WNT1诱导信号通路蛋白1在非酒精性脂肪性肝炎的作用及其机制  

Role of WNT1-induced signaling pathway protein 1 in non-alcoholic steatohepatitisand underlying mechanism

作　　者：何鑫[1] 张子龙 金鑫 黄乐 温松奇 游建[1] He Xin;Zhang Zilong;Jin Xin;Huang Le;Weng Songqi;You Jian(Department of Hepatobillary-Pancreatic and Hernia Surgery,Wuhan Fourth Hospital,Wuhan 430033,China)

机构地区：[1]武汉市第四医院肝胆胰疝外科,武汉430033

出　　处：《中华实验外科杂志》2024年第5期963-968,共6页Chinese Journal of Experimental Surgery

基　　金：吴阶平医学基金会临床科研专项资助基金(320.6750.2023-11-8)。

摘　　要：目的:探究WNT1诱导信号通路蛋白1(WISP1)在非酒精性脂肪性肝炎(NASH)的功能定位及治疗作用机制。方法:60只雄性C57BL/6J小鼠购于北京维通利华公司,采取简单随机抽样法分为5组,每组10只,正常组喂养普通饲料,通过喂养高脂高胆固醇饲料14周制备NASH小鼠模型,在造模过程中分别腹腔注射IgG单克隆抗体,0.1 g/kg WISP1高亲和力单克隆抗体,0.2 g/kg WISP-1高亲和力单克隆抗体为IgG、低剂量WISP1抗体组和高剂量WISP1抗体组,每周2次,连续给药14周。检测血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、血糖水平。苏木精-伊红(HE)、糖原(PAS)染色、油红O染色观察肝组织病理变化。实时荧光定量聚合酶链反应(Real-time PCR)检测小鼠肝组织中白细胞介素(IL)-10、IL-6、IL-1β、肿瘤坏死因子-α(TNF-α)mRNA表达水平。检测肝组织总胆固醇(TC)、甘油三脂(TG)水平,酶联免疫吸附测定法(ELISA)检测还原型谷胱甘肽(GSH)、丙二醛(MDA)、超氧化物歧化酶(SOD)活性水平,活性氧(ROS)荧光染色检测肝组织中活性氧含量;免疫组织化学(IHC)检测肝组织WISP-1、乙酰辅酶a合成酶(ACS)蛋白表达,蛋白质印迹法(Western blot)检测肝组织肝组织腺苷酸活化蛋白激酶(AMPK)、磷酸化AMPK(p-AMPK)、肉碱棕榈酰转移酶-1(CPT1)、过氧化物酶体增殖物激活受体γ辅激活因子-1(PCG-1α)蛋白表达水平。结果:模型组小鼠血清AST、ALT、血糖水平高于正常组[(245.01±57.57)U/L比(37.28±4.23)U/L、(218.96±55.27)U/L比(49.81±10.57)U/L、(25.73±2.29)mmol/L比(8.06±0.51)mmol/L,P<0.01];模型组病理表现为肝细胞脂肪变性、炎性细胞浸润、多处坏死灶;模型组小鼠肝组织炎症因子(IL-10、IL-6、IL-1β、TNF-α)mRNA水平高于正常组(7.49±059比1.00±0.38、5.57±3.22比1.00±0.59、4.86±3.86比1.00±0.34、10.18±3.24比1.00±0.17,P<0.01);MDA含量高于正常组[(3.75±0.40)nmol/ml比(2.22±0.24)nmol/ml,P<0.01],GSH、SOD活�Objective To explore the functional localization and therapeutic mechanism of WNT1-induced signaling pathway protein 1(WISP1)in non-alcoholic steatohepatitis(NASH).Methods A total of 60 male C57BL/6J mice were divided into 5 groups by simple random sampling(10 mice in each group).The normal group was fed on ordinary diet,and the NASH mouse model was prepared by feeding high-fat and high-cholesterol diet for 14 weeks.During the modeling process,the mice with intraperitoneal injection of IgG monoclonal antibody,0.1 g/kg WISP-1 high-affinity monoclonal antibody,0.2 g/kg WISP-1 high-affinity monoclonal antibodies served as IgG group,low-dose WISP-1 antibody group and high-dose WISP1 antibody group,respectively.All mice in three groups were administered twice a week for 14 weeks.Serum alanine aminotransferase(ALT),aspartate aminotransferase(AST)and blood glucose levels were detected.The pathological changes of liver tissue were observed by hematoxylin-eosin(HE),glycogen(PAS)and oil red O staining.The levels of total cholesterol(TC)and triglycerides(TG)in liver tissue were detected,and the activity levels of reduced glutathione(GSH),malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by enzyme-linked immunosorbent assay(ELISA).The mRNA expression levels of interleukin(IL)-10,IL-6,IL-1βand tumor necrosis factor-α(TNF-α)in serum of mice were detected by real-time quantitative polymerase chain reaction(Real-time PCR).The expression of WISP1 and Acetyl-CoA synthetase(ACS)in liver tissues was detected by immunohistochemistry(IHC),and the expression levels of adenosine monophosphate-actived protein kinase(AMPK),phosphorylated AMPK(p-AMPK),carnitine palmitoyltransferase(CPT1)and peroxisome proliferator-activated receptor-gamma coactivator(PGC)-1alpha(PCG-1α)in liver tissues were detected by Western blotting.Results Compared with the normal group,the serum AST,ALT and blood glucose levels in the model group were increased[(245.01±57.57)U/L vs.(37.28±4.23)U/L,(218.96±55.27)U/L vs.(49.81±10.57)U/L,(25.73±2.29

关 键 词：WNT1诱导信号通路蛋白1 非酒精性脂肪性肝炎 脂质代谢 腺苷酸活化蛋白激酶通路 脂肪过氧化 

分 类 号：R575.5[医药卫生—消化系统]