利拉鲁肽通过SIRT1/AMPK通路改善小鼠肝脏脂质变性的机制研究

Mechanism of liraglutide ameliorates fatty liver disease by regulation SIRT1/AMPK pathway in mice

目的探讨利拉鲁肽通过沉默信息调节因子1(silent information regulator 1,SIRT1)/腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)通路改善小鼠肝脏脂质变性的机制。方法将24只健康雄性6周龄C57BL/6J小鼠随机分为对照组、模型组、模型+利拉鲁肽组、模型+SIRT1组、模型+SIRT1+利拉鲁肽组、模型+SIRT1-NC组,每组4只。对照组普通饲料饲养,予以等容积生理盐水皮下注射,模型组高脂饲养12周建立代谢相关脂肪性肝病(metabolic dysfunction-associated fatty liver disease,MAFLD)模型,之后3周尾静脉注射SIRT1干扰慢病毒及利拉鲁肽。检测各组小鼠血清甘油三酯和丙氨酸氨基转移酶(alanine aminotransferase,ALT)水平,采用HE染色和油红O染色观察肝组织病理,采用实时荧光定量RT-PCR检测AMPK、SIRT1、肝激酶B1(liver kinase B1,LKB1)和去乙酰化固醇调节元件结合蛋白(sterol regulatory element binding protein-1c,SREBP-1c)基因表达水平,采用Western blot检测蛋白表达水平。结果利拉鲁肽可降低高脂喂养的C57BL/6J小鼠体质量、肝湿重、血清甘油三酯及ALT水平,油红O染色可见肝细胞脂滴减少。与模型组相比,模型+利拉鲁肽组SIRT1(0.212±0.110比0.076±0.010)、AMPK(0.518±0.051比0.248±0.023)、LKB1(1.023±0.039比0.576±0.029)基因表达量和AMPK(0.212±0.026比0.100±0.006)、LKB1(0.413±0.016比0.221±0.015)蛋白表达水平均上调,而SREBP-1c基因(0.727±0.249比9.007±1.530)和蛋白(0.187±0.008比0.824±0.114)表达水平均下调(P均<0.05)。与模型组相比,模型+SIRT1组SIRT1(0.029±0.003比0.076±0.010)、AMPK(0.105±0.013比0.248±0.023)、LKB1(0.333±0.106比0.576±0.029)基因表达量均下调(P均<0.05)。与模型+SIRT1组相比,模型+SIRT1+利拉鲁肽组LKB1(0.945±0.110比0.333±0.106;0.380±0.004比0.145±0.014)、AMPK(0.319±0.051比0.105±0.013;0.181±0.039比0.051±0.012)基因表达量和蛋白表达量均上调,而SREBP-1c基因表达量(4.239±0.554比12.740±0.976)下调(P均<0.05)。结论利拉鲁肽改善C57BL/6J小鼠肝脏脂毒性可能通过直接上调SIRT1/AMPK通路信号分子基因和蛋白表达水平,或间接激活LKB1并增强AMPK基因和蛋白表达、拮抗SREBP-1c基因和蛋白表达,从而降低脂质合成相关分子水平,而干扰SIRT1表达削弱了利拉鲁肽上调SIRT1/AMPK通路改善肝脏脂肪变性的作用。

Objective To investigate the mechanism of liraglutide ameliorates hepatic steatosis by regulation silent information regulator 1(SIRT1)/adenosine monophosphate-activated protein kinase(AMPK)pathway in mice.Methods Twenty-four healthy 6-week-old C57BL/6J male mice were randomly divided into control group,model group,model+liraglutide group,model+SIRT1 group,model+SIRT1+liraglutide group and model+SIRT1-NC group,4 mice in each group.Mice in control group were fed with regular feed and received subcutaneous injection of equal volume physiological saline.Mice in model group were fed with high-fat diet for 12 weeks to establish a metabolic dysfunction-associated fatty liver disease(MAFLD)model,followed by tail vein injection of SIRT1 interference lentivirus and liraglutide for 3 weeks.The levels of serum triglycerides and alanine aminotransferase(ALT)of mice in each group were detected.HE staining and oil red O staining were used to observe liver tissue pathology.Realtime fluorescence quantitative RT-PCR was used to detect the gene expression levels of AMPK,SIRT1,liver kinase B1(LKB1)and sterol regulatory element binding protein-1c(SREBP-1c).Western blot was used to detect the protein expression levels.Results Liraglutide could reduce body mass,liver wet weight,serum triglycerides and ALT level of C57BL/6J mice with high-fat diet,and oil red O staining showed a decrease in hepatic lipid droplets.Compared with model group,SIRT1(0.212±0.110 vs.0.076±0.010),AMPK(0.518±0.051 vs.0.248±0.023),LKB1(1.023±0.039 vs.0.576±0.029)gene expression levels and AMPK(0.212±0.026 vs.0.100±0.006),LKB1(0.413±0.016 vs.0.221±0.015)protein expression levels were upregulated of mice in the model+liraglutide group,while SREBP-1c gene(0.727±0.249 vs.9.007±1.530)and protein(0.187±0.008 vs.0.824±0.114)expression levels were downregulated(all P<0.05).Compared with model group,gene expression levels of SIRT1(0.029±0.003 vs 0.076±0.010),AMPK(0.105±0.013 vs 0.248±0.023)and LKB1(0.333±0.106 vs 0.576±0.029)were all downregulated in the model+SIRT1 group(all P<0.05).Compared with model+SIRT1 group,LKB1(0.945±0.110 vs.0.333±0.106;0.380±0.004 vs.0.145±0.014)and AMPK(0.319±0.051 vs.0.105±0.013;0.181±0.039 vs.0.051±0.012)gene expression and protein expression in model+SIRT1+liraglutide group were upregulated,while SREBP-1c gene expression was downregulated(4.239±0.554 vs.12.740±0.976)(all P<0.05).Conclusions Liraglutide may improve liver lipid toxicity in C57BL/6J mice by directly upregulating the gene and protein expression levels of SIRT1/AMPK signaling molecules,or indirectly activating LKB1 and enhancing AMPK gene and protein expression,antagonizing SREBP-1c gene and protein expression,thereby reducing lipid synthesis related molecular levels.Interference with SIRT1 expression weakens the effect of liraglutide upregulating the SIRT1/AMPK pathway on improving liver steatosis.