血吸虫病肝纤维化过程中Beclin1调节线粒体功能的初步研究  

Mitochondrial function regulated by Beclin1 in liver fibrosis in schistosomiasis

作　　者：沈双 罗军涛[2] 叶建平 SHEN Shuang;LUO Jun-tao;YE Jian-ping(Shanghai Sixth People’s Hospital East Affiliated to Shanghai University of Medicine and Health Sciences,Shanghai 201306,China;Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海健康医学院附属第六人民医院东院,上海201306 [2]上海海洋大学,上海201306

出　　处：《中国寄生虫学与寄生虫病杂志》2020年第1期41-46,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：上海市卫生和计划生育委员会青年项目(No.20174Y0124);浦东新区科技发展基金(No.PKJ2018-Y55);上海市第六人民医院医疗集团科研基金;上海健康医学院种子基金(No.SFP-18-22-14-009)。

摘　　要：目的探索Beclin1在血吸虫病肝纤维化中与线粒体功能的关系。方法将40只C57BL/6雌性小鼠随机分为感染组和健康对照组,每组20只。感染组小鼠采用腹部贴片法感染日本血吸虫尾蚴20条/鼠。感染后10周,取两组小鼠新鲜肝组织,苏木精-伊红(HE)和Masson染色观察肝组织病变情况。蛋白质印迹(Western blotting)分析肝组织中Beclin1、p-Beclin1及电压依赖型阴离子孔道蛋白1 (VDAC1)的相对表达水平。荧光定量PCR检测肝组织中胶原蛋白Col 1a1、Co1 3a1,以及肝组织中参与线粒体柠檬酸循环反应的柠檬酸合酶、异柠檬酸脱氢酶、α酮戊二酸脱氢酶和苹果酸脱氢酶mRNA的相对转录水平。取两组小鼠肝组织制备切片,电镜下观察线粒体形态。提取肝组织线粒体,利用细胞能量代谢仪依次检测加入复合物V底物二磷酸腺苷(ADP)、复合物V抑制剂寡霉素、解偶联剂FCCP、复合物Ⅱ和Ⅲ的抑制剂抗霉素A后5 min线粒体的耗氧率。结果HE和Masson染色结果显示,感染组小鼠肝脏形成肉芽肿,肉芽肿区域发生明显纤维化。Western blotting分析结果显示,感染组肝组织中VDAC1表达水平未发生改变;Beclin1和p-Beclin1的相对表达水平分别为0.65±0.05、0.78±0.03,均较健康对照组下降(P<0.05)。荧光定量PCR结果显示,感染组小鼠肝组织中Col 1a1、Col 3a1mRNA的相对转录水平分别为4.05±0.23、2.71±0.14,均较健康对照组上升(P<0.01);柠檬酸合酶、异柠檬酸脱氢酶、α酮戊二酸脱氢酶及苹果酸脱氢酶mRNA的相对转录水平分别为0.61±0.03、0.65±0.01、0.41±0.03、0.55±0.01,均较健康对照组下降(P<0.01)。电镜观察可见,感染组肝组织线粒体结构受损,嵴断裂。线粒体功能检测显示,加入复合物V底物ADP后,健康对照组和感染组耗氧率分别为(335±29)、(78±23)pmol/min,差异有统计学意义(P<0.01);加入复合物V抑制剂寡霉素后,健康对照组和感染组耗氧率分别为(80±2)、(31±Objective To explore the relationship between Beclinl and mitochondrial function in hepatic fibrosis in schistosomiasis.Methods Forty female C57 BL/6 mice were randomly divided into infected group and healthy control group(20 mice in each group).Mice in the infected group were infected with 20 Schistosoma japonicum cercariae by the abdominal patch method.Ten weeks after infection,fresh liver tissues were collected from the two groups.Liver pathology was assessed by HE and Masson staining of liver tissue sections.The relative protein levels of Beclinl,p-Beclinl and voltage-dependent anion channel protein 1(VDAC1) in liver were assessed by Western blotting.Fluorescence quantitative PCR was performed to analyze the relative mRNA expression of collagen Col lal and Col 3 a1,as well as enzymes involved in the mitochondrial citric acid cycle reaction,including citrate synthase,isocitrate dehydrogenase,alpha ketoglutarate dehydrogenase and malate dehydrogenase.Another part of liver tissues was sectioned for visualizing mitochondrion morphology by electron microscopy.To examine the mitochondria oxygen consumption rate(OCR) using cell energy metabolizer,the mitochondria extracted from liver tissues were added to the culture plates,to which complex V substrate ADP,complex V inhibitor oligomycin,uncoupler FCCP,and antimycin A(an inhibitor for complex Ⅱ plus Ⅲ) were sequentially added,allowing culture for 5 min,then the OCR was detected.Results HE and Masson staining showed that granulomas were formed in mouse liver in the infected group,and obvious fibrosis occurred around the granulomas.Western blotting showed that the protein level of VDAC1 remained unchanged in the infected liver,while Beclinl(0.65±0.05) and p-Beclinl(0.78±0.03) were decreased,compared with the control(P <0.05).The fluorescence quantitative PCR results indicated that the relative transcription levels of Col lal and Col 3 a1 mRNA in livers of the infected group were4.05±0.23 and 2.71±0.14,respectively,which were higher than those of the healthy con

关 键 词：日本血吸虫 肝纤维化 线粒体 柠檬酸循环 BECLIN1 

分 类 号：R532.21[医药卫生—内科学]