Mn-SOD通过GSK-3β影响HO-1和Drp1的抗氧化应激减轻MTX相关肝细胞损伤  

Mn-SOD alleviates methotrexate-related hepatocellular injury via GSK-3βaffecting anti-oxidative stress of HO-1 and Drp1

作　　者：李卓[1] 陈梦璇 汪苇杭 刘其摇 李耐萍[2] 贺波[2] 蒋永芳[2] 马静[2] LI Zhuo;CHEN Mengxuan;WANG Weihang;LIU Qiyao;LI Naiping;HE Bo;JIANG Yongfang;MA Jing(Department of Ophthalmology,Second Xiangya Hospital,Central South University,Changsha 410011;Department of Infectious Diseases,Second Xiangya Hospital,Central South University,Changsha 410011,China)

机构地区：[1]中南大学湘雅二医院眼科,长沙410011 [2]中南大学湘雅二医院感染科,长沙410011

出　　处：《中南大学学报（医学版）》2022年第9期1191-1199,共9页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(81500455);湖南省自然科学基金(2022JJ30825,2020JJ4793);湖南省卫生健康委员会项目(202120700992)。

摘　　要：目的:氨甲蝶呤(methotrexate,MTX)是导致药物性肝损伤最常见的药物,其致病机制与线粒体功能障碍引起的氧化应激有关。超氧化物歧化酶(superoxide dismutase,SOD)可以通过清除超氧自由基达到抗氧化应激的作用,含锰超氧化物歧化酶(manganese superoxide dismutase,Mn-SOD)属于SOD的一种。本研究拟观察Mn-SOD是否影响MTX对肝细胞的损伤,并探讨其可能的分子机制。方法:体外培养人肝细胞系L-02,并分为4组:空白对照组(加入同体积无血清培养基)、MTX组(加入40μg/孔MTX)、MTX+NC组(加入40μg/孔MTX+转染空白质粒)和MTX+SOD组(加入40μg/孔MTX+转染Mn-SOD质粒)。分别应用全自动生化仪和real-time RT-PCR法检测每组细胞培养上清的ALT、AST和miR-122的水平,评估各组肝细胞受损伤的程度;使用线粒体超氧化物(mitochondrial superoxide indicator,MitoSOX)荧光探针标记各组细胞内超氧化物,流式细胞术检测细胞凋亡情况;蛋白质印迹法检测细胞中糖原合酶激酶3β(glycogen synthase kinase-3 beta,GSK-3β)、血红素加氧酶1(heme oxygenase 1,HO-1)、线粒体分裂蛋白质(dynamin-related protein 1,Drp1)和Mn-SOD的含量。结果:与空白对照组比较,MTX组和MTX+NC组肝细胞培养上清中ALT、AST和miR-122水平明显升高(P<0.05),MTX+SOD组较MTX组和MTX+NC组显著降低(P<0.05)且与空白对照组相当。MitoSOX染色显示:MTX组和MTX+NC组的超氧化物最丰富,MTX+SOD组明显减少且与空白对照组差别不大。流式细胞术检测结果表明:与空白对照组相比,MTX组和MTX+NC组细胞凋亡明显增加,MTX+SOD组与MTX组和MTX+NC组比较细胞凋亡明显减少(P<0.05)。蛋白质印迹法检测到空白对照组和MTX+SOD组细胞Mn-SOD、p-GSK-3β和HO-1高水平表达,而MTX组和MTX+NC组细胞Mn-SOD、p-GSK-3β和HO-1比空白对照组明显降低(P<0.05);Drp1则完全相反,在MTX组和MTX+NC组中高表达,在空白对照组和MTX+SOD组中则是低表达。结论:细胞内Mn-SOD水平的降低Objective:Methotrexate(MTX)is the most common therapeutic agent that may have the risk of drug-induced liver injury.Its pathogenic mechanism is related to oxidative stress caused by mitochondrial dysfunction.Superoxide dismutase(SOD),including manganesecontaining SOD(Mn-SOD),can exert its effect of anti-oxidative stress by scavenging superoxide free radicals.Accordingly,this study is performed to explore the underlying molecular mechanism via observing whether Mn-SOD could affect the damage of MTX to hepatocytes.Methods:Human hepatocyte cell line L-02 was cultured in vitro and divided into 4 groups,including a blank group with the addition of the same volume of serum-free medium,a MTX group(40μg/well MTX drug-treatment),a MTX+NC group(40μg/well MTX drugtreatment+blank plasmid),and a MTX+SOD group(40μg/well MTX drug-treatment+MnSOD plasmid).The levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and microRNA-122(miR-122)in the supernatant of cell culture were respectively detected by automatic biochemical analytical instrument and real-time RT-PCR to evaluate the degree of hepatocyte damage in each group.MitoSOX fluorescent probe was used to label intracellular superoxide in each group,and cell apoptosis was detected by flow cytometry.Meanwhile,the contents of glycogen synthase kinase-3 beta(GSK-3β),hemeoxygenase-1(HO-1),mitochondrial fission-mediated protein of dynamin-related protein 1(Drp1),and Mn-SOD were detected by Western blotting.Results:Compared with the blank group,the levels of ALT,AST,and miR-122 in the supernatant of hepatocyte culture of the MTX group and MTX+NC group were significantly elevated(all P<0.05),and that in the MTX+SOD group were significantly decreased(P<0.05)and equivalent to that in the blank group.MitoSOX staining revealed that the MTX group and MTX+NC had the most abundant superoxide;and the amount was significantly reduced in the MTX+SOD group,without a significant difference when compared with the blank group.Furthermore,the results of flow cytometry indicat

关 键 词：含锰超氧化物歧化酶 氨甲蝶呤 肝细胞损伤 

分 类 号：R575[医药卫生—消化系统]