心脉康方调控TLR4/NLRP3焦亡通路抗动脉粥样硬化机制研究

Mechanism of Xinmaikang Recipe(心脉康方)Suppresses Pyroptosis Pathway TLR4/NLRP3 to Prevent and Treat Atherosclerosis

目的:探讨心脉康方治疗动脉粥样硬化(AS)的可能分子作用机制。方法:将46只ApoE^(-/-)小鼠进行8周高脂饲料连续喂养复制小鼠动脉粥样硬化模型,10只C57BL/6小鼠同时使用普通饲料喂养,8周后取6只ApoE^(-/-)小鼠验证造模成功后,将剩余40只小鼠按随机数字表法分为模型组、阿托伐他汀组、心脉康方低剂量组、心脉康方高剂量组,每组10只。10只C57BL/6小鼠作为空白对照组。各药物干预组小鼠给予相应的药物干预,空白对照组和模型组小鼠给予生理盐水灌胃。12周后,采用苏木素-伊红(HE)和油红O染色观察主动脉组织病理学形态改变;采用ELISA法检测血清中ox-LDL、TNF-α、IL-1β、IL-6和IL-18的水平;采用全自动生化仪检测血清中总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)含量;采用q PCR检测主动脉组织中易损斑块稳定性指标(ICAM-1 mRNA、VCAM-1 mRNA、MMP-9 mRNA、MCP-1 mRNA)及细胞焦亡相关指标(IL-1βmRNA、IL-18 mRNA、TLR4 mRNA、NLRP3 mRNA、Caspase-1)水平;采用Western blotting检测ICAM-1、VCAM-1、MMP-9、MCP-1、IL-1β、IL-18、GSDMD-N、TLR4、NLRP3、Caspase-1蛋白相对表达情况。结果:与空白对照组比较,模型组小鼠主动脉血管壁增厚,有大量斑块和脂质沉淀等病变,血清中TC、TG、LDL-C含量及ox-LDL、TNF-α、IL-1β、IL-6、IL-18水平明显升高(P<0.01),而HDL-C水平明显降低(P<0.01),同时主动脉组织中的ICAM-1、VCAM-1、MMP-9、MCP-1、IL-18、IL-1β、GSDMD-N、TLR4、NLRP3和Caspase-1蛋白和mRNA相对表达量明显升高(P<0.01)。与模型组比较,阿托伐他汀组和心脉康方高剂量组小鼠主动脉组织内脂滴和易损斑块面积均减少,血清中TC、TG、LDL-C含量及ox-LDL、TNF-α、IL-1β、IL-6、IL-18水平均降低(P<0.01),而HDL-C水平明显升高(P<0.01),主动脉组织中的ICAM-1、VCAM-1、MMP-9、MCP-1、IL-18、IL-1β、GSDMD-N、TLR4、NLRP3和Caspase-1蛋白和mRNA相对表达量均明显降低(P<0.01)。结论:心脉康方可能通过调控TLR4/NLRP3通路影响炎症反应和细胞焦亡,从而改善动脉粥样硬化。

Objective:To explore the possible molecular mechanism of Xinmaikang recipe in the treatment of atherosclerosis(AS).Methods:Totally 46 healthy male ApoE^(-/-)mice were fed with high-fat diet for 8 weeks to replicate the mouse model of atherosclerosis.A total of 10 C57BL/6 mice were fed with common feed at the same time.After 8 weeks,6 ApoE^(-/-)mice were taken to verify the success of the model.The remaining 40 mice were randomly divided into model group,atorvastatin group,low-dose Xinmaikang recipe group and high-dose Xinmaikang recipe group,with 10 mice in each group.The 10 C57BL/6 mice were used as normal control group.The mice in each drug intervention group were given corresponding drug intervention,and the mice in normal control group and model group were given normal saline by gavage.After 12 weeks,the histopathological changes of aorta were observed by hematoxylin-eosin(HE)and oil-red O staining.The levels of serum ox-LDL,TNF-α, IL-1β, IL-6 and IL-18 were detected by ELISA. The serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were detected by automatic biochemical analyzer. Real-time PCR was used to detect the vulnerable plaque stability index (expression of ICAM-1 mRNA, VCAM-1 mRNA, MMP-9 mRNA and MCP-1 mRNA) and related indicators of cell charring (expression of IL-1β mRNA, IL-18 mRNA, TLR4 mRNA, NLRP3 mRNA and Caspase-1 mRNA) in aortic tissue. Western blotting was performed to measure the protein levels of ICAM-1, VCAM-1, MMP-9, MCP-1, IL-1β, IL-18, C-GSDMD-N, TLR4, NLRP3 and Caspase-1. Results: Compared with the normal control group, aorta of mice in the model group showed thickening of vascular wall, as well as a large amount of plaque and lipid deposition. In addition, the serum levels of TC, TG, LDL-C, ox-LDL, TNF-α, IL-1β, IL-6 and IL-18 significantly increased in the model group (P<0.01), while the serum levels of HDL-C significantly decreased in model group (P<0.01). Also the protein and mRNA expression levels of ICAM-1, VCAM-1, MMP-9, MCP-1, IL-18, IL-1β, GSDMD-N, TLR4, NLRP3 and Caspase-1 in the aortic tissues significantly increased in the model group (P<0.01). Compared with the model group, the area of lipid droplets and vulnerable plaques reduced in the aorta tissues in atorvastatin group and high-dose Xinmaikang recipe group. The levels of TC, TG, LDL-C, ox-LDL, TNF-α, IL-1β, IL-6 and IL-18 in the serum decreased (P<0.01), while the levels of HDL-C in the serum were significantly increased in atorvastatin group and high-dose Xinmaikang recipe group (P<0.01). And the the protein and mRNA expression levels of ICAM-1, VCAM-1, MMP-9, MCP-1, IL-18, IL-1β, GSDMD-N, TLR4, NLRP3 and Caspase-1 in aortic tissue were significantly decreased in atorvastatin group and high-dose Xinmaikang recipe group (P<0.01). Conclusion: Xinmaikang recipe may inhibit inflammatory response and pyrosis in AS mice and improve aortic pathological damage by regulating TLR4/NLRP3 pathway.