糜烂性毒剂氮芥染毒对肝细胞线粒体结构和功能的影响

Effects of blister agent nitrogen mustard exposure on the structure and function of mitochondria in hepatocytes

目的:探讨体外和体内模型中糜烂性毒剂氮芥(HN2)染毒对肝细胞线粒体结构和功能的影响。方法:体外模型中,HepG2细胞分为正常对照组和HN2染毒组,正常对照组给予含0.1%DMSO的无血清培养基处理36 h,HN2染毒组给予8μmol/L氮芥染毒36 h,分别采用CCK-8法检测细胞活性,JC-1探针法检测线粒体膜电位,NADPH速率法检测细胞匀浆ATP含量,Seahorse细胞能量代谢分析仪检测线粒体功能;体内模型中,20只C57BL/6小鼠分为正常对照组和HN2染毒组,HN2染毒组按2 mg/kg腹腔单次注射盐酸氮芥,正常对照组注射生理盐水,染毒后3 d后采集血清和肝组织,测定血清谷丙转氨酶(ALT)和谷草转氨酶(AST)活性,进行肝组织HE染色病理检测和电镜观察,并测定肝组织ATP含量和线粒体酶复合物Ⅰ、Ⅱ和Ⅲ的活性。结果:在体外模型中,与正常对照组相比,HN2染毒组细胞活性降低了约16.2%(P<0.05),线粒体膜电位下降了约33.2%(P<0.05),线粒体能量代谢明显异常。在体内模型中,与正常对照组比较,HN2染毒后,血清ALT和AST活性显著升高(P<0.05),同时肝组织存在大量炎症细胞浸润并伴有肝实质细胞损伤,肝细胞内线粒体形态发生显著异常,表现为数量增多、体积缩小、线粒体嵴缺失或断裂,线粒体酶复合体Ⅰ、Ⅱ和Ⅲ的活性均显著降低(P<0.05)。结论:HN2染毒能够导致肝细胞发生线粒体结构损伤和功能异常,线粒体可能是HN2诱导肝损伤的主要毒作用靶点之一,本研究结果为糜烂性毒剂诱导急性肝损伤的防治策略提供了新思路。

OBJECTIVE:To investigate effects of the blister agent,nitrogen mustard(HN2),on mitochondrial structure and function of hepatocytes,based on in vitro and in vivo models.METHODS:In the in vitro model,HepG2 cells were divided into normal control and HN2 exposure groups.The normal control group was treated with serum-free medium containing DMSO(0.1%)for 36 h,and the HN2 exposure group was treated with nitrogen mustard(8μmol/L).After HN2 treatment for 36 h,cell viability was detected by CCK-8 method,mitochondrial membrane potential by JC-1 probe method,ATP content in cell homogenate and liver tissue by NADPH rate method,and mitochondrial function by Seahorse cell energy metabolism analyzer.Twenty C57BL/6 mice were divided into normal control group and HN2 exposure group.The HN2 exposure group was given a single intraperitoneal injection of nitrogen mustard hydrochloride(2 mg/kg),and the normal control group was injected with normal saline.Serum and liver tissues were used to measure activities of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST),and pathological examination of liver tissues and electron microscope observations were performed.Contents of ATP and mitochondrial enzyme complexes I,II and III activities in liver tissues were determined by corresponding kits.RESULTS:In the in vitro model,compared with the normal control group,HN2 exposure decreased cell activity by about 16.2%(P<0.05),the mitochondrial membrane potential by about 33.2%(P<0.05),and mitochondrial energy metabolism was significantly abnormal.In the in vivo model,compared with the normal control group,HN2 exposure caused significant increased activities of serum ALT and AST.At the same time,there was large number of inflammatory cell infiltrations into liver tissues accompanied by liver parenchymal cell damage.Mitochondrial morphology was significantly abnormal:manifested as increased number,decreased size,loss or fragmentation of mitochondrial cristae,and significantly decreased activity of mitochondrial enzyme complexes I,II and III(P<0.05).CONCLUSION:HN2 exposure induced mitochondrial damage in hepatocytes and this may be one of the main toxic targets of HN2 poisoning.Results from our study provide new ideas for the prevention and treatment of acute liver injury induced by blister agents such as HN2.