泛素特异性蛋白酶13(USP13)敲除对对乙酰氨基酚诱导的肝脏炎性应激反应的影响

EFFECT OF UBIQUITIN-SPECIFIC PROTEASE 13(USP13)KNOCKOUT ON ACETAMINOPHEN-INDUCED HEPATIC INFLAMMATORY STRESS IN MICE

目的研究泛素特异性蛋白酶13(ubiquitin-specific protease 13,USP13)在对乙酰氨基酚(acetaminophen,APAP)诱导的肝脏炎性应激反应中的作用。方法将Usp13^(+/+)小鼠和Usp13^(-/-)小鼠均设置为对照组(CON,APAP 0 h)和3个实验组(APAP 2 h、APAP 4 h和APAP 12 h),每组6对小鼠;饥饿18 h后实验组小鼠腹腔注射300 mg/kg APAP建立肝脏炎性应激反应模型,分别于2、4、12 h收集小鼠肝脏组织和血清;对照组在0 h收集小鼠肝脏组织和血清。HE染色观察肝脏组织病理变化;免疫组化观察肝脏组织中炎性细胞浸润情况;ELISA检测血清中丙氨酸氨基转移酶(alanine aminotransferase,ALT)和天冬氨酸氨基转移酶(aspartate aminotransferase,AST)水平;Western blot检测肝脏细胞色素P4502E1(cytochrome p4502E1,CYP2E1)蛋白表达和谷胱甘肽(glutathione,GSH)试剂盒检测肝脏GSH变化水平;Western blot检测c-Jun N端激酶(c-Jun N-terminal kinase,JNK)通路的激活情况;qPCR检测炎性因子相关基因的转录水平表达。结果与对照组相比,APAP 2 h和4 h组小鼠肝脏CYP2E1蛋白表达无明显变化、GSH显著下降(P<0.0001)、JNK通路激活和炎症因子表达升高(P<0.001)。与对照组相比,APAP 12 h组小鼠肝脏发生明显坏死,F4/80^(+)细胞数量增加(P<0.001)、Ly6G^(+)细胞数量增加(P<0.01)以及血清ALT、AST水平升高(P<0.01)。但与Usp13^(+/+)小鼠相比,Usp13敲除对APAP诱导肝脏炎性应激反应的病理生理过程并无明显影响。结论USP13缺失不影响APAP诱导的肝脏炎性应激反应过程中的各种病理生理过程,这不同于USP13参与多种肝脏疾病发生发展的报道,提示USP13在肝脏疾病作用的复杂性。

Objective To investigate the role of ubiquitin-specific protease 13(USP13)in hepatic inflammatory stress response induced by acetaminophen(APAP).Methods Usp13^(+/+)mice and Usp13^(-/-)mice were set up as the control group and three experimental groups(APAP 2 h,4 h and 12 h group),with 6 mice in each group.After being starved for 18 h,the liver inflammatory stress model was established by intraperitoneal injection of 300 mg/kg APAP in the experimental groups.Live tissues and serum were collected at 2,4,and 12 h respectively.In the control group liver tissues and serum were collected at 0 h.Histopathological changes in the liver were observed by HE staining.Inflammatory cell infiltration in liver tissue was observed by immunohistochemistry.The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the serum were measured by ELISA.Glutathione(GSH)kit was used to detect hepatic GSH changes.Western blot was used to detect hepatic cytochrome p4502E1(CYP2E1)protein expression and c-Jun N-terminal kinase(JNK)pathway activation.The expression of inflammatory factor-related genes was detected by qPCR.Results Compared with the control group,mice in the APAP 2 h and APAP 4 h groups showed no significant changes in hepatic CYP2E1 protein expression,a significant decrease in GSH(P<0.0001),an increase in JNK pathway activation and inflammatory factor expression(P<0.001).Compared with the control group,mice in the APAP 12 h group experienced significant liver necrosis,increased number of F4/80^(+)cells(P<0.001)and Ly6G^(+)cells(P<0.01),as well as increased serum ALT and AST levels(P<0.01).However,Usp13 knockout did not significantly affect the pathophysiological process of APAP-induced inflammatory stress response in the liver compared with Usp13^(+/+)mice in all experimental groups.Conclusion USP13 deletion does not affect various pathophysiological processes during the APAP-induced inflammatory stress response in the liver,which differs from previous reports showing the involvement of USP13 in the development of multiple liver diseases and suggests the complexity of the role of USP13 in liver diseases.