TNF-α/TNFR1抑制M1型Kupffer细胞自噬在三氯乙烯致敏小鼠肝损伤中的作用  

The role of TNF-α/TNFR1 inhibiting autophagy of M1-type Kupffer cells in liver injury in trichloroethylene-sensitized mice

作　　者：丁百旺 黄华 杨呓 丁雅妮 朱启星[2] 张家祥 Ding Baiwang;Huang Hua;Yang Yi;Ding Yani;Zhu Qixing;Zhang Jiaxiang(Department of Occupational Hygiene and Environmental Hygiene,School of Public Health,Anhui Medical University,Hefei 230032,China;Institute of Dermatology,the First Affiliated Hospital of Anhui Medical University,Hefei 230032,China)

机构地区：[1]安徽医科大学公共卫生学院职业卫生与环境卫生学系,合肥230032 [2]安徽医科大学第一附属医院皮肤病研究所,合肥230032

出　　处：《中华劳动卫生职业病杂志》2022年第5期321-327,共7页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：安徽省自然科学基金(2008085QH385);国家自然科学基金(81874259);安徽医科大学博士科研资助基金(XJ201941)。

摘　　要：目的:检测三氯乙烯(TCE)致敏小鼠肝脏中M1型极化与自噬相关指标表达水平,探讨肿瘤坏死因子-α(TNF-α)及肿瘤坏死因子受体1(TNFR1)调控M1型Kupffer细胞自噬在TCE致敏小鼠肝损伤中的作用。方法:于2019年11月,将45只SPF级BALB/c雌性小鼠(6~8周龄),按照单纯随机分组分为4组:空白对照组( n=5)、溶剂对照组( n=5)、TCE处理组( n=18)、TCE+R7050(抑制剂)处理组( n=17);经皮致敏小鼠,末次激发后24 h,根据皮肤反应评分情况将小鼠分为致敏组与未致敏组。取小鼠肝脏,光学及电子显微镜下观察肝脏病理变化,蛋白质印迹(Western blotting)检测TNF-α、TNFR1及自噬相关指标表达情况,免疫组化法检测M1型Kupffer细胞标志物诱导型一氧化氮合酶(iNOS)表达情况,免疫荧光双标法检测M1型Kupffer细胞自噬发生情况。 结果:TCE处理组小鼠致敏率为38.9%(7/18),TCE+R7050处理组致敏率为35.3%(6/17),两组差异无统计学意义( P=1.000)。与空白对照组比较,TCE致敏组小鼠肝细胞形态异常,肝损伤明显,肝细胞内线粒体减少,内质网断裂。Western blotting结果显示,与空白对照组比较,TCE致敏组小鼠肝脏TNF-α、TNFR1蛋白表达升高,M1型Kupffer细胞iNOS蛋白表达升高,自噬微管结合蛋白1-轻链3(LC3B)、Beclin1蛋白表达减少,差异均有统计学意义( P<0.05);免疫组化结果显示,空白对照组和溶剂对照组中iNOS未见明显表达,TCE未致敏组可见少量表达,TCE致敏组中阳性染色区域明显,iNOS表达明显升高( P<0.05);免疫荧光结果显示,空白对照组、溶剂对照组与TCE未致敏组iNOS蛋白水平较低,仅部分与P62共定位;TCE致敏组iNOS与P62的荧光信号共定位明显增加。 结论:TNF-α/TNFR1信号通路可能通过抑制M1型Kupffer细胞自噬从而诱导TCE致敏小鼠的肝损伤。Objective To detect the expression levels of M1-type polarization and autophagy-related indicators in the liver of trichloroethylene(TCE)-sensitized mice,and to explore the role of liver tumor necrosis factor-α(TNF-α)and tumor necrosis factor receptor 1(TNFR1)in regulating M1-type Kupffer cells autophagy in liver injury in TCE-sensitized mice.Methods In November 2019,according to simple random grouping,45 SPF grade BALB/c female mice(6-8 weeks old)were divided into 4 groups:blank control group(n=5),solvent control group(n=5),TCE treatment group(n=18),TCE+R7050(inhibitor)treatment group(n=17).Transdermally sensitized mice,24 h after the last challenge,the mice were divided into TCE sensitized group and TCE non-sensitized group according to the skin reaction score.The livers of mice were harvested,and the pathological changes of the livers were observed under light and electron microscopes.Western blotting was used to detect the expressions of TNF-α,TNFR1 and autophagy-related indexes.The expression of inducible nitric oxide synthase(iNOS),a marker of M1-type Kupffer cells,was detected by immunohistochemistry,and the occurrence of autophagy in M1-type Kupffer cells was detected by immunofluorescence double-labeling method.Results The sensitization rate of TCE treatment group was 38.9%(7/18),and TCE+R7050 treatment group was 35.3%(6/17),with no significant difference between the two groups(P=1.000).Compared with the blank control group,mice in the TCE sensitized group had abnormal liver ocytes,obvious liver injury,reduced mitochondria and broken endoplasmic reticulum.Western blotting results showed that the expressions of TNF-αand TNFR1 protein in the liver of the mice in the TCE sensitized group increased,the expression of iNOS protein in M1-type Kupffer cells increased,and the expressions of autophagic microtubule-associated protein 1 light-chain 3(LC3B)and Beclin1 protein were decreased(P<0.05).The results of immunohistochemistry showed that iNOS was not significantly expressed in the blank control group and

关 键 词：三氯乙烯 KUPFFER细胞 自噬 肝损伤 小鼠 

分 类 号：R575[医药卫生—消化系统]