A composition of an epha2-specific tyrosine kinase inhibitor and a statin and use thereof

Abstract

The application provides a combination of an EphA2-specific tyrosine kinase inhibitor and a statin and application thereof, and belongs to the technical field of pharmacy.The application proves that the statin can up-regulate the expression of EphA2 in macrophages at the cell and mouse levels.The EphA2-specific tyrosine kinase inhibitor ALW-II-41-27 can significantly inhibit the expression of inflammatory genes and proteins such as Nlrp3, IL-1beta and TNF-alpha of macrophages induced by the statin, and can also remodel intestinal flora, regulate the metabolic pathway of the flora, promote the generation of secondary bile acids with anti-inflammatory effect, and further prove that the combination of ALW-II-41-27 and the statin can reduce the atherosclerotic plaques of mice and increase the stability of the plaques.ALT-II-41-27 can block the pro-inflammatory effect of the statin on macrophages, and has a broad prospect in the aspect of anti-atherosclerotic combination therapy drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical technology, in particular to a combination of an EphA2-specific tyrosine kinase inhibitor and a statin drug and application thereof. BACKGROUND

[0002] The erythropoietin-induced hepatocyte receptor (Eph) gene family has the activity of receptor tyrosine kinase, mainly including two subfamilies of EphA and EphB. EphA2 is mainly highly expressed in cell lines of epithelial origin, and is highly expressed in the intestine, lung, skin and kidney, and plays a key role in the regulation of signal transduction pathways related to tumor cell growth, proliferation and metastasis.

[0003] The statin drug is a general term for a class of lipid-lowering drugs, which has the effects of lowering blood lipids, anti-atherosclerosis and anti-thrombosis, and can be roughly divided into natural compounds and synthetic compounds. Natural compounds include pravastatin, lovastatin, simvastatin, mevastatin, etc., and synthetic compounds include atorvastatin, cerivastatin, rosuvastatin, fluvastatin, etc. A large number of studies have shown that the regulation of macrophage function by statins is not completely consistent with its main lipid-lowering and anti-inflammatory properties, which is manifested as the activation of Rac1 / PI3K / PKB / caspase-1 and other inflammatory pathways in macrophages, as well as the promotion of NF-κB activation and interleukin 1β (IL-1β) release. However, there are few studies on the specific molecular regulation mechanism of statins on macrophages and the possible key targets in this process. Therefore, how to inhibit the up-regulation of pro-inflammatory factors caused by statins has become a problem to be solved. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a combination of an EphA2-specific tyrosine kinase inhibitor and a statin drug to solve the problem of up-regulation of pro-inflammatory factors in macrophages caused by taking statins alone.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0006] The present application provides a pharmaceutical composition comprising an EphA2-specific tyrosine kinase inhibitor and a statin drug.

[0007] Preferably, the EphA2-specific tyrosine kinase inhibitor comprises ALW-II-41-27.

[0008] Preferably, the statin drug comprises atorvastatin, rosuvastatin, simvastatin and fluvastatin.

[0009] The application also provides application of the pharmaceutical composition in preparation of an anti-atherosclerosis drug.

[0010] The application also provides application of the EphA2-specific tyrosine kinase inhibitor in preparation of a drug for blocking up-regulation of inflammatory gene expression in macrophages induced by statins.

[0011] Preferably, the inflammatory genes include EphA2, IL-1β, TNF-α, Nlrp3, AnxA2, Notch2 and IL-27.

[0012] By adopting the technical scheme, the application has the following beneficial effects: the application proves at the cell level and the mouse level that statins can up-regulate the expression of EphA2 genes and proteins in macrophages, and the statins can also up-regulate the expression of pro-inflammatory genes in macrophages. The EphA2-specific tyrosine kinase inhibitor ALW-II-41-27 can significantly inhibit the expression of inflammatory genes and proteins such as Nlrp3, IL-1β and TNF-α in macrophages induced by atorvastatin on one hand, and can also regulate the structure and function of intestinal flora, regulate the metabolic pathway of the flora and promote the generation of secondary bile acids with anti-inflammatory effects on the other hand. Further, the application proves that the combination of ALW-II-41-27 and statins can reduce the atherosclerotic plaques and the degree of atherosclerosis in mice. Statins, as a cornerstone drug for treating atherosclerosis, play an important role in reducing the synthesis of low-density lipoprotein cholesterol in the body, but can cause up-regulation of inflammatory gene and protein expression in macrophages. ALW-II-41-27 can block the pro-inflammatory effect of statins on macrophages, and has an important use in the combination therapy of anti-atherosclerosis drugs. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The experimental results of Example 1 are shown in the following table:

[0014] A is a gene differential volcano plot of mouse RAW264.7 cells after being intervened by atorvastatin (25 μM) for 24 hours; red indicates up-regulated genes, and blue indicates down-regulated genes;

[0015] B is the gene expression screened out according to the difference fold;

[0016] C is the result of RT-qPCR verification of the expression of RNA-seq related genes after atorvastatin (25 μM) is intervened in RAW264.7 cells, C57BL / 6J mouse bone marrow macrophages (BMDMs) and human THP-1 cells for 24 hours, respectively;

[0017] D. The results of RNA-seq related gene expression were verified by RT-qPCR after 24 hours of Rvastatin (20 μΜ), Simvastatin (10 μΜ) and Fluvastatin (20 μΜ) intervention on different macrophages described above.

[0018] Figure 2 Part of the experimental results of Example 2, wherein:

[0019] A. The number of all identified proteins in Example 2 Experiment 1;

[0020] B. The results of protein principal component analysis in Example 2 Experiment 1;

[0021] C. The subcellular localization of proteins in Example 2 Experiment 1;

[0022] D. The cluster analysis of differential proteins in Example 2 Experiment 1.

[0023] Figure 3 Part of the experimental results of Example 2, wherein:

[0024] A. The GO, KEGG and Reactome database pathway analysis of differential proteins in Example 2 Experiment 1;

[0025] B. The heat map of 29 proteins mediating NF-κΒ, MAPK and TNF signaling pathways in Example 2 Experiment 1;

[0026] C. The network interaction diagram of EphA2 and differentially expressed proteins in Example 2 Experiment 1;

[0027] D. The results of Western blot detection in Example 2 Experiment 2.

[0028] Figure 4 The experimental results of Example 3, wherein:

[0029] A. The cell proliferation activity after 24 hours of intervention of different concentrations of ALW-II-41-27 on RAW264.7 cells in Example 3 Experiment 1;

[0030] B. The results of cell inflammatory gene expression in Example 3 Experiment 2;

[0031] C. The results of Western blot detection in Example 3 Experiment 2.

[0032] D. The results of immunofluorescence staining in Example 3 Experiment 3.

[0033] Figure 5 The experimental results of Example 4, wherein:

[0034] A. The results of gross oil red O staining of mouse aorta;

[0035] B is the oil red O staining result of the mouse aortic root section;

[0036] C is the H&E staining result of the mouse aortic root section;

[0037] D is the Masson staining result of the mouse aortic root section.

[0038] Figure 6 Figure 4 is the experimental result of Example 4, wherein:

[0039] A is the CD68 and a-SMA immunofluorescence staining result of the mouse aortic root section of Example 4;

[0040] B is the plaque stability index of the mouse aorta calculated in Example 4.

[0041] Figure 7 Figure 5 is part of the experimental result of Example 5, wherein:

[0042] A is the principal component analysis of the mouse intestinal flora;

[0043] B is the enrichment result of the mouse intestinal flora at the door level;

[0044] C is the heat map of the genus level intestinal flora gene change;

[0045] D is the average abundance of the bacterial genus expressing bile salt hydrolase (BSH) involved in the synthesis of primary bile acid;

[0046] E is the average abundance of the bacterial genus expressing hydroxysteroid dehydrogenase (HSDH) involved in the synthesis of primary bile acid.

[0047] Figure 8 Figure 5 is part of the experimental result of Example 5, wherein:

[0048] A is the principal component analysis of the mouse intestinal flora;

[0049] B is the enrichment heat map of primary bile acid and secondary bile acid;

[0050] C is the enrichment of metabolic pathways in the different metabolic substances of the mouse in different intervention groups;

[0051] D is the Pearson correlation analysis of the relative abundance of secondary bile acid and aortic plaque [Note: The color scale represents the correlation strength, ranging from -0.5 (strong negative correlation) to 0.5 (strong positive correlation)]. DETAILED DESCRIPTION

[0052] The technical solutions provided by the present application will be described in detail below in combination with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0053] In the present application, mouse macrophage RAW264.7 and human monocyte-macrophage THP-1 are from China Typical Culture Collection Center, and bone marrow macrophage (BMDM) is isolated and cultured from femur and tibia of C57BL / 6J mouse.

[0054] SYBR Green Pro Taq HS premixed qPCR kit is purchased from China Aikangrui Biological Engineering Co., Ltd.; CCK-8 kit is purchased from China Biyun Tian Biological Technology Institute. Other reagents in the present application can be obtained by routine purchase, unless otherwise specified.

[0055] Example 1 proves that different types of statins can up-regulate the expression of inflammatory genes of macrophages at the gene level

[0056] Experiment 1, mouse macrophages (RAW264.7) are divided into two groups, Atorvastatin (atorvastatin, Selleck, S2077) group and control group, each group has 3 parallel holes, 1*10 6 cells are inoculated into 6-well plates, cultured with DMEM high glucose medium containing 10% fetal bovine serum in a 37°C incubator with 5% CO2, and then cultured with DMEM high glucose medium without fetal bovine serum after adhering for 8h; then the two groups of cells are intervened according to the following methods:

[0057] The Atorvastatin group is cultured with DMEM high glucose medium containing 25μM atorvastatin;

[0058] The control group is cultured with DMEM high glucose medium containing equal amount of PBS;

[0059] After 24h of continuous culture, the cells are collected to extract RNA for RNA sequencing, and the specific process includes total RNA sample detection, mRNA enrichment, double-stranded cDNA synthesis, end repair, A addition and linker, fragment selection and PCR enrichment, library quality detection and illumina sequencing (the RNA-seq part is entrusted to Wuhan Kangce Biological Technology Co., Ltd. to assist in completing). The results are shown in A-B of Figure 1 Figure 1 As shown in A of Figure 1 , atorvastatin intervention can up-regulate 588 genes and down-regulate 627 genes; further screening of genes, as shown in B of , the expression of genes related to inflammation such as EphA2, IL-1β, TNF-α, Nlrp3, AnxA2, Notch2 and IL-27 is significantly up-regulated, and it is also found that the expression of KLF family members KLF2, KLF4, KLF6 and KLF7 is significantly up-regulated.

[0060] Experiment 2, in order to verify the reliability of sequencing, mouse macrophages (RAW264.7), bone marrow macrophages (BMDMs) derived from C57BL / 6J mice femur, human monocyte macrophages (THP-1) were divided into two groups, Atorvastatin group and control group, each group had 6 parallel holes, according to 1*10 6 cells per hole into 6-well plates, cultured with DMEM high glucose medium containing 10% fetal bovine serum, cultured in 5% CO2 37℃ incubator, after adhering, use DMEM high glucose medium without fetal bovine serum to starve for 8h; then the two groups of cells were intervened according to the following method:

[0061] Atorvastatin group was cultured with DMEM high glucose medium containing 25μM atorvastatin;

[0062] The control group was cultured with DMEM high glucose medium containing equal amount of PBS;

[0063] After 24h of continuous culture, the cells were collected to extract RNA, and RT-qPCR detection was carried out according to the kit instructions, the specific process included Trizol method to extract RNA, reverse transcription and fluorescent quantitative PCR. The cDNA obtained by transcription was used as the template for fluorescent quantitative PCR, the primer sequences were shown in Table 1, the reaction system and reaction program were carried out according to the SYBR Green Pro Taq HS premixed qPCR kit instructions, and the results were shown in Figure 1 C of the middle. Figure 1 C in the middle confirmed that atorvastatin had up-regulation effect on inflammation-related genes EphA2, Nlrp3, IL-1β, TNF-α, Nlrp3 and AnxA2.

[0064] Table 1 Fluorescent quantitative PCR primers

[0065]

[0066]

[0067] Experiment 3. In order to confirm that other statins, such as Rosuvastatin, Simvastatin and Fluvastatin also have the effect of promoting macrophage inflammation, mouse macrophages (RAW264.7), bone marrow macrophages (BMDMs) and human monocyte macrophages (THP-1) were divided into 4 groups, Rosuvastatin (MCE, HY-17504), Simvastatin (MCE, HY-17502), Fluvastatin (MCE, HY-14664A) and control group. Each group had 6 parallel holes, according to 1*10 6Cells were plated into 6-well plates and cultured in DMEM high-glucose medium containing 10% fetal bovine serum in a 5% CO2 incubator at 37°C. After attachment, cells were starved for 8 hours using DMEM high-glucose medium without fetal bovine serum. The four groups of cells were then treated as follows:

[0068] Rosuvastatin group was cultured with DMEM high-glucose medium containing 20 μM rosuvastatin;

[0069] The simvastatin group was cultured with DMEM high-glucose medium containing 10 μM simvastatin;

[0070] Fluvastatin group was cultured with DMEM high-glucose medium containing 20 μM fluvastatin;

[0071] The control group was cultured with DMEM high-glucose medium containing an equal amount of PBS;

[0072] After culturing for 24 h, the cells were collected and RNA was extracted for RT-qPCR detection. The results were as follows: Figure 1 As shown in D. Figure 1 As shown in D, the use of the above three statins to intervene in macrophages also obtained results that were basically consistent with those of atorvastatin, that is, statins can significantly upregulate the expression levels of inflammatory genes in macrophages, but compared with the simvastatin and fluvastatin intervention groups, rosuvastatin's promoting effect on inflammatory genes Nlrp3, IL-1β, and TNF-α was slightly weaker.

[0073] In summary, the four commonly used statins in clinical practice, including atorvastatin, rosuvastatin, simvastatin and fluvastatin, can upregulate the expression levels of inflammatory genes in macrophages. Moreover, the regulatory effects of these four statins on macrophage function are not completely consistent with their main lipid-lowering and anti-inflammatory properties, and are manifested as activation of inflammatory pathways including IL-1β.

[0074] Example 2: Protein expression levels confirm that statins can upregulate macrophage EphA2 and inflammatory expression

[0075] Experiment 1: Mouse macrophages (RAW264.7) were divided into 4 groups, each group had 3 parallel wells, and 1*10 6Cells were seeded in 6-well plates and cultured in DMEM high glucose medium containing 10% fetal bovine serum in a 5% CO2 incubator at 37°C, and then cultured in DMEM high glucose medium without fetal bovine serum for 8 hours after adhesion. Then, the cells in the four groups were intervened according to the following methods: the atorvastatin group was cultured in DMEM high glucose medium containing 25 μM atorvastatin, the simvastatin group was cultured in DMEM high glucose medium containing 10 μM simvastatin, the rosuvastatin group was cultured in DMEM high glucose medium containing 20 μM rosuvastatin, and the control group was cultured in DMEM high glucose medium containing an equal amount of PBS. The cells were cultured for 24 hours.

[0076] Then the cells were collected to extract proteins for 4D-label-free proteomics sequencing. The specific process includes protein extraction, enzyme digestion, liquid chromatography-mass spectrometry tandem analysis, and bioinformatics analysis. The analysis process is based on the Raw file obtained by mass spectrometry detection, and the following steps are performed:

[0077] 1) According to the source of the sample, a sample-specific protein database is constructed, and then the analysis software is used for database search;

[0078] 2) Based on the results of database search, the peptide and protein level quality control analysis is performed;

[0079] 3) Quantitative analysis of proteins, including quantitative distribution and repeatability analysis, while showing the sample quantitative intensity value distribution results;

[0080] 4) Common function annotation is performed on the identified proteins, including GO, KEGG, Protein domain, COG / KOG, STRING database, Reactome, and transcription factor annotation;

[0081] 5) According to the quantitative results, the difference fold change (FC) and T-test significance Pvalue value calculation between the two groups are performed, and the difference screening is performed according to the set threshold, and the difference analysis related statistical chart is drawn;

[0082] 6) Functional classification statistical analysis of differential proteins between the two groups, including GO secondary classification, subcellular localization classification, COG / KOG classification and KEGG pathway classification statistics;

[0083] 7) Fisher's exact test method is used for enrichment analysis of differential proteins between the two groups, involving GO, KEGG, Protein domain and Reactome;

[0084] 8) When there are multiple experimental groups in the project, the functional relationship of differential proteins under different experimental conditions is compared through enrichment clustering analysis;

[0085] 9) By protein-protein interaction network (PPI) analysis, the key regulatory proteins under specific experimental conditions were screened.

[0086] Results are shown in A-D of Figure 2 and A-C of Figure 3 . As shown in A of Figure 2 , the sequencing results showed that after the intervention of atorvastatin, simvastatin and rosuvastatin, a total of 80444 peptides and 6095 proteins were significantly changed; as shown in B of Figure 2 , principal component analysis revealed that the protein level composition of the four groups was significantly different, with the largest difference between simvastatin and the control group; as shown in C of Figure 2 , the subcellular localization of the three interventions showed that the differential proteins after atorvastatin and simvastatin intervention were mainly located in the nucleus and cytoplasm, while the differential proteins after rosuvastatin intervention were mainly located in the cytoplasm and plasma membrane; Figure 2 , D shows that atorvastatin and simvastatin can significantly up-regulate the protein changes of clusters 1-3, but rosuvastatin has no significant change. Figure 3 , A shows that GO and KEGG analysis also shows that the differential pathways of atorvastatin and simvastatin intervention of macrophages are mainly concentrated in the NF-κB, MAPK and TNF signaling pathways; as shown in B of Figure 3 , of the 29 proteins involved in the NF-κB, MAPK and TNF inflammatory related pathways, EphA2, Nlrp3, Erk1, Ikkβ, IRAK2 and TRAF1 / 2 were significantly up-regulated after atorvastatin and simvastatin intervention; Figure 3 , C is protein-protein network analysis (PPI), which reveals that EphA2 can interact with a variety of inflammation-related proteins.

[0087] Experiment 2. Mouse macrophages (RAW264.7) were divided into 4 groups, each with 3 parallel holes, and 1*10 6 cells were inoculated into a 6-well plate with DMEM high glucose medium containing 10% fetal bovine serum, and cultured in a 37°C incubator with 5% CO2. After adhering, the medium was changed to DMEM high glucose medium without fetal bovine serum and starved for 8h; then the four groups of cells were intervened according to the following methods:

[0088] The atorvastatin group was cultured with DMEM high glucose medium containing 25μM atorvastatin;

[0089] The simvastatin group was cultured with DMEM high glucose medium containing 10μM simvastatin;

[0090] Rosuvastatin group was cultured with DMEM high glucose medium containing 20 μM Rosuvastatin;

[0091] Control group was cultured with DMEM high glucose medium containing equal amount of PBS;

[0092] Continue to culture for 24 h. Then collect the cells to extract protein for Western blot detection, the specific process is as follows:

[0093] (1) Extraction of total protein of cells: after washing with pre-cooled PBS, add 60 μL-100 μL of RIPA lysis buffer, use clean cell scraper to scrape as much as possible to scrape off the adherent cells, then transfer to another clean EP tube, centrifuge at 12000 g for 15 min at 4°C, then take the supernatant and store on ice for standby.

[0094] (2) BCA method for determining protein concentration: mix A reagent and B reagent according to the volume ratio of 50:1, add 200 μL per well into 96-well plate; dilute BSA protein standard to a concentration of 0.5 mg / mL, respectively take 0, 1, 2, 4, 8, 12, 16, 20 μL of the above diluted BSA standard liquid into the standard well, and then add PBS to 20 μL; add 2 μL of protein supernatant sample to the sample well, and add PBS to 20 μL; after reaction in 37°C water bath for 30 min, detect the absorbance value (OD562 nm) by enzyme label instrument, then draw the standard curve according to the absorbance value of the standard well and the known BSA protein concentration in Excel document, and calculate the corresponding sample volume according to the formula of the standard curve.

[0095] (3) PAGE gel electrophoresis: after the 10% PAGE gel is prepared, add 1x running buffer to the inside of the small glass plate, use a Pasteur pipette or a pipette to gently wash the sample well to prevent gel particles from blocking the sample well; pipette equal concentration of cell or tissue protein sample into the sample well, add protein marker to the first and last wells; set the voltage to 80V for gel loading and 120V for gel running, when the sample runs to the bottom of the separation gel, stop electrophoresis;

[0096] (4) Transfer: Pretreatment PVDF membrane: PVDF membrane of the same size as the target protein was immersed in methanol for 30 seconds, then in double distilled water for 2 minutes, and then placed in the transfer solution. After electrophoresis, the gel was cut at the corresponding position of the marker according to the molecular weight of the target protein. During the process, care should be taken to avoid drying the gel. The membrane was placed in the sandwich clamp in the following order: sponge pad → filter paper → gel → PVDF membrane → filter paper → sponge pad (negative to positive). After clamping, the transfer clamp was quickly placed in the transfer tank containing the transfer solution. The power was turned on, and the transfer time was about 1 minute per molecular weight (kDa). The transfer process was carried out on ice. After transfer, the PVDF membrane with protein blotting was taken out and washed with TBST for 3 times.

[0097] (5) Detection of target protein: The washed PVDF membrane was placed in 5% skim milk or 5% BSA in TBST for 40 minutes to 1 hour at room temperature. The specific protein primary antibody was diluted to the appropriate concentration and added to the primary antibody diluent (anti-GAPDH (Bioss, bs-2188R; 1:5000); anti-EphA2 (ABclonal, A7183; 1:1000); anti-Nlrp3 (Cell Signaling Technology, 15101S; 1:1000); cleaved-anti-IL-1β (Cell Signaling Technology, 63124; 1:1000)). After incubation at 4°C overnight, the membrane was washed with TBST for 10 minutes each time for a total of 3 times. Then the membrane was incubated with HRP-labeled corresponding rabbit or mouse secondary antibody (goat anti-rabbit / mouse secondary antibody, Hangzhou Fude Biological Technology Co., Ltd.) at 37°C for 1 hour. Exposure: discard the secondary antibody, wash the membrane with TBST for 10 minutes each time for a total of 3 times, prepare ECL luminescent solution A and B (Western blot ECL chemiluminescence detection kit, Thermo, USA, 1:1), and evenly drop the luminescent solution on the membrane to be tested in the dark room. The gel imaging system was imaged.

[0098] (6) Gray scale analysis of protein blotting: The WB band was analyzed by Image J software, and the results are shown in D of Figure 3 Figure 3 D of is the Western blot result, which further confirms the up-regulation of atorvastatin and simvastatin on EphA2.

[0099] ​In summary, the protein level experiment results again confirmed that statins can up-regulate the expression of inflammatory related proteins such as EphA2, Nlrp3, IRAK1 (interleukin-1 receptor-associated kinases), TNFAIP2 (TNFα-induced protein 2), TNFSF9 (TNF ligand superfamily, member 9), IKKβ (inhibitor of κB kinase β), NKAP (NF-κB activating protein), etc.

[0100] Example 3 ALW-II-41-27 can significantly inhibit atorvastatin-induced inflammatory gene and protein expression in macrophages

[0101] Experiment 1. Mouse macrophages (RAW264.7) were divided into 6 groups, each with 6 parallel holes, 1*10 4 cells per hole were inoculated into a 96-well plate, cultured with DMEM high glucose medium containing 10% fetal bovine serum, and incubated in a 5% CO2 37°C incubator. After adhering, the medium was changed to DMEM high glucose medium without fetal bovine serum and starved for 8h. Then the 6 groups of cells were treated with DMEM high glucose medium containing 0, 1, 2, 3, 4, 5 μM ALW-II-41-27 (Selleck Biotech, S6515) for 24h, and the proliferation activity of the cells at OD450 was detected according to the CCK-8 kit instructions, and the results are shown in Figure 4 A of FIG. 1. Figure 4 A of FIG. 1 shows that after adding ALW-II-41-27, the proliferation activity of mouse macrophages RAW264.7 decreased, and reached the lowest when the concentration was 4 μM, indicating that ALW-II-41-27 can inhibit the proliferation of mouse macrophages RAW264.7.

[0102] Experiment 2. Mouse macrophages (RAW264.7) were divided into 4 groups, namely control group (DMSO), ALW-II-41-27 group, atorvastatin group and ALW-II-41-27 + atorvastatin group. Each group has 6 parallel holes, 1*10 6 cells per hole were inoculated into a 6-well plate, cultured with DMEM high glucose medium containing 10% fetal bovine serum, and incubated in a 5% CO2 37°C incubator. After adhering, the medium was changed to DMEM high glucose medium without fetal bovine serum and starved for 8h. Then the 4 groups of cells were intervened according to the following methods:

[0103] ALW-II-41-27+atorvastatin group: After 24 h of intervention with 4 μM ALW-II-41-27, the cells were cultured in DMEM high-glucose medium containing 25 μM atorvastatin for another 24 h;

[0104] Atorvastatin group: After adding an equal amount of DMSO to the culture medium for 24 h, the cells were cultured in DMEM high-glucose medium containing 25 μM atorvastatin for another 24 h;

[0105] ALW-II-41-27 group: After 24 h of intervention with 4 μM ALW-II-41-27, the cells were cultured in DMEM high-glucose medium containing an equal amount of PBS for another 24 h;

[0106] Control group: DMSO was added to the culture medium for 24 h, and then the cells were cultured in DMEM high-glucose medium containing an equal amount of PBS for another 24 h.

[0107] Then, cells were collected to extract RNA for RT-qPCR detection, and total protein was extracted for Western blot detection. The specific procedures of RT-qPCR and Western blot were as described above, and the results were as follows. Figure 4 As shown in BC. Figure 4 As shown in B, the transcription levels of Nlrp3, IL-1β, and TNF-α inflammatory genes in the Atorvastatin group were higher than those in the blank group, indicating that atorvastatin could upregulate the transcription of Nlrp3, IL-1β, and TNF-α inflammatory genes. After combined use of ALW-II-41-27 and atorvastatin, the transcription levels of Nlrp3, IL-1β, and TNF-α inflammatory genes decreased, indicating that ALW-II-41-27 could reverse the upregulation of Nlrp3, IL-1β, and TNF-α inflammatory gene transcription levels caused by atorvastatin. Figure 4 C in the figure is the result of Western blot detection. The results of Western blot detection show that the protein expression levels of EphA2, Nlrp3, and IL-1β genes are consistent with those of RT-qPCR detection, indicating that ALW-II-41-27 can inhibit the upregulation of EphA2, Nlrp3, and IL-1β inflammatory gene expression levels caused by atorvastatin.

[0108] Experiment 3: Mouse macrophages (RAW264.7) were divided into two groups: Atorvastatin group and ALW-II-41-27 + atorvastatin group. Each group had three parallel wells, and 1*10 3Cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum in a 5% CO2 incubator at 37°C, and then starved for 8 h in DMEM high glucose medium without fetal bovine serum after adhering. Then, the two groups of cells were intervened according to the following methods:

[0109] ALW-II-41-27+atorvastatin group: 4 μM of ALW-II-41-27 was intervened for 24 h, and then 25 μM of atorvastatin was added to the DMEM high glucose medium for continuous culture for 24 h;

[0110] Atorvastatin group: an equal amount of DMSO was added to the medium for 24 h, and then 25 μM of atorvastatin was added to the DMEM high glucose medium for continuous culture for 24 h;

[0111] Then, the cell culture supernatant in the confocal dish was discarded, and the cells were washed twice with PBS, and then fixed with pre-cooled 4% paraformaldehyde at room temperature for 30 min, and then washed with PBS for 3 times for 5 min each time on a shaker. Then, Triton X-100 membrane breaker was added to PBS at a ratio of 1000:5, and then mixed well and added to the confocal dish, and then the membrane was broken for about 9 min, and then the membrane breaker was discarded, and then washed with PBS for 3 times for 5 min each time. Then, 5% BSA was added for blocking for 1 h, and then Nlrp3 primary antibody (Cell Signaling Technology, 15101S) prepared in advance with 5% BSA was added for incubation of the cells, and then the cells were incubated at 4°C overnight, and then the primary antibody was removed, and then washed with PBS for 3 times for 5 min each time; and then a fluorescent secondary antibody (Alexa Fluor 488 goat anti-rabbit fluorescent secondary antibody, China Biyun Tian Biotechnology Co., Ltd.) prepared with 5% BSA was added for incubation at room temperature for 1 h (the process needs to be operated in the dark at the beginning). After washing with PBS, DAPI was added for 10 min, and then washed with PBS buffer for 3 times, and finally, an anti-fluorescence quencher was added, and then images were collected under a confocal microscope according to different fluorescence excitation light wavelengths. The results are shown in Figure 4 As shown in (D) in FIG. 13, on the basis of atorvastatin intervention, ALW-II-41-27 can reduce the content of Nlrp3 in the cytoplasm. Figure 4 As shown in (D) in FIG. 13, on the basis of atorvastatin intervention, ALW-II-41-27 can reduce the content of Nlrp3 in the cytoplasm.

[0112] In summary, ALW-II-41-27 can greatly reverse the up-regulation of Nlrp3, IL-1β, TNF-α and other inflammation-related genes and proteins in macrophages caused by atorvastatin.

[0113] Example 4: ALW-II-41-27 can further inhibit the progression of atherosclerotic plaques on the basis of statin treatment and increase the stability of plaques

[0114] Eight-week-old male apoE- / - After 16 weeks of high-fat diet (high-fat feed containing 15% fat and 1.2% cholesterol) intervention, the mice were divided into 4 groups: control group, Atorvastatin group, ALW-II-41-27 group, ALW-II-41-27 + atorvastatin group, 8 mice in each group, and the specific intervention scheme is as follows:

[0115] Atorvastatin group: 30 mg / kg / day atorvastatin was given by gavage every day;

[0116] ALW-II-41-27 group: 30 mg / kg / day ALW-II-41-27 was injected intraperitoneally every day;

[0117] ALW-II-41-27 + atorvastatin group: 30 mg / kg / day atorvastatin was given by gavage every day + 30 mg / kg / day ALW-II-41-27 was injected intraperitoneally every day;

[0118] Control group: equal dose of PBS was given by gavage + equal dose of DMSO was injected intraperitoneally.

[0119] After 4 weeks of high-fat diet, the mice were sacrificed, and then the aorta gross and root were collected to determine the degree of atherosclerosis (AS) by staining, and the specific steps are as follows:

[0120] 1. Aorta gross oil red O staining: After being fixed in paraformaldehyde for 18-24 h, the aorta was taken out, placed neatly in a tissue embedding box, washed slowly with tap water for 45 min, then completely immersed in freshly prepared 60% isopropanol for 2-3 min, then the tissue was placed in the oil red O working solution prepared in advance and filtered for 30-45 min, then carefully taken out with forceps and placed in 60% isopropanol for 2-3 min, rinsed twice, and photographed with a Nickon digital camera according to the shape of the aorta, and the results are shown in (A) of Figure 5 From (A) of Figure 5 It can be seen from (A) that atorvastatin can reduce the plaque area of the aorta of mice, and the combination of ALW-II-41-27 administration can further reduce the plaque area on the aorta on the basis of statin administration, and the difference is statistically significant (p<0.05).

[0121] 2. Oil Red O staining of aortic root (to detect lipid droplet content in plaques): Take out the previously cut frozen sections of aortic root tissue, equilibrate at room temperature for 20 minutes, gently rinse with tap water for 45 minutes, immerse the tissue slide completely in the 60% isopropanol prepared for extraction for 2-3 minutes, then place the slide in the freshly prepared Oil Red O working solution for staining for 30 minutes, and then place it in 60% isopropanol for differentiation for 1 minute. After rinsing off excess isopropanol with running water, observe the lipid droplets under an inverted microscope, shake off the residual water on the slide, add hematoxylin, rinse with tap water after 3 minutes, and then add a few drops of 1% hydrochloric acid alcohol. The differentiation time should not exceed 5 seconds, and then rinse with tap water immediately; observe the staining of the slide under an inverted microscope. The cell nuclei in the stained slide are light blue. After air-drying at room temperature, seal the slide with a small amount of glycerol gelatin. The results are as follows: Figure 5 As shown in (B) in . Figure 5 As shown in (B), compared with the control group, the Oil Red O-positive area in the plaque of the aortic root of mice in the atorvastatin intervention group was reduced, and ALW-II-41-27 could further reduce the area of ​​lipid droplets in the plaque on the basis of atorvastatin treatment, and the difference was statistically significant (p < 0.05).

[0122] 3. H&E staining of aortic root (detection of lipid necrosis core): paraffin sections were dewaxed, stained with hematoxylin and eosin, dehydrated and sealed, and images were collected and analyzed under a microscope (blue represents nuclei and red represents cytoplasm). Figure 5 As shown in (C) in . Figure 5 As shown in (C), compared with the control group mice, atorvastatin intervention can significantly reduce the area of ​​lipid necrosis core in the plaque, and the combination of statins with ALW-II-41-27 can further reduce the area of ​​lipid necrosis core in the plaque, and the difference is statistically significant (p < 0.05).

[0123] 4. Masson staining of the aortic root (detection of collagen fiber content): Place the paraffin sections in xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, and 75% alcohol for 20 minutes, 20 minutes, 5 minutes, 5 minutes, and 5 minutes respectively, and then rinse gently with tap water; immerse all tissue blocks on the paraffin sections in Masson A solution overnight and rinse gently with tap water; immerse the paraffin sections in a mixture of Masson B solution and Masson C solution (1:1) for 1 minute, rinse gently with tap water, differentiate with 1% hydrochloric acid alcohol, and rinse gently with tap water; immerse all tissue blocks on the sections in Masson D solution for 6 minutes and rinse gently with tap water; immerse all tissue blocks on the sections in Masson E solution for 1 minute; do not rinse with water, drain the water and immerse directly in Masson again The sections were stained in F solution for 30 seconds; then rinsed and differentiated with glacial acetic acid (1%), and dehydrated twice with anhydrous ethanol; the sections after two dehydrations were immersed in anhydrous ethanol in a third cylinder for 5 minutes, and then immersed in xylene for 5 minutes to make them transparent. After drying, they were carefully sealed with neutral gum and photographed under a microscope (blue represents collagen fibers; red represents muscle fibers, cellulose, and red blood cells). The test results are as follows: Figure 5 As shown in (D) in Figure 5 As shown in (D), compared with the control group, the collagen fiber content in the plaque of the ALW-II-41-27 combined with statin intervention group was significantly increased, and the difference was statistically significant (p < 0.05).

[0124] 5. Paraffin section immunofluorescence homologous double label (detecting the content of macrophages and smooth muscle cells): First, the paraffin section was taken out and placed in xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, 85% alcohol, 75% alcohol for 15 min, 15 min, 5 min, 5 min, 5 min, 5 min, respectively, and then washed with distilled water; antigen repair: first, the paraffin section was placed in a container containing EDTA antigen repair solution, and then transferred to a microwave oven for 8 min, then stopped for 8 min, and then transferred to low heat for 7 min (this step should avoid the evaporation of the buffer to cause dry pieces). After cooling, the tissue section was placed in PBS solution and washed on a shaker at medium speed for 3 times, 5 min each time; circle, hydrogen peroxide blocking: to avoid antibody loss, a circle was drawn around the tissue imprint on the slide with a histology pen, and then the slide was incubated in 3% hydrogen peroxide for 25 min (room temperature, avoid light) to block endogenous peroxidase, and then the tissue section was placed in PBS solution and washed for 3 times, 5 min each time; serum blocking: gently shake off the residual PBS wash solution on the slide, and drop 10% donkey serum or 3% BSA, and block at room temperature for 30 min; add the first primary antibody (rabbit anti-CD68, Cell Signaling Technology, 97778S): shake off the blocking solution, and drop the pre-prepared primary antibody (according to the instructions or the proportion of the pre-experiment results) on the tissue, and then place it in a 4°C refrigerator for overnight reaction; add the corresponding HRP-labeled secondary antibody: take out the wet box, and wash the slide in PBS for 3 times, 5 min each time. After shaking dry, the HRP-labeled secondary antibody (which should be the same species as the corresponding primary antibody) was added in the circle with a pipette, and then incubated at room temperature for 40 min to 1 h; add CY3-TSA (or FITC-TSA): again, the slide was placed in PBS and washed for 3 times. After shaking the slide, TSA was added in the circle, and after 10 min of dark box or dark room reaction in the dark, the slide was placed in TBST buffer for washing for 3 times; microwave treatment: the tissue slide was placed in a repair box containing antigen repair solution, and then placed in a microwave oven according to medium heat for 8 min, stopped for 8 min, and then transferred to low heat for 7 min for treatment to remove the combined primary antibody and the corresponding secondary antibody; add the second primary antibody (rabbit anti-α-SMA, Cell Signaling Technology, 19245S): the second primary antibody was added to the circle of the slide with PBS according to the preparation, and then placed in a 4°C refrigerator for overnight incubation; add the corresponding secondary antibody (Alexa Fluor 488-conjugated goat-anti rabbit secondary antibodies / Cy3 conjugated goat-anti mouse secondary antibodies, China Biyun Tian Biotechnology Company): after taking out the slide, continue to wash as described above for 3 times, 5 min each time.After the slices were dried, the fluorescent secondary antibody (which should be the same species as the primary antibody) was added to the tissue, and incubated at room temperature for 50 minutes in the dark; nuclear staining and anti-quenching agent: 1-2 drops of DAPI staining solution were added directly to the tissue, and then reacted for 10 minutes in the dark. After nuclear staining, the slices were washed with PBS for 3 times, and then 1-2 drops of anti-fluorescence quenching agent were added to the tissue, which was reacted for 5 minutes and then washed with tap water; mounting and photographing: finally, the slices were mounted and the images were collected under a fluorescence microscope. The results are shown in Figure 6 As shown in (A) of FIG. 6, compared with the control group of mice, atorvastatin monotherapy can reduce the content of macrophages (CD68) in the plaque, and the administration of ALW-II-41-27 combined with statin intervention can further reduce the infiltration of macrophages in the plaque; however, the combination of the two drugs can significantly increase the content of smooth muscle cells (a-SMA) in the plaque, and the differences are statistically significant (p<0.05). Figure 6

[0125] 6. Based on the detection results, the plaque stability was calculated, and the calculation formula was: plaque stability = (collagen fiber content + smooth muscle cell content) / (lipid droplet content + macrophage content). The results are shown in (B) of FIG. 6. Figure 6 As shown in (B) of FIG. 6, atorvastatin can promote the plaque stability of mice fed with high-fat diet, and the administration of ALW-II-41-27 combined intervention can further increase the stability of atherosclerotic plaques and prevent plaque rupture, and the difference is statistically significant (p<0.01). Figure 6 In summary, ALW-II-41-27 can further inhibit the progression of atherosclerotic plaques and increase the stability of plaques on the basis of statin therapy.

[0126] Example 5: ALW-II-41-27 can play an anti-atherosclerotic role by remodeling the intestinal flora and promoting the synthesis of bile acids

[0127] 8-week-old male apoE - / - After 16 weeks of high-fat diet (high-fat feed containing 15% fat and 1.2% cholesterol) intervention, the mice were divided into two groups: Atorvastatin group and Atorvastatin+ALW-II-41-27 group, 5 mice in each group. The specific intervention scheme is as follows:

[0128] Atorvastatin group: 30 mg / kg / day atorvastatin was given by gavage every day;

[0129]

[0130] ​​Atorvastatin + ALW-II-41-27 group: 30 mg / kg / day atorvastatin by gavage + 30 mg / kg / day ALW-II-41-27 by intraperitoneal injection;

[0131] After continuing the high-fat diet for 4 weeks, the mouse colon feces and plasma were collected for metagenomic and non-targeted metabolomic detection, and the specific detection steps were as follows:

[0132] 1. Fecal metagenomic detection

[0133] (1) DNA extraction: 1 mL CTAB lysis solution was accurately taken into a high-pressure sterilized EP tube, and after adding lysozyme, a proper amount of mouse fecal sample was added into the lysis solution, and after repeated shaking and mixing, it was fully lysed in a 65°C water bath; after centrifugation at 12000 rpm for 10 min, the supernatant was taken with a pipette, and phenol, chloroform and isoamyl alcohol were added in proportion of 25:24:1, and after fully mixing, it was centrifuged at 12000 rpm for 10 min; the supernatant was carefully taken, and chloroform and isoamyl alcohol were added in proportion of 24:1, and after fully mixing, it was centrifuged at 12000 rpm for 10 min again; the supernatant was carefully taken and placed in a 1.5 mL centrifuge tube, then isoamyl alcohol was added, shaken and mixed, and then placed in a -20°C low-temperature refrigerator for precipitation, and then centrifuged at 12000 rpm for 10 min again, and the supernatant was discarded and the precipitate was reserved, and 75% ethanol 1 mL was added to wash the precipitate twice; the precipitate (DNA sample) was blown dry with an ultra-clean air blower; enzyme-free water was added to dissolve the DNA sample, and if necessary, the sample was heated to help dissolve; 1 μL of RNase A was taken to digest RNA, and placed at 37°C for 15 min.

[0134] (2) Library construction and sequencing:

[0135] a. Sample detection: ① Agarose gel electrophoresis (AGE) analysis was used to test the purity and integrity of DNA (parameters: genomic DNA---gel concentration: 1%, voltage: 100V, electrophoresis time: 40 min; PCR product---gel concentration: 2%, voltage: 80V, electrophoresis time: 40 min), the results showed that the dispersion was concentrated above 500 bp and there was no serious concentrated band below 500 bp. ② Nanodrop (spectrophotometer) was used to measure the purity (evaluation index OD 260 / 280) and Qubit2.0 was used to measure the concentration of the extracted DNA.

[0136] b. Library Construction and Testing: After samples pass testing, the DNA is further fragmented using an ultrasonic disruptor. Library preparation is then completed through a series of steps, including end repair, tailing, sequencing adapter addition, further purification, and PCR amplification. Once library construction is complete, to ensure library quality, preliminary quantification and dilution are performed. The insert size is then tested to ensure it meets expectations. Quantitative PCR is then used to accurately determine the effective concentration of the library.

[0137] c. Sequencing: After the library is tested and confirmed to be qualified, the sample is sequenced using the Illumina PE150 high-throughput sequencing platform to obtain the metagenomic sequences of bacteria, fungi, and viruses in the fecal sample; Quality Control:

[0138] ① KneadData software was used for data quality control and host sequence removal;

[0139] ② Species annotation and relative abundance prediction: Identify the species contained in the sample and apply it to the microbial database;

[0140] ③ Perform functional database annotation: Use HUMAnN2 software to compare the reads of each sample with the UniRef90 database content one by one. Based on the correspondence between the two, obtain the annotation information and relative abundance table of each functional database;

[0141] ④ Based on the obtained species and functional abundance table, cluster analysis, principal component analysis (PCoA) and dimensionality reduction analysis were performed; then, random forest analysis, LEfSe biomarker and difference test (DunnTest analysis) were performed based on the grouping information of the samples to find the differences in species and functional composition among the samples.

[0142] The above experiments were commissioned to Shenzhen Weishengtai Company to assist in the completion of the test and analysis results. Figure 7 As shown by Figure 7 It can be seen that in the composition of intestinal microbiota, principal component analysis showed that the degree of separation of fecal flora in the two groups was significantly different ( Figure 7 A). At the phylum level, the composition of the intestinal flora in the two groups was basically the same, but the abundance of Firmicutes in the feces of mice treated with ALW-II-41-27 combined with atorvastatin increased by nearly 1.5 times ( Figure 7B) Further analysis of Verrucomicrobia, which showed significant difference in door level, found that the relative abundance of Akkermansia muciniphila, a new generation of "star probiotics" with anti-inflammatory and anti-atherosclerotic effects, was significantly higher in the intestines of mice treated with ALW-II-41-27 in addition to atorvastatin (0.0083% vs. 0.82%, p < 0.05), compared with atorvastatin monotherapy. Figure 7 B-7C) In addition, the abundance of Enterococcus, Eggerthella, Latilactobacillus, and Thermococcus, which have anti-inflammatory effects, was higher in the intestines of mice treated with ALW-II-41-27 in addition to atorvastatin, while the abundance of Campylobacter, Mycobacterium, Ralstonia, Corynebacterium, and Prevotella, which have pro-inflammatory effects, was lower Figure 7 C) Subsequently, we found that ALW-II-41-27 can cause changes in the composition of bacteria that mediate key steps in the biosynthesis of secondary bile acids. From D-E in Figure 7 As shown in D-E in the intestines of mice treated with atorvastatin monotherapy, 38.25% of the intestinal bacteria were Bifidobacterium, Lactobacillus, Bacteroides, Clostridium, and Enterococcus, which express bile salt hydrolase (BSH) that can remove bile acids. Although no significant difference in the overall BSH was observed between the two groups, the mice treated with ALW-II-41-27 in addition to atorvastatin showed higher abundance of Lactobacillus, which is also considered a probiotic that can reduce inflammation Figure 7 E) Further studies found that intestinal microbial groups with 7a-hydroxysteroid dehydrogenase (HSDH / 7a-HSDH) activity (Bacteroides, Clostridium, Escherichia, and Eggerthella) were slightly increased in the intestines of mice treated with atorvastatin in addition to ALW-II-41-27, mainly due to the increase in Clostridium abundance (3.12% in the atorvastatin plus ALW-II-41-27 treatment group and 0.23% in the atorvastatin monotherapy group) Figure 8 E).

[0143] 2. Plasma non-targeted metabolomics detection

[0144] After thawing the mouse-derived plasma samples at 4°C or room temperature, 100 μL was pipetted from each sample into a 1.5 mL EP tube; 400 μL of pre-cooled methanol at -20°C was added to each EP tube, and the vortex was vigorously shaken for 60 s to mix thoroughly; centrifuged at 12000 rpm at 4°C for 10 min, and all the supernatant was pipetted into another new sterile EP tube and vacuum-concentrated and dried; 150 μL of 2-chlorophenylalanine was added to dissolve the precipitate, and then the sample was filtered with a 0.22 μm filter to obtain the sample to be tested; 20 μL was pipetted from each sample to be tested to mix into a quality control (QC) sample, and then the remaining sample to be tested was used for liquid chromatography-mass spectrometry (LC-MS) detection using a Thermo Fisher Ultimate 3000 liquid chromatograph. Chromatographic conditions: ACQUITY HSS T31.8 μm column (2.1 x 150 mm) was selected; the sample injection content was 2 μL; the injection temperature was 8°C, the column temperature was 40°C, and the flow rate was 0.25 mL / min in the mode of gradient elution. Mobile phase: 5 mM ammonium formate water (A)-acetonitrile (B) (negative ion); 0.1% formic acid water (C)-0.1% formic acid acetonitrile (D) (positive ion).

[0145] The elution program was performed with a gradient as follows: 2% B / D for 0-1 min; 2%-50% B / D for 1-9 min; 50%-98% B / D for 9-12 min; 98% B / D for 12-13.5 min; 98%-2% B / D for 13.5-14 min; 2% D for 14-20 min in positive mode and 2% B for 14-17 min in negative mode. 8) Mass spectrometry conditions: ThermoFisher Q Exactive Plus was used. The positive ion spray voltage was set at 3.50 kV, the negative ion spray voltage was set at 2.50 kV, the sheath gas was set at 30 arb, and the auxiliary gas was set at 10 arb. The capillary temperature was set at 325℃, the resolution was set at 70000 for full scan (scan range 81-1000), and the collision voltage was set at 30 eV for secondary fragmentation, and unnecessary secondary mass spectrum (MS / MS) information was removed. After data preprocessing, metabolite identification was performed. The identification of metabolites was first confirmed according to the accurate molecular weight (molecular weight error <= 30 ppm), and then the MS / MS fragment pattern was used to confirm the annotation of metabolites according to the human metabolome database (http: / / www.hmdb.ca), METLIN database (http: / / metlin.scripps.edu), Massbank database (http: / / www.massbank.jp / ), LipidMaps database (http: / / www.lipidmaps.org), mzClound database (https: / / www.mzcloud.org). Plasma non-targeted metabolomics detection was performed as shown in Figure 8 It can be seen from Figure 8 that PCoA shows significant differences in the metabolic profiles of plasma samples Figure 8 A). It is known that bile acids play an important role in inflammation, lipid metabolism and atherosclerosis by interacting with various host nuclear receptors such as FXR and TGR527. In this embodiment, secondary bile acids (SBAs) including deoxycholic acid (DCA), lithocholic acid (LCA) and glycochenodeoxycholic acid (GUDCA) were significantly up-regulated in ALW-II-41-27 combined with atorvastatin treatment in mice, while primary bile acids (PBAs) including chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA) and glycochenodeoxycholic acid (GCDCA) showed no significant change Figure 8B). Primary bile acids are converted to secondary bile acids by gut microbiota, so we further evaluated whether ALW-II-41-27 mediated bile acid modification by regulating the composition of gut microbiota. Finally, we found that the "Primary bile acid biosynthesis" and "Secondary bile acid biosynthesis" pathways were not different between the two groups, while the "Taurine and hypotaurine metabolism" and "Glycine, serine and threonine metabolism" two bile acid-related pathways were significantly enriched in the plasma samples of mice receiving ALW-II-41-27 combined with atorvastatin treatment Figure 8 C). To determine whether secondary bile acids have a potential association with the atherosclerotic phenotype of mice, we used SPSS software for Pearson correlation analysis. The results showed that the relative abundance of secondary bile acids DCA, LCA and GUDCA enriched in the plasma of mice treated with ALW-II-41-27 combination therapy was significantly negatively correlated with the aortic plaque area ​ D).

[0146] In summary, the anti-inflammatory and atherosclerotic protective effects of ALW-II-41-27 can be partly attributed to its regulation of gut microbiota and bile acid metabolism.

[0147] From the above examples, the application provides the use of EphA2-specific tyrosine kinase inhibitor ALW-II-41-27 in the preparation of a drug for blocking the up-regulated expression of inflammation genes caused by statins. ALW-II-41-27 can significantly inhibit the expression of inflammation genes such as Nlrp3, IL-1β and TNF-α at the level of macrophages induced by atorvastatin, and can also remodel the structure and function of gut microbiota, regulate the metabolic pathways of the microbiota, and promote the generation of secondary bile acids with anti-inflammatory effects; the combination of ALW-II-41-2 and statins can reduce atherosclerotic plaques in mice and increase plaque stability.

[0148] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises ALW-II-41-27 and statins.

2. The pharmaceutical composition according to claim 1, characterized in that The statins include atorvastatin, rosuvastatin, simvastatin and fluvastatin.

3. Use of the pharmaceutical composition according to claim 1 in the preparation of anti-atherosclerotic drugs.

4. The use according to claim 3, characterized in that ALW-II-41-27 blocks the upregulation of inflammatory genes induced by statins.

5. The use according to claim 4, characterized in that The inflammatory genes include EphA2, IL-1β, TNF-α, Nlrp3, AnxA2, Notch2 and IL-27.

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