益脉颗粒对高脂合并心肌缺血再灌注损伤大鼠心肌细胞焦亡的影响

Effects of Yimai Granules(益脉颗粒)on Myocardial Pyroptosis in Rats with Hyperlipidemia and MIRI

目的观察高脂合并心肌缺血再灌注损伤(MIRI)大鼠使用中药益脉颗粒进行干预后,大鼠心肌细胞焦亡的变化情况,阐述细胞焦亡在益脉颗粒预防治疗MIRI中的调节效应以及分子机制。方法将60只实验动物随机将其分成3个组别,即假手术组、模型组和益脉颗粒高剂量组。常规饲料喂养假手术组,高脂饲料喂养模型组和益脉颗粒高剂量组,均喂养2周。假手术组和模型组大鼠每天灌服2次10 mL/kg生理盐水,益脉颗粒组每日灌服2次10 mL/kg中药煎液,1周后进行缺血再灌注造模。MIRI造模完成后(缺血30 min再灌注2 h),大鼠腹主动脉进行取血,后分离血清备用。全自动生化分析仪检测3组大鼠血脂水平;HE染色观察各组大鼠心肌病理形态学变化;实时荧光定量PCR和Wes全自动蛋白质印迹定量分析系统检测细胞焦亡相关蛋白GSDMD、Caspase1、IL-1β、NLRP3、TLR4 mRNA及蛋白表达。结果相较于假手术组,模型组大鼠HDL-C含量显著降低(P<0.05),其余各指标血清含量(TC、TG、LDL-C)均显著升高(P<0.05);心肌组织存在间质水肿、炎性细胞的浸润,大鼠心肌GSDMD、Caspase1、IL-1β、NLRP3、TLR4 mRNA及蛋白表达显著升高(P<0.05),经中药益脉颗粒干预后,大鼠血脂相关指标显示,除HDL-C水平明显增加(P<0.05)外,TC、TG、LDL-C水平显著减少(P<0.05),大鼠心肌组织间质水肿及炎性细胞浸润有所减轻,大鼠心肌GSDMD和TLR4基因表达水平显著下调(P<0.05),大鼠心肌Caspase1、IL-1β、NLRP3基因表达水平呈明显下降趋势,但无统计学意义;大鼠心肌GSDMD、IL-1β、NLRP3蛋白表达程度有所降低(P<0.05),Caspase1与TLR4蛋白呈现显著减少的趋势,但没有差异性。结论益脉颗粒可显著改善高脂合并MIRI大鼠血脂及心肌损伤情况,其机制可能与益脉颗粒调控细胞焦亡过程有关。

Objective To observe the effect of Yimai Granules(益脉颗粒)on pyroptosis in hyperlipidemia combined with myocardial ischemia reperfusion injury(MIRI)rats and expound the regulatory role and molecular mechanism of pyrophosis in the prevention and treatment of MIRI by Yimai Granules.Methods Sixty experimental rats were randomly divided into three groups,namely sham operation group,model group and Yimai Granules high-dose group.Rats in the sham operation group were kept in routine feeding,and high-fat diet was given for the model group and Yimai Granules high-dose group for 2 weeks under other conditions being the same.Rats in the sham operation group and model group were given saline(10 mL/kg)daily,and rats in the Yimai Granules high-dose group were given Chinese medicine decoction(10 mL/kg)daily,twice a day.Ischemia reperfusion modeling was performed 1 week later.After completion of MIRI modelulation(30 minutes myocardial ischemia and 2 hours reperfusion),the serum was separated from the abdominal aorta blood of rats for later use.Blood lipid levels were detected by automatic biochemical analyzer.HE staining was used to observe the pathological changes of myocardium in each group.The mRNA and protein expressions of Gasdermin-D(GSDMD),cysteinyl aspartate specific proteinase 1(Caspase1),interleukin-1β(IL-1β),NOD-like receptor thermal protein domain associated protein 3(NLRP3)and Toll-like receptor 4(TLR4)were detected by real-time quantitative PCR and Wes automatic Western Blot quantitative analysis.Results Compared with those of the sham operation group,the high-density lipoprotein cholesterol(HDL-C)content in the model group was significantly decreased(P<0.05),and the serum contents of other indexes[total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C)]were significantly increased(P<0.05).There was interstitial edema and inflammatory cell infiltration in the myocardium,and the mRNA and protein expressions of GSDMD,Caspase1,IL-1β,NLRP3 and TLR4 in the myocardium were significantly increased(P<0.05).After the intervention of Yimai Granules,the serum lipid of rats showed that the contents of TC,TG and LDL-C were decreased significantly(P<0.05).The HDL-C content was increased significantly(P<0.05).The interstitial edema and inflammatory cell infiltration of rat myocardial tissue were alleviated.The GSDMD and TLR4 gene expression levels were significantly down-regulated(P<0.05).The gene expression levels of Caspase1,IL-1βand NLRP3 in rat myocardia were decreased significantly,but there was no statistical significance.The expressions of GSDMD,IL-1βand NLRP3 in rat myocardium were significantly down-regulated(P<0.05).The Caspase1 and TLR4 proteins showed a downward trend,with no statistical significance.Conclusions Yimai Granules can significantly improve the levels of blood lipid and myocardial injury in rats with high fat and MIRI,and the mechanism may be related to Yimai Granules regulating the process of pyroptosis.