PP2Ac去甲基化在锰暴露...肝脏炎症性损伤中的作用研究_黄丽媛.pdf

广西医科大学学报JOURNAL OF GUANGXI MEDICAL UNIVERSITY2022 Dec；39（12）PP2Ac去甲基化在锰暴露诱导大鼠肝脏炎症性损伤中的作用研究*黄丽媛1，陆建勇1，梁宁静1，袁江浪2，孔星星2，李习艺1，唐深2，陆彩玲1（1.广西医科大学公共卫生学院，南宁530021；2.广西医科大学基础医学院，南宁530021）摘要目的：研究亚急性及慢性锰暴露对SD大鼠肝脏的损伤效应，探索PP2Ac去甲基化修饰在锰暴露致肝脏炎症性损伤中的作用。方法：40只雄性SD大鼠按体重随机入组，分别设置4周（4 W）和16周（16 W）两个暴露时间点，每个时间点分两组（即4 W、16 W对照组和4 W、16 W锰暴露组），每组10只；锰暴露组腹腔注射MnCl24H2O（15 mg/kg），对照组注射同体积的生理盐水，每天1次，持续4 W、16 W。实验终点时分离大鼠肝脏，ICP-MS法检测血液及肝组织中锰的含量。苏木精伊红（HE）染色观察肝脏的病理改变。实时荧光定量聚合酶链式反应（RT-qPCR）法检测肝脏炎症相关因子肿瘤坏死因子（TNF-）、白介素6（IL-6）、趋化因子CCL-2的表达。Western blotting法检测肝组织总PP2Ac、去甲基化PP2Ac及PP2Ac甲基化调控蛋白LCMT-1及PME-1的表达。结果：与4 W对照组相比，4 W、16 W锰暴露组全血锰含量显著升高（P0.01），4 W锰暴露组肝组织锰含量显著下降（P0.05），而16 W锰暴露组肝组织锰含量显著升高（P0.01）；HE染色结果显示，与4 W对照组相比，4 W锰暴露组出现肝血窦扩张，炎症细胞浸润，16 W锰暴露组与16 W对照组相比，肝索排列紊乱，细胞间隙增大，肝窦进一步扩张充血，大量肝细胞膜破裂；与4 W对照组相比，4 W锰暴露组仅肝脏炎症因子IL-6的表达呈上升趋势，16 W锰暴露组CCL-2、IL-6的mRNA表达与16 W对照组相比呈升高趋势，并且TNF-的mRNA表达显著升高（P0.05）；4 W、16 W锰暴露组的去甲基化PP2Ac表达水平升高（P0.05），仅16W锰暴露组的甲基化PP2Ac修饰蛋白LCMT-1表达水平降低（P0.01），不同时点锰暴露组PME-1、总PP2Ac蛋白表达无明显变化。结论：锰诱导大鼠肝脏产生炎症性损伤，可能与上调肝脏PP2Ac去甲基化有关。关键词锰暴露；肝损伤；炎症；PP2Ac中图分类号：R575.1文献标志码：A文章编号：1005-930X（2022）12-1911-07DOI：10.16190/ki.45-1211/r.2022.12.007The role of PP2Ac demethylation in inflammatory liver injury induced by manganese exposure in ratsHuang Liyuan1,Lu Jianyong1,Liang Ningjing1,Yuan Jianglang2,Kong Xingxing2,Li Xiyi1,Tang Shen2,Lu Cail-ing1.(1.School of Public Health,Guangxi Medical University,Nanning 530021,China;2.School of Basic Medi-cal Sciences,Guangxi Medical University,Nanning 530021,China）AbstractObjective:To study the damage effects of subacute and chronic manganese exposure on the liver ofSD rats and explore the role of catalytic subunit of protein phosphatase 2A(PP2Ac)demethylation modificationin the inflammatory liver damage induced by manganese exposure.Methods:Forty male SD rats were randomlyenrolled according to body weight,and two exposure time points were set at 4 weeks(4 W)and 16 weeks(16W),respectively.Each time point was divided into two groups(4 W and 16 W control group,4 W and 16 W man-ganese exposure group),with 10 rats in each group.The manganese exposure group was intraperitoneally injec-ted with MnCl24H2O(15 mg/kg),while the control group was intraperitoneally injected with the same volume ofnormal saline,once a day for 4 W and 16 W.At the end of the experiment,the rat liver was isolated,and the con-tent of manganese in the blood and liver tissues wasdetected by ICP-MS.The pathological changes of theliver were observed by HE staining.The expressionsof tumor necrosis factor-(TNF-),interleukin-6(IL-*基金项目：国家自然科学基金项目（No.81760576；No.81860585；No.82160612）；广西自然科学基金项目（No.2021GXNSFAA196019）通信作者，E-mail：收稿日期：2022-10-18基础研究 1911广西医科大学学报2022 Dec；39（12）6)and chemokine CCL-2 were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qP-CR).The expressions of total PP2Ac,demethylated PP2Ac and PP2Ac methylated regulatory proteins LCMT-1and PME-1 in liver tissues were analyzed by western blotting.Results:Compared with the 4 W control group,the whole blood manganese content in 4 W and 16 W manganese exposure groups significantly increased(P0.01),the liver manganese content in 4 W manganese exposure group significantly decreased(P0.05),and theliver manganese content in 16 W manganese exposure group significantly increased(P0.01).HE staining re-sults showed that compared with the 4 W control group,the 4 W manganese exposure group showed hepatic sinu-soidal dilatation and inflammatory cell infiltration,while the 16 W manganese exposure group showed disorderedhepatic cord arrangement,enlarged intercellular space,further dilatation and congestion of hepatic sinuses,and alarge number of hepatic cell membrane rupture compared with the 16 W control group.Compared with the 4 Wcontrol group,only the expression of liver inflammatory factor IL-6 in the 4 W manganese exposure group in-creased,the mRNA of CCL-2 and IL-6 in the 16 W manganese exposure group increased compared with the 16W control group,and the mRNA expression of TNF-significantly increased(P0.05).The expression of de-methylated PP2Ac increased in the 4 W and 16 W manganese exposure groups(P0.05),while the expression ofmethylated PP2Ac modified protein LCMT-1 decreased only in the 16 W manganese exposure group(P0.01);there were no significant changes in PME-1 and total PP2Ac protein expressions in different time points of man-ganese exposure groups.Conclusion:The inflammatory damage of liver of rats induced by manganese may be re-lated to the up-regulation of liver PP2Ac demethylation.Keywordsmanganese exposure;liver injury;inflammation;PP2Ac锰是人体必需的微量元素，参与了体内重要的生物学过程。且锰在工业、农业及医学上都有广泛应用，因此人体一般不会出现锰缺乏1。但是随着国家工业化的发展，长期的锰积累导致的健康问题受到社会的广泛关注。锰易蓄积在富含线粒体的肝脏、大脑等组织中2，过量锰可诱导肝脏线粒体功能障碍，从而造成肝损伤3。锰暴露可使动物出现胆汁淤积、肝细胞核皱缩、细胞凋亡、肝细胞坏死等病理改变4-5。锰可诱导机体发生炎症反应，从而对组织产生损伤6。然而，有关锰致肝脏炎症性损伤的机制研究较少，且尚未完全阐明。蛋白磷酸酶2A（protein phosphatase 2A，PP2A）是存在于真核生物中的一种丝氨酸/苏氨酸蛋白磷酸酶，参与细胞分化、信号转导、基因转录和蛋白质翻译等多种生理学过程7。PP2A由支架亚基A、调节亚基 B、催化亚基 C 组成8。PP2A 催化亚基 C（PP2Ac）9及其甲基化修饰10在肝脏病变中发挥重要作用。本课题组前期研究发现，染锰后动物的肝组织出现病理改变。但该损伤是否与