 immunization was performed on the skin lesions at the same time Histochemical staining, the results shown in Figure 2I-J show that the epidermis of psoriatic lesions is significantly thicker than that of normal skin, and expresses a higher abundance of SERPINB3/B4. activated in skin lesions,
为进一步研究SERPINB3/B4在玫瑰痤疮发病过程中发挥的作用，本发明根据先前研究所用的造模方式，建立了抗菌肽LL37诱导的玫瑰痤疮样小鼠模型，该模型皮损表现与炎症反应模式与人类玫瑰痤疮高度相似，本发明选取7-8周BALB/c雌性小鼠，剔除小鼠背部毛发，1天后每只小鼠背部皮内注射50μl(320μM)剂量LL37，每间隔12小时注射一次，对照组小鼠给与等剂量无菌1×PBS溶液，连续注射2日，共4次，模型建立成功后观察皮肤大体表型并进行取材，其次本发明收取小鼠皮损组织并提取总量RNA，检测玫瑰痤疮样小鼠皮损中Serpinb3a的mRNA表达情况，结果可见玫瑰痤疮样小鼠皮损较实验对照组Serpinb3a在mRNA水平表达明显上调(如图2K)，差异具有统计学意义,其中Serpinb3a为SERPINB3/B4的类似物，本发明进一步通过免疫荧光检测了玫瑰痤疮样小鼠模型皮损中Serpinb3a蛋白水平表达情况，结果显示玫瑰痤疮样小鼠模型皮损中Serpinb3a表达与人类玫瑰痤疮皮损SERPINB3/B4表达类似，玫瑰痤疮样小鼠模型皮损中毛囊周围和表皮全层Serpinb3a表达明显增强，为细胞浆表达(如图2L)，综上结果表明玫瑰痤疮样小鼠皮损Serpinb3a表达水平明显上调，In order to further study the role of SERPINB3/B4 in the pathogenesis of rosacea, the present invention established a rosacea-like mouse model induced by the antimicrobial peptide LL37 according to the modeling method used in previous studies. Highly similar to human rosacea, the present invention selects 7-8 week old BALB/c female mice, removes the hair on the back of the mice, and injects 50 μl (320 μM) dose of LL37 intradermally on the back of each mouse one day later, and injects once every 12 hours , mice in the control group were given equal doses of sterile 1×PBS solution, and injected continuously for 2 days, a total of 4 times. Quantitative RNA was used to detect the mRNA expression of Serpinb3a in rosacea-like mouse skin lesions. The results showed that the expression of Serpinb3a in rosacea-like mouse skin lesions was significantly up-regulated compared with the experimental control group (as shown in Figure 2K), and the difference was statistically significant. Wherein Serpinb3a is an analogue of SERPINB3/B4, the present invention further detects the Serpinb3a protein level expression in the rosacea-like mouse model skin lesions by immunofluorescence, and the results show that Serpinb3a expression in the rosacea-like mouse model skin lesions is closely related to that of human rosacea. The expression of SERPINB3/B4 in skin lesions was similar, and the expression of Serpinb3a around the hair follicles and in the whole epidermis of the rosacea-like mouse model was significantly enhanced, and it was expressed in the cytoplasm (as shown in Figure 2L). The expression level was significantly increased,
同样，本发明根据先前研究所用的造模方式，即连续外用咪喹莫特6天，建造银屑病样小鼠模型，收取小鼠皮损进行取材，收取小鼠皮损组织提取总量RNA，通过qRT-PCR检测银屑病样小鼠皮损中Serpinb3a的mRNA表达情况，结果可见银屑病样小鼠皮损较实验对照组Serpinb3a在mRNA水平表达明显上调(如图2M)，进一步收取小鼠皮损，进行免疫荧光染色，可见银屑病样小鼠皮损中明显高表达Serpinb3a(如图2N)，Similarly, the present invention is based on the modeling method used in the previous research, that is, continuous external application of imiquimod for 6 days to build a psoriasis-like mouse model, collect mouse skin lesions for sampling, and collect mouse skin lesions to extract total RNA , the mRNA expression of Serpinb3a in the skin lesions of psoriasis-like mice was detected by qRT-PCR, and the results showed that the expression of Serpinb3a in the skin lesions of psoriasis-like mice was significantly up-regulated compared with the experimental control group (as shown in Figure 2M). Mouse skin lesions were immunofluorescently stained, and Serpinb3a was obviously highly expressed in psoriasis-like mouse skin lesions (as shown in Figure 2N).
为进一步探讨表皮中SERPINB3/B4在玫瑰痤疮及银屑病发病过程中的重要机制，本发明通过siRNA构建特异性敲降小鼠表皮Serpinb3a的小鼠模型， 并对其进行LL37皮内注射及咪喹莫特外用，建立玫瑰痤疮样及银屑病样小鼠模型，观察表皮敲降Serpinb3a后对玫瑰痤疮样小鼠及银屑病样小鼠表型的影响。In order to further explore the important mechanism of SERPINB3/B4 in the epidermis in the pathogenesis of rosacea and psoriasis, the present invention constructs a mouse model of specifically knocking down Serpinb3a in the epidermis of mice by siRNA, and injects LL37 intradermally and mites Topical administration of quimod was used to establish rosacea-like and psoriasis-like mouse models, and the effects of epidermal knockdown of Serpinb3a on the phenotypes of rosacea-like mice and psoriasis-like mice were observed.
实施例三，本发明将阴性对照序列及2条siRNA序列分别命名为Scr RNA、siRNA#1和siRNA#2进行接下来的研究，首先探讨表皮内敲降Serpinb3a对玫瑰痤疮样小鼠表型的影响，如图3A模式图所示，7-8周龄BALB/c野生型雌性小鼠，将其背部毛发剃去，前1天进行siRNA皮内注射，实验对照组皮内注射对应的阴性对照序列，随后按照前述方法进行表皮内LL37注射诱导玫瑰痤疮样小鼠模型，在注射第2次LL37前进行第2次siRNA注射，加强敲降效果，造模完成后，观察并比较三组小鼠(Scr siRNA，siRNA#1和siRNA#2)表型之间的差异并进行取材，图2B为LL37造模两日后Scr siRNA和另外2条siRNA的Control组和LL37组玫瑰痤疮样皮炎的表现，可见LL37造模后Scr siRNA组小鼠皮损部位出现明显的炎症反应，包括红斑和毛细血管扩张，而在2条siRNA敲降组小鼠皮损的炎症反应明显减轻，本发明利用Image J软件对每组玫瑰痤疮样小鼠皮损处的面积大小进行测量，结果显示Scr siRNA组LL37造模后红斑面积明显增加，2条siRNA的LL37组红斑面积较Scr的LL37组明显减小(如图3C)，其次对玫瑰痤疮样皮损红斑严重程度进行评分，结果显示Scr siRNA的LL37组造模后红斑评分明显增加，造模后2条siRNA的LL37组红斑评分明显减轻(如图3D)，Example 3, the present invention named the negative control sequence and 2 siRNA sequences as ScrRNA, siRNA#1 and siRNA#2 respectively for the next research, and first explored the effect of knocking down Serpinb3a in the epidermis on the phenotype of rosacea-like mice Effect, as shown in Figure 3A schematic diagram, 7-8 weeks old BALB/c wild-type female mice, shaved their back hair, injected siRNA intradermally one day before, and injected the corresponding negative control group intradermally Sequence, followed by intraepidermal LL37 injection to induce a rosacea-like mouse model according to the aforementioned method, and the second siRNA injection was performed before the second LL37 injection to strengthen the knockdown effect. After the modeling was completed, the three groups of mice were observed and compared (Scr siRNA, siRNA#1 and siRNA#2) differences between phenotypes were collected. Figure 2B shows the performance of rosacea-like dermatitis in the Control group and LL37 group of Scr siRNA and other 2 siRNAs two days after LL37 modeling. It can be seen that after LL37 modeling, the skin lesions of the mice in the Scr siRNA group showed obvious inflammatory reactions, including erythema and telangiectasia, while the inflammatory reactions of the mouse skin lesions in the two siRNA knockdown groups were significantly reduced. The present invention uses Image J software The area of skin lesions in each group of rosacea-like mice was measured, and the results showed that the area of erythema in the Scr siRNA group LL37 was significantly increased after modeling, and the area of erythema in the LL37 group with two siRNAs was significantly smaller than that in the Scr LL37 group (as shown in Fig. 3C), followed by scoring the erythema severity of rosacea-like skin lesions, the results showed that the erythema score of the Scr siRNA LL37 group increased significantly after modeling, and the erythema score of the 2 siRNA LL37 group was significantly reduced after modeling (as shown in Figure 3D).
此外本发明比较了LL37造模后各组小鼠皮损部位的组织学情况，取造模部位皮损，石蜡包埋切片后进行HE染色，显微镜下观察并统计小鼠真皮层炎症细胞浸润数量，如图3E可见，Scr siRNA的LL37组真皮部位浸润的炎症细胞数量较Control组明显增加，2条siRNA的LL37组造模后真皮层浸润的炎症细胞数量较Scr siRNA的LL37组明显减少，同时本发明通过Prism9软件对浸润的炎症细胞数量进行统计分析，结果可见敲降Serpinb3a后2条siRNA 的LL37组浸润的炎症细胞较Scr的LL37组明显减少(如图3F)，差异具有统计学意义，最后本发明收取小鼠皮损，提取总量RNA，通过qRT-PCR比较了上述各组小鼠皮损中玫瑰痤疮相关基因在mRNA表达水平上的差异，如图3G-H所示，Scr siRNA的LL37组较Control组的Il6和Tnf-α基因表达增加，而在敲降Serpinb3a后，2条siRNA的LL37组较Scr siRNA的LL37组上述两个基因表达水平明显回降，且差异具有统计学意义，以上结果表明，表皮内Serpinb3a的缺失从皮损炎症反应表现、组织病理学情况以及mRNA表达层面均能够明显改善LL37诱导的玫瑰痤疮样小鼠模型中玫瑰痤疮的发展。In addition, the present invention compares the histological situation of the mouse skin lesions in each group after LL37 modeling. The skin lesions of the modeling parts are taken, paraffin-embedded and sectioned, and then HE stained. The number of inflammatory cell infiltration in the dermis of the mice is observed under a microscope and counted. , as shown in Figure 3E, the number of inflammatory cells infiltrating the dermis in the Scr siRNA LL37 group was significantly higher than that in the Control group, and the number of inflammatory cells infiltrating the dermis in the 2 siRNA LL37 group was significantly lower than that in the Scr siRNA LL37 group. In the present invention, the number of infiltrating inflammatory cells was statistically analyzed by Prism9 software. The results showed that after knocking down Serpinb3a, the number of infiltrating inflammatory cells in the LL37 group with two siRNAs was significantly less than that in the Scr LL37 group (as shown in Figure 3F), and the difference was statistically significant. Finally, the present invention collects mouse skin lesions, extracts total RNA, and compares the differences in mRNA expression levels of rosacea-related genes in the skin lesions of the above-mentioned groups of mice by qRT-PCR, as shown in Figure 3G-H, Scr siRNA Compared with the Control group, the expressions of Il6 and Tnf-α genes in the LL37 group were increased, and after knocking down Serpinb3a, the expression levels of the above two genes in the LL37 group with two siRNAs were significantly lower than those in the LL37 group with Scr siRNA, and the difference was statistically significant. Significance, the above results show that the deletion of Serpinb3a in the epidermis can significantly improve the development of rosacea in the LL37-induced rosacea-like mouse model from the levels of inflammatory response, histopathology and mRNA expression.
实施例四，本发明为明确表皮内敲降Serpinb3a对银屑病样小鼠表型的影响，选择前述的两条siRNA，进行小鼠耳部皮内注射，表皮内敲降Serpinb3a，按照既往已有研究方法外用咪喹莫特进行银屑病样小鼠模型建造，如图4A模式图所示，外用咪喹莫特前1天(记作day-1)进行siRNA皮内注射，实验对照组皮内注射对应的阴性对照序列，其后每日一次咪喹莫特乳膏外用，第2天进行第2次siRNA注射，加强敲降效果，第6天造模完成后，为明确表皮内敲降Serpinb3a后对银屑病样小鼠皮损表型的影响，本发明造模第6天观察并比较三组小鼠(Scr siRNA，siRNA#1和siRNA#2)表型之间的差异并进行取材，根据既往研究[77]方法对小鼠皮损进行PASI评分及耳部厚度进行测量，可见siRNA的咪喹莫特组小鼠较Scr RNA咪喹莫特组小鼠皮损的红斑、鳞屑明显改善(如图4B)，PASI评分明显下降(如图4C)，耳部皮肤厚度亦明显下降(如图4D)，其次对各组小鼠皮损进行HE染色，结果可见，Scr siRNA咪喹莫特组表皮厚度明显增加，真皮层炎症细胞浸润明显，而两条siRNA的咪喹模特组表皮厚度及真皮层炎症细胞浸润程度明显改善(如图4E)，并对表皮层厚度、浸润的炎症细胞进行统计分析，发现二者在两条siRNA的咪喹莫特组较Scr siRNA的咪喹莫特组明显下降(如图4F-G)，为进一步明确Serpinb3a对银屑病相关基因分子水平的影响，本发明收取小鼠皮损，提取总 量RNA，通过qRT-PCR比较了上述各组小鼠皮损中银屑病相关基因在mRNA表达水平上的差异，如图4H-I所示，Scr siRNA的咪喹莫特组较Control组Il6和Il-1β基因表达增加2条siRNA的咪喹莫特组中上述两个基因表达水平较Scr siRNA的咪喹莫特组有明显回降，差异具有统计学意义，以上结果表明，耳部表皮内Serpinb3a的缺失从皮损炎症反应表现、组织病理学情况以及mRNA表达层面均能够明显改善咪喹莫特诱导的银屑病样小鼠模型中炎症疾病的发展，Example 4, in order to clarify the effect of knocking down Serpinb3a in the epidermis on the phenotype of psoriasis-like mice, the present invention selects the aforementioned two siRNAs and injects them into the ears of mice, knocking down Serpinb3a in the epidermis, according to previous methods There is a research method for constructing a psoriasis-like mouse model with topical imiquimod, as shown in Figure 4A schematic diagram, siRNA was injected intradermally 1 day before topical imiquimod (denoted as day-1), and the experimental control group The corresponding negative control sequence was injected intradermally, followed by external application of imiquimod cream once a day, and the second siRNA injection was performed on the second day to strengthen the knockdown effect. The influence of Serpinb3a on the phenotype of psoriasis-like mice skin lesions, the present invention observed and compared the differences between the phenotypes of the three groups of mice (Scr siRNA, siRNA#1 and siRNA#2) on the 6th day of modeling and According to the method of previous research [77], the PASI score and ear thickness of the mouse skin lesions were measured. It can be seen that the mice in the siRNA imiquimod group had more erythema, The scales were significantly improved (as shown in Figure 4B), the PASI score was significantly reduced (as shown in Figure 4C), and the thickness of the ear skin was also significantly decreased (as shown in Figure 4D). Secondly, HE staining was performed on the skin lesions of mice in each group. The results showed that Scr siRNA The thickness of the epidermis and the infiltration of inflammatory cells in the dermis were significantly increased in the quinomod group, while the thickness of the epidermis and the infiltration of inflammatory cells in the dermis were significantly improved in the two siRNA groups (as shown in Figure 4E). Statistical analysis of inflammatory cells showed that the two siRNA imiquimod groups were significantly lower than the Scr siRNA imiquimod group (as shown in Figure 4F-G). The present invention collects mouse skin lesions, extracts total RNA, and compares the differences in mRNA expression levels of psoriasis-related genes in the mouse skin lesions of the above-mentioned groups by qRT-PCR, as shown in Figure 4H-I The expression levels of Il6 and Il-1β genes in the Scr siRNA imiquimod group increased significantly compared with the Control group. The difference is statistically significant. The above results show that the deletion of Serpinb3a in the ear epidermis can significantly improve the imiquimod-induced psoriasis-like mouse model in terms of inflammatory response, histopathology and mRNA expression. development of inflammatory diseases,
综上所述，在小鼠表皮内敲降serpinb3a后，能够明显改善玫瑰痤疮样及银屑病样小鼠皮损处炎症反应。In summary, knocking down serpinb3a in the epidermis of mice can significantly improve the inflammatory response in skin lesions of rosacea-like and psoriasis-like mice.
实施例五，为了更进一步明确SERPINB3/B4在体外对玫瑰痤疮中具体分子作用机制，本发明体外构建SERPINB3/B4过表达质粒，并通过转染试剂将过表达质粒转染人HaCaT细胞，72小时后收取细胞RNA，进行全转录组测序，并通过体内外实验对测序结果进行验证，Example 5, in order to further clarify the specific molecular mechanism of SERPINB3/B4 on rosacea in vitro, the present invention constructs SERPINB3/B4 overexpression plasmid in vitro, and transfects the overexpression plasmid into human HaCaT cells with a transfection reagent for 72 hours Afterwards, the cellular RNA was collected, the whole transcriptome was sequenced, and the sequencing results were verified through in vivo and in vitro experiments.
本发明对过表达SERPINB3/B4的人HaCaT细胞中的差异基因表达值进行聚类分析，进而对基因表达值进行均一化处理从而得到如图5A，如图中的每一个小方格代表一个基因，蓝色表示基因表达下调，红色表示基因表达上调，颜色越深，表达的差异越明显，每一列表示每个样本不同基因的表达情况，每一行表示每个基因在不同样本中的表达情况，如图结果可见过表达SERPINB3/B4的人HaCaT细胞较正常对照之间基因表达差异显著，结果表明，在人HaCaT细胞中过表达SERPINB3/B4后基因表达较正常对照组模式发生明显变化，The present invention performs clustering analysis on the differential gene expression values in human HaCaT cells overexpressing SERPINB3/B4, and then normalizes the gene expression values to obtain as shown in Figure 5A, where each small square in the figure represents a gene , blue indicates down-regulation of gene expression, red indicates up-regulation of gene expression, the darker the color, the more obvious the difference in expression, each column indicates the expression of different genes in each sample, and each row indicates the expression of each gene in different samples, As shown in the figure, it can be seen that the gene expression of human HaCaT cells overexpressing SERPINB3/B4 is significantly different from that of the normal control.
为进一步明确过表达SERPINB3/B4的人HaCaT细胞中信号通路的变化，本发明对过表达SERPINB3/B4的人HaCaT细胞和对照组细胞所有的基因表达量进行基因富集分析(Gene Set Enrichment Analysis，GSEA)，基于玫瑰痤疮为皮肤炎症性疾病及先前研究，炎症相关信号通路在玫瑰痤疮发生发展 中发挥关键作用，所以本次研究着重关注与炎症相关联的信号通路，NF-κB信号通路几乎存在于所用的动物细胞中，参与细胞对外界刺激的各种反应，包括应激、细胞因子、自由基、微生物感染和紫外线照射等，既往研究发现此信号通路调控大量与炎症相关的基因表达，在本次研究中富集的炎症相关信号通路中NF-κB信号通路在过表达SERPINB3/B4的人HaCaT细胞中较对照组细胞系明显富集(如图5B-E)，这提示该信号通路可能参与了SERPINB3/B4调控玫瑰痤疮的发病，In order to further clarify the changes in the signaling pathways in the human HaCaT cells overexpressing SERPINB3/B4, the present invention carried out gene enrichment analysis (Gene Set Enrichment Analysis, Gene Set Enrichment Analysis, GSEA), based on rosacea is a skin inflammatory disease and previous research, inflammation-related signaling pathways play a key role in the development of rosacea, so this study focuses on signaling pathways associated with inflammation, NF-κB signaling pathway almost exists In the animal cells used, it is involved in various responses of cells to external stimuli, including stress, cytokines, free radicals, microbial infection and ultraviolet radiation. Previous studies have found that this signaling pathway regulates the expression of a large number of genes related to inflammation. Among the inflammation-related signaling pathways enriched in this study, the NF-κB signaling pathway was significantly enriched in the human HaCaT cells overexpressing SERPINB3/B4 compared with the control cell line (as shown in Figure 5B-E), which suggests that the signaling pathway may Involved in SERPINB3/B4 regulating the pathogenesis of rosacea,
为明确在人HaCaT细胞中过表达SERPINB3/B4后在蛋白水平对NF-κB信号通路的影响，本发明通过转染试剂将过表达质粒转染人HaCaT细胞系72小时后收取细胞蛋白，免疫印迹法检测在人HaCaT细胞系中表达SERPINB3/B4后对p65/NF-κB的磷酸化水平(p-p65)，同时应用Image J软件对p-p65灰度进行扫描量化，如图5F所示，本发明发现在人HaCaT细胞系中表达SERPINB3/B4后p-p65表达明显上调，p65表达无明显差异，p-p65/p65上调，结果表明NF-κB信号通路激活，同时，为了更加直观地了解表达SERPINB3/B4后对NF-κB信号通路的影响，本发明通过细胞爬片实验及免疫荧光检测过表达质粒转染人HaCaT细胞系72小时后对p65表达的影响，结果如图5G所示，在人HaCaT细胞中表达SERPINB3/B4后p65入核的细胞数目较对照组明显增多上述结果表明在在人HaCaT细胞中表达SERPINB3/B4在蛋白水平激活NF-κB信号通路，In order to clarify the effect of overexpressing SERPINB3/B4 on the NF-κB signaling pathway at the protein level in human HaCaT cells, the present invention transfected the overexpression plasmid into the human HaCaT cell line with a transfection reagent for 72 hours, and harvested the cell protein, immunoblotting The phosphorylation level of p65/NF-κB (p-p65) after SERPINB3/B4 expression in human HaCaT cell line was detected by using the method, and the gray scale of p-p65 was scanned and quantified by Image J software, as shown in Figure 5F, The present invention found that after expressing SERPINB3/B4 in the human HaCaT cell line, the expression of p-p65 was significantly up-regulated, the expression of p65 was not significantly different, and p-p65/p65 was up-regulated. The results indicated that the NF-κB signaling pathway was activated. At the same time, in order to understand The effect of expressing SERPINB3/B4 on the NF-κB signaling pathway, the present invention detected the effect on the expression of p65 after 72 hours of transfection of the overexpression plasmid into the human HaCaT cell line by cell climbing experiments and immunofluorescence, the results are shown in Figure 5G, After expressing SERPINB3/B4 in human HaCaT cells, the number of p65 nuclear cells was significantly increased compared with the control group. The above results indicate that the expression of SERPINB3/B4 in human HaCaT cells activates the NF-κB signaling pathway at the protein level,
既往研究发现，TNF-α可强烈诱导NF-κB信号的活化，为明确SERPINB3/B4对TNF-α诱导NF-κB信号活化的影响，本发明体外构建SERPINB3/B4的shRNA，建立稳定敲降SERPINB3/B4的人HaCaT细胞系，TNF-α孵育30分钟后，免疫印迹法检测敲降SERPINB3/B4后对TNF-α诱导NF-κB信号活化的影响，结果可见，构建稳定敲降SERPINB3/B4的人HaCaT细胞系对SERPINB3/B4的敲降效果明显，在正常对照组细胞中，TNF-α孵育后p-p65 表达明显上调，而敲降SERPINB3/B4后，TNF-α诱导p-p65上调的情况明显改善，同时本发明对其条带灰度进行扫描，结果统计发现差异具有明显统计学意义(如图5H-I)，结果提示，人HaCaT细胞系中SERPINB3/B4的缺失阻止了TNF-α诱导的NF-κB信号的活化，同样，本发明通过细胞爬片实验及免疫荧光检测稳定敲降SERPINB3/B4对TNF-α诱导的p65入核情况的影响，如图5J-K可见在正常对照的人HaCaT细胞系中，TNF-α孵育后30分钟后p65几乎完全入核，而敲降SERPINB3/B4后p65入核的细胞数目较对照组明显减少，同时本发明对入核细胞数目进行统计，差异明显，具有统计学意义，Previous studies have found that TNF-α can strongly induce the activation of NF-κB signaling. In order to clarify the effect of SERPINB3/B4 on the activation of NF-κB signaling induced by TNF-α, the present invention constructed SERPINB3/B4 shRNA in vitro to establish a stable knockdown of SERPINB3 /B4 human HaCaT cell line, after TNF-α incubation for 30 minutes, Western blot was used to detect the effect of knocking down SERPINB3/B4 on the activation of NF-κB signal induced by TNF-α. The results showed that the stable knockdown of SERPINB3/B4 The human HaCaT cell line has a significant knockdown effect on SERPINB3/B4. In the normal control cells, the expression of p-p65 was significantly upregulated after TNF-α incubation, and after knockdown of SERPINB3/B4, TNF-α induced p-p65 upregulation. The situation has been significantly improved. At the same time, the present invention scans the gray scale of its bands, and the statistical results show that the difference has obvious statistical significance (as shown in Figure 5H-I). The results suggest that the deletion of SERPINB3/B4 in the human HaCaT cell line prevents TNF- The activation of α-induced NF-κB signal, similarly, the present invention detects the effect of stably knocking down SERPINB3/B4 on TNF-α-induced p65 nuclear entry through cell slide experiments and immunofluorescence, as shown in Figure 5J-K. In the control human HaCaT cell line, p65 was almost completely incorporated into the nucleus after 30 minutes of TNF-α incubation, and the number of cells with p65 incorporated into the nucleus after knocking down SERPINB3/B4 was significantly reduced compared with the control group. Statistically, the difference is obvious and statistically significant,
综上结果表明，体外过表达SERPINB3/B4后诱导NF-κB信号通路下游分子上调，体外敲降SERPINB3/B4后能够阻止TNF-α诱导的NF-κB信号的活化。In conclusion, overexpression of SERPINB3/B4 in vitro induces the upregulation of downstream molecules of NF-κB signaling pathway, and knockdown of SERPINB3/B4 in vitro can prevent the activation of TNF-α-induced NF-κB signaling.
实施例六，炎症相关的趋化因子及细胞因子在炎症发生及进展中起到极其重要的作用，前期一系列的结果表明SERPINB3/B4能够调控NF-κB信号通路，为进一步明确趋化因子及细胞因子在SERPINB3/B4调控玫瑰痤疮发病中的具体作用，本发明进一步对在人HaCaT细胞中过表达SERPINB3/B4后RNA-seq的结果进行分析，如图6A-B所示，趋化因子及细胞因子信号通路在SERPINB3/B4过表达的人HaCaT细胞中高度富集，同时本发明对样本的趋化因子及细胞因子相关的差异的表达值进行聚类分析，并对基因表达值进行均一化处理得到如图6C所示的如图，与正常对照相比，SERPINB3/B4过表达的人HaCaT细胞中炎症相关的趋化因子(CCL2，CCL5，CCL20，CXCL1，CXCL2，CXCL5，CXCL8，CXCL10，CXCL11)及细胞因子(IL1A，IL1B，IL6，TNFA)表达明显上调，Example 6. Inflammation-related chemokines and cytokines play an extremely important role in the occurrence and progression of inflammation. A series of previous results show that SERPINB3/B4 can regulate the NF-κB signaling pathway. In order to further clarify the chemokines and The specific role of cytokines in regulating the pathogenesis of rosacea by SERPINB3/B4, the present invention further analyzed the results of RNA-seq after overexpressing SERPINB3/B4 in human HaCaT cells, as shown in Figure 6A-B, chemokines and Cytokine signaling pathways are highly enriched in human HaCaT cells with SERPINB3/B4 overexpression. At the same time, the present invention performs cluster analysis on the expression values of chemokine and cytokine-related differences in samples, and normalizes the gene expression values. The figure shown in Figure 6C was obtained after treatment, compared with the normal control, inflammation-related chemokines (CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL5, CXCL8, CXCL10, CXCL11) and cytokines (IL1A, IL1B, IL6, TNFA) were significantly up-regulated,
为进一步明确上述细胞因子在SERPINB3/B4过表达的人HaCaT细胞中的表达水平，本发明利用qRT-PCR的方法检测人HaCaT细胞过表达SERPINB3/B4后上述基因的表达情况，结果(如图6D)可见，在人HaCaT细胞中过表达SERPINB3/B4后，趋化因子CCL2，CCL5，CCL20，CXCL2，CXCL9，CXCL10，CXCL11 及细胞因子IL1B，IL6，TNF-α表达明显上调，结果表明在人HaCaT细胞过表达SERPINB3/B4后能够明显诱导炎症相关的趋化因子及细胞因子的表达上调，In order to further clarify the expression levels of the above-mentioned cytokines in human HaCaT cells overexpressing SERPINB3/B4, the present invention uses qRT-PCR to detect the expression of the above-mentioned genes in human HaCaT cells after overexpressing SERPINB3/B4, and the results (as shown in Figure 6D ) shows that after overexpressing SERPINB3/B4 in human HaCaT cells, the expression of chemokines CCL2, CCL5, CCL20, CXCL2, CXCL9, CXCL10, CXCL11 and cytokines IL1B, IL6, TNF-α was significantly up-regulated, the results showed that in human HaCaT cells Cells overexpressing SERPINB3/B4 can significantly induce the upregulation of inflammation-related chemokines and cytokines,
通过小鼠皮内敲降Serpinb3a能够降低LL37诱导的玫瑰痤疮相关细胞因子(Il6，Tnf-α)的表达水平，为进一步明确玫瑰痤疮样小鼠表皮内敲降Serpinb3a对炎症相关的趋化因子及细胞因子的影响，本发明应用qRT-PCR的方法检测了上述相关因子的表达水平，结果如图6E可见，敲降Serpinb3a后，LL37诱导的Ccl5，Cxcl9，Cxcl10，Cxcl11及Il1β表达水平受到明显抑制，本发明应用同样的方法检测了敲降serpinb3a后银屑病样小鼠皮损中上述趋化因子变化情况，结果如图6F所示，可见敲降Serpinb3a后，IMQ诱导的Ccl5，Ccl20，Cxcl9，Cxcl10，Cxcl11表达水平受到明显抑制，Intradermal knockdown of Serpinb3a in mice can reduce the expression level of rosacea-related cytokines (Il6, Tnf-α) induced by LL37. The influence of cytokines, the present invention used qRT-PCR to detect the expression levels of the above-mentioned related factors, the results can be seen in Figure 6E, after knocking down Serpinb3a, the expression levels of Ccl5, Cxcl9, Cxcl10, Cxcl11 and Il1β induced by LL37 were significantly inhibited , the present invention used the same method to detect the changes of the above chemokines in the skin lesions of psoriasis-like mice after knocking down serpinb3a, and the results are shown in Figure 6F. , Cxcl10, Cxcl11 expression levels were significantly inhibited,
同样的方法检测相关趋化因子及细胞因子的表达水平，结果5G如图所示，SERPINB3/B4诱导趋化因子CCL2，CCL5，CXCL9，CXCL10，CXCL11及细胞因子IL-1β，IL-6，TNF-α明显上调，而加用SC75741共同孵育后上述趋化因子及细胞因子的表达水平明显回降，上述结果表明SC75741能够通过抑制SERPINB3/B4对NF-κB信号通路的诱导激活下调相关趋化因子及细胞因子的表达水平。The same method was used to detect the expression levels of related chemokines and cytokines. As shown in the figure, SERPINB3/B4 induced chemokines CCL2, CCL5, CXCL9, CXCL10, CXCL11 and cytokines IL-1β, IL-6, TNF -α was significantly up-regulated, and the expression levels of the above chemokines and cytokines were significantly decreased after co-incubation with SC75741. The above results indicated that SC75741 could down-regulate related chemokines by inhibiting the activation of SERPINB3/B4 on the NF-κB signaling pathway and expression levels of cytokines.
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。It will be apparent to those skilled in the art that the invention is not limited to the details of the above-described exemplary embodiments, but that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Accordingly, the embodiments should be regarded in all points of view as exemplary and not restrictive, the scope of the invention being defined by the appended claims rather than the foregoing description, and it is therefore intended that the scope of the invention be defined by the appended claims rather than by the foregoing description. All changes within the meaning and range of equivalents of the elements are embraced in the present invention. Any reference sign in a claim should not be construed as limiting the claim concerned.

Claims (6)

  1. SERPINB3/B4作为靶点在玫瑰痤疮等炎症性皮肤病治疗药物中的应用，其特征在于：首先通过ELISA、免疫组化、qRT-PCR、免疫荧光等手段明确SERPINB3/B4在玫瑰痤疮患者和银屑病患者血清、皮损中的表达及Serpinb3a在LL37诱导的玫瑰痤疮样小鼠模型及咪喹莫特诱导的银屑病样小鼠模型皮损中的表达情况；The application of SERPINB3/B4 as a target in the treatment of rosacea and other inflammatory skin diseases is characterized in that: firstly, the role of SERPINB3/B4 in patients with rosacea and silver The expression of serum and skin lesions of patients with psoriasis and the expression of Serpinb3a in the rosacea-like mouse model induced by LL37 and the psoriasis-like mouse model induced by imiquimod;
    再通过siRNA皮内注射，局部敲降小鼠表皮细胞内Serpinb3a的表达，观察其对炎症模型皮损表型及组织病理学的影响，体外研究主要在人HaCaT细胞中进行，通过构建质粒在人HaCaT细胞中过表达或敲降SERPINB3/B4，并联合RNA-seq和免疫印迹、免疫荧光等方法明确其与NF-κB信号通路的关系；Then through intradermal injection of siRNA, the expression of Serpinb3a in mouse epidermal cells was locally knocked down, and its effect on the phenotype and histopathology of inflammatory model skin lesions was observed. In vitro studies were mainly carried out in human HaCaT cells. Overexpress or knock down SERPINB3/B4 in HaCaT cells, and combine RNA-seq, western blot, immunofluorescence and other methods to clarify its relationship with NF-κB signaling pathway;
    最后选择SC75741(一种强效NF-κB信号通路抑制剂)抑制小鼠表皮内NF-κB信号通路明确NF-κB信号通路在玫瑰痤疮发病过程中的作用；且运用CCK8和流式分析选择SC75741的合适浓度进行体外研究，进一步明确SERPINB3/B4与NF-κB信号通路在玫瑰痤疮及银屑病发病机制中的相互作用。Finally, SC75741 (a potent NF-κB signaling pathway inhibitor) was selected to inhibit the NF-κB signaling pathway in the mouse epidermis to clarify the role of NF-κB signaling pathway in the pathogenesis of rosacea; and use CCK8 and flow cytometry to select SC75741 In vitro studies at appropriate concentrations of SERPINB3/B4 and NF-κB signaling pathways in the pathogenesis of rosacea and psoriasis were further clarified.
  2. 根据权利要求1所述的SERPINB3/B4作为靶点在玫瑰痤疮等炎症性皮肤病治疗药物中的应用，其特征在于：本发明收集正常对照和玫瑰痤疮患者皮损，提取总RNA，检测SERPINB3/B4 mRNA水平在两组之间的表达情况，结果发现在正常对照皮损内二者几乎不表达，而玫瑰患者面部皮损中SERPINB3/B4在mRNA表达水平明显高于健康对照者(如图2 A,B)，同样，本发明对玫瑰痤疮患者皮损的丘疹或脓疱个数及皮肤红斑进行IGA评分，并与玫瑰痤疮患者皮损中SERPINB3/B4 mRNA表达水平进行相关性分析，结果显示随着SERPINB3 mRNA表达水平的增加，皮损IGA评分明显上调，结果表明SERPINB3 mRNA表达水平与IGA评分呈明显正相关(如图2 C)，此外，本发明结果显示随着SERPINB4 mRNA表达水平的增加，皮损IGA评分均明显上调，结果表明SERPINB4 mRNA表达水平与皮损严重程度呈正相关关系(如图2D)，同时本发明收集玫瑰痤疮患者面部皮损皮肤组织，进行免疫组织化学染 色，结果显示与正常健康对照者比较，玫瑰痤疮患者皮损表皮角质形成细胞全层均可检测到高丰度SERPINB3/B4表达，且其主要表达于细胞浆，而健康对照者皮肤全层几乎无表达(如图2E-F)，综上结果表明玫瑰痤疮患者皮损中SERPINB3/B4较健康对照明显高表达，According to claim 1, the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as rosacea is characterized in that: the present invention collects skin lesions of normal controls and patients with rosacea, extracts total RNA, and detects SERPINB3/B4 The expression of B4 mRNA level between the two groups, it was found that the two were almost not expressed in the normal control skin lesions, while the mRNA expression level of SERPINB3/B4 in the facial skin lesions of rose patients was significantly higher than that of healthy controls (as shown in Figure 2 A, B), similarly, the present invention carries out IGA score to the number of papules or pustules and skin erythema of skin lesions of patients with rosacea, and performs correlation analysis with SERPINB3/B4 mRNA expression level in skin lesions of patients with rosacea, the results show With the increase of SERPINB3 mRNA expression level, the IGA score of skin lesions was significantly up-regulated, and the results showed that the SERPINB3 mRNA expression level was significantly positively correlated with the IGA score (as shown in Figure 2 C). In addition, the results of the present invention showed that with the increase of SERPINB4 mRNA expression level , the IGA scores of skin lesions were significantly increased, and the results showed that the expression level of SERPINB4 mRNA was positively correlated with the severity of skin lesions (as shown in Figure 2D). At the same time, the present invention collected facial skin lesions of patients with rosacea and performed immunohistochemical staining. The results showed that Compared with normal healthy controls, the expression of high-abundance SERPINB3/B4 can be detected in the whole layer of epidermal keratinocytes of rosacea patients, and it is mainly expressed in the cytoplasm, while there is almost no expression in the whole layer of skin of healthy controls (such as Figure 2E-F), the above results show that the expression of SERPINB3/B4 in skin lesions of rosacea patients is significantly higher than that of healthy controls,
    为进一步明确SERPINB3/B4在银屑病皮损中表达情况，相似的，本发明首先收取健康对照和银屑病患者的皮损，提取皮损总量RNA，通过qRT-PCR检测皮肤中SERPINB3/B4 mRNA水平在两组之间的差异，如图2G-H可见相较健康对照者皮肤银屑病患者皮损的SERPINB3/B4在mRNA水平表达明显升高，同时对皮损进行SERPINB3/B4免疫组化染色，结果如图2I-J可见银屑病皮损较正常皮肤的表皮层明显增厚，且表达更高丰度的SERPINB3/B4，上述结果均表明，SERPINB3/B4在银屑病患者皮损中被激活，In order to further clarify the expression of SERPINB3/B4 in psoriatic skin lesions, similarly, the present invention first collects the skin lesions of healthy controls and psoriasis patients, extracts the total RNA of skin lesions, and detects SERPINB3/B4 in the skin by qRT-PCR. Differences in B4 mRNA levels between the two groups, as shown in Figure 2G-H, compared with healthy controls, the expression of SERPINB3/B4 in the skin lesions of psoriasis patients was significantly increased, and the SERPINB3/B4 immunization was performed on the skin lesions at the same time Histochemical staining, the results shown in Figure 2I-J show that the epidermis of psoriatic lesions is significantly thicker than that of normal skin, and expresses a higher abundance of SERPINB3/B4. activated in skin lesions,
    为进一步研究SERPINB3/B4在玫瑰痤疮发病过程中发挥的作用，本发明根据先前研究所用的造模方式，建立了抗菌肽LL37诱导的玫瑰痤疮样小鼠模型，该模型皮损表现与炎症反应模式与人类玫瑰痤疮高度相似，本发明选取7-8周BALB/c雌性小鼠，剔除小鼠背部毛发，1天后每只小鼠背部皮内注射50μl(320μM)剂量LL37，每间隔12小时注射一次，对照组小鼠给与等剂量无菌1×PBS溶液，连续注射2日，共4次，模型建立成功后观察皮肤大体表型并进行取材，其次本发明收取小鼠皮损组织并提取总量RNA，检测玫瑰痤疮样小鼠皮损中Serpinb3a的mRNA表达情况，结果可见玫瑰痤疮样小鼠皮损较实验对照组Serpinb3a在mRNA水平表达明显上调(如图2K)，差异具有统计学意义,其中Serpinb3a为SERPINB3/B4的类似物，本发明进一步通过免疫荧光检测了玫瑰痤疮样小鼠模型皮损中Serpinb3a蛋白水平表达情况，结果显示玫瑰痤疮样小鼠模型皮损中Serpinb3a表达与人类玫瑰痤疮皮损SERPINB3/B4表达类似，玫瑰痤疮样小鼠模型皮损中毛囊周围和表皮全层Serpinb3a表达明显增强，为细胞浆表达(如图2L)，综上结果表明玫瑰痤 疮样小鼠皮损Serpinb3a表达水平明显上调，In order to further study the role of SERPINB3/B4 in the pathogenesis of rosacea, the present invention established a rosacea-like mouse model induced by the antimicrobial peptide LL37 according to the modeling method used in previous studies. Highly similar to human rosacea, the present invention selects 7-8 week old BALB/c female mice, removes the hair on the back of the mice, and injects 50 μl (320 μM) dose of LL37 intradermally on the back of each mouse one day later, and injects once every 12 hours , mice in the control group were given equal doses of sterile 1×PBS solution, and injected continuously for 2 days, a total of 4 times. Quantitative RNA was used to detect the mRNA expression of Serpinb3a in rosacea-like mouse skin lesions. The results showed that Serpinb3a mRNA expression in rosacea-like mouse skin lesions was significantly up-regulated compared with the experimental control group (as shown in Figure 2K), and the difference was statistically significant. Wherein Serpinb3a is an analogue of SERPINB3/B4, the present invention further detects the Serpinb3a protein level expression in the rosacea-like mouse model skin lesions by immunofluorescence, and the results show that Serpinb3a expression in the rosacea-like mouse model skin lesions is closely related to that of human rosacea. The expression of SERPINB3/B4 in skin lesions was similar, and the expression of Serpinb3a around the hair follicles and in the whole epidermis of the rosacea-like mouse model was significantly enhanced, and it was expressed in the cytoplasm (as shown in Figure 2L). The expression level was significantly increased,
    同样，本发明根据先前研究所用的造模方式，即连续外用咪喹莫特6天，建造银屑病样小鼠模型，收取小鼠皮损进行取材，收取小鼠皮损组织提取总量RNA，通过qRT-PCR检测银屑病样小鼠皮损中Serpinb3a的mRNA表达情况，结果可见银屑病样小鼠皮损较实验对照组Serpinb3a在mRNA水平表达明显上调(如图2M)，进一步收取小鼠皮损，进行免疫荧光染色，可见银屑病样小鼠皮损中明显高表达Serpinb3a(如图2N)，Similarly, the present invention is based on the modeling method used in the previous research, that is, continuous external application of imiquimod for 6 days to build a psoriasis-like mouse model, collect mouse skin lesions for sampling, and collect mouse skin lesions to extract total RNA , the mRNA expression of Serpinb3a in the skin lesions of psoriasis-like mice was detected by qRT-PCR, and the results showed that the expression of Serpinb3a in the skin lesions of psoriasis-like mice was significantly up-regulated compared with the experimental control group (as shown in Figure 2M). Mouse skin lesions were immunofluorescently stained, and Serpinb3a was obviously highly expressed in psoriasis-like mouse skin lesions (as shown in Figure 2N).
    为进一步探讨表皮中SERPINB3/B4在玫瑰痤疮及银屑病发病过程中的重要机制，本发明通过siRNA构建特异性敲降小鼠表皮Serpinb3a的小鼠模型，并对其进行LL37皮内注射及咪喹莫特外用，建立玫瑰痤疮样及银屑病样小鼠模型，观察表皮敲降Serpinb3a后对玫瑰痤疮样小鼠及银屑病样小鼠表型的影响。In order to further explore the important mechanism of SERPINB3/B4 in the epidermis in the pathogenesis of rosacea and psoriasis, the present invention constructs a mouse model of specifically knocking down mouse epidermis Serpinb3a by siRNA, and injects LL37 intradermally and mites Topical administration of quimod was used to establish rosacea-like and psoriasis-like mouse models, and the effects of epidermal knockdown of Serpinb3a on the phenotypes of rosacea-like mice and psoriasis-like mice were observed.
  3. 根据权利要求1所述的SERPINB3/B4作为靶点在玫瑰痤疮等炎症性皮肤病治疗药物中的应用，其特征在于：本发明将阴性对照序列及2条siRNA序列分别命名为Scr RNA、siRNA#1和siRNA#2进行接下来的研究，首先探讨表皮内敲降Serpinb3a对玫瑰痤疮样小鼠表型的影响，如图3A模式图所示，7-8周龄BALB/c野生型雌性小鼠，将其背部毛发剃去，前1天进行siRNA皮内注射，实验对照组皮内注射对应的阴性对照序列，随后按照前述方法进行表皮内LL37注射诱导玫瑰痤疮样小鼠模型，在注射第2次LL37前进行第2次siRNA注射，加强敲降效果，造模完成后，观察并比较三组小鼠(Scr siRNA，siRNA#1和siRNA#2)表型之间的差异并进行取材，图2B为LL37造模两日后Scr siRNA和另外2条siRNA的Control组和LL37组玫瑰痤疮样皮炎的表现，可见LL37造模后Scr siRNA组小鼠皮损部位出现明显的炎症反应，包括红斑和毛细血管扩张，而在2条siRNA敲降组小鼠皮损的炎症反应明显减轻，本发明利用Image J软件对每组玫瑰痤疮样小鼠皮损处的面积大小进行测量， 结果显示Scr siRNA组LL37造模后红斑面积明显增加，2条siRNA的LL37组红斑面积较Scr的LL37组明显减小(如图3C)，其次对玫瑰痤疮样皮损红斑严重程度进行评分，结果显示Scr siRNA的LL37组造模后红斑评分明显增加，造模后2条siRNA的LL37组红斑评分明显减轻(如图3D)，According to claim 1, the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as rosacea is characterized in that: the present invention names the negative control sequence and the two siRNA sequences as ScrRNA and siRNA# 1 and siRNA#2 for the next study, first to explore the effect of knocking down Serpinb3a in the epidermis on the phenotype of rosacea-like mice, as shown in Figure 3A, 7-8 weeks old BALB/c wild-type female mice , the back hair was shaved, siRNA intradermally injected 1 day before, and the corresponding negative control sequence was intradermally injected into the experimental control group, followed by intraepidermal LL37 injection according to the aforementioned method to induce a rosacea-like mouse model. The second siRNA injection was performed before LL37 to strengthen the knockdown effect. After the modeling was completed, the differences in the phenotypes of the three groups of mice (Scr siRNA, siRNA#1 and siRNA#2) were observed and compared, and materials were collected, as shown in Fig. 2B is the expression of rosacea-like dermatitis in the Scr siRNA and other 2 siRNA Control group and LL37 group two days after LL37 modeling. It can be seen that after LL37 modeling, Scr siRNA group mice have obvious inflammatory reactions in the skin lesions, including erythema and capillary dermatitis. The blood vessels dilated, and the inflammatory response of the mouse skin lesions in the two siRNA knockdown groups was significantly reduced. The present invention used Image J software to measure the area of the rosacea-like mouse skin lesions in each group. The results showed that the Scr siRNA group LL37 After modeling, the erythema area increased significantly, and the erythema area of the 2 siRNA LL37 group was significantly smaller than that of the Scr LL37 group (as shown in Figure 3C). Secondly, the erythema severity of rosacea-like skin lesions was scored, and the results showed that the Scr siRNA LL37 group After modeling, the erythema score increased significantly, and after modeling, the erythema score of the LL37 group with two siRNAs was significantly reduced (as shown in Figure 3D).
    此外本发明比较了LL37造模后各组小鼠皮损部位的组织学情况，取造模部位皮损，石蜡包埋切片后进行HE染色，显微镜下观察并统计小鼠真皮层炎症细胞浸润数量，如图3E可见，Scr siRNA的LL37组真皮部位浸润的炎症细胞数量较Control组明显增加，2条siRNA的LL37组造模后真皮层浸润的炎症细胞数量较Scr siRNA的LL37组明显减少，同时本发明通过Prism9软件对浸润的炎症细胞数量进行统计分析，结果可见敲降Serpinb3a后2条siRNA的LL37组浸润的炎症细胞较Scr的LL37组明显减少(如图3F)，差异具有统计学意义，最后本发明收取小鼠皮损，提取总量RNA，通过qRT-PCR比较了上述各组小鼠皮损中玫瑰痤疮相关基因在mRNA表达水平上的差异，如图3G-H所示，Scr siRNA的LL37组较Control组的Il6和Tnf-α基因表达增加，而在敲降Serpinb3a后，2条siRNA的LL37组较Scr siRNA的LL37组上述两个基因表达水平明显回降，且差异具有统计学意义，以上结果表明，表皮内Serpinb3a的缺失从皮损炎症反应表现、组织病理学情况以及mRNA表达层面均能够明显改善LL37诱导的玫瑰痤疮样小鼠模型中玫瑰痤疮的发展。In addition, the present invention compares the histological situation of the mouse skin lesions in each group after LL37 modeling. The skin lesions of the modeling parts are taken, paraffin-embedded and sectioned, and then HE stained. The number of inflammatory cell infiltration in the dermis of the mice is observed under a microscope and counted. , as shown in Figure 3E, the number of inflammatory cells infiltrating the dermis in the Scr siRNA LL37 group was significantly higher than that in the Control group, and the number of inflammatory cells infiltrating the dermis in the 2 siRNA LL37 group was significantly lower than that in the Scr siRNA LL37 group. In the present invention, the number of infiltrating inflammatory cells was statistically analyzed by Prism9 software. The results showed that after knocking down Serpinb3a, the number of infiltrating inflammatory cells in the LL37 group with two siRNAs was significantly less than that in the Scr LL37 group (as shown in Figure 3F), and the difference was statistically significant. Finally, the present invention collects mouse skin lesions, extracts total RNA, and compares the differences in mRNA expression levels of rosacea-related genes in the skin lesions of the above-mentioned groups of mice by qRT-PCR, as shown in Figure 3G-H, Scr siRNA Compared with the Control group, the expressions of Il6 and Tnf-α genes in the LL37 group were increased, and after knocking down Serpinb3a, the expression levels of the above two genes in the LL37 group with two siRNAs were significantly lower than those in the LL37 group with Scr siRNA, and the difference was statistically significant. Significance, the above results show that the deletion of Serpinb3a in the epidermis can significantly improve the development of rosacea in the LL37-induced rosacea-like mouse model from the levels of inflammatory response, histopathology and mRNA expression.
  4. 根据权利要求1所述的SERPINB3/B4作为靶点在玫瑰痤疮等炎症性皮肤病治疗药物中的应用，其特征在于：本发明为明确表皮内敲降Serpinb3a对银屑病样小鼠表型的影响，选择前述的两条siRNA，进行小鼠耳部皮内注射，表皮内敲降Serpinb3a，按照既往已有研究方法外用咪喹莫特进行银屑病样小鼠模型建造，如图4A模式图所示，外用咪喹莫特前1天(记作day-1)进行siRNA皮内注射，实验对照组皮内注射对应的阴性对照序列，其后每日一次咪喹莫特乳膏外用，第2天进行第2次siRNA注射，加强敲降效果，第6天造 模完成后，为明确表皮内敲降Serpinb3a后对银屑病样小鼠皮损表型的影响，本发明造模第6天观察并比较三组小鼠(Scr siRNA，siRNA#1和siRNA#2)表型之间的差异并进行取材，根据既往研究[77]方法对小鼠皮损进行PASI评分及耳部厚度进行测量，可见siRNA的咪喹莫特组小鼠较Scr RNA咪喹莫特组小鼠皮损的红斑、鳞屑明显改善(如图4B)，PASI评分明显下降(如图4C)，耳部皮肤厚度亦明显下降(如图4D)，其次对各组小鼠皮损进行HE染色，结果可见，Scr siRNA咪喹莫特组表皮厚度明显增加，真皮层炎症细胞浸润明显，而两条siRNA的咪喹模特组表皮厚度及真皮层炎症细胞浸润程度明显改善(如图4E)，并对表皮层厚度、浸润的炎症细胞进行统计分析，发现二者在两条siRNA的咪喹莫特组较Scr siRNA的咪喹莫特组明显下降(如图4F-G)，为进一步明确Serpinb3a对银屑病相关基因分子水平的影响，本发明收取小鼠皮损，提取总量RNA，通过qRT-PCR比较了上述各组小鼠皮损中银屑病相关基因在mRNA表达水平上的差异，如图4H-I所示，Scr siRNA的咪喹莫特组较Control组Il6和Il-1β基因表达增加2条siRNA的咪喹莫特组中上述两个基因表达水平较Scr siRNA的咪喹莫特组有明显回降，差异具有统计学意义，以上结果表明，耳部表皮内Serpinb3a的缺失从皮损炎症反应表现、组织病理学情况以及mRNA表达层面均能够明显改善咪喹莫特诱导的银屑病样小鼠模型中炎症疾病的发展，According to claim 1, the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as rosacea is characterized in that: the present invention is to clarify the effects of knocking down Serpinb3a in the epidermis on the phenotype of psoriasis-like mice Influence, select the aforementioned two siRNAs, inject them intradermally into the ears of mice, knock down Serpinb3a in the epidermis, and construct a psoriasis-like mouse model with topical imiquimod according to previous research methods, as shown in Figure 4A schematic diagram As shown, siRNA was injected intradermally 1 day before topical imiquimod (denoted as day-1), and the corresponding negative control sequence was injected intradermally in the experimental control group, and then externally applied imiquimod cream once a day. The second siRNA injection was performed in 2 days to strengthen the knockdown effect. After the modeling was completed on the 6th day, in order to clarify the impact of knocking down Serpinb3a in the epidermis on the phenotype of psoriasis-like mice, the present invention modeled on the 6th day. The difference between the phenotypes of the three groups of mice (Scr siRNA, siRNA#1 and siRNA#2) was observed and compared every day, and the materials were collected. According to the method of previous research [77], the PASI score of mouse skin lesions and ear thickness were measured. Measurements showed that the erythema and scales of the mice in the imiquimod group of siRNA were significantly improved compared with the mice in the ScrRNA imiquimod group (as shown in Figure 4B), and the PASI score was significantly decreased (as shown in Figure 4C). It also decreased significantly (as shown in Figure 4D). Next, HE staining was performed on the skin lesions of mice in each group. The results showed that the thickness of the epidermis in the Scr siRNA imiquimod group increased significantly, and the infiltration of inflammatory cells in the dermis was obvious. The thickness of the epidermis and the degree of infiltration of inflammatory cells in the dermis in the model group were significantly improved (as shown in Figure 4E). Statistical analysis was performed on the thickness of the epidermis and the infiltrating inflammatory cells. The imiquimod group decreased significantly (as shown in Figure 4F-G). In order to further clarify the influence of Serpinb3a on the molecular level of psoriasis-related genes, the present invention collected mouse skin lesions, extracted the total amount of RNA, and compared the above-mentioned results by qRT-PCR. Differences in mRNA expression levels of psoriasis-related genes in skin lesions of mice in each group, as shown in Figure 4H-I, the expression of Il6 and Il-1β genes in the Scr siRNA group increased by 2 siRNAs compared with the Control group Il6 and Il-1β gene expression The expression levels of the above two genes in the imiquimod group of Scr siRNA were significantly lower than those in the imiquimod group of Scr siRNA, and the difference was statistically significant. , histopathological conditions and mRNA expression levels can significantly improve the development of inflammatory diseases in the imiquimod-induced psoriasis-like mouse model,
    综上所述，在小鼠表皮内敲降serpinb3a后，能够明显改善玫瑰痤疮样及银屑病样小鼠皮损处炎症反应。In summary, knocking down serpinb3a in the epidermis of mice can significantly improve the inflammatory response in skin lesions of rosacea-like and psoriasis-like mice.
  5. 根据权利要求1所述的SERPINB3/B4作为靶点在玫瑰痤疮等炎症性皮肤病治疗药物中的应用，其特征在于：为了更进一步明确SERPINB3/B4在体外对玫瑰痤疮中具体分子作用机制，本发明体外构建SERPINB3/B4过表达质粒，并通过转染试剂将过表达质粒转染人HaCaT细胞，72小时后收取细胞RNA，进行全转录组测序，并通过体内外实验对测序结果进行验证，According to the application of SERPINB3/B4 as a target in the treatment of rosacea and other inflammatory skin diseases according to claim 1, it is characterized in that: in order to further clarify the specific molecular mechanism of SERPINB3/B4 on rosacea in vitro, this Invented to construct the SERPINB3/B4 overexpression plasmid in vitro, and transfect the overexpression plasmid into human HaCaT cells with a transfection reagent, collect the cellular RNA after 72 hours, perform whole transcriptome sequencing, and verify the sequencing results through in vivo and in vitro experiments.
    本发明对过表达SERPINB3/B4的人HaCaT细胞中的差异基因表达值进行聚类分析，进而对基因表达值进行均一化处理从而得到如图5A，如图中的每一个小方格代表一个基因，蓝色表示基因表达下调，红色表示基因表达上调，颜色越深，表达的差异越明显，每一列表示每个样本不同基因的表达情况，每一行表示每个基因在不同样本中的表达情况，如图结果可见过表达SERPINB3/B4的人HaCaT细胞较正常对照之间基因表达差异显著，结果表明，在人HaCaT细胞中过表达SERPINB3/B4后基因表达较正常对照组模式发生明显变化，The present invention performs clustering analysis on the differential gene expression values in human HaCaT cells overexpressing SERPINB3/B4, and then normalizes the gene expression values to obtain as shown in Figure 5A, where each small square in the figure represents a gene , blue indicates down-regulation of gene expression, red indicates up-regulation of gene expression, the darker the color, the more obvious the difference in expression, each column indicates the expression of different genes in each sample, and each row indicates the expression of each gene in different samples, As shown in the figure, it can be seen that the gene expression of human HaCaT cells overexpressing SERPINB3/B4 is significantly different from that of the normal control.
    为进一步明确过表达SERPINB3/B4的人HaCaT细胞中信号通路的变化，本发明对过表达SERPINB3/B4的人HaCaT细胞和对照组细胞所有的基因表达量进行基因富集分析(Gene Set Enrichment Analysis，GSEA)，基于玫瑰痤疮为皮肤炎症性疾病及先前研究，炎症相关信号通路在玫瑰痤疮发生发展中发挥关键作用，所以本次研究着重关注与炎症相关联的信号通路，NF-κB信号通路几乎存在于所用的动物细胞中，参与细胞对外界刺激的各种反应，包括应激、细胞因子、自由基、微生物感染和紫外线照射等，既往研究发现此信号通路调控大量与炎症相关的基因表达，在本次研究中富集的炎症相关信号通路中NF-κB信号通路在过表达SERPINB3/B4的人HaCaT细胞中较对照组细胞系明显富集(如图5B-E)，这提示该信号通路可能参与了SERPINB3/B4调控玫瑰痤疮的发病，In order to further clarify the changes in the signaling pathways in the human HaCaT cells overexpressing SERPINB3/B4, the present invention carried out gene enrichment analysis (Gene Set Enrichment Analysis, Gene Set Enrichment Analysis, GSEA), based on rosacea is a skin inflammatory disease and previous research, inflammation-related signaling pathways play a key role in the development of rosacea, so this study focuses on signaling pathways associated with inflammation, NF-κB signaling pathway almost exists In the animal cells used, it is involved in various responses of cells to external stimuli, including stress, cytokines, free radicals, microbial infection and ultraviolet radiation. Previous studies have found that this signaling pathway regulates the expression of a large number of genes related to inflammation. Among the inflammation-related signaling pathways enriched in this study, the NF-κB signaling pathway was significantly enriched in the human HaCaT cells overexpressing SERPINB3/B4 compared with the control cell line (as shown in Figure 5B-E), which suggests that the signaling pathway may Involved in SERPINB3/B4 regulating the pathogenesis of rosacea,
    为明确在人HaCaT细胞中过表达SERPINB3/B4后在蛋白水平对NF-κB信号通路的影响，本发明通过转染试剂将过表达质粒转染人HaCaT细胞系72小时后收取细胞蛋白，免疫印迹法检测在人HaCaT细胞系中表达SERPINB3/B4后对p65/NF-κB的磷酸化水平(p-p65)，同时应用Image J软件对p-p65灰度进行扫描量化，如图5F所示，本发明发现在人HaCaT细胞系中表达SERPINB3/B4后p-p65表达明显上调，p65表达无明显差异，p-p65/p65上调， 结果表明NF-κB信号通路激活，同时，为了更加直观地了解表达SERPINB3/B4后对NF-κB信号通路的影响，本发明通过细胞爬片实验及免疫荧光检测过表达质粒转染人HaCaT细胞系72小时后对p65表达的影响，结果如图5G所示，在人HaCaT细胞中表达SERPINB3/B4后p65入核的细胞数目较对照组明显增多上述结果表明在在人HaCaT细胞中表达SERPINB3/B4在蛋白水平激活NF-κB信号通路，In order to clarify the effect of overexpressing SERPINB3/B4 on the NF-κB signaling pathway at the protein level in human HaCaT cells, the present invention transfected the overexpression plasmid into the human HaCaT cell line with a transfection reagent for 72 hours, and harvested the cell protein, immunoblotting The phosphorylation level of p65/NF-κB (p-p65) after SERPINB3/B4 expression in human HaCaT cell line was detected by using the method, and the gray scale of p-p65 was scanned and quantified by Image J software, as shown in Figure 5F, The present invention found that after expressing SERPINB3/B4 in the human HaCaT cell line, the expression of p-p65 was significantly up-regulated, the expression of p65 was not significantly different, and p-p65/p65 was up-regulated. The results showed that the NF-κB signaling pathway was activated. At the same time, in order to understand The effect of expressing SERPINB3/B4 on the NF-κB signaling pathway, the present invention detected the effect on the expression of p65 after 72 hours of transfection of the overexpression plasmid into the human HaCaT cell line by cell climbing experiments and immunofluorescence, the results are shown in Figure 5G, After expressing SERPINB3/B4 in human HaCaT cells, the number of p65 nuclear cells was significantly increased compared with the control group. The above results indicate that the expression of SERPINB3/B4 in human HaCaT cells activates the NF-κB signaling pathway at the protein level,
    既往研究发现，TNF-α可强烈诱导NF-κB信号的活化，为明确SERPINB3/B4对TNF-α诱导NF-κB信号活化的影响，本发明体外构建SERPINB3/B4的shRNA，建立稳定敲降SERPINB3/B4的人HaCaT细胞系，TNF-α孵育30分钟后，免疫印迹法检测敲降SERPINB3/B4后对TNF-α诱导NF-κB信号活化的影响，结果可见，构建稳定敲降SERPINB3/B4的人HaCaT细胞系对SERPINB3/B4的敲降效果明显，在正常对照组细胞中，TNF-α孵育后p-p65表达明显上调，而敲降SERPINB3/B4后，TNF-α诱导p-p65上调的情况明显改善，同时本发明对其条带灰度进行扫描，结果统计发现差异具有明显统计学意义(如图5H-I)，结果提示，人HaCaT细胞系中SERPINB3/B4的缺失阻止了TNF-α诱导的NF-κB信号的活化，同样，本发明通过细胞爬片实验及免疫荧光检测稳定敲降SERPINB3/B4对TNF-α诱导的p65入核情况的影响，如图5J-K可见在正常对照的人HaCaT细胞系中，TNF-α孵育后30分钟后p65几乎完全入核，而敲降SERPINB3/B4后p65入核的细胞数目较对照组明显减少，同时本发明对入核细胞数目进行统计，差异明显，具有统计学意义，Previous studies have found that TNF-α can strongly induce the activation of NF-κB signaling. In order to clarify the effect of SERPINB3/B4 on the activation of NF-κB signaling induced by TNF-α, the present invention constructed SERPINB3/B4 shRNA in vitro to establish a stable knockdown of SERPINB3 /B4 human HaCaT cell line, after TNF-α incubation for 30 minutes, Western blot was used to detect the effect of knocking down SERPINB3/B4 on the activation of NF-κB signal induced by TNF-α. The results showed that the stable knockdown of SERPINB3/B4 The human HaCaT cell line has a significant knockdown effect on SERPINB3/B4. In the normal control cells, the expression of p-p65 was significantly upregulated after TNF-α incubation, and after knockdown of SERPINB3/B4, TNF-α induced the upregulation of p-p65. The situation has been significantly improved. At the same time, the present invention scans the gray scale of its bands, and the statistical results show that the difference has obvious statistical significance (as shown in Figure 5H-I). The results suggest that the deletion of SERPINB3/B4 in the human HaCaT cell line prevents TNF- The activation of α-induced NF-κB signal, similarly, the present invention detects the effect of stably knocking down SERPINB3/B4 on TNF-α-induced p65 nuclear entry through cell slide experiments and immunofluorescence, as shown in Figure 5J-K. In the control human HaCaT cell line, p65 was almost completely incorporated into the nucleus after 30 minutes of TNF-α incubation, and the number of cells with p65 incorporated into the nucleus after knocking down SERPINB3/B4 was significantly reduced compared with the control group. Statistically, the difference is obvious and statistically significant,
    综上结果表明，体外过表达SERPINB3/B4后诱导NF-κB信号通路下游分子上调，体外敲降SERPINB3/B4后能够阻止TNF-α诱导的NF-κB信号的活化。In conclusion, overexpression of SERPINB3/B4 in vitro induces the upregulation of downstream molecules of NF-κB signaling pathway, and knockdown of SERPINB3/B4 in vitro can prevent the activation of TNF-α-induced NF-κB signaling.
  6. 根据权利要求1所述的SERPINB3/B4作为靶点在玫瑰痤疮等炎症性皮肤病治疗药物中的应用，其特征在于：炎症相关的趋化因子及细胞因子在炎症发生及进展中起到极其重要的作用，前期一系列的结果表明SERPINB3/B4 能够调控NF-κB信号通路，为进一步明确趋化因子及细胞因子在SERPINB3/B4调控玫瑰痤疮发病中的具体作用，本发明进一步对在人HaCaT细胞中过表达SERPINB3/B4后RNA-seq的结果进行分析，如图6A-B所示，趋化因子及细胞因子信号通路在SERPINB3/B4过表达的人HaCaT细胞中高度富集，同时本发明对样本的趋化因子及细胞因子相关的差异的表达值进行聚类分析，并对基因表达值进行均一化处理得到如图6C所示的如图，与正常对照相比，SERPINB3/B4过表达的人HaCaT细胞中炎症相关的趋化因子(CCL2，CCL5，CCL20，CXCL1，CXCL2，CXCL5，CXCL8，CXCL10，CXCL11)及细胞因子(IL1A，IL1B，IL6，TNFA)表达明显上调，According to the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as rosacea according to claim 1, it is characterized in that: inflammation-related chemokines and cytokines play an extremely important role in the occurrence and progression of inflammation A series of previous results showed that SERPINB3/B4 can regulate the NF-κB signaling pathway. In order to further clarify the specific role of chemokines and cytokines in the regulation of SERPINB3/B4 on the pathogenesis of rosacea, the present invention further investigated the human HaCaT cell Analysis of the results of RNA-seq after overexpression of SERPINB3/B4 in cells, as shown in Figure 6A-B, chemokine and cytokine signaling pathways are highly enriched in human HaCaT cells overexpression of SERPINB3/B4, and the present invention Cluster analysis was performed on the expression values of chemokine and cytokine-related differences in the samples, and the gene expression values were normalized to obtain the figure shown in Figure 6C. Compared with the normal control, SERPIN B3/B4 overexpression The expressions of inflammation-related chemokines (CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL5, CXCL8, CXCL10, CXCL11) and cytokines (IL1A, IL1B, IL6, TNFA) were significantly up-regulated in human HaCaT cells,
    为进一步明确上述细胞因子在SERPINB3/B4过表达的人HaCaT细胞中的表达水平，本发明利用qRT-PCR的方法检测人HaCaT细胞过表达SERPINB3/B4后上述基因的表达情况，结果(如图6D)可见，在人HaCaT细胞中过表达SERPINB3/B4后，趋化因子CCL2，CCL5，CCL20，CXCL2，CXCL9，CXCL10，CXCL11及细胞因子IL1B，IL6，TNF-α表达明显上调，结果表明在人HaCaT细胞过表达SERPINB3/B4后能够明显诱导炎症相关的趋化因子及细胞因子的表达上调，In order to further clarify the expression levels of the above-mentioned cytokines in human HaCaT cells overexpressing SERPINB3/B4, the present invention uses qRT-PCR to detect the expression of the above-mentioned genes in human HaCaT cells after overexpressing SERPINB3/B4, and the results (as shown in Figure 6D ) shows that after overexpressing SERPINB3/B4 in human HaCaT cells, the expression of chemokines CCL2, CCL5, CCL20, CXCL2, CXCL9, CXCL10, CXCL11 and cytokines IL1B, IL6, TNF-α was significantly up-regulated, the results showed that in human HaCaT cells Cells overexpressing SERPINB3/B4 can significantly induce the upregulation of inflammation-related chemokines and cytokines,
    通过小鼠皮内敲降Serpinb3a能够降低LL37诱导的玫瑰痤疮相关细胞因子(Il6，Tnf-α)的表达水平，为进一步明确玫瑰痤疮样小鼠表皮内敲降Serpinb3a对炎症相关的趋化因子及细胞因子的影响，本发明应用qRT-PCR的方法检测了上述相关因子的表达水平，结果如图6E可见，敲降Serpinb3a后，LL37诱导的Ccl5，Cxcl9，Cxcl10，Cxcl11及Il1β表达水平受到明显抑制，本发明应用同样的方法检测了敲降serpinb3a后银屑病样小鼠皮损中上述趋化因子变化情况，结果如图6F所示，可见敲降Serpinb3a后，IMQ诱导的Ccl5，Ccl20，Cxcl9，Cxcl10，Cxcl11表达水平受到明显抑制，Intradermal knockdown of Serpinb3a in mice can reduce the expression level of rosacea-related cytokines (Il6, Tnf-α) induced by LL37. The influence of cytokines, the present invention used qRT-PCR to detect the expression levels of the above-mentioned related factors, the results can be seen in Figure 6E, after knocking down Serpinb3a, the expression levels of Ccl5, Cxcl9, Cxcl10, Cxcl11 and Il1β induced by LL37 were significantly inhibited , the present invention used the same method to detect the changes of the above chemokines in the skin lesions of psoriasis-like mice after knocking down serpinb3a, and the results are shown in Figure 6F. , Cxcl10, Cxcl11 expression levels were significantly inhibited,
    同样的方法检测相关趋化因子及细胞因子的表达水平，结果5G如图所示，SERPINB3/B4诱导趋化因子CCL2，CCL5，CXCL9，CXCL10，CXCL11及细胞因子 IL-1β，IL-6，TNF-α明显上调，而加用SC75741共同孵育后上述趋化因子及细胞因子的表达水平明显回降，上述结果表明SC75741能够通过抑制SERPINB3/B4对NF-κB信号通路的诱导激活下调相关趋化因子及细胞因子的表达水平。The same method was used to detect the expression levels of related chemokines and cytokines. As shown in the figure, SERPINB3/B4 induced chemokines CCL2, CCL5, CXCL9, CXCL10, CXCL11 and cytokines IL-1β, IL-6, TNF -α was significantly up-regulated, and the expression levels of the above chemokines and cytokines were significantly decreased after co-incubation with SC75741. The above results indicated that SC75741 could down-regulate related chemokines by inhibiting the activation of SERPINB3/B4 on the NF-κB signaling pathway and expression levels of cytokines.
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