敲除泛素特异性蛋白酶13促进TNF-α诱导的肝实质细胞凋亡  

USP13 knockdown promotes apoptosis of hepatocytes induced by TNF-α

作　　者：刘凯 王婷[2] 陈旭 李柯颖 向慎思 任广明 杨晓明 LIU Kai;WANG Ting;CHEN Xu;LI Ke-ying;XIANG Shen-si;REN Guang-ming;YANG Xiao-ming(Institute of Lifeomics,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Anhui Medical University,Hefei 230032,China;Qingdao University,Qingdao,Shandong 266071,China)

机构地区：[1]军事科学院军事医学研究院生命组学研究所,北京100850 [2]安徽医科大学,合肥230032 [3]青岛大学,山东青岛266071

出　　处：《军事医学》2022年第9期668-672,690,共6页Military Medical Sciences

基　　金：国家自然科学基金青年科学基金(82001666)。

摘　　要：目的探究敲除泛素特异性蛋白酶13(USP13)对肿瘤坏死因子α(TNF-α)诱导下小鼠肝实质细胞凋亡的影响。方法体外分离培养小鼠原代肝实质细胞,随后用TNF-α或TNF-α和环己酰亚胺(CHX)共刺激细胞。MTS法检测细胞活力、流式细胞术检测细胞凋亡、实时荧光定量PCR(qRT-PCR)检测凋亡相关基因表达、胱天蛋白酶-3(caspase-3)凋亡试剂盒检测caspase-3的激活、Western印迹法检测核因子κB(NF-κB)和丝裂原激活蛋白激酶(MAPK)信号通路活化。结果敲除USP13的小鼠肝实质细胞在TNF-α和CHX共诱导下,细胞活力下降、凋亡增多。机制探究发现,在TNF-α和CHX共处理下,敲除USP13导致抗凋亡基因下调、促凋亡基因上调,以及caspase-3活化增强。在TNF-α单独处理下,敲除USP13导致NF-κB及MAPK信号通路活化受阻。结论敲除USP13抑制肝实质细胞中NF-κB及MAPK信号通路活化,促进TNF-α诱导的细胞凋亡。Objective To investigate the effect of ubiquitin specific proteases 13(USP13)knockdown on apoptosis of hepatocytes induced by TNF-α.Methods Primary hepatocytes of mice were isolated and cultured in vitro and treated with TNF-αor TNF-αand cycloheximide(CHX).MTS assay was used to detect cell viability while flow cytometry was performed to measure cell apoptosis.The mRNA level of apoptosis-related genes was detected by qRT-PCR.Caspase-3apoptosis kit was used to detect the levels of caspase-3 activation.Western blotting was used to detect the activation of NF-κB and MAPK signaling pathways.Results Treatment with TNF-αand CHX decreased cell viability but increased apoptosis in USP13 knockout mouse hepatocytes.Mechanism study showed that knockout of USP13 resulted in up-regulation of pro-apoptotic genes and down-regulation of anti-apoptotic genes,enhancing the activation of caspase-3 under the co-treatment with TNF-αand CHX.Moreover,knockdown of USP13 prevented TNF-α-induced activation of NF-κB and MAPK signaling pathways.Conclusion Deficiency of USP13 in mouse hepatocytes inhibits the activation of NF-κB and MAPK signaling pathways and increases hepatocyte apoptosis induced by TNF-α.

关 键 词：泛素特异性蛋白酶13 肝实质细胞 细胞凋亡 肿瘤坏死因子Α 环己酰亚胺 

分 类 号：R657.3[医药卫生—外科学]