胰高糖素样肽-1对非酒精性脂肪肝病状态下生长分化因子15表达的影响

Effects of glucagon-like peptide-1 on growth differentiation factor 15 expression in hepatocytes of non-alcoholic fatty liver disease

目的:探讨胰高糖素样肽-1(GLP-1)对非酒精性脂肪肝病(NAFLD)状态下生长分化因子15(GDF15)表达的影响。方法:共对25例NAFLD合并2型糖尿病患者26周不同药物治疗后(利拉鲁肽组9例、西格列汀组8例、甘精胰岛素组8例)进行分析。收集治疗前后体重、体重指数(BMI)及血清GDF15水平,通过磁共振成像-质子密度脂肪含量测量肝内脂肪含量(IHL)。8周龄雄性C57BL/6小鼠进行12周高脂饮食喂养,采用随机数字表法分为高脂饮食(HFD)组(6只)、艾塞那肽组(GLP-1组)(6只),另设正常饮食对照组(6只),干预8周后收取肝脏组织。HepG2细胞用300μmol/L棕榈酸钠(PA)处理,并予Exendin-4(浓度分别为0、1、20、100 nmol/L)干预,另设脱脂牛血清白蛋白对照组。使用lentiCRISPRv2-sgRNA载体构建稳定敲除GDF15的HepG2细胞,空载体转染的HepG2细胞作为阴性对照。转染的细胞用300μmol/L PA处理,并予100 nmol/L Exendin-4干预。测定血清及细胞上清中GDF15蛋白水平及肝脏组织和HepG2细胞的GDF15 mRNA水平与甘油三酯(TG)含量。采用单因素方差分析、协方差分析、Pearson相关分析等进行统计学分析。结果:在NAFLD合并糖尿病人群中,西格列汀组和甘精胰岛素组血清GDF15水平在治疗前后无明显变化。而利拉鲁肽组在26周后血清GDF15水平为(920.3±265.4)pg/ml,比治疗前的(742.2±279.0)pg/ml明显升高,差异有统计学意义(P=0.015);体重、BMI、IHL明显下降(P均<0.05)。利拉鲁肽组血清GDF15改变量与IHL改变量呈负相关(r=-0.676,P=0.045)。GLP-1显著改善高脂饮食喂养小鼠的肝脏脂质沉积及炎症状态。与HFD组相比,GLP-1组小鼠肝脏组织GDF15 mRNA明显升高。GLP-1能改善PA诱导的HepG2细胞脂质沉积,并以剂量依赖的方式上调GDF15的表达与分泌。与阴性对照组相比,敲除GDF15显著削弱了GLP-1改善细胞TG蓄积及炎症状态。结论:GLP-1可促进肝脏GDF15的表达与分泌,并改善NAFLD肝脏脂质沉积及炎症状态。

Objective To explore the effects of glucagon-like peptide-1(GLP-1)therapy on the expression of growth different factor 15(GDF15)in hepatocytes of non-alcoholic fatty liver disease(NAFLD).Methods A total of 25 patients with NAFLD and type 2 diabetes mellitus were analyzed(liraglutide group,9;insulin glargine group,8 and sitagliptin group,8).The level of serum GDF15,intrahepatic lipid(IHL),body weight,and body mass index(BMI)were collected at baseline and week 26.IHL of patients was quantified by magnetic resonance imaging-estimated proton density fat fraction.Male C57BL/6 mice challenged with a high-fat diet for 12 weeks were treated with exenatide(GLP-1 group,n=6)or normal saline(HFD group,n=6)by intraperitoneal injection.Another group with a chow diet was designed as normal control(n=6).Liver tissue were collected after 8 weeks of intervention.Human HepG2 cells were incubated in medium containing 300μmol/L sodium palmitate(PA)and treated with Exendin-4(0 nmol/L,1 nmol/L,20 nmol/L,100 nmol/L,respectively).10%bovine serum albumin was set as the control group.GDF15 knockout stable cell line constructed by using CRISPR/Cas9 system,and empty vector served as a negative control.Transfected cells were incubated in medium containing 300μmol/L PA and treated with or without 100 nmol/L Exendin-4.The levels of GDF15 released into serum or cell supernatant and relative mRNA levels of GDF15 in hepatic tissue or HepG2 cells were detected.Statistical analysis was mainly performed using one-way analysis of variance(ANOVA),covariance analysis,Pearson correlation coefficients.Results No significant changes in the level of serum GDF15 was observed in the sitagliptin and insulin glargine groups after 26-week therapy.However,the level of serum GDF15 increased significantly in the liraglutide group[(742.2±279.0)vs.(920.3±265.4)pg/ml,P=0.015],and weight,BMI and IHL decreased statistically significant[(80.4±6.3)vs.(76.9±7.2)kg;(29.0±2.2)vs.(27.8±1.9)kg/m2;(18.3±7.4)%vs.(12.2±5.5)%;respectively](all P<0.05).Furthermore,negative correlation was found between the change in serum GDF15 and IHL in the liraglutide group(r=-0.676,P=0.045).We found that hepatic steatosis and inflammation in the liver,which were improved markedly in the HFD group,reduced in GLP-1 group.Relative mRNA levels of GDF15 in liver tissue of mice were remarkably higher in the GLP-1 group than those in HFD group and control group.In addition,GLP-1 alleviated lipid deposition in HepG2 cells,which induced by PA,and elevated the expression and secretion of GDF15 in a dose-dependent manner.Compared with the negative control,GDF15 knockout abolished the effect of GLP-1 on alleviation of triglyceride accumulation and inflammation.Conclusion GLP-1 could up-regulate the expression and secretion of GDF15 and alleviatehepatic steatosis and inflammation of NAFLD.