p-ERK下调分拣蛋白表达对非酒精性脂肪肝大鼠肝损伤的机制研究

Effects of p-ERK down-regulating sortilin expression on liver injury in non-alcoholic fatty liver rats

目的:基于磷酸化细胞外信号调节激酶(p-ERK)下调分拣蛋白(sortilin)表达,分别从动物和细胞水平探讨非酒精性脂肪性肝病(NAFLD)大鼠肝损伤的形成机制。方法:40只8周龄雄性Wistar大鼠,适应性喂养1周后,随机分为:对照(control)组(n=10)、高脂饮食(HFD)组(n=10)、PD98059组(n=10)和HFD+PD98059组(n=10);control组和PD98059组给予普通饲料喂养,HFD组和HFD+PD98059组给予高脂饲料喂养,PD98059组和HFD+PD98059组给予PD98059腹腔注射。喂养6周后处死大鼠,采集血清,检测血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、天冬氨酸转氨酶(AST)和丙氨酸转氨酶(ALT)的水平;分离肝脏,计算肝脏指数;苏木精-伊红(HE)染色检测肝脏组织的病理形态学改变;免疫组化检测蛋白的表达;六胺银染色观察肝窦基底膜的病理特征;ELISA法检测血清炎症因子白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)的表达水平。同时,提取control组大鼠原代肝窦内皮细胞(LSECs),anti-CD31鉴定LSECs,油红O染色观察LSECs脂质沉积,流式细胞术检测LSECs的凋亡,RT-qPCR和Western blot技术分别从mRNA和蛋白水平检测相关蛋白的表达。结果:sortilin表达在LSECs上,ox-LDL增加了LSECs脂滴的沉积和细胞的凋亡(P<0.05);ox-LDL作用下sorti⁃lin的表达下调,抑制p-ERK(PD98059)后,sortilin表达显著升高。HFD诱导的NAFLD大鼠体重、血脂、肝功能和肝脏指数显著升高(P<0.05),炎症因子IL-6和TNF-α的释放显著增多,PD98059干预可逆转上述指标的变化(P<0.05)。结论:PD98059干预后,sortilin表达上调且p-ERK、TNF-α和IL-6的表达下调,同时NAFLD大鼠肝脏损伤、脂质蓄积、脂肪变性、炎症反应和细胞凋亡显著减轻。

AIM:To investigate the effects of phosphorylated extracellular signal-regulated kinase(p-ERK)down-regulating sortilin expression on the liver injury in non-alcoholic fatty liver disease(NAFLD)rats at the animal and cellular levels.METHODS:Eight-week-old male Wistar rats(n=40)were randomly divided into 4 groups:control group(n=10),high-fat diet(HFD)group(n=10),PD98059 group(n=10)and HFD+PD98059 group(n=10).The rats in con⁃trol group and PD98059 group were given ordinary feed,the rats in HFD group and HFD+PD98059 group were given highfat feed,and the rats in PD98059 group and HFD+PD98059 group were given PD98059 by intraperitoneal injection.After feeding for 6 weeks,the rats were weighed and killed.The serum was collected,and the levels of total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),aspar⁃tate aminotransferase(AST)and alanine aminotransferase(ALT)were measured.The liver was separated for calculating the liver index.Hematoxylin-eosin(HE)staining was used to observe the morphological changes of liver tissues,immuno⁃histochemical method was used to detect the protein expression,and hexamine silver staining was performed to evaluate the pathological characteristics of sinus basement membrane.The serum levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were measured by ELISA.At the same time,the primary liver sinusoidal endothelial cells(LSECs)were extracted from the rats of control group.Anti-CD31 was used to identify the LSECs.Oil red O staining was used to ob⁃serve the deposition of lipid accumulation.The apoptosis of LSECs was analyzed by flow cytometry.The mRNA and pro⁃tein expression levels were determined by RT-qPCR and Western blot,respectively.RESULTS:Sortilin was expressed on the LSECs.Treatment of the LSECs with ox-LDL increased the deposition of lipid droplets and apoptosis(P<0.05).The expression of sortilin was down-regulated by treatment with ox-LDL.After inhibiting p-ERK by PD98059,the expres⁃sion of sortilin was significantly increased(P<0.05).The HFD-induced NAFLD rats had significantly increased body weight,blood lipids,liver function,liver index,and the release of inflammatory factors IL-6 and TNF-α(P<0.05).After PD98059 intervention,all above indexes were reversed(P<0.05).CONCLUSION:After PD98059 intervention,the expression of sortilin is up-regulated and the protein levels of p-ERK,TNF-αand IL-6 are down-regulated.At the same time,liver injury,lipid accumulation,steatosis,inflammation and apoptosis are significantly attenuated.