祛痰活血方调控PINK1/Parkin信号通路诱导NASH大鼠肝细胞自噬的实验研究

Study on the mechanism of Qutan Huoxue Recipe regulating PINK1/Parkin signaling pathway to induce autophagy of NASH rat hepatocytes

目的:探讨祛痰活血方通过调控PINK1/Parkin信号通路诱导肝细胞自噬改善非酒精性脂肪性肝炎(NASH)大鼠肝损伤的作用机制。方法:雄性SD大鼠30只,随机分为对照组、模型组和祛痰活血方低、中、高剂量组,每组6只。对照组大鼠给予普通饲料,其余各组大鼠予高脂饮食,造模8周。祛痰活血方低、中、高剂量组大鼠连续给药4周后,处死各组大鼠,称量并记录大鼠体质量及肝质量,测定肝功能及血脂水平,观察各组大鼠肝组织病理变化和肝内脂质沉积情况,透射电镜下记录各组大鼠肝组织脂滴和自噬小体数量,检测各组大鼠肝组织自噬蛋白PINK1、Parkin、SQSTM1表达以及PINK1/Parkin信号通路相关蛋白表达。结果:与对照组相比,模型组大鼠体重增加,行动迟缓;祛痰活血方治疗后大鼠一般情况好转,体重、肝重减轻。模型组大鼠肝功能指标和血脂水平高于对照组,祛痰活血方治疗后大鼠ALT、AST、TG、TC下降。透射电镜观察,对照组和模型组大鼠肝组织均未见明显自噬体,模型组大鼠肝组织可见大量脂滴,祛痰活血方治疗后大鼠肝组织自噬小体数量增加、脂滴数量减少且体积变小。免疫组织化学法检测,对照组大鼠肝组织SQSTM1无阳性表达,PINK1、Parkin阳性表达较低;与对照组相比,模型组大鼠肝组织PINK1、Parkin无阳性表达,SQSTM1表达较高;与模型组相比,祛痰活血方治疗后,大鼠肝组织PINK1、Parkin阳性表达增加,SQSTM1表达降低。蛋白免疫印迹分析,模型组大鼠肝组织自噬蛋白PINK1、Parkin、SQSTM1表达减低,祛痰活血方治疗后大鼠肝组织PINK1、Parkin、SQSTM1表达增加。结论:祛痰活血方可能通过调控PINK1/Parkin信号通路诱导肝细胞自噬,减轻肝细胞炎症及肝组织脂质沉积,从而发挥治疗NASH的作用。

Objective:To investigate the mechanism of expectorant blood revitalization in improving NASH liver injury by regulating the PINK1/Parkin signaling pathway to induce autophagy in hepatocyte.Methods:Thirty male SD rats were randomly divided into control group,model group,Qutan Huoxue Recipe low、medium and high dose groups,with 6 rats in each group.The control group was given ordinary feed,and the remaining groups were given a high-fat diet for 8 weeks.After 4 weeks of continuous administration,rats were sacrificed.The body weight and liver weight of the rats were weighed and recorded,and the liver function and blood lipid levels were measured.The pathological changes of liver tissue and intrahepatic lipid deposition were observed in each group.The expressions of Parkin,PINK1,SQSTM1 and PINK1/Parkin signaling pathway related proteins in liver tissue were detected.Results:Compared with the control group,the rats in the model group gained weight and acted slowly.After treatment with Qutan Huoxue Recipe,the general condition of rats was improved,and the body weight and liver weight were reduced.The enzymatic indexes and blood lipids of liver function in the model group were higher than those in the control group,and ALT,AST,TG,and TC decreased after the administration of Qutan Huoxue Recipe.Transmission electron microscope observation showed that there was no obvious autophagosome in the liver tissue of the control group and the model group,and a large number of lipid droplets in the liver tissue of the model group.After treatment with Qutan Huoxue Recipe,the number of autophagosomes in the liver tissue of the rats increased,and the number of lipid droplets decreased and the volume became smaller.Immunohistochemical staining showed that SQSTM1 had no positive expression in liver tissues of the control group,and the positive expressions of PINK1 and Parkin were low.Compared with the control group,the model group had no positive expression of PINK1 and Parkin in liver tissue,and the expression of SQSTM1 was higher.Compared with the model group,Qutan Huoxue Recipe treatment increased the positive expressions of PINK1 and Parkin in liver tissue,and decreased the expression of SQSTM1.Western blot showed that the expression of autophagy proteins PINK1、Parkin and SQSTM1 in the model group decreased,and the expression of PINK1,Parkin and SQSTM1 increased after the treatment of Qutan Huoxue Recipe.Conclusion:Qutan Huoxue Recipe activation may induce autophagy in hepatocytes,reduce hepatocyte inflammation and lipid deposition in liver tissues by regulating the PINK1/Parkin signaling pathway,and thus play a role in the treatment of NASH.