Use of nod1 inhibitor ml130 in hypoxia-induced macrophage polarization

Abstract

This invention relates to the field of cardiovascular disease research technology, and particularly to the application of the NOD1 inhibitor ML130 in hypoxia-induced macrophage polarization, including the use of ML130 in the preparation of drugs that regulate hypoxia-induced macrophage polarization. These drugs inhibit the NOD1 / RIP2 signaling axis and can be used to intervene in the formation and progression of atherosclerotic plaques. This invention creatively combines the NOD1 inhibitor ML130 with the hypoxic microenvironment of atherosclerotic plaques, specifically inhibiting the NOD1 / RIP2 signaling pathway and directly regulating macrophage polarization balance; reducing the accumulation of pro-inflammatory cells in unstable plaque areas, inhibiting foam cell formation and necrotic core expansion, significantly improving plaque stability, and reducing the risk of serious cardiovascular events such as acute coronary syndrome; it clarifies that ML130 exerts its effect by blocking the NOD1 / RIP2-NF-κB / MAPK signaling axis, exhibiting high target specificity and avoiding the side effects of broad-spectrum inhibition by traditional anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to the field of cardiovascular disease research technology, and in particular to the application of the NOD1 inhibitor ML130 in hypoxia-induced macrophage polarization. Background Technology

[0002] Atherosclerosis is the leading pathological basis of cardiovascular disease worldwide, and its core mechanism is closely related to chronic inflammation and macrophage polarization imbalance within plaques. The characteristic hypoxic microenvironment formed within plaques due to luminal narrowing, intimal thickening, and active metabolism of inflammatory cells can activate the NOD1 / RIP2 signaling pathway, driving macrophages to transform into the M1 pro-inflammatory phenotype, thereby exacerbating plaque progression and increasing the risk of rupture.

[0003] Existing technologies, such as the application of PCSK9 in macrophage M2 polarization and related diseases disclosed in CN115094134B, have shown that downregulating PCSK9 in colon cancer cells or using PCSK9 inhibitors can promote the M2-to-M1 phenotype transformation of co-cultured macrophages and inhibit colon cancer cell invasion and metastasis. Another example is the application of DDX3X inhibitors in the preparation of drugs for treating aseptic loosening of artificial joints, disclosed in CN118649238B. This approach reduces mitochondrial homeostasis imbalance in macrophages and decreases the expression levels of mitochondrial stress-related proteins by inhibiting the oligomerization level of ASC protein in macrophages. While these technologies demonstrate the effectiveness of specific target inhibition, they do not address the regulatory relationship between the NOD1 / RIP2 signaling pathway and macrophage polarization under hypoxic microenvironments in atherosclerosis. Therefore, existing technologies cannot meet the precise needs of 'targeting the microenvironment-regulating cell phenotype-stabilizing plaques' in AS treatment, lacking specific intervention methods for macrophage polarization balance under hypoxic conditions. Summary of the Invention

[0004] The purpose of this invention is to address the problem of difficulty in regulating macrophage polarization under hypoxic conditions in the prior art, and to propose the application of the NOD1 inhibitor ML130 in hypoxia-induced macrophage polarization.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The application of NOD1 inhibitor ML130 in hypoxia-induced macrophage polarization, including its use in the preparation of drugs that regulate hypoxia-induced macrophage polarization: The drug regulates macrophage polarization towards the M2 anti-inflammatory phenotype by inhibiting the NOD1 / RIP2 signaling axis, reducing the secretion levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α, and increasing the expression levels of anti-inflammatory factors IL-10 and TGFβ. The low-oxygen conditions are defined as an oxygen concentration of 1%-5%. The macrophages include M1 macrophages derived from Ly6Chi mononuclear cells and M2 macrophages derived from Ly6Clo mononuclear cells; The drug can be used to intervene in the formation and progression of atherosclerotic plaques.

[0006] Preferably, the drug is used to prepare a drug for treating atherosclerosis.

[0007] Preferably, the drug can reduce the expression of CXCL9, CXCL10, and CXCL5 in macrophages.

[0008] Preferably, the drug can increase the expression levels of CCL17 and CCL24 in macrophages.

[0009] Preferably, the drug is used to prepare a drug for stabilizing atherosclerotic plaques.

[0010] Preferably, the drug comprises an effective dose of ML130 and a pharmaceutically acceptable carrier.

[0011] Preferably, the carrier includes one or more of a buffer, excipient, or stabilizer.

[0012] Preferably, the drug can inhibit the interaction between NOD1 and RIP2.

[0013] Preferably, the drug can block the activation of NF-κB and MAPK signaling pathways.

[0014] Preferably, the drug is used to prepare a drug that improves the hypoxic microenvironment of atherosclerotic plaques.

[0015] Compared with the prior art, the present invention has the following advantages: 1. This invention creatively combines the NOD1 inhibitor ML130 with the hypoxic microenvironment of atherosclerotic plaques, specifically inhibiting the NOD1 / RIP2 signaling pathway, directly regulating macrophage polarization balance, and directly targeting the core target of AS inflammation progression. This fills the technical gap in existing treatments that do not target macrophage polarization regulation under hypoxic conditions, thereby achieving precise targeting of the pathological mechanism.

[0016] 2. This invention significantly reduces local inflammatory infiltration in plaques and decreases the amplification effect of inflammatory factor networks by inhibiting the activation of M1 macrophages and the secretion of pro-inflammatory factors, while promoting the anti-inflammatory phenotype transformation of M2 macrophages, thereby effectively delaying plaque progression and achieving powerful regulation of inflammatory responses.

[0017] 3. This invention enhances plaque stability by regulating the expression of macrophage polarization-related chemokines, reducing the aggregation of pro-inflammatory cells in unstable plaque areas, inhibiting foam cell formation and necrotic core expansion, significantly improving plaque stability, and reducing the risk of serious cardiovascular events such as acute coronary syndrome.

[0018] 4. This invention clarifies that ML130 exerts its effects by blocking the NOD1 / RIP2-NF-κB / MAPK signaling axis, exhibiting high target specificity and avoiding the side effects caused by the broad-spectrum inhibition of traditional anti-inflammatory drugs, thus providing a precise and clear direction for the treatment of atherosclerotic diseases.

[0019] 5. This invention can be used alone as a treatment for atherosclerosis, or in combination with existing lipid-lowering and antiplatelet drugs to further optimize the treatment effect and provide personalized treatment plans for patients with AS at different stages, which has important clinical translational value. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the experimental control of the application of the NOD1 inhibitor ML130 proposed in this invention in hypoxia-induced macrophage polarization; Figure 2 CD86 + Schematic diagram of large macrophages and Ly6Chi monocytes; Figure 3 This is a diagram showing the comparison of experimental results; Figure 4 CD86 + Type 1 macrophages, C206 + Schematic diagram of the distribution of M2 and M2 macrophages. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0022] Reference Figures 1-4 The application of NOD1 inhibitor ML130 in hypoxia-induced macrophage polarization includes its use in the preparation of drugs that regulate hypoxia-induced macrophage polarization. ML130 specifically targets the NOD1 / RIP2 signaling pathway, directly inhibiting the protein-protein interaction between NOD1 and RIP2, preventing the recruitment and activation of RIP2 after NOD1 oligomerization, and blocking the NOD1 / RIP2-NF-κB / MAPK signaling axis from the source, thus achieving an overall shift from pro-inflammatory to anti-inflammatory.

[0023] The drug inhibits the NOD1 / RIP2 signaling axis, that is, it blocks the NOD1 / RIP2-NF-κB / MAPK signaling axis by inhibiting the protein-protein interaction between NOD1 and RIP2. This interferes with the in situ differentiation of monocytes after recruitment and the phenotypic reprogramming of established macrophages, regulates the polarization of macrophages towards the M2 anti-inflammatory phenotype, reduces the secretion levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α, and increases the expression levels of anti-inflammatory factors IL-10 and TGFβ.

[0024] Hypoxic conditions, defined as oxygen concentrations of 1%-5%, precisely simulate the pathophysiological oxygen gradient from the necrotic core to the peripheral area of ​​atherosclerotic plaques. Severe hypoxia exists in the core area of ​​atherosclerotic plaques, with oxygen partial pressures as low as 0.5%-2%, and this is positively correlated with the degree of inflammation. By explicitly defining hypoxic conditions as 1%-5%, this precisely simulates the pathophysiological oxygen gradient from the necrotic core to the peripheral area of ​​the plaque. In vitro experiments were conducted using a 1% oxygen concentration. As an extreme hypoxia model, it aims to maximize the stress response of the NOD1 / RIP2 signaling pathway, thereby clearly verifying the intervention efficacy of ML130 in the most severe pathological microenvironment.

[0025] Macrophages include M1 macrophages derived from Ly6Chi monocytes and M2 macrophages derived from Ly6Clo monocytes. Ly6Chi monocytes exhibit strong inflammatory tropism and are the main precursors of M1 macrophages within plaques; while Ly6Clo monocytes are associated with tissue repair and anti-inflammation. ML130 achieves a holistic transformation of macrophages from pro-inflammatory to anti-inflammatory by intervening in the in situ differentiation of recruited monocytes and the phenotypic reprogramming of established macrophages.

[0026] Drugs can be used to intervene in the formation and progression of atherosclerotic plaques. Regulation is not only reflected in surface markers (CD86↓, CD206↑) and enzyme markers (iNOS↓, Arg-1↑), but also extends to the remodeling of the chemokine spectrum.

[0027] ML130 specifically targets the NOD1 / RIP2 signaling pathway, directly inhibiting the protein-protein interaction between NOD1 and RIP2, preventing the recruitment and activation of RIP2 after NOD1 oligomerization, and blocking the NOD1 / RIP2-NF-κB / MAPK signaling axis at its source. Activation of this signaling axis can lead to IκBα degradation, NF-κB p65 subunit phosphorylation (p-p65), and ERK1 / 2 phosphorylation (p-ERK1 / 2) levels. ML130 can inhibit these phosphorylation events in a concentration-dependent manner, thereby reversing the hypoxia-induced M1 polarization tendency of macrophages and promoting their conversion to the M2 anti-inflammatory phenotype.

[0028] The drug is used to prepare medications for treating atherosclerosis.

[0029] The drug can reduce the expression of CXCL9, CXCL10, and CXCL5 in macrophages.

[0030] The drug increased the expression levels of CCL17 and CCL24 in macrophages. ML130 decreased the expression of CXCL9, CXCL10, and CXCL5 (all M1-related, capable of recruiting pro-inflammatory cells such as effector T cells), while increasing the expression of CCL17 and CCL24 (M2-related, capable of recruiting regulatory T cells, etc.). This dual regulatory strategy fundamentally alters the chemotaxis and aggregation patterns of local immune cells in plaques, providing a direct explanation for the inflammation reduction and increased stability observed in in vivo experiments.

[0031] The drug is used to prepare agents for stabilizing atherosclerotic plaques.

[0032] The drug comprises an effective dose of ML130 and a pharmaceutically acceptable carrier. As a specific small molecule inhibitor, ML130's core function is to directly inhibit the protein-protein interaction between NOD1 and RIP2, preventing the recruitment and activation of RIP2 after NOD1 oligomerization, thereby blocking signal transduction at its source. The blocked signaling axis is NOD1 / RIP2-NF-κB / MAPK. Experimental data show that hypoxia activation of this axis leads to a significant increase in IκBα degradation, NF-κB p65 subunit phosphorylation (p-p65), and ERK1 / 2 phosphorylation (p-ERK1 / 2). After ML130 intervention, these phosphorylation events were inhibited in a concentration-dependent manner. The NF-κB and MAPK pathways are key hubs regulating the transcription of inflammatory factors (IL-1β, IL-6, TNF-α) and polarization-related genes; therefore, blocking this axis can globally reverse inflammation and polarization imbalance at the transcriptional level.

[0033] The carrier includes one or more of buffers, excipients, or stabilizers.

[0034] Marker alteration: ML130 achieves an overall transformation of macrophages from pro-inflammatory to anti-inflammatory by intervening in the in situ differentiation of monocytes after recruitment and the phenotypic reprogramming of established macrophages; Cytokine regulation: Reduces the secretion of pro-inflammatory factors IL-1β, IL-6, and TNF-α, and increases the expression of anti-inflammatory factors IL-10 and TGFβ; Chemokine spectrum remodeling: Reduced expression of CXCL9, CXCL10, and CXCL5 (recruiting pro-inflammatory cells), increased expression of CCL17 and CCL24 (recruiting anti-inflammatory cells), altering the local immune cell aggregation pattern in plaques.

[0035] The drug can inhibit the interaction between NOD1 and RIP2. It is clear that ML130 regulates polarization balance through a cascade reaction of inhibiting NOD1-RIP2 interaction, blocking the NF-κB / MAPK axis, and reshaping the chemokine spectrum, rather than broad-spectrum anti-inflammatory. It has high target specificity and fewer side effects.

[0036] The drug can block the activation of NF-κB and MAPK signaling pathways.

[0037] The drug is used to prepare a drug that improves the hypoxic microenvironment of atherosclerotic plaques. It not only improves the inflammatory microenvironment within the plaque, but also regulates the recruitment of immune cells through chemokine remodeling, thereby enhancing plaque stability and overcoming the shortcomings of traditional anti-inflammatory drugs that only suppress inflammation but do not maintain stability.

[0038] To verify the effects, in vitro and in vivo experiments were designed: I. Experimental Materials 1) Cells: Mouse bone marrow-derived macrophages, RAW264.7 macrophage line, ApoE Mice (6-8 weeks old, male) 2) Reagents: NOD1 inhibitor ML130 (purity ≥98%), oxLDL, low-glucose DMEM medium, fetal bovine serum (FBS, Gibco), penicillin-streptomycin mixture, PBS buffer, RIPA lysis buffer, BCA protein quantification kit, NOD1 antibody, RIP2 antibody, p-NF-κBp65 antibody, IκBα antibody, ERK1 / 2 antibody, p-ERK1 / 2 antibody, IL-1β, IL-6, TNF-α, IL-10, TGFβ ELISA kit, CD86 (M1 marker), CD206 (M2 marker) flow cytometry antibody, qPCR primers Consumables: Cell culture flasks, 6-well / 24-well / 96-well cell culture plates, flow cytometry tubes, PVDF membranes, ELISA plates, centrifuge tubes 3) Instruments: Hypoxia incubator (Thermo, 1%) 3% 5% The equipment includes: a concentration-controlled flow cytometer (BDFACS Canto II), a real-time quantitative PCR instrument (Roche Light Cycler 480), a Western blot electrophoresis and transfer system (Bio-Rad), an ELISA reader (Thermo Multiskan Sky), an ultrasonic homogenizer, a refrigerated centrifuge, a pathological sectioning machine, and a fluorescence microscope (Olympus). II. In vitro experiments: The regulatory effect of ML130 on hypoxia-induced macrophage polarization 1) Experimental Grouping ①Normoxia control group (21%) ): A complete culture medium containing only macrophages.

[0039] ② Hypoxia model group: Macrophages + hypoxic environment (1%) ) + oxLDL (50 μg / mL).

[0040] ③ ML130 low-concentration intervention group: hypoxia model group + ML130 (1μM).

[0041] ④ML130 medium concentration intervention group: hypoxia model group + ML130 (5μM).

[0042] ⑤ High concentration of ML130 intervention group: hypoxia model group + ML130 (10μM).

[0043] 2) Experimental methods ① Macrophage culture and treatment: BMDM isolation and culture: Bone marrow from 6-8 week old C57BL / 6 mice was collected. After removing red blood cells with red blood cell lysis buffer, the marrow was cultured in DMEM medium containing 10% FBS, 1% penicillin-streptomycin and M-CSF (20 ng / mL) for 7 days to obtain mature BMDM.

[0044] RAW264.7 cell culture: cultured in low-glucose DMEM medium containing 10% FBS at 37°C and 5% C. ,twenty one% Cultured under specific conditions and passaged for 3-5 generations for experimental use.

[0045] Hypoxia induction and drug intervention: Macrophages were seeded in 6-well plates (5 × 10⁶ cells / well). 5 After 24 hours of adhesion, the culture medium was replaced with oxLDL and placed in a hypoxic incubator (1%). The cells were cultured and treated with different concentrations of ML130 for 48 hours; the normal oxygen control group had an oxygen content of 21%. Synchronous cultivation under specific conditions.

[0046] ②Indicator testing: qPCR detection of polarization markers and inflammatory factor mRNA expression: Total RNA was extracted from cells and reverse transcribed into cDNA. The mRNA levels of CD86, iNOS (M1 marker), CD206, Arg-1 (M2 marker), IL-1β, IL-6, TNF-α, IL-10, and TGFβ were detected by qPCR, with GAPDH as an internal reference gene.

[0047] Flow cytometry was used to detect the polarization ratio of macrophages: cells were collected, washed twice with PBS, and CD86-FITC and CD206-PE antibodies were added. The cells were incubated at 4°C in the dark for 30 minutes. The proportion of CD86⁺M1 and CD206⁺M2 macrophages was detected and analyzed by flow cytometry.

[0048] Western blot detection of NOD1 / RIP2 signaling pathway-related proteins: Total cellular protein was extracted, quantified using the BCA method, and then subjected to SDS-PAGE electrophoresis, membrane transfer, and blocking. Primary antibodies (NOD1, RIP2, p-NF-κBp65, IκBα, p-ERK1 / 2, ERK1 / 2) were added and incubated overnight at 4°C. Secondary antibodies were incubated at room temperature for 1 hour. ECL staining was performed, and the gray values ​​of the bands were quantified using ImageJ software.

[0049] ELISA detection of inflammatory factor content in cell supernatant: Cell culture supernatant from each group was collected, and the protein concentrations of IL-1β, IL-6, TNF-α, IL-10, and TGFβ were detected according to the ELISA kit instructions.

[0050] 3) Experimental Results ① Effects of ML130 on macrophage polarization markers under hypoxia: Compared with the normoxic control group, the expression of CD86 and iNOS mRNA and the proportion of CD86⁺ cells were significantly increased in the hypoxia model group (P<0.001), while the expression of CD206 and Arg-1 mRNA and the proportion of CD206⁺ cells were significantly decreased (P<0.001). After ML130 intervention, the above indicators showed a concentration-dependent reversal, and the high concentration group (10 μM) was significantly different from the hypoxia model group (P<0.01).

[0051] ② Effects of ML130 on macrophage inflammatory factor secretion under hypoxic conditions: In the hypoxia model group, the secretion of IL-1β, IL-6, and TNF-α was significantly increased (P<0.001), while the secretion of IL-10 and TGFβ was significantly decreased (P<0.001); After ML130 intervention, the secretion of pro-inflammatory factors decreased, while the secretion of anti-inflammatory factors increased, and the higher the concentration, the more significant the effect (P<0.05 or P<0.01).

[0052] ③ Effects of ML130 on the NOD1 / RIP2 signaling pathway under hypoxia: NOD1 and RIP2 protein expression and p-NF-κBp65 and p-ERK1 / 2 phosphorylation levels were significantly increased in the hypoxia model group (P<0.001), while IκBα protein expression was significantly decreased (P<0.001); ML130 could inhibit NOD1 / RIP2 expression and downstream NF-κB and MAPK pathway activation in a concentration-dependent manner (P<0.05 or P<0.01).

[0053] 4) Statistical analysis of key in vitro experimental indicators Group CD86⁺M1 percentage (%) CD206⁺M2 percentage (%) IL-1β (pg / mL) IL-10 (pg / mL) p-NF-κB p65 / β-actin NOD1 / β-actin Normal oxygen control group 12.3±1.5 38.6±2.8 45.2±5.1 126.8±8.5 0.21±0.03 0.35±0.04 Hypoxia model group 47.8±3.2*** 15.3±1.9*** 289.5±12.6*** 42.3±4.8*** 1.68±0.12*** 1.24±0.09*** ML130 low concentration group (1μM) 35.6±2.5# 24.7±2.1# 198.7±9.8# 78.5±6.3# 1.12±0.08# 0.89±0.07# ML130 medium concentration group (5μM) 22.4±2.0## 31.2±2.5## 112.3±7.2## 105.6±7.1## 0.65±0.05## 0.62±0.05## ML130 high concentration group (10μM) 16.7±1.8### 36.9±2.7### 78.5±6.5### 118.9±8.2### 0.38±0.04### 0.42±0.04### Note: Compared with the normal oxygen control group, ***P<0.001; compared with the hypoxia model group, #P<0.05, ##P<0.01, ###P<0.001.

[0054] 5) Using four techniques—qPCR (mRNA level), flow cytometry (protein level and cell population), Western blot (signaling pathway proteins), and ELISA (secretory proteins)—a complete chain of evidence was constructed from gene transcription, protein expression, cell phenotype to functional secretion, fully demonstrating the regulatory role of ML130 in macrophage polarization and inflammatory phenotype.

[0055] III. In vivo experiments: ML130's effect on ApoE under hypoxic conditions Intervention effect on atherosclerosis in mice 1) Experimental Grouping ① Normal control group: C57BL / 6 mice (6-8 weeks old, male), fed with a normal diet and intraperitoneal injection of physiological saline, and kept in a normal oxygen environment.

[0056] ②AS model group: ApoE Mice were fed a high-fat diet (containing 21% fat and 0.15% cholesterol) plus intraperitoneal injection of saline, and subjected to a hypoxic environment (1%). They are fed for 8 hours a day.

[0057] ③ ML130 low-dose group: ApoE Mice were fed a high-fat diet and received intraperitoneal injections of ML130 (5 mg / kg) in a hypoxic environment.

[0058] ④ ML130 high-dose group: ApoE Mice were fed a high-fat diet and received intraperitoneal injections of ML130 (10 mg / kg) in a hypoxic environment.

[0059] Each group consisted of 10 mice, and the experiment lasted for 12 weeks. The mice were weighed weekly, and the dosage was adjusted accordingly.

[0060] 2) Experimental methods ① Animal model construction and drug administration: After 1 week of adaptive feeding, the AS model group and the ML130 intervention group were fed a high-fat diet and placed in a hypoxic environment (8 hours a day, with normal oxygen environment for the rest of the time). At the same time, the corresponding drugs or physiological saline were injected into the peritoneum three times a week for 12 weeks.

[0061] ② Specimen collection: After the experiment, the mice were fasted for 12 hours, anesthetized by intraperitoneal injection of sodium pentobarbital, and blood samples were collected (serum was separated by centrifugation). The aorta and heart tissue were then separated.

[0062] ③Indicator testing: a. Serum lipid and inflammatory factor detection: The levels of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were detected by a fully automated biochemical analyzer; the concentrations of serum IL-1β, IL-6, TNF-α, IL-10, and TGFβ were detected by ELISA.

[0063] b. Aortic plaque analysis: The aorta was isolated, rinsed with PBS, longitudinally cut, stained with Oil Red O, and photographed. ImageJ software was used to calculate the percentage of plaque area to aortic intima area. Paraffin sections were prepared from the aortic root, and HE staining was used to observe plaque morphology. Masson staining was used to detect collagen content in the plaque (reflecting plaque stability).

[0064] c. Detection of macrophage polarization in plaques: Paraffin sections from the aortic root were dewaxed to water, and after antigen retrieval, CD86 and CD206 antibodies were added for immunofluorescence staining. The number of CD86⁺M1 and CD206⁺M2 macrophages in the plaques was observed and counted under a fluorescence microscope.

[0065] d. Detection of NOD1 / RIP2 signaling pathway in aortic tissue: Total protein was extracted from aortic tissue, and the expression levels of NOD1, RIP2, p-NF-κBp65, and p-ERK1 / 2 proteins were detected by Western blot.

[0066] 3) Experimental Results ①ML130 for ApoE Effects on serum lipids and inflammatory factors in mice: Compared with the normal control group, the serum TC, TG and LDL-C levels in the AS model group were significantly increased (P<0.001), the HDL-C level was significantly decreased (P<0.001), the secretion of pro-inflammatory factors increased and the secretion of anti-inflammatory factors decreased (P<0.001); after ML130 intervention, there was no significant change in serum lipid levels, but the levels of inflammatory factors were significantly improved (P<0.05 or P<0.01).

[0067] ②ML130 vs. ApoE Effects on aortic plaques in mice: The proportion of aortic plaque area was significantly increased in the AS model group (P<0.001), and the collagen content in the plaque was significantly decreased (P<0.001); the proportion of plaque area was significantly decreased in the high-dose ML130 group (P<0.01), the collagen content was significantly increased (P<0.01), and the plaque stability was improved.

[0068] ③ Effects of ML130 on macrophage polarization and the NOD1 / RIP2 pathway in plaques: In the AS model group, the number of CD86⁺M1 macrophages in plaques was significantly increased (P<0.001), the number of CD206⁺M2 macrophages was significantly decreased (P<0.001), and the NOD1 / RIP2 pathway was activated (P<0.001); after ML130 intervention, the macrophage polarization balance shifted towards M2 type (P<0.05 or P<0.01), and the activation of the NOD1 / RIP2 pathway was inhibited (P<0.05 or P<0.01).

[0069] 4) Statistical analysis of key in vivo experimental indicators (x±s, n=10) Group Patch area percentage (%) Collagen content (%) Serum IL-6 (pg / mL) CD86⁺ cell count within the plaque (cells / field of view) CD206⁺ cell count within the plaque (cells / field of view) normal control group 0.8±0.2 32.5±3.1 35.6±4.2 12.4±2.1 45.8±3.6 AS model group 28.6±2.5*** 15.3±1.8*** 189.7±10.5*** 89.6±5.3*** 18.7±2.3*** ML130 low-dose group (5 mg / kg) 21.3±2.1# 20.7±2.0# 132.4±8.6# 65.4±4.8# 27.5±3.1# ML130 high-dose group (10mg / kg) 14.5±1.7## 28.9±2.6## 76.8±6.9## 38.7±3.5## 39.6±3.4## Note: Compared with the normal control group, ***P<0.001; compared with the AS model group, #P<0.05, ##P<0.01.

[0070] 5) In vivo experiments clearly showed that serum lipid levels did not change significantly after ML130 intervention. This finding is of great value, indicating that the anti-AS effect of ML130 is independent of the lipid-regulating pathway, and its target is the immune inflammatory disorder itself. This provides a clear pharmacological basis for its combined use with existing statins, which can work synergistically through different mechanisms (lipid-lowering vs. anti-inflammatory / plaque stabilization) to achieve dual pathological intervention, consistent with the personalized treatment strategy of benefit point 5.

[0071] It can be known that: In vitro experiments have confirmed that hypoxia can activate the NOD1 / RIP2 signaling pathway in macrophages, promote their polarization toward the M1 type, and exacerbate the inflammatory response. ML130 can regulate the balance of hypoxia-induced macrophage polarization in a concentration-dependent manner by inhibiting the NOD1 / RIP2-NF-κB / MAPK signaling axis, thereby reducing the secretion of pro-inflammatory factors and increasing the release of anti-inflammatory factors. In vivo experiments have shown that ML130 can improve ApoE under hypoxic conditions. Disruption of macrophage polarization in atherosclerotic plaques in mice inhibits plaque inflammation, increases collagen content, and improves plaque stability without affecting serum lipid metabolism.

[0072] In summary, the above results fully demonstrate that ML130 can intervene in the progression of atherosclerosis by regulating macrophage polarization mediated by the NOD1 / RIP2 signaling pathway under hypoxic conditions, providing new experimental evidence for the treatment of atherosclerosis.

[0073] It should be noted that the NOD1 inhibitor ML130 described in this invention is a representative compound, and its structural analogues or other NOD1 inhibitors with the same inhibitory effect are also applicable to this invention. Their effects can be verified through conventional experiments in the field.

[0074] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of NOD1 inhibitor ML130 in hypoxia-induced macrophage polarization, including the use of NOD1 inhibitor ML130 in the preparation of drugs that regulate hypoxia-induced macrophage polarization, characterized in that: The drug regulates macrophage polarization towards the M2 anti-inflammatory phenotype by inhibiting the NOD1 / RIP2 signaling axis, reducing the secretion levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α, and increasing the expression levels of anti-inflammatory factors IL-10 and TGFβ. The low-oxygen conditions are defined as an oxygen concentration of 1%-5%. The macrophages include M1 macrophages derived from Ly6Chi mononuclear cells and M2 macrophages derived from Ly6Clo mononuclear cells; The drug can be used to intervene in the formation and progression of atherosclerotic plaques.

2. The application of the NOD1 inhibitor ML130 according to claim 1 in hypoxia-induced macrophage polarization, characterized in that, The drug is used to prepare a medicine for treating atherosclerosis.

3. The application of the NOD1 inhibitor ML130 according to claim 2 in hypoxia-induced macrophage polarization, characterized in that, The drug can reduce the expression of CXCL9, CXCL10, and CXCL5 in macrophages.

4. The application of the NOD1 inhibitor ML130 according to claim 3 in hypoxia-induced macrophage polarization, characterized in that, The drug can increase the expression levels of CCL17 and CCL24 in macrophages.

5. The application of the NOD1 inhibitor ML130 according to claim 4 in hypoxia-induced macrophage polarization, characterized in that, The drug is used to prepare agents for stabilizing atherosclerotic plaques.

6. The application of the NOD1 inhibitor ML130 according to claim 5 in hypoxia-induced macrophage polarization, characterized in that, The drug comprises an effective dose of ML130 and a pharmaceutically acceptable carrier.

7. The application of the NOD1 inhibitor ML130 according to claim 6 in hypoxia-induced macrophage polarization, characterized in that, The carrier includes one or more of a buffer, excipient, or stabilizer.

8. The application of the NOD1 inhibitor ML130 according to claim 7 in hypoxia-induced macrophage polarization, characterized in that, The drug can inhibit the interaction between NOD1 and RIP2.

9. The application of the NOD1 inhibitor ML130 according to claim 8 in hypoxia-induced macrophage polarization, characterized in that, The drug can block the activation of NF-κB and MAPK signaling pathways.

10. The application of the NOD1 inhibitor ML130 according to claim 9 in hypoxia-induced macrophage polarization, characterized in that, The drug is used to prepare a drug that improves the hypoxic microenvironment of atherosclerotic plaques.

Images