Effect Of Apple Polyphenols On Hepatic Steatosis In High-fat Diet Mice By Regulating Endoplasmic Reticulum Stress

Objective:To investigate the effect of apple polyphenol(APE)on hepatic steatosis in high-fat diet mice and its possible mechanism.Method:1.Animal grouping and intervention methods: After adaptive feeding for 1 week,32C57BL/6J mice were randomly divided into control group(CON,n = 8),model group(HFD,n = 8),lovastatin group(LOV,n = 8)and apple polyphenol group(APE,n = 8).The CON group fed a normal diet(Research Diets Catalog Number D12450J)and given a daily oral gavage of distilled water(0.2 m L/20 g).The HFD group fed a HFD(Research Diets Catalog Number D12492)and given a daily oral gavage of distilled water(0.2 m L/20 g).The HFD-fed mice in LOV group were given an oral gavage of 5 mg/kg lovastatin daily.The HFD-fed mice in APE group were given an oral gavage of 800 mg/kg APE daily.After8 weeks of intervention,blood was collected through the orbital vein,and liver and epididymal adipose tissue were retained for subsequent detection of indicators.2.Weight change monitoring: Weight of mice was measured weekly.3.Blood biochemical index detection: The level of Triacylglycerol(TG),Total cholesterol(TC),High-density lipoprotein cholesterol(HDL-C),Low density lipoprotein cholesterol(LDL-C),Alanine aminotransferase,ALT),Glutamic oxalacetic transaminase(AST)in serum were detected by automatic biochemical analyzer.4.Pathological of liver tissue and adipose tissue: Hematoxylin-eosin(HE)staining was used to observe the pathological changes of liver tissue and epididymal adipose tissue.5.Detection of liver lipid levels,oxidative stress and inflammatory indicators: Elisa was used to detect free fatty acid(FFA),TG,TC,Malondialdehyde(MDA)and Glutathione peroxidase(GSH-Px),Superoxide dismutase(SOD),Catalase(CAT),Interleukin-6(IL-6),IL-6),Tumor necrosis factor-α(TNF-α),Interleukin-18(IL-18)and Interleukin-1β(IL-1β)levels in liver.6.Expression of endoplasmic reticulum stress(ERS)and lipid synthesis related proteins in liver tissue: Western blot(WB)was used to detect the proteins expression level of hepatic endoplasmic reticulum stress including Glucose-regulated protein 78(GRP78),Inositol-requiring kinase 1α(IRE1α),Phosphorylation of IRE1α(p-IRE1α),Protein kinase RNA-like ER kinase(PERK),Phosphorylation of PERK(p-PERK),Activating transcription factor 6(ATF6),X box-binding protein-1(XBP1),Eukaryotic initiation factor2α(e IF2α),Phosphorylation of e IF2α(p-e IF2α),C/EBP homologous protein(CHOP),and proteins related to lipid synthesis including Sterol regulatory element-binding protein-1c(SREBP-1c),Acetyl-Co A carboxylase 1(ACC1),Fatty acid synthase(FAS)and Peroxisome proliferator-activated receptor-γ(PPAR-γ)protein expression levels.7.Detection of ERS related proteins and lipolysis related proteins in adipose tissue:Endoplasmic reticulum stress proteins GRP78,IRE1α,p-IRE1α,XBP1,PERK,p-PERK,e IF2α,p-e IF2α,ATF6 and lipolysis related proteins adipose triglyceride lipase(ATGL)and hormone-sensitive lipase(HSL)expression levels Results:1.After 8 weeks of intervention,the body weight of mice in HFD group was significantly higher than that in CON group from the second week(P < 0.05);The body weight of mice in LOV group and APE group was significantly lower than that in HFD group from the 4th week(P < 0.05).2.The liver weight,liver index,epididymal fat weight and epididymal fat index of mice in HFD group were significantly higher than those in CON group(P < 0.05).The liver weight,liver index,epididymal fat weight and epididymal fat index of mice in LOV and APE groups were significantly lower than those in HFD group(P < 0.05).3.The serum TC,LDL-C,HDL-C,ALT and AST levels in HFD group were significantly higher than those in CON group(P < 0.05),and the levels of serum TG,TC,LDL-C,HDL-C and ALT in LOV group and APE group were significantly lower than those in HFD group(P < 0.05).4.HE staining results of liver tissue and white adipose tissue showed that hepatic steatosis and epididymal adipose tissue expansion were significantly improved in LOV group and APE group compared with HFD group.5.The levels of hepatic FFA,TG and TC in HFD group were significantly higher than those in CON group(P < 0.05),and the hepatic FFA,TG and TC levels in LOV group and APE group were significantly lower than those in HFD group(P < 0.05).6.The content of hepatic MDA in HFD group was significantly higher than CON group,and the contents of hepatic SOD,CAT and GSH-Px in HFD group were significantly lower than those in CON group(P < 0.05).The hepatic MDA content in LOV and APE were significantly lower than HFD group,and the hepatic SOD,CAT and GSH-Px contents were significantly higher than those in HFD group(P < 0.05).7.The levels of hepatic IL-6,TNF-α,IL-18 and IL-1β in HFD group were higher than CON group(P < 0.05),and the hepatic IL-6,TNF-α,IL-18 and IL-1β levels in LOV and APE were significantly lower than those in HFD group(P < 0.05).8.The expressions of ERS related proteins GRP78,IRE1α,p-IRE1α,XBP1,PERK,p-PERK,e IF2α,p-e IF2α,CHOP and ATF6 in liver of mice in HFD group were significantly higher than those in CON group(P < 0.05).The expressions of GRP78,IRE1α,p-IRE1α,XBP1,PERK,p-PERK,e IF2α,p-e IF2α,CHOP and ATF6 in liver of mice in LOV and APE groups were significantly lower than those in HFD group(P < 0.05).9.The expression levels of SREBP-1c,FAS,ACC1 and PPAR-γ in liver tissue of mice in HFD group were significantly higher than those in CON group(P < 0.05).The expression levels of SREBP-1c,FAS,ACC1 and PPAR-γ of mice in LOV and APE groups were significantly lower than those in HFD group(P < 0.05).10.The expression of ERS related proteins GRP78,IRE1α,p-IRE1α,XBP1,PERK,p-PERK,e IF2α,p-e IF2α and ATF6 in adipose tissue of mice in HFD group was significantly higher than that in CON group(P < 0.05).The expression of GRP78,IRE1α,p-IRE1α,XBP1,PERK,p-PERK,e IF2α,p-e IF2α,and ATF6 in adipose tissue of mice in LOV and APE groups were significantly lower than those in HFD group(P < 0.05).11.The expression of lipolysis related proteins HSL and ATGL in adipose tissue of mice in HFD group was significantly higher than that in CON group(P < 0.05).The expression of HSL and ATGL in adipose tissue of mice in LOV and APE groups were significantly lower than those in HFD group(P < 0.05).Conclusion:Apple polyphenol reduced lipid synthesis by inhibiting ERS in the liver on the one hand,and reduced lipolysis by inhibiting ERS in the adipose tissue on the other hand,thereby reducing the accumulation of TG in the liver,and ultimately improving hepatic steatosis in high-fat diet mice.