基于线粒体途径探索马兰草脐灸促肝纤维化模型大鼠活化肝星状细胞凋亡机制研究  

Mechanism on The Apoptosis of Activated Hepatic Stellate Cells in Rats with Hepatic Fibrosis by Ammophila Arenaria Umbilical Moxibustion Based on Mitochondrial Pathway

作　　者：李嘉 高玲 施杨婉玲 田源红 杨孝芳 陈迎龙 LI Jia;GAO Ling;SHI-YANG Wan-ling;TIAN Yuan-hong;YANG Xiao-fang;CHEN Ying-long(Guizhou University of Chinese Medicine,Guiyang 550001,China)

机构地区：[1]贵州中医药大学,贵阳550025

出　　处：《中国中医基础医学杂志》2021年第5期770-776,共7页JOURNAL OF BASIC CHINESE MEDICINE

基　　金：国家自然科学基金资助项目(81860887)-基于水通道蛋白8调控活化肝星状细胞线粒体凋亡途径探讨马兰贴敷脐疗法治疗肝硬化腹水的作用及机制研究;国家自然科学基金资助项目(81560771)-基于毒损肝络理论从线粒体途径研究健脾活血祛湿方调控AQP8、9对H22荷瘤小鼠、肝癌细胞凋亡的影响;贵州省教育厅青年科技人才成长项目(黔教和KY[2016]184)-马兰草脐灸抗肝纤维的作用及机制研究;贵州省2017年度高层次创新型人才项目(黔人领发〔2018〕5号)-苗药-马兰结合中医外治法干预肝络疾病的机理研究;贵州省科技厅合作计划项目(黔科合LH字[2015]7293号)-马兰草脐灸对肝硬化腹水大鼠肝AQP8、9和线粒体的影响;贵州省中医药、民族医药科学技术专项课题项目(QZYY-2018-013)-基于水通道蛋白8调控活化肝星状细胞线粒体凋亡途径探讨马兰贴敷脐疗法对肝硬化腹水大鼠的影响及机制研究。

摘　　要：目的:研究马兰草脐灸通过线粒体途径促进肝纤维化(hepatic fibrosis,HF)模型大鼠活化肝星状细胞(hepatic stellate cells,HSCs)凋亡的作用机制。方法:将40只Wistar大鼠随机分为空白组、模型组、马兰草脐灸组、秋水仙碱组,除空白组外均建立HF模型,然后给予马兰草脐灸和秋水仙碱片水溶液灌胃,干预后取材检测。观察大鼠一般情况及大鼠肝脏形态行肝组织苏木精-伊红染色法(hematoxylin-eosin staining,HE)染色,测定肝功能、细胞色素c氧化酶(cytochrome c oxidase,COX)活性、羟脯氨酸(hydroxyproline,Hyp)含量,免疫印迹法(western blotting,WB)和免疫荧光检测肝组织B细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 Associated X protein,Bax)蛋白表达,dUTP缺口末端标记方法(TdT-mediated dUTP Nick-End Labeling,Tunel)-α-平滑肌动蛋白(smooth muscle actin,α-SMA)荧光双染检测活化HSCs凋亡情况。结果:与空白组比较,模型组大鼠毛色晦暗,体质量饮食减少,肝脏鲜红、干硬,大量炎症细胞浸润,脂肪变性,谷丙转氨酶(ALT)、谷草转氨酶(AST)、Hyp、COX蛋白表达水平均显著增加,Bcl-2表达升高,Bax表达下降且荧光不显著,并有少量不显著的Tunel阳性细胞和大量显著的α-SMA阳性细胞;与模型组比较,药物干预组大鼠的一般情况均有好转,马兰草脐灸组大鼠肝脏形态呈深褐色较润泽,秋水仙碱组大鼠肝脏形态呈半紫红半深褐色,脂肪细胞和炎症细胞均减少,ALT、AST、Hyp、COX均显著降低,α-SMA阳性细胞减少、荧光减弱,Tunel阳性细胞增加,其中马兰草脐灸组Bcl-2表达显著下降,Bax表达显著上升,秋水仙碱组Bcl-2、Bax表达均显著上升。结论:马兰草灸脐可以有效改善模型大鼠HF程度,其机制可能与马兰草脐灸法通过线粒体细胞凋亡途径促进活化的HSC凋亡有关。Objective:To study the mechanism of Ammophila Arenaria umbilical moxibustion promoting the apoptosis of hepatic stellate cells(HSCs)in rats with hepatic fibrosis(HF)through mitochondrial pathway.Methods:Forty Wistar rats were randomly divided into three groups of blank group,model group,Ammophila Arenaria umbilical moxibustion group and colchicine group.The HF model was established in all groups except the blank group,and rats were given gavage administration of Ammophila Arenaria umbilical moxibustion and Colchicine Tablets water solution,respectively.The general situation of rats and hepatic morphology of rats were observed,followed by hematoxylin-eosin staining(HE)of hepatic tissue.Further measurement was carried out on liver function,the activity of cytochrome c oxidase(COX)and the content of hydroxyproline(Hyp).Western Blotting(WB)and immunofluorescence were used to detect the protein expression of B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax)in hepatic tissue.The TdT-mediated dUTP-biotin nick endlabeling(Tunel)-α-smooth muscle actin(α-SMA)dual fluorescence staining was performed to detect the apoptosis of activated HSCs.Results:Compared with the blank group,rats in the model group were slow in response,dark in hair color,and reduction in body weight and diet,with bright red,dry and hard liver,massive inflammatory cells infiltration and fatty degeneration of hepatic cells.Meanwhile,there were obvious increase in expression levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),Hyp,COX and Bcl-2,increased expression of Bax with no significant change of fluorescence,accompanied by a few TUNEL positive cells and a large number ofα-SMA positive cells in the model group.Furthermore,compared with the model group,the general situation of rats was improved in drug intervention groups.Rats in the Ammophila Arenaria umbilical moxibustion group showed dark brown and more moist hepatic morphology,and those of the colchicine group revealed half purple and half dark brown hepatic morpholo

关 键 词：线粒体途径 马兰草脐灸 肝纤维化大鼠 HSCS 

分 类 号：R285.5[医药卫生—中药学]