基于NLRP3炎症小体探讨地奥心血康抗动脉粥样硬化作用机制

Investigation on Anti-atherosclerosis Mechanism of Di’ao Xinxuekang Based on NLRP3 Inflammasome

目的:研究地奥心血康(Di’ao Xinxuekang,DXXK)对小鼠RAW264.7巨噬细胞和动脉粥样硬化(Atherosclerosis,AS)大鼠胸主动脉NOD样受体3(NLRP3)炎症小体的影响,探讨其抗AS的作用机制。方法:氧化低密度脂蛋白(ox-LDL)刺激RAW264.7细胞建立体外模型,MCC950和DXXK干预,酶联免疫吸附测定法(ELISA)检测肿瘤坏死因子-α(TNF-α),白细胞介素-1β(IL-1β)含量,实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)检测NLRP3,衔接蛋白凋亡相关斑点样蛋白(ASC)及半胱氨酸天冬氨酸蛋白水解酶-1(Caspase-1)mRNA和蛋白的表达。60只雄性SD大鼠随机分为正常组,模型组,阿托伐他汀组(2 mg·kg^(-1)),DXXK高、中、低剂量组(100,30,10 mg·kg^(-1)),每组10只。采用高脂饲料+维生素D2建立AS模型,全自动生化分析仪检测血脂水平,计算AS指数(AI);ELISA检测血清TNF-α,IL-1β含量;Real-time PCR和Western blot检测胸主动脉NLRP3,ASC,Caspase-1 mRNA和蛋白的表达;苏木素-伊红(HE)染色和天狼星红染色观察腹主动脉及主动脉窦病理形态学变化。结果:与正常组比较,模型组RAW264.7细胞TNF-α,IL-1β与NLRP3,ASC,Caspase-1mRNA和蛋白表达显著升高(P<0.01);与模型组比较,各药物组上述指标明显降低(P<0.05,P<0.01)。与模型组比较,DXXK组大鼠胆固醇(TC),甘油三酯(TG),低密度脂蛋白胆固醇(LDL-C),AI显著降低,高密度脂蛋白胆固醇(HDL-C)明显升高(P<0.05,P<0.01),血清TNF-α,IL-1β与胸主动脉NLRP3,ASC,Caspase-1 mRNA和蛋白表达明显降低(P<0.05,P<0.01),腹主动脉病变及主动脉窦纤维增生明显改善。结论:DXXK具有显著的抗AS作用,抑制NLRP3炎症小体可能是其作用机制之一。

Objective: To investigate the effects of Di’ao Xinxuekang(DXXK) on NLRP3 inflammasome in mouse RAW264.7 macrophages and thoracic aorta of rats with atherosclerosis(AS),so as to explore its anti-AS mechanism. Method: RAW264.7 cells were stimulated with oxidized low density lipoprotein(ox-LDL)and then intervened with MCC950 and DXXK. The contents of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were determined by enzyme linked immunosorbent assay(ELISA). The mRNA and protein expression levels of Nod-like receptor protein 3(NLRP3),inflammasome adaptor protein apoptosisassociated speck-like protein containing CARD(ASC),and cysteine-dependent aspartate-directed protease-1(Caspase-1)were detected by real-time polymerase chain reaction(Real-time PCR)and Western blotting. Sixty male SD rats were randomly divided into the normal group,model group,atorvastatin group(2.0 mg·kg^(-1)),as well as high-,medium-,and low-dose(100,30,and 10 mg·kg^(-1))DXKK groups,with 10 rats in each group.The rats were exposed to the high-fat diet and vitamin D2 for inducing AS. The blood lipid level was measured using an automatic biochemical analyzer,followed by the calculation of AS index(AI). The contents of serum TNF-α and IL-1β were determined by ELISA,and the mRNA and protein expression levels of NLRP3,ASC,and Caspase-1 in thoracic aorta were assayed by Real-time PCR and Western blotting. HE staining and Sirius red staining were conducted to observe the pathomorphological changes in the abdominal aorta and aortic sinus.Result: Compared with the normal group,the model group exhibited significantly increased TNF-α and IL-1βcontents and up-regulated NLRP3,ASC,and Caspase-1 mRNA and protein expression in RAW264.7 cells(P<0.01). The above indexes in each drug administration group were significantly reduced in contrast to those in the model group(P<0.05, P<0.01). The comparison with the model group showed that cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C),and AI in each DXXK group significantly declined,while the high-density lipoprotein cholesterol(HDL-C)was significantly elevated(P<0.05,P<0.01).The levels of serum TNF-α and IL-1β and the mRNA and protein expression levels of NLRP3,ASC,and Caspase-1 in the thoracic aorta were decreased(P<0.05, P<0.01). Abdominal aortic lesions and fibrous hyperplasia of aortic sinus were significantly improved. Conclusion: DXXK has a significant anti-AS effect,which is possibly related to the inhibition of NLRP3 inflammasome.