Application of probiotic vesicles in the preparation of drugs for treating atherosclerosis

Abstract

The present invention discloses the application of probiotic vesicles in the preparation of drugs for treating atherosclerosis, and belongs to the field of biomedical technology. The present invention confirms that probiotic vesicles have good foam macrophage targeting and regulating capabilities, and further elaborates the mechanism of regulating macrophages: ① Upregulating transcription factor NR1H3, promoting ABCA1 expression, and promoting lipid outflow; ② Promoting macrophage polarization to M2 type. In short, the foam macrophage targeting capability provided by probiotic vesicles is combined with the regulation of macrophage function, which has a significant therapeutic effect on atherosclerosis. The present invention provides an ideal nanocarrier and a new method for treating atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of probiotic vesicles in the preparation of drugs for treating atherosclerosis. Background Art

[0002] Atherosclerosis (AS) is a lipid-driven chronic vascular inflammatory disease. It is a common cause and pathophysiological basis of cardiovascular and cerebrovascular diseases. Myocardial infarction and stroke caused by AS are the main causes of death in humans. In recent years, great progress has been made in the prevention and treatment of AS and its related diseases. However, due to the complex pathogenesis of AS, its early diagnosis and treatment is still a severe challenge facing modern medicine.

[0003] Macrophages are the most important and abundant immune cells during AS injury. Macrophages play a vital role in every link from AS injury to AS plaque rupture. Macrophages have multiple differentiation sources, and monocytes in circulating blood are the main source of macrophages that pass through the vascular wall and enter the tissue. The pathogenesis hypothesis is as follows: factors such as elevated blood lipids can cause endothelial damage, endothelial cells secrete inflammatory factors and chemokines, such as monocyte chemoattractant factor (MCP), monocytes adhere, aggregate and migrate to the subendothelium, and differentiate into macrophages; mediated by scavenger receptors SR-A, CD36, etc., macrophages take up oxidized low-density lipoprotein (ox-LDL) and accumulate in the cells; increased intracellular cholesterol load leads to lipid accumulation, macrophages are transformed into foam cells, and pathological changes such as lipid streaks and atherosclerotic plaques are formed. Therefore, macrophages are the "key driving force" in the development of AS, affecting plaque stability and AS outcomes.

[0004] Theoretically, if macrophages can be regulated, the progression of atherosclerosis can be effectively inhibited. The CANTOS study proved for the first time that anti-inflammatory drugs have great application value in the treatment of atherosclerotic cardiovascular disease. However, the results of the CIRT study recently published in the New England Journal of Medicine showed that the use of the broad-spectrum anti-inflammatory drug methotrexate did not reduce the mortality rate of atherosclerotic cardiovascular disease. Therefore, finding drugs that target and regulate macrophages can serve as a new breakthrough, leading to a new direction for future research on the prevention and treatment of AS, which has important scientific value and social significance.

[0005] Nanoparticles have been widely used in the treatment of tumors in the past few decades. Since atherosclerotic plaques show high permeability and retention effects similar to solid tumors, nanoparticle-based solutions provide new ideas for the treatment of atherosclerosis. Extensive studies have shown that nanoparticles significantly improve the pharmacokinetic characteristics and chemical stability of drug therapeutics, such as small molecule drugs, peptides, etc. Compared with free drugs alone, nanotherapeutics can also reduce off-target and systemic side effects.

[0006] Extracellular vesicles have been shown to be ideal nanocarriers and have entered a number of clinical trials. Extracellular vesicles from different sources have different functions. Probiotic-derived vesicles (OMVs) are believed to have anti-inflammatory functions and immunomodulatory properties. For example, OMVs can regulate intestinal immune cells to inhibit inflammatory bowel disease. In addition, some studies have preliminarily clarified that OMVs can regulate macrophages to inhibit inflammation. Specifically, OMVs induce the secretion of anti-inflammatory cytokines IL-10 and immunomodulatory cytokines IL-1β and GM-CSF, correcting the imbalance between M1 and M2 macrophages, thereby improving allergic dermatitis. Unlike common inflammatory diseases, macrophages in atherosclerotic diseases usually have lipid metabolism disorders, and the role of OMVs in atherosclerotic macrophages is still unclear. Summary of the invention

[0007] The present invention uses vesicles (OMV) from the probiotic strain Lactobacillus rhamnosus to explore the application and function of OMV as a carrier in atherosclerosis. In vitro experiments and NIRF imaging are used to detect the ability of OMV to target macrophages, and bioinformatics and molecular biology experiments preliminarily explain the mechanism by which OMV regulates macrophages to inhibit atherosclerosis. The present invention will provide new carriers and ideas for the diagnosis and treatment of atherosclerotic diseases.

[0008] The present invention provides application of probiotic vesicles in preparing medicine for treating atherosclerosis.

[0009] Preferably, the probiotic vesicles are derived from probiotics.

[0010] More preferably, the probiotic is Lactobacillus rhamnosus.

[0011] More preferably, the preparation process of the probiotic vesicles is as follows: after centrifugation of the 48h MRS broth culture of Lactobacillus rhamnosus, the supernatant is washed in PBS, and the obtained vesicles are resuspended in PBS, filtered, and stored in PBS at -80°C.

[0012] More preferably, the Lactobacillus rhamnosus 48h MRS broth culture is centrifuged at 600 g for 30 min, and then centrifuged at 100,000×g for 70 min.

[0013] More preferably, the washing conditions in the PBS are: 4° C., 100,000×g.

[0014] More preferably, the filtering is performed using a 0.22 μm filter.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention confirms that OMV has good foam macrophage targeting and regulation capabilities, and further elaborates on the mechanism of regulating macrophages: ① Upregulating transcription factor NR1H3, promoting ABCA1 expression, and promoting lipid efflux; ② Promoting macrophage polarization to M2 type. In short, the foam macrophage targeting capability provided by OMV combined with macrophage function regulation has a significant therapeutic effect on atherosclerosis. The present invention provides an ideal nanocarrier and new method for treating atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Characteristic diagram of OMV in Example 1, where (A) Schematic diagram of separating probiotic vesicles. (B) Transmission electron microscope image and hydrated particle size of OMV. (C) Average hydrated particle size of OMV in saline within 7 days (n=3). (D) Average zeta potential of OMV in saline within 7 days (n=3). (E) Schematic diagram of OMV loading ICG. (FI) Flow cytometry analysis of OMV@ICG incubated with THP-1+PMA or THP-1+PMA+oxLDL at different concentrations for different times. (J) Confocal imaging of OMV@ICG after incubation with THP-1+PMA and THP-1+PMA+oxLDL, respectively. (n=3, scale bar=50μm).

[0018] Figure 2 The results of multiple tests on the targeted delivery of OMV in atherosclerotic mice in Example 1, including (A) a schematic diagram of imaging of atherosclerotic mice. (B) Near-infrared fluorescence (NIRF) imaging of various tissues after tail vein injection of ICG or OMV@ICG. (C) Near-infrared fluorescence imaging of the aorta after tail vein injection of ICG or OMV@ICG. (D) HE staining and Oil Red O staining of atherosclerotic lesions, and fluorescent images of accumulated ICG in atherosclerotic plaques. (E) Co-localization image of the fluorescent signal of accumulated ICG in atherosclerotic plaques and FITC-labeled macrophages.

[0019] Figure 3The results of the efficacy of OMV on atherosclerotic mice in Example 1 are shown, wherein (A) is a schematic diagram of the grouping and treatment process of atherosclerotic mice. (B) In vitro NIRF image of aortic tissue after treatment. (C) Quantitative data of ICG fluorescence signals accumulated in the aorta 2 hours after injection. (D) Oil red O staining of the aortic sinus. (EF) HE staining and Oil red O staining of atherosclerotic lesions in the aortic sinus. (G) Quantitative analysis of Oil red staining. (H) Fluorescent image of accumulated ICG in atherosclerotic plaques in aortic root sections of mouse models. (I) Masson staining of aortic sections. (J) Changes in T-CHO, TG, HDL-C and LDL-C in mouse blood after different treatments. (n=5, *P<0.05; **P<0.01; ***P<0.001).

[0020] Figure 4 The results of in vivo safety testing of OMV treatment in Example 1, including (A) changes in mouse body weight within 30 days. (BE) blood biochemistry of mice after different treatments. (FH) monitoring of liver and kidney function of mice after treatment. (I) H&E stained sections of important organs of mice (n=5, scale bar=200μm).

[0021] Figure 5 Bioinformatics analysis of the mechanism of OMV inhibition of atherosclerosis in Example 1. (A) Schematic diagram of transcriptome sequencing grouping. (B) Sequencing quality control analysis. (C) Genes that changed after OMV treatment. (D) Top 50 genes that changed significantly after OMV treatment. (E) Biological functions that changed significantly after OMV treatment. (F) Biological processes that changed significantly after OMV treatment, including lipid efflux and macrophage regulation.

[0022] Figure 6 The results of the experiment in Example 1 in which OMV promoted lipid efflux through the expression of ABCA1, where (A) the signal pathways that changed significantly after OMV treatment, including the lipid and atherosclerosis pathway. (B) Genes with significant changes in the "lipid and atherosclerosis" pathway. (C) The intersection of differentially expressed genes in the "lipid and atherosclerosis" pathway and genes regulating lipid efflux. (D) Microscopic images of foamy macrophages treated with different concentrations of OMV after Oil Red O staining. (EF) Western blot and RT-qPCR analysis of ABCA1 expression in foamy macrophages treated with different concentrations of OMV. (G) Immunohistochemical analysis of ABCA1 expression in aortic tissue after treatment. (n=5, *P<0.05; **P<0.01; ***P<0.001).

[0023] Figure 7The results of OMV increasing the expression of ABCA1 by upregulating the NR1H3 transcription factor in Example 1, where (A) the intersection of differentially expressed genes and transcription factors regulating ABCA1 was taken to determine that OMV regulated ABCA1 through NR1H3. (BC) Western blot and RT-qPCR were used to detect the expression of NR1H3 regulated by OMVs. (DF) OMVs treatment or NR1H3 overexpression increased the protein level of ABCA1. (GI) After NR1H3 knockdown, OMV treatment reversed the expression of NR1H3 and ABCA1. (J) Schematic diagram of the mechanism by which OMV promotes lipid efflux. (n=5, *P<0.05; **P<0.01; ***P<0.001).

[0024] Figure 8 The results of the experiment in Example 1 in which OMV promotes macrophage polarization, wherein (A) OMV promotes the mechanism of macrophage transformation to M2 type. (B) M1 markers, including CD86, NOS2, TLR4, were downregulated. (C) M2 markers, including CD209, IL1R2, TGM2, were upregulated. (DF) Cytokine expression in foam cells before and after OMV treatment. (F) Morphological changes of foam cells before and after OMV treatment. (G) Western blot analysis of M1 (CD86) and M2 (CD206) 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 of the expression of macrophage (F4 / 80), M1 macrophage (CD86) and M2 macrophage (CD206) markers in aortic tissue after various treatments. (n=5, *P<0.05; **P<0.01; ***P<0.001). DETAILED DESCRIPTION

[0025] Example 1

[0026] Experimental procedures

[0027] 1. Cell Culture and Induction of Foam Cells

[0028] Human THP-1 cells were obtained from the American Type Culture Collection (ATCC; USA) and cultured at 0.5×10 6 / mL density was inoculated in RPMI 1640 medium containing 10% FBS and cultured at 37°C for use. 100ng / mL PMA was added to THP-1 cells and cultured for 48h, and THP-1 cells were induced into macrophages. Subsequently, the cell culture medium was replaced with serum-free RPMI 1640 medium containing 50μg / mL oxLDL and incubated for 48h, and the macrophages were induced into foamy macrophages.

[0029] 2. Animals

[0030] The animal experiment protocol was approved by the Animal Care Committee of Tongji Medical College, Huazhong University of Science and Technology. - / - Deficient mice (6 weeks old) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., China, and were maintained in a specific pathogen-free environment. C57BL / 6 mice (female, 6 weeks old) were purchased from Beijing Huafukang Biotechnology Co., Ltd., China, and were maintained in a specific pathogen-free environment.

[0031] 3. Preparation of OMVs from Lactobacillus rhamnosus

[0032] Lactobacillus rhamnosus was purchased from BeNa Culture Collection (BNCC136673, China) and cultured in MRS medium. The culture supernatant was collected and centrifuged at 600 g for 30 min. The supernatant was centrifuged again at 4°C and 100,000 × g. The obtained precipitate was washed twice with PBS (4°C, 100,000 × g). The final precipitate was resuspended in PBS, filtered through a 0.22 μm filter (Millipore, USA), quantified using a BCA protein detection kit, and then stored at -80°C for future use.

[0033] 4. OMV characteristics

[0034] Transmission electron microscopy (TEM, Hitachi, Japan) and dynamic light scattering (DLS, Malvern Instruments Ltd., Worcestershire, UK) were used to examine the morphology, hydrated particle size, and potential of OMVs. To monitor the stability of OMVs in vitro, DLS was used to monitor the hydrated particle size and potential for 7 days.

[0035] 5. In vitro cell binding ability

[0036] Flow cytometry and confocal imaging were used to detect in vitro cell binding ability. Dimethyl sulfoxide (DMSO, Solarbio, China) was used as a penetration enhancer to increase the solubility and permeability of ICG on the lipid membrane of OMVs. ICG was encapsulated with 4% (v / v) dimethyl sulfoxide (DMSO) in saline. OMVs were then mixed with ICG and cultured in the dark (4°C) for 6 hours with stirring to obtain OMV@ICG. The samples were purified using a PDG-25 desalting column and stored at 4°C. OMV@ICG was incubated with macrophages or foam cells at 37°C for different time points (0h, 1h, 6h, 12h) and different concentrations (0μg / mL, 1μg / mL, 5μg / mL, 10μg / mL and 20μg / mL), and the fluorescence signal was detected by flow cytometer. OMV@ICG (ICG 5μg / mL) and ICG (5μg / mL) were then incubated with macrophages or foam cells at 37°C for 24 hours. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, Boster, Wuhan, China), and the cytoskeleton was stained with FITC-labeled phalloidin (Proteintech, Wuhan, China). After fixation with paraformaldehyde, the cells were observed under a fluorescence microscope (Leica, Germany).

[0037] 6. In vitro NIRF Imaging

[0038] 6w old male ApoE - / - Mice were fed a high-fat diet for 3 months and randomly divided 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 attached to the heart was removed. Static NIRF images were collected using an IVIS spectral imaging system (excitation 750 nm, emission 790 nm).

[0039] 7. Treatment options

[0040] 6w old male ApoE - / - Mice were fed a high-fat diet for 4 weeks and randomly divided into two groups, namely the OMV group and the PBS group (n=5), and then OMV (5 mg / mL, 0.2 mL) or PBS (0.2 mL) was injected into the mice through the tail vein once a week for 8 weeks. At week 19, all model mice were injected with OMV@ICG and the mice were killed.

[0041] 8. Blood Biochemical Analysis

[0042] Blood was collected using EDTA-coated tubes and immediately analyzed for hematological parameters using an automated hematology analyzer (Sysmex KX-21, Sysmex Co., Japan), 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, 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 measured, and serum total cholesterol concentration was measured.

[0043] 9. Histological Analysis

[0044] ApoE - / - The aorta of mice was stained with gross Oil Red O. AS lesions were evaluated using hematoxylin and eosin (H&E) staining, and the plaque and lumen areas were measured under a microscope. Arterial frozen sections were stained with Oil Red O or Sudan IV to evaluate the lipid content in plaques. Masson trichrome staining was used to evaluate the collagen content in AS plaques. Immunohistochemistry (IHC) staining and immunofluorescence staining were used to analyze specific indicators, such as ABCA1 expression, M1 macrophages, M2 macrophages, and accumulation of fluorescent signals in plaques. In addition, major organs (heart, liver, spleen, lungs, and kidneys) were stained with H&E and observed under an optical microscope (Olympus IX73, Japan).

[0045] 10. Transcriptome Sequencing

[0046] THP-1 cells were treated with PMA (100 ng / mL) for 48 hours, then cultured with oxLDL (50 μg / mL) for 48 hours to obtain foamy macrophages, which were then treated with OMV (10 μg / mL). Transcriptome sequencing of foamy macrophages before and after OMV treatment was performed.

[0047] 11. Western Blot Analysis

[0048] THP-1, THP-1+PMA (TP), THP-1+PMA+oxLDL (TO), and THP-1+PMA+oxLDL+OMV (TV) cells were collected and lysed on ice for 30 minutes with lysis buffer containing phosphatase inhibitors and 1% protease. Then, the cell lysate was centrifuged at 12000g, 4°C for 15 minutes, and the supernatant was collected. The 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 and then incubated with the primary antibody overnight at 4°C. The next day, the membrane was washed with 1TBST for 30 minutes 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 are as follows: NR1H3 antibody (#14351-1-AP, 1:1000), CD86 antibody (#13395-1-AP, 1:1000), CD206 antibody (#60143-1-Ig, 1:1000. GAPDH antibody (#ab8245, 1:3000) was purchased from Abcam. ABCA1 (#54186, 1:1000) was purchased from Signalway Antibody.

[0049] 12. Quantitative RT-qPCR Analysis

[0050] Total RNA of cells was extracted using Trizol reagent (#15596026, Invitrogen). The extracted RNA samples were reverse transcribed using PrimeScriptTMRT kit (#RR047A, TAKARA, JPN). Quantitative real-time PCR was performed using TB GreenTM Fast qPCR Mix kit (#RR430A, TAKARA, JPN). GAPDH was used as a reference gene.

[0051] 13. RNA interference

[0052] 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.

[0053] 14. Bioinformatics Analysis

[0054] Bioinformatics analysis was performed using R. The DESeq2 package was used for differential analysis and to obtain the average values ​​of different genes. The ggplot2 and pheatmap packages were used to draw volcano plots and heat maps. The ClusterProfiler package was used for gene set enrichment analysis (GSEA). The GOplot package was used to draw Gocircle diagrams.

[0055] 15. Statistical Analysis

[0056] Statistical analysis was performed using t-test for comparisons between groups, and one-way analysis of variance or two-way analysis of variance 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.

[0057] Experimental Results

[0058] 1. OMV characteristics

[0059] OMVs were isolated from Lactobacillus rhamnosus MRS broth as described in Figure 1 A. Transmission electron microscopy (TEM) showed that OMVs were irregular spherical in shape. DLS analysis showed that the average diameter of the isolated OMVs was 149.67±2.90nm ( Figure 1 B). The average hydrated particle size and zeta potential of OMVs did not change significantly over a period of 7 days, indicating their good stability ( Figure 1 C and Figure 1 D) ICG was loaded into OMVs for flow cytometry and confocal microscopy ( Figure 1 E). As shown in macrophages (THP-1+PMA) and foamy macrophages (THP-1+PMA+oxLDL), the fluorescence signal increased with the increase of OMV@ICG concentration and reached a peak at 5 μg / mL ( Figure 1 F and Figure 1 H); and the fluorescence signals in macrophages and foam cells increased with the extension of culture time ( Figure 1 G and Figure 1 I). In addition, the fluorescence signal in foamy macrophages was higher than that in macrophages, and similar results were found in confocal images ( Figure 1 J).

[0060] 2. OMVs specifically target foamy macrophages

[0061] To investigate the targeting ability of OMVs to foamy macrophages, OMV@ICG and ICG were injected into ApoE cells through the tail vein. - / -Mice (n=3). Mice were killed at different time points (1, 2, and 3 h), and heart and aorta tissues were obtained for NIRF imaging ( Figure 2 A). The OMV@ICG-treated group showed stronger fluorescence than the free ICG-treated group in aortic tissue and aortic root sections ( Figure 2 B-2D). Oil red staining of aortic tissue and oil red and H&E staining of aortic root sections showed that AS models were successfully established in both groups, and the plaque areas were similar ( Figure 2 C and Figure 2 D) Figure 2 As shown in E, the fluorescence signal of ICG is well consistent with the fluorescence signal of the macrophage marker F4-80.

[0062] 3. OMV treatment of atherosclerosis model mice

[0063] We verified the efficacy of OMV on AS plaques, and the specific grouping and treatment regimen were as follows Figure 3 As shown in A. After treatment, the aorta was isolated, and the efficacy was evaluated by NIRF imaging, pathological sections, and immunohistochemistry. Figure 3 As shown in B, the aorta of the OMV-treated group showed weaker fluorescence signals than that of the control group. Fluorescence quantitative analysis was consistent with the NIRF image ( Figure 3 C).

[0064] The aorta was grossly stained with Oil Red O, and the red area was the plaque area ( Figure 3 D) The aortic root pathological sections were then subjected to histological analysis. Figure 3 E and Figure 3 F shows H&E and Oil Red staining of the aortic root showing atherosclerotic plaques. The Oil Red area in the OMV-treated group was significantly reduced by more than 10% ( Figure 3 G). In addition, the ICG fluorescence signal in the control group was stronger ( Figure 3 H). Masson staining showed that the OMV-treated group had high collagen concentration and fibrous cap thickness ( Figure 3 I). 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. Serum high-density lipoprotein cholesterol (HDL-C) levels were increased in the OMV-treated group ( Figure 3 J).

[0065] 4. In vivo safety of OMV therapy

[0066] To systematically evaluate the safety of OMV treatment, C57BL / 6 mice were injected with 100 mg of OMV every 10 days for a total of 3 injections. Mice treated with PBS served as the control group. There was no significant change in the body weight of mice in each group ( Figure 4 A). In addition, there were no significant changes in blood routine tests between the groups ( Figure 4 B-4E). In addition, no significant liver or kidney toxicity was observed in either group ( Figure 4 F-4H). H&E staining also did not reveal any clear evidence of damage to major organs (heart, liver, spleen, lung, kidney, or intestine). Figure 4 I).

[0067] 5. Bioinformatics analysis of the protective mechanism of OMV against AS development

[0068] To further explore the potential mechanism of OMV inhibition of AS, transcriptome sequencing was performed on foam macrophages (THP-1+PMA+oxLDL) before and after OMV treatment ( Figure 5 A). Principal component analysis (PCA) was used to analyze the quality control of RNA sequencing ( Figure 5 B) Differentially expressed genes after OMV treatment Figure 5 C, including 1705 up-regulated genes and 1510 down-regulated genes. The top 50 significantly differentially expressed genes are as follows Figure 5 D, including NCF1, HMOX1, IL23A and NCF1B. GO analysis showed that OMV-mediated AS inhibition involves many biological functions, including cytokine-mediated signaling and active regulation of responses to external stimuli ( Figure 5 E). Many biological processes, including leukocyte migration, macrophage activation, upregulation of lipid transport, and lipid export from cells were significantly upregulated ( Figure 5 F) Based on this, we speculate that OMVs inhibit AS mainly by promoting lipid efflux and regulating macrophages.

[0069] 6. OMVs promote lipid efflux from foamy macrophages

[0070] Several signaling pathways were identified by KEGG analysis, including TNF signaling pathway, cytokine-cytokine receptor interaction, and lipid and atherosclerosis signaling pathway. Among them, the pathway most closely associated with lipid efflux was the “lipid and atherosclerosis” pathway ( Figure 6 A). The differentially expressed genes obtained by sequencing were intersected with all genes in the “lipid and atherosclerosis” pathway to obtain the significantly differentially expressed genes in this pathway ( Figure 6 B), and then these genes were intersected with genes regulating lipid efflux, and ABCA1 was found to be the only gene involved in OMV-regulated lipid efflux ( Figure 6 C). To further confirm whether OMV can promote lipid efflux, foamy macrophages (TO and TV) were stained with Oil Red O before and after OMV treatment. The results showed that the higher the OMV concentration, the lower the intracellular lipid content ( Figure 6D). To further verify that OMVs can upregulate ABCA1 to promote lipid efflux, we used Western blot and RT-qPCR analysis to detect the expression levels of ABCA1 protein and mRNA in foam cells. Foam macrophages were treated with different concentrations of OMVs (0, 10, 20, and 50 μg / mL). The results showed that both protein and mRNA expression levels increased proportionally with the OMV concentration ( Figure 6 E and 6F). In addition, immunohistochemical analysis showed that the level of ABCA1 in aortic plaques of the OMV-treated group was higher than that of the control group ( Figure 6 G).

[0071] To further elucidate the mechanism of ABCA1 upregulation, we intersected the differentially expressed genes with the transcription factors that regulate ABCA1 and obtained two transcriptional genes, NR1H3 and FOXA1 ( Figure 7 A). It has been reported that upregulation of NR1H3 promotes ABCA1 expression, whereas upregulation of FOXA1 inhibits ABCA1 expression. Therefore, we speculated that OMVs promote ABCA1 expression by upregulating NR1H3. Western blot and RT-qPCR analysis demonstrated that NR1H3 protein and mRNA expression increased proportionally with OMV concentration ( Figure 7 B and 7C). To further demonstrate that OMVs regulate ABCA1 expression through NR1H3, NR1H3 was overexpressed or knocked out in foamy macrophages. The results showed that NR1H3 overexpression increased the protein level of ABCA1 ( Figure 7 D-7F), while NR1H3 knockout reduced ABCA1 protein levels in foamy macrophages (see Figure 7 G-7I), Figure 7 J is a schematic diagram of the mechanism by which OMVs promote lipid efflux. In addition, the combined use of NR1H3 overexpression and OMVs did not enhance the expression of ABCA1, while NR1H3 knockout could be reversed by OMV treatment. In conclusion, OMVs can upregulate ABCA1 by increasing the expression of NR1H3.

[0072] 7. OMVs promote macrophage polarization

[0073] In addition to promoting lipid efflux, OMVs can also promote macrophage polarization ( Figure 8 A). According to bioinformatics analysis, markers of M1 macrophages were downregulated after OMV treatment, such as CD86, NOS2, and TLR4 ( Figure 8 B). In addition, several markers of M2 macrophages were upregulated, including CD209, IL1R2, and TGM2 ( Figure 8C). After OMV treatment, the levels of some inflammatory factors also changed significantly; anti-inflammatory factors (IL10, IL11, IL1RN) increased, and the pro-inflammatory factor IL13 decreased ( Figure 8 D and 8E). After OMV treatment, the morphology of foamy macrophages also changed from round to long spindle ( Figure 8 F). Western blot and RT-qPCR analysis showed that CD86 (M1 macrophage marker) was downregulated and CD206 (M2 macrophage marker) was upregulated after OMV treatment ( Figure 8 G). In addition, in vitro confocal imaging results showed that after OMV treatment, CD86 decreased and CD206 increased ( Figure 8 H). In vitro immunohistochemical analysis showed downregulation of F4 / 80 and CD86, and upregulation of CD206 ( Figure 8 I).

[0074] The present invention provides a multifunctional drug delivery carrier for inhibiting atherosclerosis. Different from the existing PEGylation and antibody-based targeted delivery strategies, OMV can target foam macrophages in atherosclerotic plaques due to chemotaxis. This biological nanocarrier provides a broad-spectrum functionalization strategy driven by cell function, and can achieve targeted delivery without specific targeting molecules or complex biocoupling processes. After OMV accumulates in targeted foam macrophages as a nanocarrier, it can promote the lipid outflow and polarization of foam macrophages, thereby effectively inhibiting atherosclerosis.

[0075] At present, many nanoparticles have been used in the study of targeting foam macrophages to treat atherosclerosis, including polymer nanoparticles, lipid nanoparticles, magnetic nanoparticles, recombinant high-density lipoprotein nanoparticles and cell membrane biomimetic nanoparticles. However, these nanoparticles are mainly used as drug delivery carriers and do not have therapeutic functions themselves. They require complex morphological size control, chelation targets, drug loading or control of external magnetic fields to obtain specific targeting or therapeutic capabilities. The complex synthesis steps, difficult batch quality control, and potential toxicity of some nanoparticles limit the clinical application of these nanoparticles. In this study, OMV was obtained from the supernatant of orally available probiotic culture, which has the advantages of simple preparation, high safety, strong carrying capacity and high repeatability. More interestingly, OMV itself has good foam macrophage targeting and regulatory capabilities, which can inhibit the progression of atherosclerosis.

[0076] Probiotic vesicles have good foamy macrophage targeting ability. According to the results of this study, OMV can specifically target foamy macrophages. OMVs have a suitable size and potential, which helps OMVs to better passively target and penetrate atherosclerotic plaques. On the other hand, foamy macrophages are phagocytes that engulf dead cells, foreign particles or microorganisms. In in vitro flow cytometry and confocal experiments, the strongest fluorescence signal can be observed after incubation of OMVs with foamy macrophages, confirming that OMVs have the best binding ability with foamy macrophages. In the in vitro fluorescence imaging experiment, it can also be observed that the aortic tissue and aortic root pathological sections of the OMV@ICG group have stronger fluorescence signals, and on the pathological sections, the fluorescence signal of ICG is consistent with the distribution of macrophages, confirming that OMVs have good foamy macrophage targeting ability.

[0077] Probiotic vesicles have the function of regulating foam macrophages to inhibit atherosclerosis. Foam macrophages are an important target of atherosclerotic plaques. At present, studies have confirmed that foam macrophages can be used to treat atherosclerotic plaques, including promoting the lipid outflow of foam macrophages, promoting the differentiation of foam macrophages into M2 macrophages, and promoting the apoptosis of foam macrophages. This study confirmed that OMV can inhibit atherosclerosis based on biological indicators such as in vitro tissue imaging, gross oil red staining, aortic sinus section oil red and four blood lipids. Subsequently, the regulatory effect of OMV on macrophages was analyzed by bioinformatics, which mainly includes two aspects: promoting the lipid outflow of foam macrophages and promoting the differentiation of foam macrophages into M2 macrophages. This study found that ABCA1 was significantly upregulated by taking the intersection of lipid outflow-related genes and differential genes. ABCA1 has been widely confirmed to be an important channel protein regulating lipid outflow. This study also used molecular biological methods such as western-blot to verify that OMV can indeed upregulate ABCA1. To further study the transcription factors that upregulate ABCA1, we intersected the transcription factors that regulate ABCA1 reported in the literature with the differentially expressed genes, and determined that OMV regulates the expression of ABCA1 through the NR1H3 transcription factor. Subsequently, we overexpressed and knocked down NR1H3, combined with OMV treatment, and further confirmed that OMV upregulated the expression of ABCA1 by upregulating the NR1H3 transcription factor, thereby promoting lipid efflux from foam macrophages. In addition, this study found through bioinformatics that M1 macrophage markers were downregulated and M2 macrophage markers were upregulated, and then western-blot, immunofluorescence, and immunohistochemistry further confirmed the results.

[0078] The present invention preliminarily describes the mechanism by which probiotic vesicles regulate macrophages to inhibit atherosclerosis. Probiotic vesicles are complex biological nanocarriers, including many miRNAs, lncRNAs and proteins, etc., and the specific composition or multiple compositions of macrophages that can be regulated need further study. In addition, probiotic vesicles are a nanocarrier that can load multiple drugs or combine multiple treatment methods including sonodynamic therapy and Cherenkov therapy to improve the efficacy of atherosclerosis.

[0079] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of probiotic vesicles in the preparation of a drug for treating atherosclerosis, characterized in that: The probiotic vesicles are derived from Lactobacillus rhamnosus BNCC136673.

2. The use according to claim 1, characterized in that: The preparation process of the probiotic vesicles is as follows: after centrifugation of the 48h MRS broth culture of Lactobacillus rhamnosus, the supernatant is washed in PBS, and the obtained vesicles are resuspended in PBS, filtered, and stored in PBS at -80°C.

3. The use according to claim 2, characterized in that: The Lactobacillus rhamnosus 48h MRS broth culture was centrifuged at 600g for 30min and then at 100000×g for 70min.

4. The use according to claim 3, characterized in that: The washing conditions in the PBS are: 4° C., 100,000×g.

5. The use according to claim 4, characterized in that: The filtration was performed using a 0.22 μm filter.

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