ADAM8的表达对急性酒精性肝损伤小鼠的影响  

The effect of ADAM8 expression on acute alcoholic liver injury in mice

作　　者：杨孟利 李三强[1] 张凯杰 崔钦奕 冯家阳 李依林 李昊远 齐婧含 YANG Mengli;LI Sanqiang;ZHANG Kaijie;CUI Qinyi;FENG Jiayang;LI Yilin;LI Haoyuan;QI Jinghan(College of Basic Medicine and Forensic Medicine,Henan University of Science and Technology,Luoyang 471000,China)

机构地区：[1]河南科技大学基础医学与法医学院,471000

出　　处：《天津医药》2024年第4期346-350,共5页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(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 探究CRISPR/Cas9技术靶向抑制去整合素-金属蛋白酶8(ADAM8)在小鼠急性酒精性肝损伤中的作用及分子机制。方法 18只6~8周龄健康雌性昆明小鼠根据随机数字表法分为正常组、酒精组和质粒组,每组6只。正常组不做处理,质粒组采用水流动力学注射法尾静脉注射利用CRISPR/Cas9技术抑制ADAM8基因的三合一重组质粒ADAM8-sgRNA3(3 g/kg),酒精组尾静脉注射等量生理盐水。酒精组和质粒组采用50%(V/V)分析纯乙醇一次性灌胃(14 mL/kg)诱导急性肝损伤。禁食16 h后进行眼球取血,检测丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)活性;苏木素-伊红(HE)和过碘酸-雪夫(PAS)染色检测肝脏损伤和糖原变化情况;蛋白免疫印迹法检测肝组织中ADAM8、细胞色素CYP450 2E1(CYP2E1)、热休克蛋白70(HSP70)及丝裂原活化蛋白激酶(MAPK)通路相关因子的表达。结果 与正常组相比,酒精组ALT和AST升高,肝损伤评分增加,糖原含量减少,ADAM8、CYP2E1、磷酸化的细胞外调节蛋白激酶1/2(p-ERK1/2)、磷酸化的p38(p-p38)MAPK以及磷酸化的c-Jun氨基未端激酶(p-c-Jun)的表达升高,而HSP70的表达降低(P<0.05)。与酒精组相比,质粒组ALT和AST降低,肝损伤评分降低(P<0.05),糖原含量增多;ADAM8、CYP2E1、p-ERK1/2、p-p38 MAPK以及p-c-Jun的表达降低,HSP70的表达增加(P<0.05)。结论 利用CRISPR/Cas9技术靶向抑制ADAM8的表达,可以通过MAPK信号通路改善小鼠急性酒精性肝损伤。Objective To investigate the role and molecular mechanism of CRISPR/Cas9 technology targeting the inhibition of disintegrin and metalloprotease 8(ADAM8)in acute alcoholic liver injury in mice.Methods Eighteen healthy Kunming female mice aged 6-8 weeks were randomly divided into the normal group,the alcohol group and the plasmid group,with 6 mice in each group.The normal group was given no treatment,and the plasmid group was injected with three-in-one recombinant plasmid ADAM8-sgRNA3(3 g/kg)by CRISPR/Cas9 technology to inhibit the ADAM8 gene using hydrodynamic injection tail vein method.The alcohol group was injected with an equal amount of physiological saline through tail vein.After 3 days of regular feeding,the alcohol group and the plasmid group were subjected with 50%(V/V)alcohol analytical grade ethanol by a one-time gavage(14 mL/kg)to induce acute liver injury.After fasting for 16 hours,eyeball blood sample was taken to detect activities of alanine aminotransferase(ALT)and aspartate aminotransferase(AST).Mice were euthanized and liver tissue was separated and extracted.Hematoxylin-Eosin staining(HE staining)and Periodic Acid-Schiff staining(PAS staining)were used to detect liver injury and glycogen changes in each group of mice.The expression levels of ADAM8,cytochrome CYP4502E1(CYP2E1),heat shock protein 70(HSP70)and mitogen-activated protein kinase(MAPK)pathway related factors were detected by Western blot assay.Results Compared with the normal group,ALT and AST were increased,liver injury score was increased,glycogen content was decreased,ADAM8,CYP2E1,phosphorylated extracellular regulated kinase 1/2(p-ERK1/2),phosphorylated p38(p-p38)MAPK and phosphorylated c-Jun aminoterminal kinase(p-c-Jun)were increased in the alcohol group.The expression of HSP70 was decreased(P<0.05).Compared with alcohol group,ALT and AST were decreased,liver injury score was decreased in the plasmid group(P<0.05).Glycogen content was increased.ADAM8,CYP2E1,p-ERK1/2,p-P38 MAPK and p-c-Jun expression levels were decreased,while HSP

关 键 词：ADAM蛋白质类 急性酒精性肝损伤 丝裂原激活蛋白激酶类 CRISPR/Cas9 

分 类 号：R364.5[医药卫生—病理学]