Study On The Ameliorative Effects And The Mechanisms Of Apple Polyphenol Extract On Hepatic Steatosis In Aged Mice

BackgroundNon-alcoholic fatty liver disease(NAFLD)is one of the most predominant chronic liver diseases.With the development of modern civilization,people’s dietary habits have changed dramatically,and irrational diets have made more and more people are suffering from NAFLD.The prevalence,incidence and annual mortality rates associated with NAFLD in China are among the highest in the world.Epidemiological studies have shown that the prevalence of NAFLD increases with age and ageing is closely related to liver lipid metabolism.With the dramatic increase in China’s ageing population,the burden of NAFLD and its associated complications is accumulating in the ageing population.Hepatic steatosis,the initial stage in the progression of NAFLD,manifests as excessive deposition of hepatic lipids and is usually benign and reversible;therefore,inhibition/improvement of hepatic steatosis may reverse and improve the poor outcome of NAFLD.Recent studies have found that polyphenolic compounds in phytochemicals can modulate lipid metabolism,and some dietary polyphenol extracts can improve hepatic steatosis in specific animal models.Apple polyphenol extract(APE)is a polyphenol mixture that extracted from fresh and whole apples,and all monomers in APE have a benzene ring and are bound to multiple hydroxyl groups,which include many active monomers and have been shown to have strong antioxidant,anti-inflammatory and other bioactive effects.However,studies on the role of APE in regulating the lipid metabolism of aging animals are still few and further research is needed.ObjectivesHigh-fat diet fed C57BL/6 male aged mice at 12 months of age were used as a study model,with 2-month-old young mice as controls,and baseline groups of young and aged mice were set,to investigate the effects of the natural ageing process and highfat diet feeding on the development of hepatic steatosis and whether APE could prevent and improve hepatic steatosis in aged mice by modulating lipid metabolism,and to elucidate other potential mechanisms.Methods1.Animal grouping.Twenty-four young 2-month-old C57BL/6 male mice were randomly divided into 4 groups,and thirty-five aged 12-month-old C57BL/6 male mice were randomly divided into 5 groups.Next,one group of young mice and one group of aged mice were randomly selected as the baseline groups,and were executed after one week of adaptive feeding.The remaining mice were divided into 7 groups of 6-8 mice each group according to different dietary patterns and interventions and fed for 12 weeks as follows:① Young control group(YCON):fed with standard diet and gavaged with sterile water;②Young high-fat group(YHFD):fed with 60%high-fat diet and gavaged with sterile water;③Young APE group(YAPE):fed with standard diet and gavaged with 500 mg/(kg·bw·day)APE.④ Aged control group(CON):fed with standard diet and gavaged with sterile water;⑤ Aged high-fat group(HFD):fed with 60%high-fat diet and gavaged with sterile water;⑥ Aged APE group(APE):fed with standard diet and gavaged with 500 mg/(kg·bw·day)APE.⑦ Aged HAP group(HAP):fed with 60%high-fat diet and gavaged with 500 mg/(kg·bw·day)APE.2.The amount of food consumed was recorded daily and their body weight was weighed weekly.Oral glucose tolerance tests were performed at the end of the 12th week of intervention.12 weeks later,mice were fasted overnight,anaesthetised and executed.Blood was collected from the retro-orbital plexus to measure total cholesterol(TC),triglyceride(TG),alanine aminotransferase(ALT)and aspartate aminotransferase(AST).Liver,subcutaneous fat,perirenal fat,epididymal fat and brown adipose tissue were also weighed.The livers.were stained with HE to observe histomorphological changes.The liver was scored for steatosis,inflammation and ballooning,and NAFLD activity scores(NAS)was calculated.RT-qPCR and western blot were used to detect the mRNA and protein expression levels of lipid metabolism and autophagy-related indicators in the liver,respectively.The colon contents were taken and the gut microbiota was sequenced using the 16S rDNA amplicon method.Results1.Basic indices(1)Body weight:The final body weight and weight gain of HFD-fed mice were significantly higher than those of the control group,and the weight gain of aged mice was more significant than that of young mice.(2)Food intake and energy intake:Compared to the CON,the HFD-fed mice showed a decrease in food intake but a significant increase in energy intake.The APE treatment significantly reduced the increase in liver and white adipose tissue weight in HFD-fed young and aged mice,and tended to increase the brown fat content.2.Glucose tolerance and serum TC,TG levels and indicators of liver damage(1)The results of oral glucose tolerance test showed that glucose tolerance was severely impaired in young and aged mice fed HFD compared to CON,and APE treatment improved impaired glucose tolerance in aged mice.(2)High-fat feeding induced an increase in serum TC levels in young and aged mice and serum TG levels in aged mice,and APE was able to reduce serum TC and TG levels,but did not have a significant effect on TG.(3)Serum ALT and AST levels were elevated in high-fat fed mice,and the elevation in AST was more pronounced in aged mice,while APE treatment significantly reduced AST levels.3.Histological analysis of the liverHistopathological sections showed that NAS scores in the young and aged CON groups were not significantly different compared to the corresponding baseline groups,and that HFD induced typical hepatic steatosis compared to CON,especially in aged mice with higher NAS scores,suggesting that a high-fat diet has a more pronounced effect on hepatic steatosis than natural ageing.APE significantly improved hepatic steatosis and ballooning,but had no significant effect on inflammation scores.4.Expression of genes related to hepatic lipid metabolism4.1 Hepatic lipid metabolism-related gene mRNA expression(1)In young mice,mRNA levels of Srebp-1c,Cyp7α1,Cyp27α1,Abcα1 and Abcg1 were elevated in HFD-fed mice compared to controls,and mRNA levels of Lkb1 and Pgc-1α were significantly higher after APE treatment.(2)In aged mice,compared with controls,the mRNA levels of Sirt1,Ampk,Hsl and Abcgl were significantly decreased and the levels of Srebp-1c were increased in HFD-fed mice,and the expression levels of Acc,Hmgcr and Cd36 were higher in aged control mice than in young mice,and after APE treatment the mRNA levels of Sirtl,Lkb1,Ampk,Hsl,Pgc-1α and Cyp7a1 levels were significantly increased after APE treatment.4.2 Protein expression of genes related to hepatic lipid metabolism(1)In young mice,compared to controls,HFD-fed mice had decreased protein expression of SIRT1 and HSL,and significantly decreased protein expression of ACC,SCD1 and FAS.p-AMPK,CP1 A,PGC-1α and CYP7A1 had a trend of decrease and p-ACC had a trend of increase.APE treatment down-regulated the expression of AMPK and HSL,and up-regulated the expression levels of ACC.(2)In aged mice,compared with the control group,the protein expression of SIRT1,PGC-1α and HSL was significantly decreased in HFD-fed mice,and the protein levels of ACC,SCD1 and FAS were also significantly decreased.p-AMPK had a tendency to decrease,and CPT1A,p-ACC and CYP7A1 had a tendency to increase,and the aged mice in the high-fat group had a tendency to increase the expression of SIRT1,PGC-lα,ACC,FAS and HMGCR,which were lower than in young mice.APE treatment reduced ACC levels in standard diet-fed aged mice and significantly increased SIRT1,PGC-1α and HSL levels in aged mice fed high-fat diet,and APE reversed the decrease in ACC,SCD1 and FAS protein expression and down-regulated the protein expression level of HMGCR.5.Expression of genes related to hepatic autophagy5.1 Hepatic autophagy-related gene mRNA expression(1)In young mice,mRNA expression levels of LC3a,Atg7,TFEB,Lamp1,Ulkl and Becnl were increased in HFD-fed mice compared to controls,and mRNA levels of TFEB,Lamp1 and Becn1 were significantly increased after APE treatment.(2)In aged mice,mRNA levels of LC3a,LC3b,Atg7,Lamp2,Rab7,Ulk1 and Becnl were significantly decreased and levels of Lamp1 were increased in HFD-fed mice compared to control mice,and mice in the aged HFD group exhibited lower expression levels of Atg7,Lamp2 and Becnl than young mice,and APE treatment significantly reduced the expression of Foxol,LC3a,LC3b,Atg7,Atg5,TFEB,Lamp2,Rab7 and Becnl in aged mice fed standard diet,and upregulated the expression levels of LC3a,Atg7,Atg5,Rab7,Ulk1 and Becnl in aged mice fed high-fat diet,with a tendency to elevate the expression levels of LC3b,TFEB,Lamp1 and Lamp2.5.2 Hepatic autophagy-related gene protein expression(1)In young mice,the protein expression levels of FOXO1,LC3I and LC3II,ATG5 and RAB7 were decreased in HFD-fed mice compared to controls,and the expression level of LAMP2 was significantly increased after APE treatment.(2)In aged mice,compared to controls,ATG5 protein levels were significantly decreased in HFD-fed mice and mice in the aged high-fat group exhibited higher levels of FOXO1 and P62 than young mice.APE treatment significantly upregulated the expression of LC3I and LC3II,ATG5,RAB7 and LAMP1 in aged mice fed standard diet,decreased FOXO1 and increased ATG5 and LAMP1 levels in aged mice fed highfat diet,while there was a trend towards increased LC3I and LC3II levels and a trend towards decreased P62.6.Analysis of gut microbiota(1)Gut microbiota in colonic contents analysis showed that HFD feeding significantly reduced the alpha diversity of gut microbiota,and it was further reduced after APE treatment,especially in aged mice.PCoA analysis showed that there was clustering of mice of different ages with the same dietary pattern and separation between different dietary patterns,and that APE significantly altered the beta diversity of the microbiota compared to the HFD group.(2)At the phylum level,the HFD group showed a decrease in the abundance of the Bacteroides,an increase in the abundance of the Firmicutes and an increase in the F/B ratio compared to the CON group.APE reduced the abundance of the Firmicutes and the F/B ratio and significantly increased the relative abundance of the Verrucomicrobia compared to the HFD group.(3)At the genus level,HFD reduced Muribaculaceae unclassified in young and aged mice,and its abundance was further reduced after APE treatment.HFD increased the abundance of Eubacterium_coprostanoligenes_group.The abundance of Akkermansia significantly increased after APE treatment in the HAP group,while APE reduced the relative abundance of Eubacterium_coprostanoligenes_group.(4)LEfSe analysis showed an increase in the relative abundance of Clostridiale and Oscillospirales in young mice fed a high-fat diet,while aged mice were characterised by Firmicutes.In the HAP group,the relative abundance of Verrucomicrobiales was highest,while Akkermansia was the most dominant bacteria.(5)Analysis of bacterial phenotypes showed that APE treatment affected the levels of anaerobic,aerobic,facultatively anaerobic,Gram-positive,Gram-negative,contains mobile elements bacteria and stress tolerant bacteria.Among these,APE significantly increased the relative abundance of facultatively anaerobic,Gram-negative,forms biofilms and stress tolerant bacteria,while decreasing the abundance of anaerobic and Gram-positive bacteria in high-fat fed aged mice.7.Correlation analysis between gut microbiota and other indicatorsSpearman’s correlation analysis showed that there was a significant correlation between the abundance of Bacteroidetes and the indicators.Akkermansia showed a positive correlation with fatty acid β-oxidation and lipolysis,cholesterol metabolism and autophagy,while Clostridium and Eubacterium]_coprostanoligenes_group showed the opposite.The abundance of Bacteroidetes showed a positive correlation with autophagy and a negative correlation with cholesterol synthesis,while Firmicutes and Lachnospiraceae_NK4A136_group showed the opposite.The abundance of Muribaculaceae showed a negative correlation with basic indices,biochemical parameters,and autophagy.ConclusionsBy comparing with aged baseline mice and aged control mice,high-fat fed aged mice exhibiting more severe hepatic steatosis than young mice,which suggested that irrational dietary patterns have a more significant effect on hepatic steatosis in mice than natural aging.APE can alleviate high-fat induced hepatic steatosis in aged mice by activating SIRT1 to regulate lipid metabolism,promoting autophagy and remodeling the gut microbiota.