基于线粒体自噬及Pink1/Parkin信号通路探讨柴胡疏肝散治疗功能性消化不良大鼠的作用机制

Mechanism of Chaihu Shugansan in Treatment of Functional Dyspepsia in Rats Based on Mitophagy and Pink1/Parkin Signaling Pathway

目的:基于线粒体自噬及PTEN诱导激酶1(Pink1)/E3泛素连接酶(Parkin)信号通路探讨柴胡疏肝散治疗功能性消化不良大鼠的作用机制。方法:将40只SD大鼠按照随机分组原则分为正常组、模型组、柴胡疏肝散组、阳性药物组(多潘立酮),每组10只。正常组大鼠不予造模,其他各组采用夹尾刺激法制备功能性消化不良大鼠模型,每次造模结束后,正常组及模型组灌胃生理盐水,柴胡疏肝散组和多潘立酮组则分别用柴胡疏肝散(4.8 g·kg^(-1))及多潘立酮(4.5 mg·kg^(-1))灌胃。造模用药28 d后取材,检测每组大鼠胃排空率及小肠推进率;苏木素-伊红(HE)染色观察胃窦组织损伤情况;透射电子显微镜(TEM)观察胃间质细胞(ICCs)超微结构;免疫荧光共定位法观察细胞色素C氧化酶(COXⅣ)与Parkin的表达;蛋白免疫印迹法(Western blot)检测微管相关蛋白1轻链3(LC3)、线粒体自噬相关蛋白PHB2、Pink1、Parkin、USP30的表达水平。结果:与正常组比较,模型组大鼠胃排空率及小肠推进率均明显下降(P<0.05);与模型组比较,柴胡疏肝散组胃排空率及小肠推进率均明显提高(P<0.05)。透射电镜结果显示,柴胡疏肝散可减轻胃窦组织线粒体肿胀程度。免疫荧光结果显示,与正常组比较,Parkin在线粒体的荧光表达显著提高(P<0.01);与模型组比较,Parkin在线粒体的荧光表达显著减少(P<0.01)。Western blot结果显示,与正常组比较,LC3、Pink1、Parkin及PHB2蛋白表达均明显提高(P<0.05,P<0.01),USP30蛋白表达显著降低(P<0.01);与模型组比较,LC3、Pink1、Parkin及PHB2蛋白表达均明显降低(P<0.05,P<0.01),USP30蛋白表达显著提高(P<0.01)。结论:柴胡疏肝散治疗功能性消化不良的作用机制可能与抑制Pink1/Parkin信号通路的激活,从而抑制ICCs线粒体过度自噬有关。

Objective:To investigate the mechanism of Chaihu Shugansan in the treatment of functional dyspepsia in rats based on mitophagy and PTEN-induced kinase 1(Pink1)/E3 ubiquitin ligase(Parkin).signaling pathway.Method:According to the principle of random grouping,40 SD rats were assigned into a normal group,a model group,a Chaihu Shugansan group,and a positive drug(domperidone)group,with 10rats in each group.The rats in other groups except the normal group received tail-clamping stimulation to replicate the model of functional dyspepsia.After each time of stimulation,the rats in the normal,model,Chaihu Shugansan,and positive drug groups were administrated with normal saline,normal saline,Chaihu Shugansan(4.8 g·kg^(-1)),and an aqueous solution of domperidone(4.5 mg·kg^(-1)),respectively.After 28 days of modeling,the gastric emptying rate and the small intestine propulsion rate of the rats in each group were measured and the tissue samples were collected.Hematoxylin-eosin(HE)staining was employed for observation of damage in gastric antrum tissue,and transmission electron microscopy(TEM)for ultrastructural observation of gastric interstitial cells of Cajal(ICCs).Immunofluorescence co-localization was adopted to observe the expression of cytochrome c oxidase(COXⅣ)and Parkin.Western blot was employed to determine the expression levels of microtubule-associated protein 1,light chain 3(LC3),and the mitophagy-associated proteins prohibitin2(PHB2),Pink1,Parkin,and ubiquitin-specific protease 30(USP30).Result:Compared with the normal group,the modeling decreased the gastric emptying rate and the small intestine propulsion rate(P<0.05).Compared with the model group,Chaihu Shugansan increased the gastric emptying rate and the small intestine propulsion rate(P<0.05).The results of TEM showed that Chaihu Shugansan reduced the swelling degree of mitochondria in gastric antrum tissue.Compared with the normal group,the modeling increased the fluorescence intensity of Parkin in mitochondria(P<0.01),while such increase can be alleviated by Chaihu Shugansan(P<0.01).Western blotting results showed that compared with the normal group,the modeling upregulated the protein levels of LC3,Pink1,Parkin,and PHB2(P<0.05,P<0.01)and down-regulated the protein level of USP30(P<0.01).Compared with the model group,Chaihu Shugansan down-regulated the protein levels of LC3,Pink1,Parkin,and PHB2(P<0.05,P<0.01)and up-regulated the protein level of USP30(P<0.01).Conclusion:Chaihu Shugansan may treat functional dyspepsia by blocking the Pink1/Parkin signaling pathway to inhibit excessive mitochondrial autophagy in ICCs.