Mechanism of metformin in alleviating non-alcoholic steatohepatitis in mice
Objective To investigate the role and mechanism of metformin in ameliorating non-al-coholic fatty hepatitis(NASH)in mice.Methods A total of 18 8-week C57BL/6J male mice were divid-ed into three groups:normal diet group(ND group),high-fat-high-sugar plus cholesterol diet(HFF)mod-el 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 ex-amined.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 struc-tural domain-related protein 3(NLRP3),and inducible nitric oxide synthase(iNOS)were analyzed by re-al-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 bioinformat-ics.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 1 A/1B-light chain 3(LC3-Ⅰ/Ⅱ),autophagy-associat-ed 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 in-flammatory 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 pro-tein expression levels of ACC,FASN,and SREBP1c in the liver tissues of mice in the Met group were low-er 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±0.05vs.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±0.12vs.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±0.06vs.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 LC3 Ⅱ/LC3 Ⅰ ratio 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 ex-pression 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 steato-sis and inflammation in NASH mice by inhibiting the PI3K/Akt pathway,enhancing autophagy,and pro-moting polarization from M1 macrophages to M2 macrophages.
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