蛭龙活血通瘀胶囊通过NLRP3炎症小体抗动脉粥样硬化的机制研究

目的:探究蛭龙活血通瘀胶囊通过NLRP3信号途径抗动脉粥样硬化的分子机制。方法:将45只新西兰大白兔随机分为正常对照组、模型对照组、蛭龙活血通瘀胶囊3.12 g/kg组、阿托伐他汀钙0.51 mg/kg组、蛭龙活血通瘀胶囊3.12 g/kg+阿托伐他汀钙组0.51 mg/kg,每组9只,适应性喂养一周后除正常对照组外其余行颈动脉空气干燥术,并予高脂饲料饲喂养,正常对照组予普通饲料喂养。在实验的第2 w、4 w行颈动脉彩超;第4 w末处死家兔取血清及颈动脉组织进行血脂、HE染色、油红染色、免疫组化、免疫印迹、ELISA实验检测相关指标并进行统计分析。结果:与正常对照组比较,模型对照组血脂各项指标、颈动脉内中膜厚度、颈动脉斑块数量均显著增高(P<0.05或P<0.01),表明造模成功;与模型对照组比较,各用药组的血脂各项指标(TC、TG、LDL-C含量)、颈动脉内中膜厚度、颈动脉斑块数量均明显改善(P<0.01);HE染色提示颈动脉细胞显著增生,可见由大量泡沫细胞构成的脂质斑块,斑块内有炎症细胞浸润,治疗后炎症细胞明显减少,其中蛭龙活血通瘀胶囊3.12 g/kg+阿托伐他汀钙0.51 mg/kg组效果最佳。NLRP3免疫组化提示模型对照组颈动脉病变组织中有大量NLRP3阳性表达,比正常对照组中显著增多(P<0.01);经治疗后,蛭龙活血通瘀胶囊3.12 g/kg组、阿托伐他汀钙0.51 mg/kg组和蛭龙活血通瘀胶囊3.12 g/kg+阿托伐他汀钙0.51 mg/kg组NLRP3阳性表达明显减少,其中蛭龙活血通瘀胶囊3.12 g/kg+阿托伐他汀钙0.51 mg/kg组NLRP3阳性表达最少(P<0.01)。免疫印迹实验结果显示,与模型对照组比较,蛭龙活血通瘀胶囊3.12 g/kg、阿托伐他汀钙0.51 mg/kg组和蛭龙活血通瘀胶囊3.12 g/kg+阿托伐他汀钙0.51 mg/kg组中p-NF-κB、NLRP3、Caspase-1、pro-IL-1β、IL-1β、pro-IL-18和IL-18蛋白水平均明显下调(P<0.05或P<0.01);ELISA实验结果显示,与模型对照组比较,各用药组在血清IL-1β、NF-κB及IL-18含量均明显下降(P<0.05或P<0.01)。结论:蛭龙活血通瘀胶囊能够改善高脂血症颈动脉粥样硬化家兔血脂及减少颈动脉斑块生成,且与阿托伐他汀钙联用效果更佳,其机制与通过抑制NF-κB的磷酸化进而抑制NLRP3炎症小体活化及其下游炎症因子的释放有关。

Research on Molecular Mechanism of Zhilonghuoxuetongyu Capsule on Anti-Atherosclerosis Based on NLRP3 Inflammasome Signaling Pathway

Objective:To investigate the molecular mechanism of Zhilonghuoxuetongyu Capsule on anti-atherosclerosis based on NLRP3 Inflammasome Signaling Pathway.Methods:45 New Zealand white rabbits were randomly divided into the control group,the model group,3.12 g/kg Zhilonghuoxuetongyu Capsule group,0.51 mg/kg Atorvastatin group and 3.12 g/kg ZL+0.51 mg/kg Atorvastatin group,9 rabbits in each group.After one week for rabbits to acclimatize to the new surroundings,carotid air drying was performed except for the control group.High-fat diet was given,while the control group was given normal diet.Doppler ultrasound examination of carotid artery was performed at week 2 and 4 of the experiment.At the end of the 4 th week,the blood serum and carotid artery tissues were taken and detected in the related experiments.Blood lipid,H&E staining,oil red O staining,immunohistochemistry,western blot and ELISA indexes were detected.Results:Compared with the control group,the lipids,intracarotid media thickness and numbers of carotid plaque quality in the model group were significantly increased,which indicated that the model was successfully established.Compared with the model group,the indexes of blood lipids,intracarotid media thickness and the number of carotid plaque in each medication group were improved,and the differences were statistically significant(P<0.01).HE staining suggested that the cells were significantly proliferated,showing lipid plaques composed of a large number of foam cells,inflammatory cells infiltrated into the plaque and decreased after treatment,the results showed the combination of ZL+Atorvastatin was most effective.Immunohistochemistry of NLRP3 suggested that there was a large number of positive NLRP3 expression in carotid artery lesions in the model group than that in the control group,which suggested a successful modeling.After treatment,the positive expression of NLRP3 was decreased in ZL group,Atorvastatin group and ZL+Atorvastatin group,while the ZL+Atorvastatin combination group had the least positive expression of NLRP3(P<0.01.Western blot Results showed,compared with the model group,p-NF-κB,NLRP3,Caspase-1,pro-IL-1β,IL-1β,pro-IL-18 and IL-18 protein levels in ZL group,Atorvastatin group and ZL+Atorvastatin group were significantly decreased(P<0.05 or P<0.01).ELISA results showed,compared with the model group,the expressions of serum IL-1β,NF-κB and IL-18 proteins in ZL group,Atorvastatin group and ZL+Atorvastatin group were significantly decreased(P<0.05 or P<0.01).Conclusion:Zhilonghuoxuetongyu Capsule can improve the metabolism of serum lipids and reduce the formation of carotid atherosclerotic plaques in hyperlipidemia and carotid atherosclerosis rabbits.It works better when combined with Atorvastatin.The mechanism may be related to inhibiting the activation of NLRP3 inflammasome and the release of downstream inflammatory factors by inhibiting the phosphorylation of NF-κB.