SIRT1/AMPK通路在利拉鲁肽早期干预缓解高脂饮食导致的大鼠非酒精性脂肪性肝病中的作用

目的:探讨胰高血糖素样肽1(GLP-1)受体激动剂利拉鲁肽(Lira)早期干预对高脂饮食(HFD)诱导的非酒精性脂肪性肝病(NAFLD)大鼠的影响及沉默信息调节因子1(SIRT1)/AMP活化蛋白激酶(AMPK)通路在其中的作用。方法:SPF级雄性SD大鼠随机分为普通饮食(ND)组、HFD组和HFD+Lira组,每组8只。适应性饲养1周后,按不同分组给药,HFD+Lira组大鼠每日固定时间皮下注射Lira(200μg/kg),其余2组注射等体积生理盐水。干预期间注意观察大鼠体重、毛发、食欲、大小便及活动情况,以便及时调整药量。每周记录体重、进食量和血糖,第16周行葡萄糖耐量实验;第18周末麻醉后行高胰岛素-正葡萄糖钳夹实验,该实验结束后颈动脉取血,处死后取肝脏及不同部位的脂肪组织。血清检测丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)等指标;HE染色法观察肝组织病理损伤变化;油红O染色法观察肝组织脂质蓄积程度;马松染色和天狼星红染色法观察肝脏纤维化程度;活性氧簇(ROS)染色观察肝脏氧化应激情况;免疫荧光染色观察肝脏GLP-1受体表达情况;免疫组织化学染色法观察SIRT1和第172位苏氨酸磷酸化的AMPKp-AMPK(Thr172)]的表达及定位;Western blot法检测肝组织AMPK、p-AMPK(Thr172)、SIRT1、第372位丝氨酸磷酸化的固醇调节元件结合蛋白1cp-SREBP-1c(Ser372)]、第79位磷酸化的乙酰辅酶A羧化酶p-ACC(Ser79)]、肉毒碱棕榈酰转移酶1A(CPT1A)和脂肪酸合成酶(FAS)的蛋白水平。结果:HE和油红O染色结果证实HFD组肝组织结构紊乱,脂质蓄积严重,马松和天狼星红染色显示纤维化程度严重,提示NAFLD大鼠模型建立成功。与ND组相比,HFD组血清总胆固醇(TC)、甘油三酯(TG)、AST和ALT,以及肝组织丙二醛(MDA)、TC、TG和ROS水平均显著升高(P<0.01),超氧化物歧化酶(SOD)活性显著降低(P<0.01),肝组织p-AMPK(Thr172)、SIRT1、p-SREBP-1c(Ser372)、p-ACC(Ser79)和CPT1A蛋白水平显著降低(P<0.05或P<0.01),FAS表达显著增加(P<0.01);与HFD组比较,HFD+Lira组大鼠肝组织脂质蓄积和纤维化程度明显减轻,血清TG、TC、AST和ALT,以及肝组织MDA、TC、TG和ROS水平均显著降低(P<0.05或P<0.01),SOD活性增强(P<0.05),肝组织p-AMPK(Thr172)、SIRT1、p-SREBP-1c(Ser372)、p-ACC(Ser79)和CPT1A蛋白水平显著升高(P<0.05或P<0.01),FAS表达显著减少(P<0.01)。结论:Lira能够减轻HFD诱导的NAFLD大鼠胰岛素抵抗、肝纤维化和氧化应激程度,并改善肝脏脂质代谢,其作用可能与SIRT1/AMPK通路有关。

Role of SIRT1/AMPK pathway in early intervention of Lira alleviating non-alcoholic fatty liver disease caused by high-fat diet in rats

AIM:To investigate the effect of early intervention of glucagon-like peptide-1(GLP-1)receptor agonist liraglutide(Lira)on oxidative stress,glucose tolerance,hepatic steatosis and insulin resistance of the rats with high-fat diet(HFD)-induced non-alcoholic fatty liver disease(NAFLD),and to explore the role of silent information regulator 1(SIRT1)/AMP-activated protein kinase(AMPK)signaling pathway in this process.METHODS:Twenty-four male SD rats were randomly divided into normal diet(ND)group,HFD group and HFD+Lira group,with 8 rats in each group.After 1 week of adaptive feeding,the rats in HFD+Lira group were subcutaneously injected with Lira(200μg/kg)per day at a fixed time point,while the rats in the remaining 2 groups were injected with normal saline at the same volume.During the intervention,the body weight,hair,appetite,defecation and activity of the rats were observed to adjust the dosage timely.The body weight,food intake and blood glucose were recorded weekly.Glucose tolerance test was performed at the end of the 16th week.At the end of the 18th week,hyperinsulinemic euglycemic clamp test was conducted after anesthesia.Blood was taken from the carotid artery.The liver and adipose tissues from different parts were taken after death.The serum alanine aminotransferase(ALT),aspartate aminotransferase(AST)and other indicators were detected.HE staining was used to observe the pathological changes of the liver tissue.Lipid accumulation in the liver tissues was observed by oil red O staining.Liver fibrosis was observed by Masson staining and Sirius red staining.Fluorescence staining for reactive oxygen species(ROS)was used to observe the oxidative stress in the liver.The expression of GLP-1 receptor in the liver was observed by immunofluorescence staining.The expression and localization of SIRT1 and phosphorylated AMPK at Thr172p-AMPK(Thr172)]were observed by immunohistochemical staining.The protein levels of AMPK,p-AMPK(Thr172),SIRT1,phosphorylated sterol regulatory element binding protein-1c at Ser372p-SREBP-1c(Ser372)],phosphorylated acetyl coenzyme A carboxylase at Ser79p-ACC(Ser79)],carnitine palmitoyltransferase 1A(CPT1A)and fatty acid synthase(FAS)in liver tissues were determined by Western blot.RESULTS:The results of HE and oil red O staining of rat liver tissues in HFD group confirmed the structural disorder and serious lipid accumulation,while Masson and Sirius red staining showed severe fibrosis,suggesting the successful establishment of NAFLD rat model.Compared with ND group,the levels of total cholesterol(TC),triglyceride(TG),AST and ALT in serum,and the levels of malondialdehyde(MDA),TC,TG and ROS in liver tissues in HFD group were significantly increased(P<0.01),while the activity of superoxide dismutase(SOD)was decreased(P<0.01).The protein levels of p-AMPK(Thr172),SIRT1,p-SREBP-1c(Ser372),p-ACC(Ser79)and CPT1A in the liver tissues were significantly reduced(P<0.05 or P<0.01),while the expression of FAS was increased(P<0.01).Compared with HFD group,lipid accumulation and fibrosis in the liver tissues of the rats in HFD+Lira group were significantly attenuated,the serum levels of TC,TG,AST and ALT,and MDA,TC,TG and ROS in liver tissues were markedly reduced(P<0.05 or P<0.01),while SOD activity was increased(P<0.05).The protein levels of p-AMPK(Thr172),SIRT1,p-SREBP-1c(Ser372),p-ACC(Ser79)and CPT1A in the liver tissues were significantly increased(P<0.05 or P<0.01),while the expression of FAS was decreased(P<0.01).CONCLUSION:Lira attenuates insulin resistance,oxidative stress and fibrosis,and improves liver lipid metabolism in the rats with NAFLD induced by HFD,which may be mediated by SIRT1/AMPK signaling pathway.