孙菁,王世卉
SUN Jing,WANG Shihui

• 1:中国医科大学附属第一医院

摘要(Abstract)：

目的:探究白藜芦醇(Res)对刀豆素A(Con A)诱导小鼠自身免疫性肝炎(AIH)的保护作用及机制。方法:C57BL/6小鼠随机均分为对照组、AIH组、AIH+低剂量Res组和AIH+高剂量Res组,AIH+低剂量Res组和AIH+高剂量Res组小鼠每天分别以5 mg/(kg·d)及20 mg/(kg·d)Res灌胃,持续5 d;对照组小鼠以相同体积生理盐水灌胃,AIH组小鼠不灌胃;灌胃3 d后,AIH组、AIH+Res低剂量组及AIH+Res高剂量组经尾静脉单次注射Con A 20 mg/kg建立AIH模型;建模48 h时处死各组小鼠,收集肝脏组织,Real-time PCR检测Foxp3、RORγt、Bax、Bcl-2、Caspase-3及Caspase-9 mRNA相对表达水平,Western blot检测Foxp3、RORγt、Bax、Bcl-2、Cleaved Caspase-3及Cleaved Caspase-9蛋白相对表达水平,肝组织HE染色观察肝脏组织学变化、TUNEL染色观察肝细胞凋亡情况;同时收集外周血,检测血清中AST、ALT/GPT、IL-6、IL-10与IL-17A水平。结果:与对照组小鼠相比,AIH组小鼠血清AST、ALT/GPT、IL-6、IL-17A水平,肝组织中肝细胞凋亡水平、Bax、Caspase-3、Caspase-9、RORγt mRNA表达水平、Bax、Cleaved Caspase-3、Cleaved Caspase-9、RORγt蛋白显著上升,血清IL-10,肝脏组织Bcl-2、Foxp3 mRNA和蛋白表达水平显著下降(P<0.05),HE染色结果显示肝脏组织损伤程度与淋巴细胞浸润程度显著增加;与AIH组小鼠相比,AIH+高剂量Res组小鼠血清AST、ALT/GPT、IL-6、IL-17A,肝脏组织中肝细胞凋亡水平、Bax、Caspase-3、Caspase-9、RORγt mRNA表达水平、Bax、Cleaved Caspase-3、Cleaved Caspase-9、RORγt蛋白显著下降,血清IL-10、肝脏组织Bcl-2、Foxp3 mRNA和蛋白表达水平显著升高(P<0.05),HE染色结果显示肝脏组织损伤程度与淋巴细胞浸润程度显著减轻;AIH+低剂量Res组小鼠上述指标介于AIH组及AIH+Res高剂量组之间。结论:Res可能通过减少血清炎症因子的表达,抑制Con A诱导的AIH小鼠肝脏细胞的凋亡,而这种保护作用随Res剂量的增加而增强。
Objective: To explore the protective effects and mechanisms of resveratrol on concanavalin-A induced mouse autoimmune hepatitis. Methods: C57 BL/6 mice were randomly divided into 4 groups: the control group,AIH group,AIH + Res low dose group and AIH + Res high dose group.AIH + Res low dose group and AIH + Res high dose group were given intragastric administration of resveratrol at a dose of 5 mg/kg/d and 20 mg/kg/d daily respectively for 5 days. The control group were given intragastric administration with the same volume of saline daily for 5 days. The AIH group were not given intragastric administration. AIH group,AIH + Res low dose group and AIH + Res high dose group received single injection with concanavalin-A at a dose of 20 mg/kg into the tail vein to establish the AIH model three days after intragastric administration. Each group was killed 48 hours after the establishment of AIH models to collect liver tissues and peripheral blood. Partial liver tissues were fixed in 4% paraformaldehyde for HE staining and TUNLE detection and partial livers were frozen in liquid nitrogen at-80 ℃ for Real-time PCR detection and Western blot detection. Peripheral blood remained at room temperature for 2 h,centrifuged at 3 000 rpm/min for 15 min. Then serum was collected at-20 ℃ for AST,ALT/GPT and Elisa detection. Results: Compared with the control group,the serum levels of AST,ALT/GPT,IL-6 and IL-17 A,apoptosis of liver tissue,mRNA expression levels of Bax,Caspase-3,Caspase-9 and RORγt in liver tissues,protein expression levels of Bax,Cleaved Caspase-3,Cleaved Caspase-9 and RORγt in liver tissues in AIH group increased significantly. The serum level of IL-10,mRNA and protein expression levels of Bcl-2 and Foxp3 in liver tissues of AIH group significantly decreased,and their hematoxylin-eosin staining showed that the liver tissues were damaged and lymphocyte infiltration were significantly enhanced. Compared with AIH group,the apoptosis of liver tissues,mRNA expression levels of Bax and RORγt in liver tissues,protein expression level of Cleaved Caspase-3 of AIH + Res low dose group significantly decreased,and their protein expression level of Bcl-2 increased significantly,while the HE staining showed that the degree of hepatic tissue damage and lymphocyte infiltration in liver tissue were alleviated to a certain degree. Compared with AIH group,the serum levels of AST,ALT/GPT,IL-6 and IL-17 A,apoptosis of liver tissue,mRNA expression levels of Bax,Caspase-3,Caspase-9 and RORγt in liver tissues,protein expression levels of Bax,Cleaved Caspase-3,Cleaved Caspase-9 and RORγt in liver tissues of AIH +Res high dose group significantly decreased,and their serum level of IL-10,mRNA and protein expression levels of Bcl-2 and Foxp3 in liver tissues increased significantly,while hematoxylin-eosin staining showed that liver tissue damage and lymphocyte infiltration significantly reduced. Compared with the AIH + Res low dose group,the serum levels of AST,ALT/GPT,IL-6 and IL-17 A,apoptosis of liver tissues,mRNA expression level of Caspase-9,protein expression level of Bax,Cleaved Caspase-3,Cleaved Caspase-9 and RORγt in liver tissues of AIH + Res high dose group significantly decreased,and their protein expression level of Foxp3,mRNA and protein expression level of Bcl-2 increased significantly,while HE staining showed that the extent of liver tissue injury and lymphocyte infiltration were significantly reduced. Conclusion: Resveratrol promotes the expression of blood inflammatory factors,inhibits the apoptosis of liver tissues,inhibits the differentiation of Treg cells,promotes the differentiation of Th17 cells,protects the liver tissues of con A-induced AIH mice. The protective effect of resveratrol on con A-induced AIH mice increases with increasing dose.

关键词(KeyWords)： 白藜芦醇;刀豆素A;自身免疫性肝炎;炎性因子;凋亡
resveratrol;concanavalin-A;autoimmune hepatitis;inflammatory factors;apoptosis

Abstract:

Keywords:

基金项目(Foundation): 吴阶平医学基金会肝病医学部肝硬化门脉高压并发症科研项目(02016004)

作者(Author): 孙菁,王世卉
SUN Jing,WANG Shihui

DOI: 10.19367/j.cnki.1000-2707.2018.07.006

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