核因子E2相关因子2基因启动子多态性对酒精性肝病小鼠感染创伤弧菌的影响

The effects of NF-E2-related factor-2 prompter polymorphism on alcoholic liver disease with Vibrio vulniflcus sepsis

目的探讨核因子E2相关因子2（nrf2）基因启动子336位点多态性对酒精性肝病小鼠感染创伤弧菌的影响。方法取C5786雄性小鼠，简单随机抽样法分为正常喂养组（A组，10只）、酒精性肝病组（B组，10只）、正常喂养且腹腔注射感染创伤弧菌组（C组，8只）、酒精性肝病感染创伤弧菌组（D组，110只），以基因测序法检测D组小鼠nrf2基因启动子336位点多态性，分为非突变组（336T）（D1组，7只）及突变组（336C）（D2组，10只）。应用RT-PCR、Western-blotting、ELISA检测各组小鼠肝组织，啦、肿瘤坏死因子-α（TNF-α）、白介素-10（IL-10）及高迁移率族蛋白B1（HMGB1）的基因及蛋白水平，观察小鼠肝组织病理变化。结果A、B、C、D1、D2组小鼠在感染创伤弧菌48h后肝组织n啦基因的mRNA表达量中位数分别为0．115、0．173、0．211、0．764、0．352（χ2 =40．64，P〈0．05）；IL-10基因的mRNA表达量中位数分别为0．338、0．637、1．002、1．825、1．403（χ2 =41．05，P〈0．05）；TNF-α基因的mRNA表达量中位数分别为0．140、0．254、0．372、0．399、0．699（χ2=38．16，P〈0．05）；HMGBl基因的mRNA表达量中位数分别为0．230、0．410、0．668、0．508、1．021（χ2=31．45，P〈0．05）。A、B、C、D1、D2组小鼠在感染创伤弧菌48h后肝组织nrf2基因的蛋白表达量中位数分别为0．908、1．461、2．061、3．982、2．243（X。=33．72，P〈0．05）；IL-IO基因的蛋白表达量中位数分别为13．97、22．54、30．14、57．98、41．53（f=37．31，P〈n05）；TNF-α基因的蛋白表达量中位数分别为114．07、142．94、175．44、174．60、266．11（χ2 =32．29，P〈0．05）；HMGBl基因的蛋白表达量中位数分别为2．01、6．05、9．62、6．24、12．89（χ2 =36．94．P〈0．05）。与A组比较，B组可见大量散在脂肪滴，呈空泡样改变，c组可见炎症细胞浸润，肝细胞条索紊乱，D组可见肝细胞充血水肿，肝小叶完整性破坏，汇管区可见扩张的肝管和静脉。结论nrf2基因启动子336位点T→C突变使酒精性肝病C5786小鼠体内nrf2表达明显降低，并加剧酒精性肝病C5786小鼠在感染创伤弧菌时的炎症反应及脏器损害。

Objective To investigate the influence of genetic polymorphism in NF-E2-related factor-2（nrf2） gene promoter locus at 336 in alcoholic liver disease （ ALD ） with Vibrio vulnificus （ VV ） sepsis. Methods Through the simple random sampling method,C57B6 male mice were divided into normal feeding group（group A, 10 mice）, alcoholic liver disease group （group B, 10 mice ）, normal feeding group infected with W through intraperitoneal injection （ group C, 8 mice ）, alcoholic liver disease group infected with VV（ group D,110 mice）. Through gene sequencing method,nrf2 gene promoter 336 polymorphism in D group was analyzed and grouped into： non-mutation group（336T） （ group D1,7 mice） and mutation group （336C） （ group D2,10 mice ）. Through RT-PCR, Western-blotting and ELISA method, expressions of nrf2, tumor necrosis factor-α （TNF-α）, i nterleukin-10 （IL-10）, high mobility group protein 1 （HMGB1）gene and protein of liver were measured. The pathological changes in liver were recorded with light microscope. Results After infected with VV for 48 hours for A, B, C, DI, D2 group, the expression medians of nrf2 mRNA in liver were 0. 115,0. 173,0. 211,0. 764,0. 352, respectively（ χ2 = 40. 64, P 〈 O. 05 ） , the expression medians of IL-10 mRNA in liver were O. 338, O. 637,1. 002,1. 825,1. 403, respectively （ χ2 = 41.05, P 〈 0. 05 ）, the expression medians of TNF- α mRNA in liver were 0. 140, O. 254, O. 372, O. 399, O. 699, respectively（x= =38.16,P 〈0. 05） ,the expression medians of HMGB1 mRNA in liver were 0. 230,0. 410, 0. 668,0. 508,1. 021, respectively （χ2 = 31.45,P 〈 O. 05）. After infected with VV 48 hours for mice in A, B, C,D1 ,D2 group,the expression medians of nrf2 protein in liver were 0. 908,1. 461,2. 061,3. 982,2. 243, respectively（ χ2 = 33.72 ,P 〈 O. 05 ） , the expression medians of IL-10 protein in liver were 13.97,22. 54, 30. 14,57.98,41.53, respectively（ χ2 = 37.31, P 〈 0. 05 ） ,the expression medians of TNF-ot protein in liver were 114. 07,142. 94,175.44,174. 60,266. 11, respectively（ χ2 = 32. 29, P 〈 0. 05 ） , the expi＇ession medians of HMGB1 protein in liver were 2. 01,6. 05,9. 62,6. 24, 12. 89, respectively （ χ2 = 36. 94, P 〈 0. 05 ）. Compared with group A, there were large amount of fat drops, fatty changes in group B, inflammatory cell infiltration, disorder of hepatic cell in group C, and extension of hepatic duct and vein, edema of liver cells and disorder of hepatic cells in group D. Conclusion The nrf2 gene promoter of T336C mutation in C57B6 mouse of ALD can significantly decrease the expression of nrf2, and intensify organ inflammation and damage when they were infected by VV.