LU504282B1 - Application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as acne rosacea - Google Patents
Application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as acne rosacea Download PDFInfo
- Publication number
- LU504282B1 LU504282B1 LU504282A LU504282A LU504282B1 LU 504282 B1 LU504282 B1 LU 504282B1 LU 504282 A LU504282 A LU 504282A LU 504282 A LU504282 A LU 504282A LU 504282 B1 LU504282 B1 LU 504282B1
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- LU
- Luxembourg
- Prior art keywords
- serpinb3
- expression
- obviously
- acne rosacea
- mouse
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Abstract
The present invention discloses the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as acne rosacea, the expression of SERPINB3/B4 in skin lesions of these patients are defined through ELISA, immunohistochemistry and qRT-PCR and other means, and the expression of the Serpinb3a in the skin lesions of LL37-induced acne rosacea-like and imiquimod-induced psoriasis-like mouse models, through the siRNA intradermal injection, the expression of Serpinb3a in mouse epidermal cells is locally knocked down, SERPINB3/B4 is over-expressed or knocked down in human HaCaT cells by constructing plasmids, combined with RNA-seq and immunoblotting, immunofluorescence and other methods to further define the specific molecules mechanism of action of the SERPINB3/B4 in inflammatory skin diseases in vitro.
Description
Application of SERPINB3/B4 as a target in the treatment of 0004282 inflammatory skin diseases such as acne rosacea
The present invention relates to the technical field of the inflammatory skin diseases, specifically relates to the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as acne rosacea.
Background technology
The acne rosacea and the psoriasis are the more common chronic inflammatory skin diseases, the incidence rate is higher, and in the clinic is prone to repeat attacks, which brings great pain to patients, the pathogenesis of both has not been fully elucidated, most studies think that immune system dysfunction plays an important role in the pathogenesis of both; currently, treatment solutions for acne rosacea and psoriasis still need to be improved, therefore, it is of great significance to further clarify the pathogenesis of the two diseases and find new treatment solutions, protease function plays an important role in maintaining skin structure and regulating skin response to pathogens and allergens,in the process of previous research on the pathogenesis of acne rosacea,whole transcriptome sequencing is performed to total RNA of skin lesions in acne rosacea patients, it found that the SERPINB3/B4 is obviously up-regulated in the skin lesions of patients with acne rosacea,therefore, it is of great significance to discuss the specific mechanism of SERPINB3/B4 in the pathogenic process of inflammatory skin diseases.
The shortcomings of the prior art in the application of inflammatory skin diseases treatment medicines are: 1.the application of the traditional medicines does not have the concurrency that occurs along the inhibition quality in the process,which is not convenient for users to judge the response of inflammatory attack.
Object of the present invention proposes the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as acne rosacea, in order to solve the problems proposed in the background techniques described above-mentioned.
In order to achieve the objects,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 acne rosacea, the expression of SERPINB3/B4 in the serum and skin lesions of patients with acne rosacea and psoriasis are defined firstly through the ELISA, immunohistochemistry, qRT-PCR and immunofluorescence and other means, and the expression situation of Serpinb3a in the skin lesions of LL37-induced acne rosacea-like the mouse model and imiquimod-induced psoriasis-like the mouse model; and then intradermal injection through the siRNA, the expression of Serpinb3a in mouse epidermal cells is locally knocked down, to observe its effect on the skin lesion phenotype and histopathology in the inflammatory model, in vitro study is conducted mainly in human HaCaT cells; SERPINB3/B4 is overexpressed or knocked down in human HaCaT cells by constructing plasmids, the relationship with NF-kB signaling pathway is defined by combining with RNA-seq and immunoblotting,immunofluorescence and other methods; finally, SC75741(a potent NF-kB signaling pathway inhibitor) is selected to inhibit the
NF-kB signaling pathway in the epidermis of the mouse to clarify the role of NF-kB signaling pathway in the pathogenic process of acne rosacea; and CCK8 and flow analysis are used to select the appropriate concentration of SC75741 for in vitro study to further clarify the interaction between SERPINB3/B4 and NF-kB signaling pathway in the pathogenesis of acne rosacea and psoriasis.
Preferably, the present invention collects skin lesions of normal controls and acne rosacea patients, extracting total RNA, and detecting the expression situation of SERPINB3/B4 mRNA level between the two groups, the results showed that they are hardly expressed in normal control lesions, while the mRNA expression level of SERPINB3/ B4 in facial lesions of acne. pace rosacea patients is obviously higher than that in healthy controls (as shown in FIG.2 A B),at the same time, the present invention performs IGA score on the number of papules or pustules and skin erythema in patients with acne rosacea, and correlation analysis with SERPINB3/B4 mRNA expression levels in skin lesions of acne rosacea patients, the results show that with the increase of SERPINB3 mRNA expression levels, the IGA score of the lesion is obviously increased,the results show that the expression level of SERPINB3 mRNA are positively correlated with IGA score (as shown in FIG.2C); in addition, the results of the present invention show that with an increase in the level of SERPINB4 mRNA expression, the IGA score of skin lesions is obviously upgraded, the results show that the expression level of SERPINB4 mRNA is positively correlated with the severity of skin lesions (as shown in FIG.2D); at the same time, the present invention collects the skin tissue of the facial lesions of acne rosacea patients for immunohistochemical staining, the results show that compared with normal healthy controls, the high abundance of SERPINB3/B4 expression can be detected in the whole thickness of epidermal keratinocytes in patients with acne rosacea, and it is mainly expressed in the cytoplasm, there is almost no expression in the whole skin layer of healthy controls (as shown in FIG.2E-F),the results show that SERPINB3/B4 are obviously higher in the skin lesions of acne rosacea patients than the healthy controls;
In order to further define the expression situation of SERPINB3/B4 in psoriasis skin lesions, similarly, firstly, the present invention collects healthy controls and skin lesions of psoriasis patients, total RNA is extracted from skin lesions, the difference of SERPINB3/B4 mRNA level in skin between the two groups are detected through the qRT-PCR, as shown in FIG.2G-H, it can be seen that the expression of SERPINB3/B4 in skin lesions of healthy controls with the skin psoriasis patient is obviously increased at the mRNA level, at the same time,SERPINB3/B4 immunohistochemical staining is carried out the skin lesions, the results are shown in FIG.2I-J that the psoriasis skin lesions are obviously thicker than the epidermis layer of normal skin,and express higher abundance SERPINB3/B4, the above-mentioned results all indicate that
SERPINB3/B4 1s activated in the skin lesions of patients with psoriasis; in order to further study the role of the SERPINB3/B4 in the pathogenic process of the acne rosacea,the present invention 1s based on the modeling mode used in previous research,a mouse model of acne rosacea induced by antimicrobial peptide LL37 is established, the skin lesions of this model is highly similar to the inflammatory response pattern and the human acne rosacea, the present invention selects 7-8 weeks BALB/c female mice and removes the back hair of mice, one day later, SOUL (320uM) dose of LL37 is injected into the back of each mouse,the injections are given at 12-hour intervals, then the mice in the control group are given an equal dose of sterile 1xPBS solution, consecutive injection for 2 days,a total of 4 times,after the model is successfully established, the general phenotype of the skin is observed and the material is taken, secondly, the present invention collects mouse skin lesion tissue and extracts total RNA the mRNA expression situation of Serpinb3a in skin lesions of acne rosacea-like the mouse is detected, the results show that the skin lesions of acne rosacea-like the mouse are obviously up-regulated compared with the experimental control group Serpinb3a at the mRNA level (as shown in FIG.2K), the difference is statistically significant, among the serpinb3a is an analogue of SERPINB3/B4, the present invention further detects the expression situation of Serpinb3a protein level in acne rosacea-like the mouse lesions through the immunofluorescence, the results show that the expression of Serpinb3a in the skin lesions of acne rosacea-like the mouse model are similar to the expression of SERPINB3/B4 in human rosacea skin lesions,the expression of
Serpinb3a around hair follicles and in the whole layer epidermis of acne rosacea-like mouse model skin lesions is obviously enhanced,the expression for cytoplasm (as shown in FIG.2L), in summary, the results show that the expression level of Serpinb3a in acne rosacea-like the mouse are obviously up-regulated.
at the same time, the present invention is based on the modeling mode used in previous research, the consecutive topical imiquimod for 6 days to establish a psoriasis -like mouse model, the skin lesions of mice are collected and the total RNA 1s extracted from the skin lesion tissues of mice, the mRNA expression of Serpinb3a in skin lesions of psoriasis-like mouse is detected 5 through qRT-PCR,the results show that the skin lesions of psoriasis-like the mouse are obviously up-regulated compared with the experimental control group Serpinb3a at the mRNA level (as shown in FIG.2M),to further the mouse skin lesions is collected for immunofluorescent staining,it can be seen that Serpinb3a is highly expressed in the skin lesions of psoriasis-like mice (as shown in FIG.2N); in order to further explore the SERPINB3/B4 in the epidermis in the acne rosacea and pathogenic process of the psoriasis,the present invention constructs a mouse model of Serpinb3a of the epidermis of a specific knockdown mouse through siRNA and intradermal injection of
LL37 and external application of imiquimod are performed to establish the acne rosacea-like and psoriasis-like mouse models,to observe the influence of epidermal knockdown of Serpinb3a on the phenotype of rosacea-like the mouse and psoriasis-like the mouse.
Preferably, the present invention names the negative control sequence, and two siRNA sequences respectively names as Scr RNA, siRNA #1 and siRNA #2 for subsequent research, firstly, the influence of intra-epidermal knockdown Serpinb3a on the phenotype of acne rosacea-like the mouse is discussed,as shown in FIG.3A pattern diagram, 7-8 weeks old BALB/c wild-type female mice are shaved off their back hair, the intradermal injection of siRNA is performed 1 day before, the negative control sequence corresponding to intradermal injection in the experimental control group,subsequently, intra-epidermal LL37 injection induced acne rosacea-like mouse model is performed according to the above method,the second siRNA injection is performed before the second LL37 injection, enhances knock-down influence, after the mold is completed, the differences in phenotypes among the three groups of mice (Scr siRNA,
siRNA # 1 and siRNA # 2) are observed and compared, and the material is taken,FIG.2B shows the LL37 modeling two days after Scr siRNA, the manifestations of acne rosacea-like dermatitis in the control group and LL37 group of the other two siRNAs,it can be seen that after LL37 modeling, there is a significant inflammatory response in the skin lesions of the Scr siRNA group in the mouse lesions,comprising the erythema and telangiectasia, but the inflammatory response of the skin lesions in the two siRNA knockdown groups is obviously reduced, the present invention uses Image J software to measure the area size of each group of acne rosacea-like mouse skin lesions, the results-show that the erythema area of Scr siRNA group is obviously increased after LL37 modeling, the erythema area of the LL37 group of the two siRNAs are obviously lower than that of the LL37 group of Scr (as shown in FIG.3C), secondly, the severity degree of erythema of the acne rosacea-like skin lesion is scored, the results show that the erythema score of LL37 group of Scr siRNA are obviously increased after modeling, the erythema score of LL37 group of two of the Scr siRNA are obviously reduced after modeling,(as shown in FIG.3D);
in addition,the present invention compares the histology situation of the mouse skin lesions in each group after LL37 modeling,to take the skin lesions of the molding part, after paraffin embedded sections to perform HE staining, the number of the inflammatory cell infiltrates in the corium layer of the mouse is observed and counted under the microscope, as shown in FIG.3E, the number of inflammatory cells infiltrate in the corium part that the Scr siRNA LL37 group is obviously increased than the Control group, LL37 group of 2 siRNAs after modeling, the number of inflammatory cells infiltrate in the corium layer that is obviously reduced compared with that in the LL37 group of Scr siRNA, at the same time, the present invention performs a statistical analysis of the number of infiltrated inflammatory cells through Prism9 software, the results show that the number of inflammatory cells infiltrated by the LL37 group of the two siRNAs after knocking down Serpinb3a are obviously reduced compared with that in the LL37 group of Scr(as shown in FIG.3F), the difference is statistically significant, finally, the present. SER invention collects the mouse skin lesions and extracts total RNA, the differences in mRNA expression levels of acne rosacea-related genes in the skin lesions of mice in the above groups are compared through qRT-PCR as shown in FIG.3G-H, the expression of 116 and Tnf-agenes in the LL37 group of Scr siRNA is increased compared to the Control group, and after knocking down Serpinb3a, the expression levels of the above two genes in the LL37 group of 2 siRNA are obviously reduced compared to the LL37 group of Scr siRNA, and the difference is statistically significant,the above results indicate that the absence of Serpinb3a in the intra-epidermis can obviously improve the development of acne rosacea in the LL37-induced acne rosacea-like mouse model from the aspects of skin inflammatory response, histopathology and mRNA expression layer.
Preferably, the present invention aims to clarify the effect of knocking down Serpinb3a in the intra-epidermis on the phenotype of psoriasis-like the mouse, selects the two siRNA above, the ear of the mouse intra-dermal injection is carried out, the intra-epidermal knocks down the
Serpinb3a, the external application imiquimod is used to construct a psoriasis-like the mouse model according to previous research methods,as shown in FIG.4A pattern diagram, intra-dermal injection of siRNA is performed 1 day before external application imiquimod (denoted as day-1), the negative control sequence corresponding to the intradermal injection in the experimental control group, thereafter imiquimod emulsifiable paste external application once a day, the second siRNA injection is performed on the second day to enhance the knockdown influence, after the modeling is completed on the 6th day, in order to define the influence of intra-epidermal knockdown after Serpinb3a on the phenotype of psoriasis-like mice skin lesions,on day 6 of modelling of the present invention, the differences in phenotypes among the three groups of mice (Scr siRNA, siRNA # 1 and siRNA # 2) are observed and compared, and the material is taken, according to previous research [77] ,the PASI score and ear thickness of the skin lesions in mice are measured, it can be seen that the erythema and scales of the mice in the siRNA imiquimod group are obviously improved compared with the mice in the Scr RNA imiquimod group (as shown in FIG.4B), the PASI score is obviously decreased(as shown in FIG.4C), the thickness of the ear and skin are also obviously decreased (as shown in FIG.4D), secondly, HE staining is performed on the skin lesions of each group of mice, as a result, it can be seen that Scr siRNA imiquimod group epidermal thickness is obviously increased, skin corium inflammatory cell infiltration is obvious, and the imiquimod group of two siRNAs epidermal thickness and the degree of infiltration of inflammatory cells in the corium layer are obviously improved (as shown in FIG.4E), and statistical analysis of epidermal thickness and infiltrated inflammatory cells are performed, it was found that the imiquimod group of two siRNAs are obviously decreased compared with the Scr siRNA (as shown in FIG.4F-G), in order to further define the influence of Serpinb3a on the molecular level of psoriasis related-genes, the present invention collects the mouse skin lesions and extracts total RNA, the differences in mRNA expression levels of psoriasis-related genes in the skin lesions of mice in the above groups are compared through the qRT-PCR as shown in FIG 4H-I, the imiquimod group of Scr siRNA increases to 2 compared with control group 116 and II-1B gene expression, and the expression levels of the above two genes in the miquimod group are obviously decreased compared with the imiquimod group of Scr siRNA, the difference is statistically significant, the above results indicate that the absence of Serpinb3a in the ear epidermis can obviously improve the development of inflammatory diseases in the imiquimod-induced psoriasis-like the mouse model from the aspects of inflammatory response of skin lesions, histopathology situations and mRNA expression layers. in conclusion, knockdown of serpinb3a in mouse intra-epidermis can obviously improve the inflammatory response in skin lesions of acne rosacea-like and psoriasis-like of the mouse.
Preferably, in order to further define the specific molecular mechanism action of
SERPINB3/B4 on acne rosacea in vitro,the present invention constructs SERPINB3/B4 over-expression plasmids in vitro,and human HaCaT cells are transfected with over-expressed plasmids through transfection reagent, after 72 hours, the cell RNA is collected and whole transcriptome sequencing is performed, the sequencing results are verified by in vivo and in vitro experiments; the present invention performs cluster analysis of differential gene expression values in human HaCaT cells over-expressing SERPINB3/B4, then, the gene expression values are homogenized treatment to obtain FIG.5A, as shown in the FIG, each small square represents a gene, blue represents down-regulation gene expression,red represents up-regulation gene expression,the deeper of the color, the difference of expression is more obvious, each column represents the expression situation of different genes in each sample, and each row represents the expression situation of each gene in different samples,as shown in the fig, the results can be seen that the gene expression of human HaCaT cells over-expressing SERPINB3/B4 are obviously different compared with the normal controls the results show that the gene expression after over-expressing of SERPINB3/B4 in human HaCaT cells are obviously changed compared with the normal control pattern; in order to further define the changes in signaling pathways in human HaCaT cells over-expressing SERPINB3/B4, the present invention performs gene set enrichment analysis (GSEA) on all gene expression amounts of human HaCaT cells and control cells over-expressing
SERPINB3/B4,based on the skin inflammatory diseases of the acne rosacea and previous research,the inflammation-related signaling pathways play a key role in the development of acne rosacea, therefore, the present research focuses on the signaling pathways associated with inflammation, the NF-kB signaling pathway is existed in almost all animal cells used and is involved in various cellular responses to external stimuli, including stress, cytokines, free radicals, microbial infections, and ultraviolet radiation, previous studies have found that this signaling pathway regulates the expression of a large number of genes related to the inflammation, among the inflammation-related signaling pathways of the set enrichment in this study, the NF-kB signaling pathway is obviously enriched in human HaCaT cells over-expressing SERPINB3/B4 compared with the control group (as shown in FIG.5B-E), this indicates that the signaling pathway may be involved in SERPINB3/B4 regulation of the pathogenesis of acne rosacea; in order to define the influence of SERPINB3/B4 over-expressing on NF-kB signaling pathway at the protein level in human HaCaT cells, the present invention transfects an over-expressing plasmid into a human HaCaT cell line through transfection reagent, the cell protein is collected after 72 hours, the phosphorylation level of p65/ NF-KkB (p-p65) is detected by immunbolting after SERPINB3 / B4 expression in human HaCaT cell line, at the same time,
Image J software is used to scan and quantify the p-p65 gray scale, as shown in FIG.SF, the present invention found that the expression of p-p65 is obviously up-regulation after the expression of the SERPINB3/B4 in human HaCaT cell line, there is no significant difference in p65 expression, p-po5/p6S is up-regulation, the results show that the NF-KB signaling pathway are activated, in order to more intuitively understand the influence of the expression of the
SERPINB3/B4 on NF-kB signaling pathway, in the present invention, the influence of p65 expression in human HaCaT cell line 72 hours after transfection of over-expression plasmid is detected through the cell round coverslip experiment and immunofluorescence the results are shown in FIG.5G, after expression SERPINB3/B4 in people's HaCaT cell, the number of cells in p65 nucleation are obviously increased compared with the control group,the above results indicate that the expression of SERPINB3/B4 in human HaCaT cells activates the NF-kB signaling pathway at the protein level, the previous studies have found that TNF-acan strongly induce the activation of NF-kB signal, in order to define the influence of SERPINB3/B4 on TNF-a-induced NF-kB signal activation, the present invention constructs shRNA of the SERPINB3/B4 in vitro and establishes a human HaCaT cell line that stably knocks down SERPINB3/B4, after TNF-ais incubated for 30 minutes, immunoblotting method detects the influence after knockdown SERPINB3/B4 on
TNF-a-induced NF-kB signaling activation, the results can be seen that the construction of human HaCaT cell line with stable knockdown of SERPINB3/B4 has obvious knockdown influence on SERPINB3/B4,in normal control cells, after TNF-a incubation, the expression of p-p65 is obviously up-regulated, and after knocking down SERPINB3/B4, the situation of the
TNF-ainduction up-regulation of p-p65 is obviously improve,at the same time, the present invention scans the strip gray scale, as a result statistics finds that the difference has obvious statistical significance (as shown in FIG.SH-I), the results show that the absence of
SERPINB3/B4 in human HaCaT cell line prevents the activation of TNF-a-induced NF-KB signaling, similarly, the present invention detects the influence of stable knockdown
SERPINB3/B4 on TNF-a-induced p65 nucleation through the cell round coverslip experiment and immunofluorescence,as shown in FIG. 5J-K, it can be seen in the normal control human
HaCaT cell line,after incubation with TNF-a, for 30 minutes, p65 almost completely entered the nucleus, after knocking down SERPINB3/B4,the number of cells nucleated by p65 are obviously decreased compared with the control group,at the same time, the present invention counts the number of nuclear cells, and the difference is obvious and statistically significant; in conclusion, the results show that after over-expression of SERPINB3/B4 in vitro induces molecular up-regulation downstream of NF-xB signaling pathway, after knockdown of
SERPINB3/B4 in vitro can prevent TNF-a-induced NF-kB activation.
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 the
SERPINB3/B4 can regulate the NF-kB signaling pathway,in order to further define the specific role of chemokines and cytokines in SERPINB3/B4 regulating the pathogenesis of acne rosacea,
the present invention further analyzes the results of RNA-seq after over-expression of
SERPINB3/B4 in human HaCaT cells, as shown in FIG.6A-B,chemokines and cytokines signaling pathways are highly enriched in SERPINB3/B4 over-expression human HaCaT cells, at the same time, the present invention performs cluster analysis on the expression values of the difference of the chemokines and cytokines-related in samples,and the gene expression values are homogenized treatment to obtain as shown in FIG.6C, compared with normal controls, the expression of inflammation-related chemokines (CCL2, CCLS, CCL20, CXCL1, CXCL2,
CXCLS, CXCL8, CXCL10, CXCL11 ) and cytokines (IL1 A, IL1 B, IL6, TNFA ) in human
HaCaT cells over-expressing SERPINB3/B4 are obviously up-regulated; in order to further define the expression level of the above cytokines in human HaCaT cells over-expressing in the SERPINB3/B4, the present invention uses the qRT-PCR method that detects the expression of the above genes after SERPINB3/B4 of the over-expression in human
HaCaT cells,the results can be seen as shown in FIG.6D, after the SERPINB3/B4of the over-expression in human HaCaT cells, the expression of chemokines CCL2, CCLS, CCL20,
CXCL2 , CXCL9, CXCL10, CXCL11 and cytokines ILIB, IL6 TNF-a are obviously up-regulated, the results show that after the SERPINB3/B4 of the over-expression in human
HaCaT cells can obviously induce the up-regulation of inflammation-related chemokines and cytokines; the intra-dermal knockdown of Serpinb3a through the mouse can able to reduce the expression levels of LL37-induced acne rosacea-related cytokines( 116, Tnf-a), in order to further define the influence of intra-epidermal knockdown of Serpinb3a in acne rosacea-like the mouse on inflammation-related chemokines and cytokines, the present invention applies the method of qRT-PCR that the expression levels of the above related factors are detected, the results can be seen as shown in FIG.6E, after knocking down the Serpinb3a, the expression levels of Ccl5,
Cxcl9, Cxcl10, Cxcl11 and 111 induced by LL37 are obviously inhibited, the present invention applies the same method to detect the changes situations of above chemokines in the skin lesions of psoriasis-like the mouse after knocking down serpinb3a, the results are shown in FIG.6F,it can be seen that the expression levels of Ccl5, Ccl20, Cxcl9, Cxcl10 and Cxcl11 induced by IMQ are obviously inhibited after knocking down Serpinb3a; the expression levels of related chemokines and cytokines are detected by the same method, the results are shown in FIG.5G, the SERPINB3/B4 induced chemokines CCL2, CCLS, CXCL9,
CXCL10, CXCLI1 and cytokines IL-1P, IL-6, TNF-a are obviously up-regulated,after co-incubation with SC75741, the expression levels of the above chemokines and cytokines are obviously decreased, the above results show that SC75741 can down-regulate the expression levels of related chemokines and cytokines by inhibiting the induced activation of NF-kB signaling pathway by SERPINB3/B4.
Compared with the prior art, the present invention has the following beneficial effects: 1. Firstly, the present invention reports the expression situation of SERPINB3/B4 in skin lesions of patients with acne rosacea and psoriasis,and its correlation with disease severity; in this study of the present invention, two chronic inflammatory skin diseases, acne rosacea and psoriasis are used to illustrate that SERPINB3/B4 promotes inflammatory response through
NF-kB signaling pathway,the present invention find that the SC75741(potent inhibitor of the
NF-KB signaling pathway)can obviously improve the LL37-induced inflammatory response in the skin lesions of acne rosacea-like the mouse, and it obviously inhibited the activation of
NF-kB signaling pathway induced by SERPINB3/B4. Therefore, the present invention provides a new explanation for the pathogenesis of acne rosacea and psoriasis, and provides a new idea for the treatment of two chronic inflammatory skin diseases.
Description of attached drawings
FIG.1 is a work flow diagram of the present invention.
FIG.2 is the expression level of SERPINB3 mRNA in the skin lesions of A healthy control and acne rosacea patients of the present invention; correlation analysis between IGA score and SERPINB3 mRNA expression levels in skin lesions of patients with B acne rosacea; the expression level of SERPINB3 mRNA in the skin lesions of C healthy control and acne rosacea patients; correlation analysis between IGA score and SERPINB3 mRNA expression levels in skin lesions of patients with D acne rosacea,
E-F immunohistochemical staining (IHC) detects the expression situation of SERPINB3/B4 in acne rosacea lesions of patients skin relative healths collator's normal skin slice; Epi indicates epidermis;Der indicates corium,; the mRNA expression level of SERPINB3 and SERPINB4 in the skin of G-H healthy controls and psoriasis patients; HS is a healthy control group, and psoriasis is a psoriasis group;
I-J immunohistochemistry detects the expression situation of SERPINB3 in the normal skin of psoriasis patients and healthy controls; D immunohistochemistry detects the expression situation of SERPINB4 in the normal skin of psoriasis patients and healthy controls; the mRNA expression result of Serpinb3a in the skin lesions of mice in the LL37 group and the K control group; immunofluorescence staining results of Serpinb3a in skin lesions of L control mice and
LL37 mice, Serpinb3a (red) is localized in the cytoplasm, and DAPI staining (blue) is nuclear localization; the mRNA expression result of Serpinb3a in the skin lesions of mice in the IMQ group and the M control group; immunofluorescence staining results of Serpinb3a in skin lesions of N control mice and
IMQ mice, Serpinb3a (red) is localized in the cytoplasm, and DAPI staining (blue) is nuclear localization; ruler: SOum.
FIG.3 is an experimental mode diagram of the À mouse of the present invention;
B the upper fig is a representative gross view of LL37-induced acne rosacea-like skin lesions in a mouse model; the fig below is the enlarged result view of the stereoscope;
C each group mouse erythema area statistics result;
D the statistical analysis result after scoring the severity degree of erythema in each group of mice;
E is the result of HE staining of skin lesion tissue corresponding to D fig;
F the inflammatory cells infiltrated in the skin lesions of each group are statistically analyzed;
G -HqRT-PCR detects the mRNA expression of 116 and Tnf-a in the skin lesions of mice in each group.
FIG .4 is an experimental mode diagram of the A mouse;
B the enlarged result diagram of the stereoscope of skin lesions in each group mouse model the IMQ is imiquimod group;
C PASI score at skin lesions in each group mouse;
D the statistical results of ear thickness of each group mouse, length unit: mm;
E is the result of HE staining of skin lesion tissue corresponding to C chart, ruler: SOum,;
F the inflammatory cells infiltrated in the skin lesions of each group mouse is statistically analyzed;
G the thickness of the epidermis layer is measured by HE staining results in each group mouse under the microscope, and statistical analysis, length unit: micron; the result diagram of the qRT-PCR of the mRNA expression level of 116 and Il-1P in the skin lesions of each group mouse.
FIG.S is a cluster analysis of the transcriptome data difference gene of the over-expressing
SERPINB3/B4 of the AHaCaT cells in the present invention compared with the blank control group cells,each group of 3 transfected cells ;among them, red represents high gene expression, blue represents low gene expression; the results of the B-EGSEA analysis show that the ranking of KEGG pathways enriched by overlapping differentially expressed genes is compared with SERPINB3/B4 and VECTOR groups in human HaCaT cells, among them, NF-kB signaling pathways are indicated by red boxes;
F immunblotting detects changes situations in p-p65 protein levels 72 hours after human
HaCaT cells are transfected with SERPINB3/B4 over-expressing plasmids;quantitative analysis of protein levels through the Image] software;
G left view shows the expression and localization of p65 after 72 hours of immunofluorescence detection of human HaCaT cells transfected with SERPINB3/B4 over-expressing plasmids, an arrow out indicates that p65 is in the nucleus, DAPI staining (blue) indicates the position of the nucleus, ruler: SOum; right view: statistical analysis is performed on the positive cells of p65 into the nucleus, and the difference is statistically significant;
H-I left view shows the establishment of human HaCaT cells with knockdown of
SERPINB3/B4 in vitro,cell lines are incubated with 100 ng/ml TNF-o for 30 minutes, the expression variations of p-p65 in the knockdown group are detected through the immunoblotting compared with the control group; right view is carried out the statistical analysis of p-p65 gray scale by ImageJ software, the difference is statistically significant;
J-K shows the establishment of human HaCaT cells with knockdown of SERPINB3/B4 in vitro,cell lines are incubated with 100 ng/ml TNF-afor 30 minutes, the expression and localization of p65 at the protein level in the knockdown group are detected through the immunoblotting compared with the control group, the difference is statistically significant, p65 (red) membrane expression is activated that enters the nucleus, which is nuclear expression,
DAPI staining (blue) indicates nuclear localization, scale: S0um.
FIG.6 shows the A-BGSEA analysis results of the present invention: the over-expression of
SERPINB3/B4 in human HaCaT cells is compared with VECTOR blank control group,Chemokines and cytokines signaling pathways are highly enriched in human HaCaT cells over-expressing SERPINB3/B4; the CHaCaT cell is a cluster analysis of the transcriptome data difference gene of the over-expressing SERPINB3/B4 of the AHaCaT cells in the present invention compared with the blank control group cells,each group of 3 transfected cells; among them, red represents high gene expression, blue represents low gene expression;
DqRT-PCR detects the expression levels of chemokines CCL2, CCLS, CCL20, CXCL2,
CXCL9, CXCL10, CXCL11 and cytokines IL1B, IL6, TNF-q,
E-FqRT-PCR detects changes of chemokines in skin lesions in acne rosacea-like mouse models and psoriasis-like mouse models after knocking down Serpinb3a; the mRNA levels of inflammatory factors are detected by GqRT-PCR.
Specific embodiments
The following will be combined with the accompanying drawings in the examples of the present invention, the technical solutions in the examples of the present invention will be clearly and completely described, it is clear that the described examples are only a part of the examples of the present invention, and not all of the examples. Based on the examples in the present invention, all other examples obtained by ordinary technicians in the field without making creative results, are belonged the scope of protection of the present invention.
In the description of the present invention, it is necessary to understand that the terms “top”, “bottom”, “inner”, “outer”, “front end” “rear end” “two ends” “one end” “the other end” etc, they are based on the orientation or position relationship shown in the attached drawings, it is only to facilitate the description of the present invention or to simplify the description, but it is not to indicate or imply that the device or element referred to must have a particular orientation,
it should be constructed and operated in a particular orientation, and therefore, it cannot be understood as a limitation to the present invention. In addition, the terms "first" "second” are only used for distinguishing description, and they can not be understood as indicating or implying the relative importance.
In the description of the present invention, it should be noted that unless expressly specified and limited, the terms “installation”, “adjacent”, “connection” should be understood in a broad sense, for example, it can be a fixed connection, a removable connection, or an integrated connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly through an intermediate medium, or it can be a connection connection between the inside of two elements. For these general technicians in this field, the specific meaning of the above terms in the present invention can be understood on a case-by-case basis.
As shown in FIG.1-FIG.6, the present invention provides the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as acne rosacea;
Example 1, the application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as acne rosacea,characterized by:firstly the expression of SERPINB3/B4 in the skin lesions of acne rosacea patients and psoriasis patients and the expression of Serpinb3a in the skin lesions of LL37-induced acne rosacea-like mouse model and imiquimod-induced psoriasis-like mouse model are determined through the ELISA the immunocytochemistry,the qRT-PCR and the immunofluorescence; and then intradermal injection through the siRNA the expression of Serpinb3a in mouse epidermal cells is locally knocked down, to observe its effect on the skin lesion phenotype and histopathology in the inflammatory model, in vitro study is conducted mainly in human HaCaT cells; SERPINB3/B4 is overexpressed or knocked down in human HaCaT cells by constructing plasmids, the relationship with NF-kB signaling pathway is clarified by combining with RNA-seq and immunoblotting,immunofluorescence and other methods;
finally, SC75741(a potent NF-kB signaling pathway inhibitor)is selected to inhibit the
NF-kB signaling pathway in the epidermis of the mouse to clarify the role of NF-kB signaling pathway in the pathogenic process of acne rosacea, and CCK8 and flow analysis are used to select the appropriate concentration of SC75741 for in vitro study to further clarify the interaction between SERPINB3/B4 and NF-KB signaling pathway in the pathogenesis of acne rosacea and psoriasis.
Example 2, the present invention collects skin lesions of normal controls and acne rosacea patients, extracting total RNA, and detecting the expression situation of SERPINB3/B4 mRNA level between the two groups,the results showed that they are hardly expressed in normal control lesions, while the mRNA expression level of SERPINB3/ B4 in facial lesions of acne rosacea patients is obviously higher than that in healthy controls (as shown in FIG.2 A B),at the same time,the present invention performs IGA score on the number of papules or pustules and skin erythema in patients with acne rosacea, and correlation analysis with SERPINB3/B4 mRNA expression levels in skin lesions of acne rosacea patients,the results show that with the increase of SERPINB3 mRNA expression levels,the IGA score of the lesion is obviously increased,the results show that the expression level of SERPINB3 mRNA is positively correlated with IGA score (as shown in FIG.2C); in addition, the results of the present invention show that with an increase in the level of SERPINB4 mRNA expression, the IGA score of skin lesions is obviously upgraded, the results show that the expression level of SERPINB4 mRNA is positively correlated with the severity of skin lesions (as shown in FIG.2D); at the same time, the present invention collects the skin tissue of the facial lesions of acne rosacea patients for immunohistochemical staining,the results show that compared with normal healthy controls, the high abundance of SERPINB3/B4 expression can be detected in the whole thickness of epidermal keratinocytes in patients with acne rosacea, and it is mainly expressed in the cytoplasm, there is almost no expression in the whole skin layer of healthy controls (as shown in FIG.2F-F),the results show that SERPINB3/B4 are obviously higher in the skin lesions of acne rosacea patients than the healthy controls; in order to further define the expression situation of SERPINB3/B4 in psoriasis skin lesions,similarly,firstly,the present invention collects healthy controls and skin lesions of psoriasis patients,total RNA is extracted from skin lesions, the difference of SERPINB3/B4 mRNA level in skin between the two groups are detected through the qRT-PCR,as shown in
FIG.2G-H, it can be seen that the expression of SERPINB3/B4 in skin lesions of healthy controls with the skin psoriasis patient is obviously increased at the mRNA level,at the same time, SERPINB3/B4 immunohistochemical staining is carried out the skin lesions,the results are shown in FIG.2I-J that the psoriasis skin lesions are obviously thicker than the epidermis layer of normal skin,and express higher abundance SERPINB3/B4, the above-mentioned results all indicate that SERPINB3/B4 1s activated in the skin lesions of patients with psoriasis; in order to further study the role of the SERPINB3/B4 in the pathogenic process of the acne rosacea,the present invention is based on the modeling mode used in previous research,a mouse model of acne rosacea induced by antimicrobial peptide LL37 is established, the skin lesions of this model is highly similar to the inflammatory response pattern and the human acne rosacea,the present invention selects 7-8 weeks BALB/c female mice and removes the back hair of mice, one day later, SOUL (320uM) dose of LL37 is injected into the back of each mouse, the injections are given at 12-hour intervals,then the mice in the control group are given an equal dose of sterile 1xPBS solution, consecutive injection for 2 days, a total of 4 times, after the model is successfully established, the general phenotype of the skin is observed and the material is taken, secondly, the present invention collects mouse skin lesion tissue and extracts total RNA, the mRNA expression situation of Serpinb3a in skin lesions of acne rosacea-like the mouse is detected, the results show that the skin lesions of acne rosacea-like the mouse are obviously up-regulated compared with the experimental control group Serpinb3a at the mRNA level (as shown in FIG.2K),the difference is statistically significant, among the serpinb3a is an analogue of
SERPINB3/B4, the present invention further detects the expression situation of Serpinb3a protein level in acne rosacea-like the mouse lesions through the immunofluorescence,the results show that the expression of Serpinb3a in the skin lesions of acne rosacea-like the mouse model are similar to the expression of SERPINB3/B4 in human rosacea skin lesions,the expression of
Serpinb3a around hair follicles and in the whole layer epidermis of acne rosacea-like mouse model skin lesions is obviously enhanced,the expression for cytoplasm (as shown in FIG.2L),in summary, the results show that the expression level of Serpinb3a in acne rosacea-like the mouse are obviously up-regulated. at the same time, the present invention is based on the modeling mode used in previous research, the consecutive topical imiquimod for 6 days to establish a psoriasis -like mouse model, the skin lesions of mice are collected and the total RNA 1s extracted from the skin lesion tissues of mice, the mRNA expression of Serpinb3a in skin lesions of psoriasis-like mouse is detected through qRT-PCR, the results show that the skin lesions of psoriasis-like the mouse are obviously up-regulated compared with the experimental control group Serpinb3a at the MRNA level (as shown in FIG.2M), to further the mouse skin lesions is collected for immunofluorescent staining,it can be seen that Serpinb3a is highly expressed in the skin lesions of psoriasis-like mice (as shown in FIG.2N); in order to further explore the SERPINB3/B4 in the epidermis in the acne rosacea and pathogenic process of the psoriasis, the present invention constructs a mouse model of Serpinb3a of the epidermis of a specific knockdown mouse through siRNA and intradermal injection of
LL37 and external application of imiquimod are performed to establish the acne rosacea-like and psoriasis-like mouse models,to observe the influence of epidermal knockdown of Serpinb3a on the phenotype of rosacea-like the mouse and psoriasis-like the mouse.
Example 3, the present invention names the negative control sequence, and two siRNA sequences respectively names as Scr RNA, siRNA #1 and siRNA #2 for subsequent research, firstly, the influence of intra-epidermal knockdown Serpinb3a on the phenotype of acne rosacea-like the mouse is discussed, as shown in FIG.3A pattern diagram, 7-8 weeks old
BALB/c wild-type female mice are shaved off their back hair, the intradermal injection of siRNA is performed 1 day before, the negative control sequence corresponding to intradermal injection in the experimental control group, subsequently, intra-epidermal LL37 injection induced acne rosacea-like mouse model is performed according to the above method, the second siRNA injection is performed before the second LL37 injection, enhances knock-down influence, after the mold is completed, the differences in phenotypes among the three groups of mice (Scr siRNA, siRNA # 1 and siRNA # 2) are observed and compared, and the material is taken, FIG.2B shows the LL37 modeling two days after Scr siRNA, the manifestations of acne rosacea-like dermatitis in the control group and LL37 group of the other two siRNAs, it can be seen that after
LL37 modeling, there is a significant inflammatory response in the skin lesions of the Scr siRNA group in the mouse lesions, comprising the erythema and telangiectasia, but the inflammatory response of the skin lesions in the two siRNA knockdown groups is obviously reduced, the present invention uses Image J software to measure the area size of each group of acne rosacea-like mouse skin lesions, the results show that the erythema area of Scr siRNA group are obviously increased after LL37 modeling the erythema area of the LL37 group of the two siRNAs are obviously lower than that of the LL37 group of Scr (as shown in FIG.3C), secondly, the severity degree of erythema of the acne rosacea-like skin lesion is scored, the results show that the erythema score of LL37 group of Scr siRNA are obviously increased after modeling, the erythema score of LL37 group of two of the Scr siRNA are obviously reduced after modeling,(as shown in FIG.3D); in addition, the present invention compares the histology situation of the mouse skin lesions in each group after LL37 modeling,to take the skin lesions of the molding part, after paraffin embedded sections to perform HE staining, the number of the inflammatory cell infiltrates in the corium layer of the mouse is observed and counted under the microscope, as shown in
FIG.3E,the number of inflammatory cells infiltrate in the corium part that the Scr siRNA LL37 group is obviously increased than the Control group, LL37 group of 2 siRNAs after modeling, the number of inflammatory cells infiltrate in the corium layer that is obviously reduced compared with that in the LL37 group of Scr siRNA, at the same time, the present invention performs a statistical analysis of the number of infiltrated inflammatory cells through Prism9 software, the results show that the number of inflammatory cells infiltrated by the LL37 group of the two siRNAs after knocking down Serpinb3a are obviously reduced compared with that in the
LL37 group of Scr(as shown in FIG.3F),the difference is statistically significant, finally, the present invention collects the mouse skin lesions and extracts total RNA, the differences in mRNA expression levels of acne rosacea-related genes in the skin lesions of mice in the above groups are compared through qRT-PCR, as shown in FIG.3G-H, the expression of 116 and
Tnf-agenes in the LL37 group of Scr siRNA is increased compared to the Control group,and after knocking down Serpinb3a,the expression levels of the above two genes in the LL37 group of 2 siRNA are obviously reduced compared to the LL37 group of Scr siRNA and the difference is statistically significant, the above results indicate that the absence of Serpinb3a in the intra-epidermis can obviously improve the development of acne rosacea in the LL37-induced acne rosacea-like mouse model from the aspects of skin inflammatory response, histopathology and mRNA expression layer.
Example 4, the present invention aims to clarify the effect of knocking down Serpinb3a in the intra-epidermis on the phenotype of psoriasis-like the mouse,selects the two siRNA above, the ear of the mouse intra-dermal injection is carried out, the intra-epidermal knocks down the
Serpinb3a,the external application imiquimod is used to construct a psoriasis-like the mouse model according to previous research methods, as shown in FIG.4A pattern diagram, intradermal injection of siRNA is performed 1 day before external application imiquimod (denoted as day-1), the negative control sequence corresponding to the intradermal injection in the experimental control group, thereafter imiquimod emulsifiable paste external application once a day, the second siRNA injection is performed on the second day to enhance the knockdown influence, after the modeling is completed on the 6th day, in order to clarify the influence of intra-epidermal knockdown after Serpinb3a on the phenotype of psoriasis-like mice skin lesions,on day 6 of modelling of the present invention, the differences in phenotypes among the three groups of mice (Scr siRNA, siRNA # 1 and siRNA # 2) are observed and compared, and the material is taken, according to previous research [77], the PASI score and ear thickness of the skin lesions in mice are measured, it can be seen that the erythema and scales of the mice in the siRNA imiquimod group are obviously improved compared with the mice in the Scr RNA imiquimod group (as shown in FIG.4B), the PASI score is obviously decreased(as shown in
FIG.4C), the thickness of the ear and skin are also obviously decreased (as shown in FIG.4D), secondly, HE staining is performed on the skin lesions of each group of mice,as a result,it can be seen that Scr siRNA imiquimod group epidermal thickness is obviously increased,skin corium inflammatory cell infiltration is obvious, and the imiquimod group of two siRNAs epidermal thickness and the degree of infiltration of inflammatory cells in the corium layer are obviously improved (as shown in FIG.4E), and statistical analysis of epidermal thickness and infiltrated inflammatory cells are performed, it was found that the imiquimod group of two siRNAs are obviously decreased compared with the Scr siRNA (as shown in FIG.4F-G), in order to further clarify the influence of Serpinb3a on the molecular level of psoriasis related-genes, the present invention collects the mouse skin lesions and extracts total RNA, the differences in mRNA expression levels of psoriasis-related genes in the skin lesions of mice in the above groups are compared through the qRT-PCR,as shown in FIG.4H-L the imiquimod group of Scr siRNA increases to 2 compared with control group Il6 and Il-1ß gene expression, and the expression levels of the above two genes in the miquimod group are obviously decreased compared with the imiquimod group of Scr siRNA, the difference is statistically significant,the above results indicate that the absence of Serpinb3a in the ear epidermis can obviously improve the development of inflammatory diseases in the imiquimod-induced psoriasis-like the mouse model from the aspects of inflammatory response of skin lesions, histopathology situations and mRNA expression layers; in conclusion, knockdown of serpinb3a in mouse intra-epidermis can obviously improve the inflammatory response in skin lesions of acne rosacea-like and psoriasis-like of the mouse.
Example 5, in order to further clarify the specific molecular mechanism action of
SERPINB3/B4 on acne rosacea in vitro,the present invention constructs SERPINB3/B4 over-expression plasmids in vitro,and human HaCaT cells are transfected with over-expressed plasmids through transfection reagent, after 72 hours, the cell RNA is collected and whole transcriptome sequencing is performed, the sequencing results are verified by in vivo and in vitro experiments; the present invention performs cluster analysis of differential gene expression values in human HaCaT cells over-expressing SERPINB3/B4, then, the gene expression values are homogenized treatment to obtain FIG.5A, as shown in the FIG, each small square represents a gene, blue represents down-regulation gene expression, red represents up-regulation gene expression, the deeper of the color, the difference of expression is more obvious,each column represents the expression situation of different genes in each sample, and each row represents the expression situation of each gene in different samples, as shown in the fig, the results can be seen that the gene expression of human HaCaT cells over-expressing SERPINB3/B4 are obviously different compared with the normal controls, the results show that the gene expression after over-expressing of SERPINB3/B4 in human HaCaT cells are obviously changed compared with the normal control pattern;
in order to further define the changes in signaling pathways in human HaCaT cells over-expressing SERPINB3/B4, the present invention performs gene set enrichment analysis (GSEA) on all gene expression amounts of human HaCaT cells and control cells over-expressing
SERPINB3/B4, based on the skin inflammatory diseases of the acne rosacea and previous research, the inflammation-related signaling pathways play a key role in the development of acne rosacea, therefore, the present research focuses on the signaling pathways associated with inflammation, the NF-kB signaling pathway is existed in almost all animal cells used and is involved in various cellular responses to external stimuli, including stress, cytokines, free radicals, microbial infections, and ultraviolet radiation,previous studies have found that this signaling pathway regulates the expression of a large number of genes related to the inflammation, among the inflammation-related signaling pathways of the set enrichment in this study, the NF-kB signaling pathway is obviously enriched in human HaCaT cells over-expressing SERPINB3/B4 compared with the control group (as shown in FIG.5B-E) this indicates that the signaling pathway may be involved in SERPINB3/B4 regulation of the pathogenesis of acne rosacea; in order to define the influence of SERPINB3/B4 over-expressing on NF-kB signaling pathway at the protein level in human HaCaT cells, the present invention transfects an over-expressing plasmid into a human HaCaT cell line through transfection reagent, the cell protein is collected after 72 hours, the phosphorylation level of p65/ NF-kB (p-p65) is detected by immunbolting after SERPINB3 / B4 expression in human HaCaT cell line, at the same time,
Image J software is used to scan and quantify the p-p65 gray scale,as shown in FIG.SF, the present invention found that the expression of p-p65 is obviously up-regulation after the expression of the SERPINB3/B4 in human HaCaT cell line, there is no significant difference in p65 expression, p-p65/p65 is up-regulation, the results-show that the NF-kB signaling pathway is activated, in order to more intuitively understand the influence of the expression of the
SERPINB3/B4 on NF-kB signaling pathway,in the present invention, the influence of p65 expression in human HaCaT cell line 72 hours after transfection of over-expression plasmid is detected through the cell round coverslip experiment and immunofluorescence, the results are shown in FIG.5G,after expression SERPINB3/B4 in people's HaCaT cell, the number of cells in p65 nucleation are obviously increased compared with the control group,the above results indicate that the expression of SERPINB3/B4 in human HaCaT cells activates the NF-KB signaling pathway at the protein level, the previous studies have found that TNF-acan strongly induce the activation of NF-kB signal, in order to define the influence of SERPINB3/B4 on TNF-a-induced NF-kB signal activation,the present invention constructs ShRNA of the SERPINB3/B4 in vitro and establishes a human HaCaT cell line that stably knocks down SERPINB3/B4, after TNF-ais incubated for 30 minutes, immunoblotting method detects the influence after knockdown SERPINB3/B4 on
TNF-a-induced NF-kB signaling activation, the results can be seen that the construction of human HaCaT cell line with stable knockdown of SERPINB3/B4 has obvious knockdown influence on SERPINB3/B4, in normal control cells, after TNF-a incubation, the expression of p-p65 is obviously up-regulated, and after knocking down SERPINB3/B4, the situation of the
TNF-oinduction up-regulation of p-p65 is obviously improve, at the same time, the present invention scans the strip gray scale,as a result statistics finds that the difference has obvious statistical significance (as shown in FIG.SH-I),the results show that the absence of
SERPINB3/B4 in human HaCaT cell line prevents the activation of TNF-a-induced NF-kB signaling, similarly, the present invention detects the influence of stable knockdown
SERPINB3/B4 on TNF-a-induced p65 nucleation through the cell round coverslip experiment and immunofluorescence,as shown in FIG. 5J-K, it can be seen in the normal control human
HaCaT cell line,after incubation with TNF-a for 30 minutes, p65 almost completely entered the nucleus,after knocking down SERPINB3/B4, the number of cells nucleated by p65 are obviously decreased compared with the control group, at the same time, the present invention counts the number of nuclear cells, and the difference 1s obvious and statistically significant; in conclusion, the results show that after over-expression of SERPINB3/B4 in vitro induces molecular up-regulation downstream of NF-kB signaling pathway, after knockdown of
SERPINB3/B4 in vitro can prevent TNF-a-induced NF-KB activation.
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 the
SERPINB3/B4 can regulate the NF-kB signaling pathway, in order to further define the specific role of chemokines and cytokines in SERPINB3/B4 regulating the pathogenesis of acne rosacea, the present invention further analyzes the results of RNA-seq after over-expression of
SERPINB3/B4 in human HaCaT cells,as shown in FIG.6A-B, chemokines and cytokines signaling pathways are highly enriched in SERPINB3/B4 over-expression human HaCaT cells, at the same time, the present invention performs cluster analysis on the expression values of the difference of the chemokines and cytokines-related in samples, and the gene expression values are homogenized treatment to obtain as shown in FIG.6C, compared with normal controls, the expression of inflammation-related chemokines ( CCL2, CCLS, CCL20, CXCL1, CXCL2,
CXCLS, CXCL8, CXCL10, CXCL11 ) and cytokines (IL1 A, IL1 B, IL6, TNFA ) in human
HaCaT cells over-expressing SERPINB3/B4 are obviously up-regulated; in order to further define the expression level of the above cytokines in human HaCaT cells over-expressing in the SERPINB3/B4, the present invention uses the qRT-PCR method that detects the expression of the above genes after SERPINB3/B4 of the over-expression in human
HaCaT cells, the results can be seen as shown in FIG.6D, after the SERPINB3/B4 of the over-expression in human HaCaT cells, the expression of chemokines CCL2, CCLS, CCL20,
CXCL2, CXCL9, CXCL10, CXCL11 and cytokines IL1B,IL6 , TNF-a are obviously up-regulated, the results show that after the SERPINB3/B4 of the over-expression in human
HaCaT cells can obviously induce the up-regulation of inflammation-related chemokines and cytokines; the intra-dermal knockdown of Serpinb3a through the mouse can able to reduce the expression levels of LL37-induced acne rosacea-related cytokines(Il6, Tnf-a),in order to further define the influence of intra-epidermal knockdown of Serpinb3a in acne rosacea-like the mouse on inflammation-related chemokines and cytokines, the present invention applies the method of qRT-PCR that the expression levels of the above related factors are detected, the results can be seen as shown in FIG.6E, after knocking down the Serpinb3a, the expression levels of Ccl5,
Cxcl9, Cxcl10, Cxcl11 and IN1P induced by LL37 are obviously inhibited, the present invention applies the same method to detect the changes situations of above chemokines in the skin lesions of psoriasis-like the mouse after knocking down serpinb3a, the results are shown in FIG.6F,it can be seen that the expression levels of Ccl5, Ccl20, Cxcl9, Cxcl10 and Cxcl11 induced by IMQ are obviously inhibited after knocking down Serpinb3a; the expression levels of related chemokines and cytokines are detected by the same method, the results are shown in FIG.5G, the SERPINB3/B4 induced chemokines CCL2, CCLS, CXCL9,
CXCL10, CXCL11 and cytokines IL-1B, IL-6, TNF-a are obviously up-regulated, after co-incubation with SC75741, the expression levels of the above chemokines and cytokines are obviously decreased, the above results show that SC75741 can down-regulate the expression levels of related chemokines and cytokines by inhibiting the induced activation of NF-kB signaling pathway by SERPINB3/B4.
For these ordinary technicians in this field, it is obvious that the present invention is not limited to the details of above examples, and can be realized in other specific forms without deviating from the spirit or basic characteristics of the present invention. Therefore, no matter from which point of view, the example should be regarded as exemplary, and it's non-restrictive. The scope of the present invention is limited by the claim rather than the above a a ; LE ; LU504282 description.
Therefore, it aims to include all changes within the meaning and scope of the equivalent elements of the claim.
Any accompanying markings in the claim should not be regarded as limiting the claim involved.
Claims (6)
1. An application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as acne rosacea, characterized in that the expression of SERPINB3/B4 in the serum and skin lesions of patients with acne rosacea and psoriasis are defined firstly through the ELISA, immunohistochemistry, qRT-PCR and immunofluorescence and other means, and the expression situation of Serpinb3a in the skin lesions of LL37-induced acne rosacea-like the mouse model and imiquimod-induced psoriasis-like the mouse model; and then intra-dermal injection through the siRNA, the expression of Serpinb3a in mouse epidermal cells is locally knocked down, to observe its effect on the skin lesion phenotype and histopathology in the inflammatory model, in vitro study is conducted mainly in human HaCaT cells; SERPINB3/B4 is over-expressed or knocked down in human HaCaT cells by constructing plasmids, the relationship with NF-kB signaling pathway is defined by combining with RNA-seq and immunoblotting, immunofluorescence and other methods; finally, SC75741(a potent NF-kB signaling pathway inhibitor) is selected to inhibit the NF-kB signaling pathway in the epidermis of the mouse to define the role of NF-kB signaling pathway in the pathogenic process of acne rosacea, and CCK8 and flow analysis are used to select the appropriate concentration of SC75741 for in vitro study to further define the interaction between SERPINB3/B4 and NF-kB signaling pathway in the pathogenesis of acne rosacea and psoriasis.
2. The application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as acne rosacea according to claim 1, characterized in that the present invention collects skin lesions of normal controls and acne rosacea patients, extracting total RNA, and detecting the expression situation of SERPINB3/B4 mRNA level between the two groups,the results show that they are hardly expressed in normal control lesions, while the mRNA expression level of SERPINB3/ B4 in facial lesions of acne rosacea patients 1s obviously higher than the healthy controls (as shown in FIG.2 A, B), at the same time, the present invention performs IGA score on the number of papules or pustules and skin erythema in patients with acne rosacea, and correlation analysis with SERPINB3/B4 mRNA expression levels in skin lesions of acne rosacea patients, the results show that with the increase of SERPINB3 mRNA expression levels, the IGA score of the lesion 1s obviously increased, the results show that the expression level of SERPINB3 mRNA is positively correlated with IGA score (as shown in
FIG.2C); in addition, the results of the present invention show that with an increase in the level of SERPINB4 mRNA expression, the IGA score of skin lesions is obviously upgraded, the results show that the expression level of SERPINB4 mRNA is positively correlated with the severity of skin lesions (as shown in FIG.2D); at the same time, the present invention collects the skin tissue of the facial lesions of acne rosacea patients for immunohistochemical staining, the results show that compared with normal healthy controls, the high abundance of SERPINB3/B4 expression can be detected in the whole thickness of epidermal keratinocytes in patients with acne rosacea, and it is mainly expressed in the cytoplasm, there is almost no expression in the whole skin layer of healthy controls (as shown in FIG.2E-F), the results show that SERPINB3/B4 are obviously higher in the skin lesions of acne rosacea patients than the healthy controls; in order to further define the expression situation of SERPINB3/B4 in psoriasis skin lesions, similarly, firstly, the present invention collects healthy controls and skin lesions of psoriasis patients total RNA is extracted from skin lesions, the difference of SERPINB3/B4 mRNA level in skin between the two groups are detected through the qRT-PCR, as shown in FIG.2G-H, it can be seen that the expression of SERPINB3/B4 in skin lesions of healthy controls with the skin psoriasis patient is obviously increased at the mRNA level, at the same time, SERPINB3/B4 immunohistochemical staining is carried out the skin lesions, the results are shown in FIG.2I-J that the psoriasis skin lesions are obviously thicker than the epidermis layer of normal skin,and express higher abundance SERPINB3/B4, the above-mentioned results all indicate that
SERPINB3/B4 1s activated in the skin lesions of patients with psoriasis; in order to further study the role of the SERPINB3/B4 in the pathogenic process of the acne rosacea, the present invention is based on the modeling mode used in previous research, the mouse model of acne rosacea induced by antimicrobial peptide LL37 is established, the skin lesions of this model is highly similar to the inflammatory response pattern and the human acne rosacea, the present invention selects 7-8 weeks BALB/c female mice and removes the back hair of mice, one day later, SOUL (320uM) dose of LL37 is injected into the back of each mouse, the injections are given at 12-hour intervals, then the mice in the control group are given an equal dose of sterile 1xPBS solution, consecutive injection for 2 days, a total of 4 times, after the model is successfully established, the general phenotype of the skin is observed and the material is taken, secondly, the present invention collects mouse skin lesion tissue and extracts total RNA, the mRNA expression situation of Serpinb3a in skin lesions of acne rosacea-like the mouse is detected, the results show that the skin lesions of acne rosacea-like the mouse are obviously up-regulated compared with the experimental control group Serpinb3a at the mRNA level (as shown in FIG.2K), the difference is statistically significant, among the serpinb3a is an analogue of SERPINB3/B4, the present invention further detects the expression situation of Serpinb3a protein level in acne rosacea-like the mouse lesions through the immunofluorescence, the results show that the expression of Serpinb3a in the skin lesions of acne rosacea-like the mouse model are similar to the expression of SERPINB3/B4 in human rosacea skin lesions, the expression of Serpinb3a around hair follicles and in the whole layer epidermis of acne rosacea-like mouse model skin lesions is obviously enhanced, the expression for cytoplasm (as shown in FIG.2L), in summary, the results show that the expression level of Serpinb3a in acne rosacea-like the mouse are obviously up-regulated;
at the same time, the present invention is based on the modeling mode used in previous research, the consecutive topical imiquimod for 6 days to establish a psoriasis-like mouse model, the skin lesions of mice are collected and the total RNA 1s extracted from the skin lesion tissues of mice, the mRNA expression of Serpinb3a in skin lesions of psoriasis-like mouse is detected through qRT-PCR,the results-show that the skin lesions of psoriasis-like the mouse is obviously up-regulated compared with the experimental control group Serpinb3a at the mRNA level (as shown in FIG.2M), to further the mouse skin lesions is collected for immunofluorescent staining,it can be seen that Serpinb3a is highly expressed in the skin lesions of psoriasis-like mice (as shown in FIG.2N); in order to further explore the SERPINB3/B4 in the epidermis in the acne rosacea and pathogenic process of the psoriasis, the present invention constructs a mouse model of Serpinb3a of the epidermis of a specific knockdown mouse through siRNA, and intradermal injection of LL37 and external application of imiquimod are performed to establish the acne rosacea-like and psoriasis-like mouse models,to observe the influence of epidermal knockdown of Serpinb3a on the phenotype of rosacea-like the mouse and psoriasis-like the mouse.
3. The application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as acne rosacea according to claim 1, characterized in that the present invention names the negative control sequence, and two siRNA sequences respectively names as Scr RNA, siRNA #1 and siRNA #2 for subsequent research, firstly, the influence of intra-epidermal knockdown Serpinb3a on the phenotype of acne rosacea-like the mouse is discussed, as shown in
FIG.3A pattern diagram, 7-8 weeks old BALB/c wild-type female mice are shaved off their back hair, the intradermal injection of siRNA is performed 1 day before, the negative control sequence corresponding to intradermal injection in the experimental control group, subsequently, intra-epidermal LL37 injection induced acne rosacea-like mouse model is performed according to the above method, the second siRNA injection is performed before the second LL37 injection,
enhances knock-down influence, after the mold is completed, the differences in phenotypes among the three groups of mice (Scr siRNA, siRNA # 1 and siRNA # 2) are observed and compared, and the material is taken, FIG.2B shows the LL37 modeling two days after Scr siRNA, the manifestations of acne rosacea-like dermatitis in the control group and LL37 group of the other two siRNAs, it can be seen that after LL37 modeling, there is a significant inflammatory response in the skin lesions of the Scr siRNA group in the mouse lesions, comprising the erythema and telangiectasia, but the inflammatory response of the skin lesions in the two siRNA knockdown groups is obviously reduced, the present invention uses Image J software to measure the area size of each group of acne rosacea-like mouse skin lesions, the results show that the erythema area of Scr siRNA group are obviously increased after LL37 modeling,the erythema area of the LL37 group of the two siRNAs are obviously lower than that of the LL37 group of Scr (as shown in FIG.3C), secondly, the severity degree of erythema of the acne rosacea-like skin lesion is scored, the results show that the erythema score of LL37 group of Scr siRNA are obviously increased after modeling, the erythema score of LL37 group of two of the Scr siRNA are obviously reduced after modeling (as shown in FIG.3D); in addition, the present invention compares the histology situation of the mouse skin lesions in each group after LL37 modeling, to take the skin lesions of the molding part, after paraffin embedded sections to perform HE staining, the number of the inflammatory cell infiltrates in the corium layer of the mouse is observed and counted under the microscope, as shown in FIG.3E, the number of inflammatory cells infiltrate in the corium part that the Scr siRNA LL37 group is obviously increased than the Control group, LL37 group of 2 siRNAs after modeling, the number of inflammatory cells infiltrate in the corium layer that is obviously reduced compared with that in the LL37 group of Scr siRNA, at the same time, the present invention performs a statistical analysis of the number of infiltrated inflammatory cells through Prism9 software, the results show that the number of inflammatory cells infiltrated by the LL37 group of the two siRNAs after knocking down Serpinb3a are obviously reduced compared with that in the LL3T ee group of Scr(as shown in FIG.3F), the difference is statistically significant, finally, the present invention collects the mouse skin lesions and extracts total RNA, the differences in mRNA expression levels of acne rosacea-related genes in the skin lesions of mice in the above groups are compared through qRT-PCR, as shown in FIG.3G-H, the expression of 116 and Tnf-agenes in the LL37 group of Scr siRNA is increased compared to the Control group, and after knocking down Serpinb3a, the expression levels of the above two genes in the LL37 group of 2 siRNA are obviously reduced compared to the LL37 group of Scr siRNA, and the difference is statistically significant, the above results indicate that the absence of Serpinb3a in the intra-epidermis can obviously improve the development of acne rosacea in the LL37-induced acne rosacea-like mouse model from the aspects of skin inflammatory response, histopathology and mRNA expression layer.
4. The application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as acne rosacea according to claim 1, characterized in that the present invention aims to clarify the effect of knocking down Serpinb3a in the intra-epidermis on the phenotype of psoriasis-like the mouse, selects the two siRNA above, the ear of the mouse intra-dermal injection is carried out, the intra-epidermal knocks down the Serpinb3a, the external application imiquimod is used to construct a psoriasis-like the mouse model according to previous research methods,as shown in FIG.4A pattern diagram, intradermal injection of siRNA is performed 1 day before external application imiquimod (denoted as day-1), the negative control sequence corresponding to the intradermal injection in the experimental control group, thereafter imiquimod emulsifiable paste external application once a day, the second siRNA injection is performed on the second day to enhance the knockdown influence, after the modeling is completed on the 6th day, in order to clarify the influence of intra-epidermal knockdown after Serpinb3a on the phenotype of psoriasis-like mice skin lesions, on day 6 of modelling of the present invention, the differences in phenotypes among the three groups of mice (Scr siRNA, siRNA # 1 and siRNA # 2) are observed and compared, and the material 1s taken, according to previous research [77], the PASI score and ear thickness of the skin lesions in mice are measured, it can be seen that the erythema and scales of the mice in the siRNA imiquimod group are obviously improved compared with the mice in the Scr RNA imiquimod group (as shown in
FIG.4B), the PASI score is obviously decreased (as shown in FIG.4C), the thickness of the ear and skin are also obviously decreased (as shown in FIG.4D); secondly, HE staining is performed on the skin lesions of each group of mice, as a result, it can be seen that Scr siRNA imiquimod group epidermal thickness is obviously increased, skin corium inflammatory cell infiltration is obvious, and the imiquimod group of two siRNAs epidermal thickness and the degree of infiltration of inflammatory cells in the corium layer are obviously improved (as shown in
FIG.4E), and statistical analysis of epidermal thickness and infiltrated inflammatory cells are performed, it was found that the imiquimod group of two siRNAs are obviously decreased compared with the Scr siRNA (as shown in FIG.4F-G), in order to further clarify the influence of Serpinb3a on the molecular level of psoriasis related-genes, the present invention collects the mouse skin lesions and extracts total RNA the differences in mRNA expression levels of psoriasis-related genes in the skin lesions of mice in the above groups are compared through the qRT-PCR, as shown in FIG.4H-I, the imiquimod group of Scr siRNA increases to 2 compared with control group Il6 and Il-1B gene expression, and the expression levels of the above two genes in the miquimod group are obviously decreased compared with the imiquimod group of Scr siRNA the difference is statistically obviously, the above results indicate that the absence of Serpinb3a in the ear epidermis can obviously improve the development of inflammatory diseases in the imiquimod-induced psoriasis-like the mouse model from the aspects of inflammatory response of skin lesions, histopathology situations and mRNA expression layers; in conclusion, knockdown of serpinb3a in mouse intra-epidermis can obviously improve the inflammatory response in skin lesions of acne rosacea-like and psoriasis-like of the mouse.
5. The application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as acne rosacea according to claim 1, characterized in that in order to further clarify the specific molecular mechanism action of SERPINB3/B4 on acne rosacea in vitro, the present invention constructs SERPINB3/B4 over-expression plasmids in vitro, and human HaCaT cells are transfected with over-expressed plasmids through transfection reagent, after 72 hours, the cell RNA is collected and whole transcriptome sequencing is performed, the sequencing results are verified by in vivo and in vitro experiments; the present invention performs cluster analysis of differential gene expression values in human HaCaT cells over-expressing SERPINB3/B4, then, the gene expression values are homogenized treatment to obtain FIG.5A, as shown in the FIG,each small square represents a gene,blue represents down-regulation gene expression,red represents up-regulation gene expression,the deeper of the color, the difference of expression is more obvious,each column represents the expression situation of different genes in each sample, and each row represents the expression situation of each gene in different samples,as shown in the fig the results can be seen that the gene expression of human HaCaT cells over-expressing SERPINB3/B4 is obviously different compared with the normal controls, the results show that the gene expression after over-expressing of SERPINB3/B4 in human HaCaT cells are obviously changed compared with the normal control pattern; in order to further define the changes in signaling pathways in human HaCaT cells over-expressing SERPINB3/B4,the present invention performs gene set enrichment analysis (GSEA) on all gene expression amounts of human HaCaT cells and control cells over-expressing SERPINB3/B4, based on the skin inflammatory diseases of the acne rosacea and previous research, the inflammation-related signaling pathways play a key role in the development of acne rosacea, therefore, the present research focuses on the signaling pathways associated with inflammation, the NF-kB signaling pathway is existed in almost all animal cells used and is involved in various cellular responses to external stimuli, including stress, cytokines, free radicals, microbial infections, and ultraviolet radiation,previous studies have found that this signaling pathway regulates the expression of a large number of genes related to the inflammation, among the inflammation-related signaling pathways of the set enrichment in this study, the NF-kB signaling pathway is obviously enriched in human HaCaT cells over-expressing SERPINB3/B4 compared with the control group (as shown in FIG.5B-E) this indicates that the signaling pathway may be involved in SERPINB3/B4 regulation of the pathogenesis of acne rosacea;
in order to define the influence of SERPINB3/B4 over-expressing on NF-kB signaling pathway at the protein level in human HaCaT cells, the present invention transfects an over-expressing plasmid into a human HaCaT cell line through transfection reagent, the cell protein is collected after 72 hours, the phosphorylation level of p65/ NF-kB (p-p65) is detected by immunbolting after SERPINB3 / B4 expression in human HaCaT cell line, at the same time,
Image J software is used to scan and quantify the p-p65 gray scale,as shown in FIG.5F, the present invention found that the expression of p-p65 is obviously up-regulation after the expression of the SERPINB3/B4 in human HaCaT cell line, there is no significant difference in p65 expression, p-p65/p6S is up-regulation, the results show that the NF-kB signaling pathway are activated, in order to more intuitively understand the influence of the expression of the
SERPINB3/B4 on NF-kB signaling pathway,in the present invention, the influence of p65 expression in human HaCaT cell line 72 hours after transfection of over-expression plasmid is detected through the cell round coverslip experiment and immunofluorescence, the results are shown in FIG.5G, after expression SERPINB3/B4 in people's HaCaT cell, the number of cells in p65 nucleation are obviously increased compared with the control group, the above results indicate that the expression of SERPINB3/B4 in human HaCaT cells activates the NF-kB signaling pathway at the protein level; the previous studies have found that TNF-acan strongly induce the activation of NF-kB signal, in order to define the influence of SERPINB3/B4 on TNF-a-induced NF-kB signal activation, the present invention constructs shRNA of the SERPINB3/B4 in vitro and establishes a human HaCaT cell line that stably knocks down SERPINB3/B4, after TNF-ais incubated for 30 minutes, immunoblotting method detects the influence after knockdown SERPINB3/B4 on TNF-a-induced NF-kB signaling activation, the results can be seen that the construction of human HaCaT cell line with stable knockdown of SERPINB3/B4 has obvious knockdown influence on SERPINB3/B4, in normal control cells, after TNF-a incubation, the expression of p-p65 is obviously up-regulated, and after knocking down SERPINB3/B4, the situation of the TNF-oinduction up-regulation of p-p65 is obviously improve, at the same time, the present invention scans the strip gray scale, as a result statistics finds that the difference has obvious statistical significance (as shown in FIG.SH-I), the results show that the absence of SERPINB3/B4 in human HaCaT cell line prevents the activation of TNF-a-induced NF-KB signaling, similarly, the present invention detects the influence of stable knockdown SERPINB3/B4 on TNF-a-induced p65 nucleation through the cell round coverslip experiment and immunofluorescence, as shown in FIG. 5J-K, it can be seen in the normal control human HaCaT cell line,after incubation with TNF-a for 30 minutes, p65 almost completely entered the nucleus,after knocking down SERPINB3/B4, the number of cells nucleated by p65 are obviously decreased compared with the control group, at the same time, the present invention counts the number of nuclear cells, and the difference is obvious and statistically significant; in conclusion, the results show that after over-expression of SERPINB3/B4 in vitro induces molecular up-regulation downstream of NF-kB signaling pathway, after knockdown of SERPINB3/B4 in vitro can prevent TNF-a-induced NF-kB activation.
6. The application of SERPINB3/B4 as a target in the treatment of inflammatory skin diseases such as acne rosacea according to claim 1, characterized in that inflammation-related pace chemokines and cytokines play an extremely important role in the occurrence and progression of inflammation, a series of previous results show that the SERPINB3/B4 can regulate the NF-kB signaling pathway,in order to further define the specific role of chemokines and cytokines in
SERPINB3/B4 regulating the pathogenesis of acne rosacea, the present invention further analyzes the results of RNA-seq after over-expression of SERPINB3 / B4 in human HaCaT cells, as shown in FIG.6A-B,chemokines and cytokines signaling pathways are highly enriched in SERPINB3/B4 over-expression human HaCaT cells, at the same time, the present invention performs cluster analysis on the expression values of the difference of the chemokines and cytokines-related in samples, and the gene expression values are homogenized treatment to obtain as shown in FIG.6C, compared with normal controls, the expression of inflammation-related chemokines ( CCL2, CCLS, CCL20, CXCL1, CXCL2, CXCLS, CXCLS, CXCL10, CXCL11 ) and cytokines (IL1 A, IL1 B, IL6, TNFA ) in human HaCaT cells over-expressing SERPINB3/B4 are obviously up-regulated,
in order to further define the expression level of the above cytokines in human HaCaT cells over-expressing in the SERPINB3/B4, the present invention uses the qRT-PCR method that detects the expression of the above genes after SERPINB3/B4 of the over-expression in human HaCaT cells the results can be seen as shown in FIG.6D, after the SERPINB3/B4of the over-expression in human HaCaT cells, the expression of chemokines CCL2, CCLS, CCL20,
CXCL2, CXCL9, CXCL10, CXCLI11 and cytokines IL1B, IL6, TNF-o are obviously up-regulated, the results show that after the SERPINB3/B4 of the over-expression in human HaCaT cells can obviously induce the up-regulation of inflammation-related chemokines and cytokines;
the intra-dermal knockdown of Serpinb3a through the mouse can able to reduce the expression levels of LL37-induced acne rosacea-related cytokines (116, Tnf-a), in order to further define the influence of intra-epidermal knockdown of Serpinb3a in acne rosacea-like the mouse on inflammation-related chemokines and cytokines, the present invention applies the method of qRT-PCR that the expression levels of the above related factors are detected, the results can be seen as shown in FIG.6E, after knocking down the Serpinb3a, the expression levels of CclS, Cxcl9, Cxcl10, Cxcl11 and II1P induced by LL37 are obviously inhibited,the present invention applies the same method to detect the changes situations of above chemokines in the skin lesions of psoriasis-like the mouse after knocking down serpinb3a, the results are shown in FIG.6F, it can be seen that the expression levels of CclS, Ccl20, Cxcl9, Cxcl10 and Cxcl11 induced by IMQ are obviously inhibited after knocking down Serpinb3a;
the expression levels of related chemokines and cytokines are detected by the same method, the results are shown in FIG.5G, the SERPINB3/B4 induced chemokines CCL2, CCLS, CXCL9, CXCL10, CXCLI11 and cytokines IL-1P, IL-6, TNF-a are obviously up-regulated, after co-incubation with SC75741, the expression levels of the above chemokines and cytokines are obviously decreased, the above results show that SC75741 can down-regulate the expression levels of related chemokines and cytokines by inhibiting the induced activation of NF-kB signaling pathway by SERPINB3/B4.
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