细胞自噬在亚慢性锰染毒致C57BL/6小鼠肝脏损伤中的作用研究  

Role of cell autophagy in liver injury in C57BL/6 mice after subchronic manganese exposure

作　　者：申向丽 刘春燕 张建彬 刘启玲 SHEN Xiang-li;LIU Chun-yan;ZHANG Jian-bin;LIU Qi-ling(School of Public Health,Shaanxi University of Chinese Medicine,Xianyang,Shaanxi 712046,China;Department of Preventive Medicine,Air Force Medical University,Xi'an,Shaanxi 710032,China)

机构地区：[1]陕西中医药大学公共卫生学院,陕西咸阳712046 [2]空军军医大学预防医学系,陕西西安710032

出　　处：《环境与职业医学》2020年第2期168-173,共6页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金青年项目(81602815);陕西省自然科学基金青年项目(2018JQ8005).

摘　　要：[背景]近年来研究发现锰与肝脏疾病的发生发展密切相关,但其发病机制一直未明。自噬在肝脏疾病中发挥的作用一直具有争议。3-甲基腺嘌呤(3-MA)是自噬功能阻断剂,可通过调控自噬水平为锰致肝损伤的机制研究提供依据。[目的]揭示亚慢性锰染毒对C57BL/6小鼠肝脏的损伤效应,阐明细胞自噬在其中的关键作用及机制。[方法]利用C57BL/6小鼠建立亚慢性锰染毒模型,随机分为4组,每组14只,分别为对照组、锰染毒组、3-MA处理组、锰染毒联合3-MA干预组。对照组腹腔注射生理盐水(0.01 mg·kg^-1),锰染毒组腹腔注射剂量为15 mg·kg^-1的氯化锰(每周4次),3-MA处理组腹腔注射7.33 mg·kg^-1的3-MA(每周3次),锰染毒联合3-MA干预组先注射15 mg·kg^-1的氯化锰,30 min后注射7.33 mg·kg^-1的3-MA。造模结束后随即进行相关指标检测。运用HE染色法观察小鼠肝脏形态学的改变;透射电子显微镜下观察各组小鼠肝细胞的损伤及自噬溶酶体的改变;运用原子吸收分光光度计对各组小鼠血锰含量进行检测;使用全自动生化分析仪检测各组小鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)水平的变化;使用细胞凋亡(TUNEL)染色技术评估各组肝细胞的凋亡水平;使用Western blotting检测自噬相关蛋白LC3-II、Beclin1的表达情况。[结果]HE染色发现与对照组相比,锰染毒组肝细胞间隙有较多炎性细胞浸润,且双核细胞数量增多;3-MA干预后,锰染毒对肝脏的炎性浸润及损伤效应有所降低。透射电镜观察发现锰染毒组小鼠肝细胞内线粒体呈现肿胀空泡化现象,并发现较多待降解的自噬溶酶体;锰染毒联合3-MA干预后线粒体空泡化减少,自噬溶酶体数量降低。与对照组相比,锰染毒组血锰、ALT、AST水平增高(P<0.01),注射3-MA干预后能够降低锰染毒对ALT、AST水平的增高效应(P<0.01)。Western blotting检测发现与对照组相比,锰染毒组自噬相关蛋白LC3-[Background]Recent studies have found that manganese is closely related to the development of liver disease,but its pathogenesis has not been elucidated.The role of autophagy in liver diseases is controversial.As an autophagy blocker,3-methyladenine(3-MA)can regulate autophagy levels,which provides clues for revealing the mechanism of manganese-induced liver injury.[Objective]This study is designed to reveal the damage effect of subchronic manganese exposure on the liver of C57 BL/6 mice,and to elucidate the key role and mechanism of subsequent autophagy in cells.[Methods]C57 BL/6 mice were used to establish a subchronic manganese exposure model.The animals were randomly divided into four groups with 14 animals in each group:control group,manganese exposure group,3-MA treatment group,and manganese exposure combined with 3-MA intervention group.The control group was intraperitoneally injected with normal saline(0.01 mL·g-1);the manganese group was intraperitoneally injected with 15 mg·kg^-1 manganese chloride(four times per week);the 3-MA treatment group was intraperitoneally injected with 7.33 mL·g-13-MA(three times per week);the manganese exposure combined with 3-MA intervention group was injected with 15 mg·kg^-1 manganese chloride for 30 min before 7.33 m L·g-13-MA.Relevant indicators were tested immediately after modeling.Liver histological changes were observed with HE staining.The injury of hepatocytes and the changes of autophagic lysosomes in liver tissues were observed under transmission electron microscope.Blood manganese level was detected using atomic absorption spectrophotometer.The changes of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were detected using automatic biochemical analyzer.The apoptosis level of hepatocytes was evaluated with TdT-mediated dUTP nick end labeling(TUNEL)staining.The expressions of autophagy related proteins LC3-II and Beclin1 were detected by Western blotting.[Results]The HE staining results showed that compared with the control group,

关 键 词：锰染毒 自噬 肝损伤 3-甲基腺嘌呤 

分 类 号：R575[医药卫生—消化系统]