糜烂性毒剂氮芥染毒诱导急性肝损伤中自噬的变化及作用

Changes and Role of Autophagy in Acute Liver Injury Induced by Blister Agent Nitrogen Mustard

目的建立糜烂性毒剂氮芥(nitrogenmustard,HN2)诱导小鼠急性肝损伤模型和HN2染毒HepG2细胞(人源性肝癌细胞系)损伤模型,探讨自噬在糜烂性毒剂致急性肝损伤中的作用及机制。方法体内模型中,将20只C57BL/6雄性小鼠随机分为正常对照组和HN2染毒组;HN2染毒组小鼠给予腹腔注射盐酸氮芥(2mg/kg),正常对照组小鼠给予腹腔注射等体积的生理盐水。染毒3天后收集血清和肝组织,测定血清天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)和丙氨酸氨基转移酶(alanineaminotransferase,ALT)活性,进行肝组织苏木精-伊红(hematoxy-lin-eosin,HE)染色病理检测,电镜观察线粒体等细胞器形态,Westernblot检测肝组织自噬相关蛋白LC3-Ⅱ/Ⅰ比值和p62的蛋白表达。体外模型中,HepG2细胞分为正常对照组和HN2染毒组;正常对照组细胞给予含二甲基亚砜(dime-thylsulfoxide,DMSO)(0.1%)的无血清培养基处理36h。HN2染毒组细胞给予盐酸氮芥(8μmol/L)处理36h。采用细胞计数试剂盒(cellcountingkit8,CCK-8)检测细胞活性,单丹磺酰尸胺(monodansylcadaverine,MDC)探针法检测线粒体自噬改变,自噬双标腺病毒(mRFP-GFP-LC3)检测肝细胞自噬流的改变;在细胞模型中,给予自噬激动剂雷帕霉素(rapamycin,RAPA)和自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)预处理干预,检测细胞活力水平。结果在体内模型中,与正常对照组比较,HN2染毒组小鼠血清ALT和AST活性显著升高(P均<0.05),同时肝组织存在大量炎细胞浸润并伴有肝实质细胞损伤;肝实质细胞内出现大量自噬体,肝组织LC3-Ⅱ/Ⅰ比值升高(P<0.05),同时p62的蛋白表达显著下调(P<0.05)。在体外模型中,与正常对照组相比,HN2染毒组细胞活性显著降低(P<0.05),MDC荧光强度显著升高,自噬体数量增加(P均<0.05)。在HN2染毒细胞模型中,给予自噬激动剂RAPA预处理后细胞活力显著升高(P<0.05),而给予自噬抑制剂3-MA预处理后细胞活力显著降低(P<0.05)。结论HN2染毒能够诱导肝细胞发生自噬,细胞自噬可能是糜烂性中毒的关键性保护性反应,本研究为糜烂性毒剂诱导急性肝损伤的有效防治提供了新方向。

Objective To establish a mouse model of acute liver injury induced by blister agent nitrogen mustard(HN2)and the injury model of HepG2 cells(human hepatocellular carcinoma cell lines)exposed to HN2,and to explore the role and mechanism of autophagy in acute liver injury induced by blister agent.Methods In in vivo model,20 C57BL/6 male mice were randomly divided into the normal control group and the HN2 exposed group;the mice in the HN2 exposed group were intraperitoneally injected with nitrogen mustard hydrochloride(2 mg/kg)once time,and the mice in the normal control group were intraperitoneally injected with the same volume of saline.Three days after the exposure,serum and hepatic tissue were collected,the activities of serum aspartate aminotransferase(AST)and alanine aminotransferase(ALT)were measured,hematoxylin-eosin(HE)staining was performed for pathological examination of hepatic tissue,the morphology of organelle such as mitochondria was observed under electron microscope,and the autophagy related protein LC3-Ⅱ/Ⅰratio,protein expression of p62 in hepatic tissue were detected by Western blot.In the in vitro model,HepG2 cells were divided into a normal control group and HN2 exposed group;the cells in normal control group were treated with serum-free medium containing dimethyl sulfoxide(DMSO)(0.1%)for 36 h,while the cells in HN2 exposed group were treated with 8μmol/L nitrogen mustard hydrochloride of treatment after 36 h.Cell viability was detected by cell counting kit 8(CCK-8),the changes of mitophagy were detected by monodansylcadaverine(MDC)probe method,the changes in liver cell autophagy flow were detected by double labeled adenovirus(mRFP-GFP-LC3);in the cell model,pretreatment intervention with autophagy agonist rapamycin(RAPA)and autophagy inhibitor 3-methyladenine(3-MA)was administered to detect the cell viability level.Results In the in vivo model,compared with the normal control group,the serum ALT and AST viability of mice in HN2 exposed group significantly increased(all P<0.05),at the same time,there was large amount of inflammatory cell infiltration in hepatic tissue accompanied by hepatic parenchymal cell injury;a large number of autophagosomes appeared in hepatic parenchymal cells,and the LC3-Ⅱ/Ⅰratio in hepatic tissue increased(P<0.05),meanwhile,the protein expression of p62 was significantly downregulated(P<0.05).In the in vitro model,compared with the normal control group,the cells in HN2 exposed group showed a significant decrease in cell viability(P<0.05),the fluorescence intensity of MDC and the number of autophagosomes sign;ficantly increased(all P<0.05).In the HN2 exposed cell model,the cell viability significantly increased after pretreatment with autophagy agonists RAPA(P<0.05),while the cell viability was significantly reduced after pretreatment with autophagy inhibitor 3-MA(P<0.05).Conclusion HN2 exposure can lead to cellular autophagy in hepatocytes,which may be a key protective effects in blister agents poisoning.This study provides a new direction for the effective prevention and treatment of acute liver injury induced by blister agents.