抑制解整合素金属蛋白酶8表达对酒精性肝纤维化小鼠炎症损伤的机制  

Mechanism of inhibiting a disintegrin and metalloprotease 8 expression on inflammatory damage in alcoholic liver fibrosis mice

作　　者：杨孟利 李三强[1,2] 张凯杰 冯家阳 李昊远 许畅 YANG Meng-li;LI San-qiang;ZHANG Kai-jie;FENG Jia-yang;LI Hao-yuan;XU Chang(Molecular Medicine Key Laboratory of Liver Injury and Repair,College of Basic Medicine and Forensic Medicine,He’nan University of Science and Technology,He’nan Luoyang 471000,China;He’nan Center for Engineering and Technology Research on Prevention and Treatment of Liver Diseases,He’nan Luoyang 471000,China)

机构地区：[1]河南科技大学基础医学与法医学院肝脏损伤与修复分子医学重点实验室,河南洛阳471000 [2]河南省肝病防治工程技术研究中心,河南洛阳471000

出　　处：《解剖学报》2024年第6期746-752,共7页Acta Anatomica Sinica

基　　金：国家自然科学基金(82170606);河南省高等学校重点科研项目计划基础研究专项(23ZX006)。

摘　　要：目的探讨抑制解整合素金属蛋白酶8(ADAM8)的表达对酒精性肝纤维化小鼠炎症损伤的机制。方法C57BL/6N雄性小鼠随机分为对照组、酒精组和质粒组,每组各10只。酒精组和质粒组日常喂养酒精液体饲料,并灌胃31.5%乙醇(5 g/kg,2次/周),对照组喂养对照液体饲料,并灌胃等量生理盐水;质粒组小鼠尾静脉注入抑制ADAM8基因的有效质粒ADAM8-小向导RNA3(sgRNA3)(2 g/kg,2次/周),酒精组尾静脉注射等量生理盐水,持续诱导8周进行小鼠酒精性肝纤维化模型制作。眼球取血后处死小鼠并分离肝脏,天狼星红和HE染色检测肝纤维化和损伤情况;免疫组织化学法检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(collagenⅠ)与ADAM8的表达;Real-time PCR法检测ADAM8 mRNA的表达;Western blotting检测ADAM8、转化生长因子β1(TGF-β1)、p-p38 MAPK及热休克蛋白27(HSP27)的表达;生化法检测谷丙转氨酶(ALT)和谷草转氨酶(AST)的活性;ELISA法检测肿瘤坏死因子α(TNF-α)与白细胞介素1(IL-1)浓度。结果酒精组胶原纤维容积分数和肝损伤积分增加,α-SMA和collagen-Ⅰ的阳性面积率升高;ADAM8 mRNA和ADAM8蛋白的表达增加,其阳性面积率升高;AST、ALT、TNF-α和IL-1水平升高;TGF-β1、p-p38MAPK及HSP27的蛋白表达增加。质粒组胶原纤维容积分数和肝损伤积分减少,α-SMA和collagenⅠ的阳性面积率降低;ADAM8 mRNA和ADAM8蛋白的表达减少,其阳性面积率降低;AST、ALT、TNF-α和IL-1水平降低;TGF-β1、p-p38MAPK及HSP27的蛋白表达减少。结论下调ADAM8的表达可以通过抑制TGF-β1/p38MAPK信号通路减轻炎症损伤,从而改善小鼠酒精性肝纤维化。Objective To explore the mechanism of inhibiting a disintegrin and metalloprotease 8(ADAM8)expression on inflammatory damage in alcoholic liver fibrosis mice.Methods C57BL/6N male mice were randomly divided into the control group,the alcohol group,and the plasmid group,with 10 mice in each group.The alcohol group and plasmid group were fed alcohol liquid feed on a daily basis and gavaged with 315%ethanol(5 g/kg,twice a week);The control group was fed control liquid feed and gavaged with an equal amount of saline.The plasmid group was injected with the effective plasmid ADAM8⁃small guide RNA3(sgRNA3)(2 g/kg,twice a week)to inhibit the ADAM8 gene through the tail vein,while the alcohol group was injected with an equal amount of saline through the tail vein for 8 weeks to induce alcoholic liver fibrosis.After eyeball blood collection,the mice were euthanized,and their liver was separated and extracted.Sirius red and HE staining were employed to assess liver fibrosis and damage;Western blotting was used to determine the expression ofα⁃smooth muscle actin(α⁃SMA),collagenⅠ,and ADAM8;Real⁃time PCR was used to measure the expression of ADAM8 mRNA;the expression of ADAM8,transforming growth factor⁃β1(TGF⁃β1),p⁃p38 MAPK and heat shock protein 27(HSP27)proteins was detected by Western blotting,Biochemical detection were used to detect alanine aminotransferase(ALT)and aspartate transaminase(AST)activity;tumor necrosis factor⁃α(TNF⁃α)and interleukin⁃1(IL⁃1)levels were determined by ELISA.Results The alcohol group had increased collagen fiber volume fraction,liver injury scores,and positive area rates ofα⁃SMA and collagenⅠ;the expression of ADAM8 mRNA and ADAM8 protein increased,with increased positive area rate;and levels of AST,ALT,TNF⁃α,and IL⁃1 were higher,along with increased expression of TGF⁃β1,p⁃p38MAPK,and HSP27 proteins.In the plasmid group,the collagen fiber volume fraction,liver injury scores,and positive area rates ofα⁃SMA and collagenⅠwas reduced;the expression of

关 键 词：解整合素金属蛋白酶8 酒精性肝纤维化 炎症 损伤 转化生长因子β1/p38丝裂原活化蛋白激酶信号通路 免疫印迹法 小鼠 

分 类 号：R364.5[医药卫生—病理学]