CN115475183A - Application of probiotic vesicles in preparation of medicine for treating atherosclerosis - Google Patents
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Abstract
The invention discloses an application of probiotic vesicles in preparation of a medicament for treating atherosclerosis, and belongs to the technical field of biological medicines. The invention proves that the probiotic vesicle has good foam macrophage targeting ability and regulating ability, and further elaborates the mechanism for regulating the macrophages: (1) up-regulating transcription factor NR1H3 to promote ABCA1 expression and lipid outflow; (2) promote the polarization of macrophages to M2 type. In a word, the foam macrophage targeting ability provided by the probiotic vesicles is combined with macrophage function regulation, so that the probiotic vesicles have a remarkable treatment effect on atherosclerosis. The invention provides an ideal nano-carrier and a new method for treating atherosclerosis.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of probiotic vesicles in preparation of a medicine for treating atherosclerosis.
Background
Atherosclerosis (AS) is a lipid-driven chronic inflammatory vascular disease, a common etiology and pathophysiological basis of cardiovascular and cerebrovascular diseases, and myocardial infarction and stroke caused by it are major causes of human death. In recent years, great progress has been made in the prevention and treatment of AS and related diseases, but due to the complex pathogenesis of AS, early diagnosis and treatment of AS is still a serious challenge facing modern medicine.
Macrophages are the most important and abundant immune cells during AS injury. Macrophages play a crucial role in every link from AS injury to AS plaque rupture. Macrophages are of diverse differentiation origin, and monocytes in the circulating blood are the primary source of macrophages entering the tissue through the vessel wall. The pathogenesis hypothesis is that endothelial injury is caused by factors such as blood fat increase, endothelial cells secrete inflammatory factors and chemotactic factors such as monocyte chemotactic factor (MCP), and monocytes adhere, aggregate and migrate to the subcutaneous space to be differentiated into macrophages; under the mediation of scavenger receptor SR-A, CD and the like, macrophages take up oxidized low-density lipoprotein (ox-LDL) and accumulate in cells; the increase of cholesterol load in cells causes lipid accumulation, and macrophages are converted into foam cells to form pathological changes such as lipid stripes, atherosclerotic plaques and the like. Thus, macrophages are the "key driver" for AS development, affecting plaque stability and AS outcome.
Theoretically, if macrophages could be regulated, they could effectively inhibit the progression of atherosclerosis. The CANTOS research proves that the anti-inflammatory drug has great application value in the field of treatment of arteriosclerotic cardiovascular diseases for the first time. However, recent results of the CIRT study published in the journal of Wassel a 'h' y 'gu't New England showed that the use of methotrexate, a broad-spectrum anti-inflammatory drug, did not reduce the mortality of arteriosclerotic cardiovascular diseases. Therefore, the search for the medicine for targeting and regulating the macrophage can be used AS a new breakthrough to lead a new direction of future AS prevention and treatment research, and has important scientific value and social significance.
Nanoparticles have been widely used in the treatment of tumors over the last decades, and since atherosclerotic plaque sites exhibit high permeability and retention effects similar to those of solid tumors, nanoparticle-based solutions provide a new approach to the treatment of atherosclerosis. Extensive research has shown that nanoparticles significantly improve the pharmacokinetic profile and chemical stability of pharmacotherapeutic agents, such as small molecule drugs, polypeptides, etc. Nano-therapeutics can also reduce off-target and systemic side effects compared to free drugs alone.
Extracellular vesicles have proven to be ideal nanocarriers and have now entered into a number of clinical trials. Different sources of extracellular vesicles have different functions, and probiotic-derived vesicles (OMVs) are believed to have anti-inflammatory and immunomodulatory properties, such as OMVs that regulate intestinal immune cells to suppress inflammatory bowel disease. In addition, several studies have preliminarily demonstrated that OMVs can modulate macrophages to inhibit inflammation. Specifically, OMVs induce the secretion of the anti-inflammatory cytokine IL-10 and immunoregulatory cytokines IL-1 β and GM-CSF, correct the imbalance between M1 and M2 macrophages, and thereby ameliorate allergic dermatitis. Unlike common inflammatory diseases, macrophages in atherosclerotic diseases often present with disorders of lipid metabolism, and the role OMVs play in atherosclerotic macrophages is unclear.
Disclosure of Invention
The invention uses vesicles (OMVs) from the probiotic strain lactobacillus rhamnosus to explore the application and function of OMVs as vectors in atherosclerosis. In vitro experiments and NIRF imaging were used to examine the ability of OMVs to target macrophages, bioinformatics and molecular biology experiments initially explained the mechanism by which OMVs regulate macrophage inhibition of atherosclerosis. The invention provides a new vector and a new idea for diagnosing and treating the atherosclerosis disease.
The invention provides an application of probiotic vesicles in preparation of a medicament for treating atherosclerosis.
Preferably, the probiotic vesicles are derived from probiotics.
More preferably, the probiotic is lactobacillus rhamnosus.
More preferably, the probiotic vesicles are prepared as follows: after centrifugation of lactobacillus rhamnosus 48 hmmrs broth cultures, the supernatants were washed in PBS and the vesicles obtained were resuspended in PBS, filtered and stored in PBS at-80 ℃.
More preferably, the Lactobacillus rhamnosus 48hMRS broth culture is centrifuged at 600g for 30min followed by 100000 Xg for 70min.
More preferably, the washing conditions in PBS are: 100000 Xg at 4 ℃.
More preferably, the filtration is filtration using a 0.22 μm filter.
Compared with the prior art, the invention has the following beneficial effects:
the invention proves that OMV has good foam macrophage targeting ability and regulating ability, and further elaborates the mechanism for regulating macrophages: (1) up-regulating transcription factor NR1H3, promoting ABCA1 expression and lipid efflux; (2) promote the polarization of macrophages to M2 type. In a word, the foam macrophage targeting ability provided by the OMV is combined with macrophage function regulation and control, so that the composite nano-carrier has a remarkable treatment effect on atherosclerosis, and an ideal nano-carrier and a new method are provided for treating atherosclerosis.
Drawings
FIG. 1 is a characteristic diagram of OMVs of example 1, in which (A) a schematic of probiotic vesicle isolation. (B) Transmission Electron microscopy images and hydrated particle size of OMV. (C) Mean hydrated particle size of OMV in physiological saline over 7 days (n = 3). (D) Average zeta potential of OMVs in physiological saline over 7 days (n = 3). (E) schematic ICG Loading of OMVs. (F-I) flow analysis OMV @ ICG was incubated with THP-1+ PMA or THP-1+ PMA + oxLDL at different concentrations for different periods of time. (J) Confocal images of the incubation of OMV @ ICG with THP-1+ PMA, THP-1+ PMA + oxLDL, respectively. (n =3, scale bar =50 μm).
FIG. 2 is a graphical representation of the results of multiple assays of the targeted delivery of OMVs in atherosclerotic mice in example 1, wherein (A) is an image of an atherosclerotic mouse. (B) Near infrared fluorescence (NIRF) imaging of tissues following tail vein injection of ICG or OMV @ ICG. (C) Near-infrared fluorescence imaging of aorta after tail vein injection of ICG or OMV @ ICG. (D) HE staining and oil red O staining of atherosclerotic lesions, and fluorescence images of accumulated ICG in atherosclerotic plaques. (E) The fluorescence signal of the accumulated ICG in atherosclerotic plaques colocalizes the image with FITC-labeled macrophages.
FIG. 3 is a graph of the results of the therapeutic effect of OMVs on atherosclerotic mice in example 1, wherein (A) the grouping and treatment process of the atherosclerotic mice are shown schematically. (B) in vitro NIRF images of post-treatment aortic tissue. (C) Quantitative data on ICG fluorescence signal accumulated in aorta 2 hours after injection. And (D) staining the aortic sinus part with oil red O. (E-F) HE staining and oil red O staining of aortic Dou Yuyang sclerosing lesions. (G) quantitative analysis of oil red staining. (H) Fluorescence images of ICG accumulated in atherosclerotic plaques of aortic root sections of mouse model. (I) Masson staining of aortic sections. (J) Changes in T-CHO, TG, HDL-C and LDL-C in the blood of mice after different treatments. (n = 5; P <0.05; P <0.01; P < 0.001).
FIG. 4 is the results of in vivo safety tests of OMV treatment in example 1, wherein (A) the change in body weight of mice over 30 days. (B-E) blood biochemistry of mice after different treatments. (F-H) monitoring of liver and kidney function in mice after treatment. (I) H & E stained sections of mouse vital organs (n =5, scale bar =200 μm).
FIG. 5 is a bioinformatic analysis of the mechanism by which OMVs inhibit atherosclerosis in example 1. (A) schematic representation of transcriptome sequencing packets. And (B) sequencing quality control analysis. (C) genes which changed after OMV treatment. (D) the first 50 genes that changed significantly after OMV treatment. (E) significantly altered biological function following OMV treatment. (F) Biological processes that vary significantly following OMV treatment include lipid efflux and macrophage regulation.
Fig. 6 is the results of the OMV-facilitated lipid efflux assay by ABCA1 expression in example 1, in which (a) the signaling pathways, including lipid and atherosclerotic pathways, changed significantly after OMV treatment. (B) Genes with significantly altered "lipid and atherosclerosis" pathways. (C) The differential genes in the "lipid and atherosclerosis" pathway intersect with genes that regulate lipid efflux. (D) Foam macrophages treated with different concentrations of OMVs, oil red O stained under-lens images. (E-F) Western blot and RT-qPCR analysis of ABCA1 expression in OMV-treated foam macrophages at different concentrations. (G) Immunohistochemical analysis expression of ABCA1 in aortic tissue after treatment. (n = 5; P <0.05; P <0.01; P < 0.001).
FIG. 7 shows the results of increasing ABCA1 expression by OMV through up-regulation of NR1H3 transcription factor in example 1, where (A) the differential gene intersects with the transcription factor regulating ABCA1, confirming that OMV regulates ABCA1 through NR1H 3. (B-C) detecting the expression of NR1H3 regulated by OMVs through Western blot and RT-qPCR. (D-F) OMVs treatment or NR1H3 overexpression increases the protein levels of ABCA1. (G-I) NR1H3 knockdown, OMV treatment can reverse NR1H3 and ABCA1 expression. (J) schematic representation of the mechanism by which OMVs promote lipid efflux. (n = 5; < 0.05;. P < 0.01;. P < 0.001).
FIG. 8 is the results of the macrophage polarization promotion assay of OMVs in example 1, wherein (A) the mechanism by which OMVs promote macrophage conversion to M2-type is shown schematically. (B) The expression of M1 markers, including CD86, NOS2, TLR4, is down-regulated. (C) Expression of M2 markers, including CD209, IL1R2, TGM2, is upregulated. (D-F) cytokine expression in foam cells before and after OMV treatment. (F) morphological changes of foam cells before and after OMV treatment. (G) Westernblot analysis of M1 (CD 86) and M2 (CD 206) macrophage markers in foam cells before and after OMV treatment. (H) Confocal imaging of M1 and M2 macrophages in foam cells before and after OMV treatment. (I) Immunohistochemical analysis macrophage (F4/80), M1 macrophage (CD 86) and M2 macrophage (CD 206) marker expression in aortic tissues after various treatments. (n = 5; < 0.05;. P < 0.01;. P < 0.001).
Detailed Description
Example 1
Procedure of experiment
1. Cell culture and foam cell induction
Human THP-1 cells were obtained from American type culture Collection (ATCC; USA) at 0.5X 10 6 The cells were inoculated in RPMI 1640 medium containing 10% FBS at a density of/mL and cultured at 37 ℃ for use. THP-1 cells were cultured for 48h with 100ng/mL PMA, and induced to become macrophages. The cell culture medium was then replaced with serum-free RPMI 1640 medium containing 50. Mu.g/mL oxLDL and incubated for 48h, and macrophages were induced to macrophage spuhaga cellsAnd (4) cells.
2. Animal(s) production
The animal protocol was approved by the animal care committee of the college of peer-relief medical college of science and technology university in huazhong. Male ApoE -/- Defect mice (6 weeks old) were purchased from Experimental animals technology, inc., weitongli, beijing, china, and were housed in a specific pathogen-free environment. C57BL/6 mice (female, 6 weeks old) were purchased from Beijing Huafukang Biotech, inc., china and maintained in a specific pathogen-free environment.
3. Preparation of OMV (OMV) derived from lactobacillus rhamnosus
Lactobacillus rhamnosus was purchased from bera Culture Collection (BNCC 136673, china), cultured in MRS medium, culture supernatant was collected, after centrifugation at 600g for 30min, the supernatant was again centrifuged at 4 ℃ and 100000 × g, the obtained precipitate was washed 2 times with PBS (4 ℃, 100000 × g), the finally obtained precipitate was resuspended in PBS, filtered through a 0.22 μm filter (Millipore, USA), quantified using BCA protein assay kit, and stored at-80 ℃ for future use.
OMV characteristics
The morphology, hydrated particle size and potential of OMVs were examined using transmission electron microscopy (TEM, hitachi, japan) and dynamic light scattering (DLS, malvern Instruments ltd., worcestershire, uk). To monitor the stability of OMVs in vitro, DLS was used to monitor hydrated particle size and potential for 7 days.
5. In vitro cell binding capacity
In vitro cell binding capacity was examined using flow analysis and confocal imaging. Dimethyl sulfoxide (DMSO, solarbio, china) as a permeation enhancer increases the solubility and permeability of ICG on OMV lipid membranes. ICG was encapsulated in 4% (v/v) dimethyl sulfoxide (DMSO) in physiological saline. OMV was then mixed with ICG and cultured with stirring in the dark (4 ℃) for 6 hours to obtain OMV @ ICG. Samples were purified using a PDG-25 desalting column and stored at 4 ℃. OMV @ ICG was incubated with macrophages or foam cells at 37 ℃ for various time points (0 h, 1h, 6h, 12 h) and at various concentrations (0. Mu.g/mL, 1. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, and 20. Mu.g/mL), and fluorescence signals were detected by flow cytometry. OMV @ ICG (ICG 5. Mu.g/mL) and ICG (5. Mu.g/mL) were then incubated with macrophages or foam cells for 24 hours at 37 ℃. The nuclei were counterstained with 4', 6-diamino-2-phenylindole (DAPI, boster, wuhan, china) and the cytoskeleton was stained with FITC-labeled phalloidin (Proteintech, wuhan, china). The paraformaldehyde is fixed and observed under a fluorescence microscope (lycra, germany).
6. In vitro NIRF imaging
Male ApoE of 6w size -/- Mice received a high fat diet for 3 months and were randomized into two groups, injected with OMV @ ICG and free ICG, respectively. Mice were anesthetized with 2% isoflurane at different time points (1, 2, and 3 h) and the aorta with attached heart was removed. The IVIS spectral imaging system acquires static NIRF images (excitation 750nm, emission 790 nm).
7. Treatment regimens
Male ApoE of 6w size -/- Mice were fed a high fat diet for 4 weeks and randomized into two groups, OMV group and PBS group (n = 5), followed by injection of OMV (5 mg/mL,0.2 mL) or PBS (0.2 mL) into the mice via the tail vein of the mice, respectively, once a week for 8 weeks. At week 19, all model mice were injected with OMV @ ICG and the mice were sacrificed.
8. Blood biochemical analysis
Blood was collected with EDTA-coated tubes and immediately analyzed with an automatic hematology analyzer (Sysmex KX-21, sysmex Co., japan) for hematological parameters including analysis of Red Blood Cells (RBC), white Blood Cells (WBC), platelets (PLT), hemoglobin (HGB), hematocrit (HCT), mean Corpuscular Volume (MCV), mean hemoglobin (MCH), and Mean Corpuscular Hemoglobin Concentration (MCHC). Plasma alanine Aminotransferase (ALT), aspartate Aminotransferase (ASP), alkaline phosphatase (ALP), creatinine (CRE), blood Urea Nitrogen (BUN), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and Triglyceride (TG) concentrations were determined, and serum total cholesterol concentrations were determined.
9. Histological analysis
ApoE -/- The aorta of the mice was stained with gross oil red O. Using hematoxylin and eosin (H)&E) The AS lesions were assessed by staining and plaque and luminal area were measured under a microscope, respectively. Oil red O staining or Sudan IV staining of frozen sections of arteries to assess plaqueLipid content. Collagen content in AS plaques was assessed using Masson trichrome staining. Immunohistochemical (IHC) staining and immunofluorescence staining analyze specific indexes, such as ABCA1 expression level, M1 type macrophages, M2 type macrophages, fluorescent signal accumulation conditions in plaques and the like. In addition, H is used for major organs (heart, liver, spleen, lung and kidney)&E staining and observation under light microscope (olympus IX73, japan).
10. Transcriptome sequencing
THP-1 cells were treated with PMA (100 ng/mL) for 48 hours and then incubated with oxLDL (50. Mu.g/mL) for 48 hours to obtain foam macrophages, which were then treated with OMV (10. Mu.g/mL). Foam macrophages before and after OMV treatment were transcriptome sequenced.
11. Western blot analysis
THP-1, THP-1+ PMA (TP), THP-1+ PMA + oxLDL (TO) and THP-1+ PMA + oxLDL + OMV (TV) cells were collected and lysed with lysis buffer containing phosphatase inhibitor and 1% protease on ice for 30 minutes. Then, the cell lysate was centrifuged at 12000g,4 ℃ for 15 minutes, and the supernatant was collected. Protein concentration was determined using a protein quantification kit (# P0012S, beyotime). The gel was transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour at room temperature, then incubated with primary antibody overnight at 4 ℃. The following day, membranes were washed with 1TBST for 30min and incubated with the corresponding secondary antibody for 1h. After incubation, the membrane was washed 3 times with PBS and exposed to X-ray film using ECL detection reagent (# WP20005, thermoFisher). The antibodies used in this experiment were as follows: (ii) a NR1H3 antibody (# 14351-1-AP,1, 1000), CD86 antibody (# 13395-1-AP, 1.
12. Quantitative RT-qPCR analysis
Total RNA from cells was extracted using Trizol reagent (# 15596026, invitrogen). The extracted RNA samples were reverse transcribed using PrimeScriptTM RT kit (# RR047A, TAKARA, JPN). Quantitative real-time PCR was performed using the TB GreenTM Fast qPCR Mix kit (# RR430A, TAKARA, JPN). GAPDH was used as a reference gene.
RNA interference
siControl and gene-specific siRNA were purchased from Sigma Aldrich. THP-1 cells were transfected with siControl or siRNA in Lipofectamine 2000 (# 11668019, thermo Fisher). 12 hours after transfection, the transfection medium was replaced with DMEM containing 10% FBS.
14. Biological information analysis
Bioinformatics analysis was performed using the R language. The DESeq2 software package was used for differential analysis and the mean values of the different genes were obtained. ggplot2 and pheatmap packages were used to plot volcanic and thermal maps. The ClusterProfiler software package was used for Gene Set Enrichment Analysis (GSEA). The GOplot package is used to draw a Gocircle drawing.
15. Statistical analysis
Statistical analysis of comparisons between groups was performed using the t-test, and one-way anova or two-way anova was performed for multiple comparisons. Statistical significance was assessed using GraphPad Prism 8 software (GraphPad software, inc.). P <0.05 was considered statistically significant. All values are expressed as mean ± standard deviation.
Results of the experiment
OMV characteristics
OMVs were isolated from lactobacillus rhamnosus MRS broth with the protocol shown in figure 1A. Transmission Electron Microscopy (TEM) showed that OMVs were in an irregular spherical morphology. DLS analysis showed that the average diameter of the isolated OMVs was 149.67 ± 2.90nm (fig. 1B). The mean hydrated particle size and zeta potential of OMVs did not change significantly for up to 7 days, indicating good stability (fig. 1C and 1D). ICG was loaded into OMVs for flow analysis and confocal experiments (fig. 1E). As shown by macrophages (THP-1 + PMA) and foam macrophages (THP-1 + PMA + oxLDL), the fluorescence signal increases with increasing concentration of OMV @ ICG, peaking at 5 μ g/mL (FIG. 1F and FIG. 1H); and the fluorescence signals in both macrophages and foam cells increased with the time of culture (FIG. 1G and FIG. 1I). In addition, the fluorescence signal in foam macrophages was higher than macrophages, similar results were also found in confocal images (fig. 1J).
OMV-specific targeting of foam macrophages
To investigate the targeting ability of OMVs on foam macrophages, OMV @ ICG and ICG were injected separately with ApoE via their tail vein -/- Mouse (n = 3). Mice were sacrificed at different time points (1, 2, and 3 h) and heart and aortic tissues were obtained for NIRF imaging (fig. 2A). This omv @ ICG treated group showed stronger fluorescence on aortic tissue and aortic root sections than the free ICG treated group (fig. 2B-2D). Oil red staining of aortic tissue and oil red and H of aortic root sections&E staining showed that both groups successfully constructed AS models and the plaque areas were similar (fig. 2C and 2D). As shown in FIG. 2E, the fluorescence signal of ICG is in good agreement with the fluorescence signal of macrophage marker F4-80.
OMV treatment of atherosclerotic model mice
We verified the efficacy of OMVs on AS plaque, and the specific grouping and treatment protocol is shown in fig. 3A. Aorta isolation after treatment, NIRF imaging, pathological section and immunohistochemical assessment of efficacy. As shown in fig. 3B, the aorta of OMV treated group showed weaker fluorescence signal than the control group. The fluorescence quantification was consistent with NIRF images (fig. 3C).
The aorta was stained with gross oil red O using oil red O staining, and the resulting red region was the plaque region (fig. 3D). The pathological section of the aortic root was then histologically analyzed. As shown in fig. 3E and 3F, H & E and oil red staining of the aortic root revealed atherosclerotic plaques. The oil red area was significantly reduced by more than 10% in the OMV treated group (fig. 3G). In addition, the ICG fluorescence signal was stronger in the control group (fig. 3H). Masson staining determined OMV treated groups had high collagen concentration and fibrous cap thickness (fig. 3I). Consistent with these observations, serum total cholesterol (T-CHO), triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) were also lower in the OMV treated group. Elevated high density lipoprotein cholesterol (HDL-C) levels in serum of OMV treated groups (fig. 3J).
In vivo safety of OMV treatment
To systematically assess the safety of OMV treatment, C57BL/6 mice were injected with 100mgOMV every 10 days for a total of 3 injections. PBS treated mice served as control group. The body weight of each group of mice did not change significantly (fig. 4A). Furthermore, there was no significant change in blood routine measurements between groups (fig. 4B-4E). In addition, no significant liver or kidney toxicity was observed in either group (FIGS. 4F-4H). No obvious evidence of major organ (heart, liver, spleen, lung, kidney or intestinal) damage was observed with H & E staining (fig. 4I).
Bioinformatics analysis of mechanisms of protective action of OMV on AS development
To further investigate the potential mechanism of OMV to inhibit AS, transcriptome sequencing of foam macrophages (THP-1 + PMA + oxLDL) before and after OMV treatment was performed (FIG. 5A). Principal Component Analysis (PCA) was used to analyze quality control of RNA sequencing (fig. 5B). The differential genes expressed after OMV treatment are shown in figure 5C, including 1705 up-regulated genes and 1510 down-regulated genes. The first 50 significantly differentially expressed genes are shown in FIG. 5D, including NCF1, HMOX1, IL23A, and NCF1B, among others. GO analysis indicated that OMV-mediated AS inhibition was involved in many biological functions, including cytokine-mediated signaling and positive modulation of external stimuli response (fig. 5E). Many biological processes, including leukocyte migration, macrophage activation, upregulation of lipid transport, and significant upregulation of lipid output by cells (fig. 5F). Accordingly, we speculate that OMVs inhibit AS mainly by promoting lipid efflux and regulating macrophages.
OMV stimulation of foam macrophage lipid efflux
Several signaling pathways were identified by KEGG analysis, including TNF signaling pathways, cytokine-cytokine receptor interactions, lipid and atherosclerotic signaling pathways. Among the most closely related pathways to lipid efflux are the "lipid and atherosclerosis" pathways (fig. 6A). Sequencing the obtained differential genes intersected all the genes in the "lipid and atherosclerosis" pathway, obtaining significantly different genes in this pathway (fig. 6B), and subsequently intersecting these genes with genes regulating lipid efflux, finding ABCA1 to be the only relevant gene for OMV to regulate lipid efflux (fig. 6C). TO further confirm whether OMVs promote lipid efflux, foam macrophages before and after OMV treatment (TO and TV) were stained with oil red O. The results indicate that the higher the OMV concentration, the lower the intracellular lipid content (fig. 6D). To further verify that OMVs were able to upregulate ABCA1 to promote lipid efflux, we tested the expression levels of ABCA1 protein and mRNA in foam cells using Western blot and RT-qPCR analysis, respectively. Foam macrophages were treated with different concentrations of OMV (0, 10, 20 and 50. Mu.g/mL). The results show that the protein and mRNA expression levels increase proportionally with OMV concentration (fig. 6E and 6F). In addition, immunohistochemical analysis showed that ABCA1 levels in aortic plaques were higher in OMV treated groups than in control groups (fig. 6G).
To further elucidate the mechanism of ABCA1 upregulation, we intersected differentially expressed genes with transcription factors that regulate ABCA1, obtaining two transcriptional genes NR1H3 and FOXA1 (fig. 7A). It is reported that NR1H3 upregulation can promote ABCA1 expression, while FOXA1 upregulation can inhibit ABCA1 expression. Therefore, we speculate that OMVs promote ABCA1 expression by upregulating NR1H 3. Western blot and RT-qPCR analysis demonstrated that NR1H3 protein and mRNA expression increased proportionally with OMV concentration, respectively (FIGS. 7B and 7C). To further demonstrate that OMVs regulate ABCA1 expression through NR1H3, NR1H3 is overexpressed or knocked out in foam macrophages. The results show that NR1H3 overexpression increased the protein levels of ABCA1 (fig. 7D-7F), whereas NR1H3 knock-out decreased the protein levels of ABCA1 in foam macrophages (fig. 7G-7I), and fig. 7J is a schematic diagram of the mechanism by which OMVs promote lipid efflux. Furthermore, the combined use of NR1H3 overexpression and OMV did not enhance ABCA1 expression, whereas NR1H3 knock-out was reversed by OMV treatment. In summary, OMVs can up-regulate ABCA1 by increasing expression of NR1H 3.
OMV promotion of macrophage polarization
In addition to promoting lipid efflux, OMVs can also promote macrophage polarization (fig. 8A). According to bioinformatic analysis, markers of M1 macrophages were down-regulated following OMV treatment, such as CD86, NOS2 and TLR4 (fig. 8B). In addition, several markers of M2 macrophages were upregulated, including CD209, IL1R2, and TGM2 (fig. 8C). Some inflammatory factor levels also changed significantly after OMV treatment; the anti-inflammatory factors (IL 10, IL11, IL1 RN) were increased and the pro-inflammatory factors IL13 were decreased (fig. 8D and 8E). Following OMV treatment, foam macrophage morphology also changed from round to long spindle (fig. 8F). Western blot and RT-qPCR analysis showed that CD86 (M1 macrophage marker) was down-regulated and CD206 (M2 macrophage marker) was up-regulated after OMV treatment (FIG. 8G). In addition, in vitro confocal imaging results showed a decrease in CD86 and an increase in CD206 following OMV treatment (fig. 8H). Ex vivo immunohistochemical analysis showed down-regulation of F4/80 and CD86, up-regulation of CD206 (FIG. 8I).
The invention provides a multifunctional drug delivery carrier for inhibiting atherosclerosis. Unlike existing pegylation and antibody-based targeted delivery strategies, OMVs can target foam macrophages in atherosclerotic plaques due to chemotaxis. The biological nano-carrier provides a broad-spectrum functionalization strategy driven by cell functions, and targeted delivery can be realized without specific targeting molecules or complex biological coupling processes. After OMV is used as a nano-carrier to accumulate in the targeted foam macrophage, the OMV can promote the outflow and polarization of foam macrophage lipid, thereby effectively inhibiting atherosclerosis.
At present, more nanoparticles are applied to the research of treating atherosclerosis by targeting foam macrophages, and the nanoparticles comprise polymer nanoparticles, lipid nanoparticles, magnetic nanoparticles, recombinant high-density lipoprotein nanoparticles, cell membrane bionic nanoparticles and the like. However, these nanoparticles are mainly used as drug delivery carriers, do not have a therapeutic function, and require complex morphological size control, chelating targets, drug loading or control of an exogenous magnetic field, etc. to obtain specific targeting or therapeutic capabilities. The clinical application of the nanometer is limited by the defects of complex synthetic steps, difficult control of batch quality, potential toxicity of part of the nanometer and the like. In the research, OMV is obtained from the culture supernatant of the orally-taken probiotics, and has the advantages of simple preparation, high safety, strong carrying capacity, high repeatability and the like. More interestingly, OMVs have good foam macrophage targeting and regulating capabilities and can inhibit the development of atherosclerosis.
The probiotic vesicles have good foam macrophage targeting ability. According to the results of this study, OMVs can specifically target foam macrophages. OMVs are of suitable size and potential, which helps OMVs better passively target and penetrate atherosclerotic plaques. Foam macrophages, on the other hand, are phagocytic cells that phagocytose dead cells, foreign particles, or microorganisms. In the in vitro flow and confocal experiments, the strongest fluorescence signal can be observed after the OMV is incubated with the foam macrophage, and the optimal binding capacity of the OMV and the foam macrophage is proved. In an isolated fluorescence imaging experiment, the aorta tissue of an OMV @ ICG group and an aorta root pathological section can be observed to have stronger fluorescence signals, and on the pathological section, the fluorescence signals of the ICG and macrophage distribution have consistency, so that the OMV is proved to have better foam macrophage targeting capability.
The probiotic vesicles have the function of regulating foam macrophages to inhibit atherosclerosis. The foam macrophage is an important target of the atherosclerotic plaque, and researches prove that the foam macrophage can be used for treating the atherosclerotic plaque, and specifically comprise the steps of promoting the lipid outflow of the foam macrophage, promoting the differentiation of the foam macrophage to an M2 type macrophage, promoting the apoptosis of the foam macrophage and the like. The research proves that the OMV can inhibit atherosclerosis according to biological indexes such as isolated tissue imaging, gross oil red staining, aorta Dou Qiepian oil red and blood fat. The macrophage regulating effect of OMVs was subsequently analyzed by bioinformatics, mainly including two aspects, promoting foam macrophage lipid efflux and promoting foam macrophage differentiation to M2-type macrophages. The research discovers that the ABCA1 is obviously up-regulated by taking intersection of the lipid efflux related gene and the differential gene, the ABCA1 is widely proved to be an important channel protein for regulating and controlling lipid efflux, and the research also adopts molecular biology modes such as western-blot and the like to verify that the OMV can actually up-regulate the ABCA1. To further study the transcriptional factors that up-regulate ABCA1, we intersected the transcriptional factors that have been reported in the literature to regulate ABCA1 with the differential genes, confirming that OMVs regulate ABCA1 expression via the NR1H3 transcriptional factor. We subsequently overexpress NR1H3 and knock-down, in combination with OMV treatment, further demonstrated that OMVs upregulate ABCA1 expression by upregulating NR1H3 transcription factors, promoting foam macrophage lipid efflux. In addition, the research discovers that M1 type macrophage markers are down-regulated and M2 type macrophage markers are up-regulated through bioinformatics, and then western-blot, immunofluorescence, immunohistochemistry and the like further confirm the result.
The invention preliminarily elaborates a mechanism for regulating macrophages to inhibit atherosclerosis by using the probiotic vesicles, wherein the probiotic vesicles are complex biological nano-carriers and comprise a plurality of miRNA (micro ribonucleic acid), lncRNA (long ribonucleic acid), protein and the like, and specific certain composition or a plurality of compositions of the macrophages can be regulated and need further research. In addition, the probiotic vesicles are nano-carriers, can be loaded with various medicaments, or are combined with various treatment modes such as sonodynamic treatment and Cherenkov treatment, so that the curative effect of atherosclerosis is improved.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (7)
1. Application of probiotic vesicles in preparation of medicines for treating atherosclerosis.
2. The use according to claim 1, wherein the probiotic vesicles are derived from probiotics.
3. Use according to claim 2, characterized in that the probiotic bacteria are Lactobacillus rhamnosus.
4. The use according to claim 3, characterized in that the probiotic vesicles are prepared as follows: after centrifugation of the lactobacillus rhamnosus 48h MRS broth culture, the supernatant was washed in PBS and the vesicles obtained were resuspended in PBS, filtered and stored in PBS at-80 ℃.
5. The use according to claim 4, characterized in that the Lactobacillus rhamnosus 48h MRS broth culture is centrifuged at 600g for 30min and then 100000 Xg for 70min.
6. Use according to claim 5, wherein the washing conditions in PBS are: 100000 Xg at 4 ℃.
7. Use according to claim 6, wherein the filtration is with a 0.22 μm filter.
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| WO2013159234A1 (en) * | 2012-04-27 | 2013-10-31 | The Governors Of The University Of Alberta | Glycoengineered outer membrane vesicles and use thereof as vaccines |
| CN114990038A (en) * | 2022-05-24 | 2022-09-02 | 郑州大学 | Bacterial outer membrane vesicle and application thereof in preparation of preeclampsia treatment medicine |
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| WO2013159234A1 (en) * | 2012-04-27 | 2013-10-31 | The Governors Of The University Of Alberta | Glycoengineered outer membrane vesicles and use thereof as vaccines |
| CN114990038A (en) * | 2022-05-24 | 2022-09-02 | 郑州大学 | Bacterial outer membrane vesicle and application thereof in preparation of preeclampsia treatment medicine |
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