二甲双胍减轻小鼠非酒精性脂肪肝炎的作用机制

Mechanism of metformin in alleviating non-alcoholic steatohepatitis in mice

目的探讨二甲双胍改善小鼠非酒精性脂肪肝炎(NASH)脂肪代谢和炎症的作用机制。方法将18只8周C57BL/6J雄性小鼠按随机数字法分为3组:正常饮食组、高糖高脂加胆固醇饮食(HFF)模型(HFF组)和HFF模型二甲双胍处理组(Met组),每组6只小鼠,喂养16周,第12周起Met组每天给予一次二甲双胍药物(150 mg/kg)灌胃干预,持续4周,正常饮食组和HFF组给予等体积生理盐水灌胃处理。造模结束后,麻醉并称取小鼠体重后,取小鼠血清和肝组织,计算肝脏指数(肝重/体重),检测谷丙转氨酶(ALT)、谷草转氨酶(AST)及血清甘油三酯(TG)、肝脏TG;通过苏木精-伊红(HE)染色和油红O染色观察肝脏的病理变化;蛋白质印迹法(Western blot)检测脂质合成相关蛋白甾醇调节元件结合蛋白(SREBP1c)、乙酰辅酶A羧化酶(ACC)、脂肪酸合成酶(FASN)的蛋白表达水平;实时定量反转录聚合酶链反应(RT-qPCR)检测Acc、Fasn、Srebf1、白细胞介素-1β(IL-1β)、淋巴细胞抗原6(Ly6g)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)、诱导型一氧化氮合酶(iNOS)mRNA相对表达水平。其次,利用HFF组和Met组小鼠肝组织进行表达谱高通量测序并分析相关信号通路的变化。最后,Western blot检测自噬底物p62、微管相关蛋白1A/1B-轻链3(LC3-Ⅰ/Ⅱ)、自噬相关基因5(ATG5)、磷脂酰肌醇3-激酶(PI3K)和蛋白激酶B(Akt)及其磷酸化水平,以及M1型巨噬细胞标志物[IL-1β、iNOS、单位细胞趋化蛋白1(MCP1)、CD86]和M2型巨噬细胞标志物(CD163、CD206)的蛋白水平。两组间采用t检验进行比较。结果HE染色和油红O染色显示,与正常饮食组比较,HFF组小鼠肝组织脂滴累积,炎性细胞浸润增加;HFF组小鼠肝组织ACC、SREBP1c以及iNOS、MCP1的蛋白表达水平高于正常饮食组(1.03±0.15比0.60±0.15,t=3.277,P<0.05;1.04±0.08比0.70±0.10,t=4.467,P<0.05;1.16±0.02比0.54±0.22,t=3.772,P<0.05;1.20±0.15比0.51±0.09,t=6.908,P<0.05)。Met组小鼠体重、肝重、肝脏指数低于HFF组[(27.45±4.26)g比(35.51±1.69)g,t=4.306,P<0.05;(1.20±0.21)g比(1.74±0.17)g,t=4.839,P<0.05;0.04±0.01比0.05±0.01,t=2.147,P<0.05];Met组小鼠血清ALT和AST对比HFF组无明显变化[(21.33±4.32)U/L比(30.33±10.84)U/L,t=1.889,P>0.05;(106.70±17.33)U/L比(112.30±14.33)U/L,t=0.617,P>0.05)]。Met组小鼠血清TG、肝脏TG水平低于HFF组[(0.72±0.27)mmol/L比(1.33±0.41)mmol/L,t=3.004,P<0.05;(0.02±0.01)mmol/L比(0.05±0.03)mmol/L,t=2.350,P<0.05)];HE染色和油红O染色结果观察到Met组小鼠肝细胞内未见明显空泡和脂滴;Met组小鼠肝组织中ACC、FASN、SREBP1c蛋白表达水平低于HFF组(0.37±0.13比0.66±0.09,t=3.174,P<0.05;0.61±0.03比1.11±0.14,t=6.069,P<0.05;0.48±0.05比0.88±0.01,t=12.310,P<0.05);Met组小鼠肝组织中ACC、FASN、SREBP1c mRNA表达水平低于HFF组(2.21±0.08比8.13±3.45,t=2.974,P<0.05;1.06±0.12比1.39±0.05,t=4.310,P<0.05;1.03±0.06比1.50±0.26,t=3.055,P<0.05)。Met组小鼠IL-1β、Ly6g、NLRP3、iNOS mRNA表达水平低于HFF组(0.47±0.02比1.40±0.08,t=21.030,P<0.05;0.16±0.06比0.65±0.19,t=4.276,P<0.05;0.67±0.04比1.17±0.16,t=5.082,P<0.05;0.82±0.52比4.49±0.78,t=6.784,P<0.05)。Met组小鼠肝组织p-Akt/Akt、p-PI3K/PI3K比值低于HFF组(1.05±0.02比1.58±0.04,t=15.420,P<0.05;0.47±0.11比1.07±0.10,t=6.906,P<0.05);Met组小鼠肝组织ATG5、CD163蛋白表达和LC3II/LC3I比值高于HFF组(1.05±0.11比0.52±0.04,t=7.690,P<0.05;0.99±0.02比0.64±0.04,t=14.470,P<0.05;1.58±0.05比1.34±0.03,t=7.091,P<0.05);Met组小鼠肝组织P62、IL-1β、iNOS、MCP1蛋白表达低于HFF组(0.72±0.06比1.07±0.08,t=6.047,P<0.05;0.17±0.10比0.56±0.10,t=4.310,P<0.05;0.42±0.03比0.78±0.15,t=4.038,P<0.05;0.39±0.10比0.72±0.02,t=4.253,P<0.05);Met组小鼠肝组织CD86、CD206蛋白表达对比HFF组无明显变化(0.65±0.04比0.68±0.08,t=0.600,P>0.05;0.72±0.33比0.88±0.21,t=0.664,P>0.05)。结论二甲双胍治疗通过抑制PI3K/Akt通路,增强自噬,促进M1型巨噬细胞向M2型巨噬细胞极化,从而减轻小鼠NASH的肝脂肪变性和炎症。

Objective To investigate the role and mechanism of metformin in ameliorating non-alcoholic fatty hepatitis(NASH)in mice.Methods A total of 188-week C57BL/6J male mice were divided into three groups:normal diet group(ND group),high-fat-high-sugar plus cholesterol diet(HFF)model group(HFF group),and HFF model treated with metformin group(Met group)for 16 weeks,6 mice per group.The Met group was given metformin drug(150 mg/kg)by gavage intervention once per day for 4 weeks starting from the 12th week,and the ND group and the HFF group were given an equal volume of saline gavage treatment.At the end of modeling,mice were anesthetized and dissected to weigh their body weight,liver tissue weight and calculate the liver index(liver weight/body weight).Serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglyceride(TG),and hepatic TG were examined.The pathological changes in the liver were observed by hematoxylin and eosin(HE)and oil red O staining.The protein levels of sterol regulatory element binding protein(SREBP1c),acetyl coenzyme A carboxylase(ACC),and fatty acid synthase(FASN)were detected by Western blotting,and the mRNA levels of Acc,Fasn,Srebf1,interleukin-1β(IL-1β),Ly6g,and NOD-like receptor thermoprotein structural domain-related protein 3(NLRP3),and inducible nitric oxide synthase(iNOS)were analyzed by real-time quantitative reverse transcription-polymerase chain reaction(RT-qPCR).Secondly,transcriptional profiling of liver tissues from HFF and Met groups were performed and then further analyzed by bioinformatics.The protein levels of phosphatidylinositol 3-kinase(PI3K),and protein kinase B(Akt)and their phosphorylations were verified by Western blotting.Finally,the protein levels of autophagy associated genes such as p62,microtubule-associated protein 1A/1B-light chain 3(LC3-Ⅰ/Ⅱ),autophagy-associated gene 5(ATG5),as well as M1-type macrophage markers(IL-1β,iNOS,MCP1,CD86)and M2 type macrophage markers(CD163,CD206)were detected by Western blotting.A t-test was used to compare the two groups.Results HE staining and oil red O staining showed that compared with the normal diet group,mice in the HFF group had an accumulation of lipid droplets in liver tissue and an increase in inflammatory cell infiltration;mice in the HFF group had a higher level of protein expression in liver tissue of ACC,SREBP1c as well as iNOS and MCP1 than that of the normal diet group(1.03±0.15 vs.0.60±0.15,t=3.277,P<0.05;1.04±0.08 vs.0.70±0.10,t=4.467,P<0.05;1.16±0.02 vs.0.54±0.22,t=3.772,P<0.05;1.20±0.15 vs.0.51±0.09,t=6.908,P<0.05).Body weights,liver weights,and liver indices of the mice in the Met group were lower than those in the HFF group[(27.45±4.26)g vs.(35.51±1.69)g,t=4.306,P<0.05;(1.20±0.21)g vs.(1.74±0.17)g,t=4.839,P<0.05;0.04±0.01 vs.0.05±0.01,t=2.147,P<0.05];the serum ALT and AST did not change significantly compared to the HFF group[(21.33±4.32)U/L vs.(30.33±10.84)U/L,t=1.889,P>0.05;(106.70±17.33)U/L vs.(112.30±14.33)U/L,t=0.617,P>0.05)].Mice in the Met group had lower serum TG,liver TG levels were lower than those in the HFF group[(0.72±0.27)mmol/L vs.(1.33±0.41)mmol/L,t=3.004,P<0.05;(0.02±0.01)mmol/L vs.(0.05±0.03)mmol/L,t=2.350,P<0.05)];the results of H&E staining and oil red O staining were observed that no obvious vacuoles and lipid droplets were seen in the hepatocytes of mice in the Met group;the protein expression levels of ACC,FASN,and SREBP1c in the liver tissues of mice in the Met group were lower than those in the HFF group(0.37±0.13 vs.0.66±0.09,t=3.174,P<0.05;0.61±0.03 vs.1.11±0.14,t=6.069,P<0.05;0.48±0.05 vs.0.88±0.01,t=12.310,P<0.05);ACC,FASN,and SREBP1c mRNA expression levels in the liver tissues of mice in the Met group were lower than those in the HFF group(2.21±0.08 vs.8.13±3.45,t=2.974,P<0.05;1.06±0.12 vs.1.39±0.05,t=4.310,P<0.05;1.03±0.06 vs.1.50±0.26,t=3.055,P<0.05).mice in the Met group had lower levels of IL-1β,Ly6g,NLRP3,and iNOS mRNA expression than those in the HFF group(0.47±0.02 vs.1.40±0.08,t=21.030,P<0.05;0.16±0.06 vs.0.65±0.19,t=4.276,P<0.05;0.67±0.04 vs.1.17±0.16,t=5.082,P<0.05;0.82±0.52 vs.4.49±0.78,t=6.784,P<0.05).Liver tissue p-AKT/AKT and p-PI3K/PI3K ratios were lower in the Met group than in the HFF group(1.05±0.02 vs.1.58±0.04,t=15.420,P<0.05;0.47±0.11 vs.1.07±0.10,t=6.906,P<0.05);Met group mice had lower liver tissue ATG5,CD163 protein expression and LC3Ⅱ/LC3Ⅰratio were higher than those in the HFF group(1.05±0.11 vs.0.52±0.04,t=7.690,P<0.05;0.99±0.02 vs.0.64±0.04,t=14.47,P<0.05;1.58±0.05 vs.1.34±0.03,t=7.091,P<0.05);The protein expression of p62,IL-1β,iNOS,and MCP1 in liver tissue of Met group mice was lower than that of HFF group(0.72±0.06 vs.1.07±0.08,t=6.047,P<0.05;0.17±0.10 vs.0.56±0.10,t=4.310,P<0.05;0.42±0.03 vs.0.78±0.15,t=4.038,P<0.05;0.39±0.10 vs.0.72±0.02,t=4.253,P<0.05);there were no significant changes in the protein expression of CD86 and CD206 in liver tissues of the Met group of mice compared to the HFF group(0.65±0.04 vs.0.68±0.08,t=0.600,P>0.05;0.72±0.33 vs.0.88±0.21,t=0.664,P>0.05).Conclusion Met treatment reduces hepatic steatosis and inflammation in NASH mice by inhibiting the PI3K/Akt pathway,enhancing autophagy,and promoting polarization from M1 macrophages to M2 macrophages.