Study On The Effects Of Ferulic Acid On Improving Oxidative Stress In Rat Liver And Lipid Metabolic Disorders Induced By High-fat Diet

Objective: The present study aimed to indentify the key gut microbiota for ferulic acid to ameliorate oxidative stress induced by high-fat diet in rats,and elucidate the regulation mechanism of the hepatic Kelch-like ECH-associated protein-1(Keap1)/transcription factor NF-E2 related factor 2(Nrf2)/antioxidant response element(ARE)pathway by key metabolites.The results of the present study can provide a scientific basis for the development of precise antioxidants targeting the gut microbiota,and provide a reference for the prevention and intervention of metabolic diseases such as hyperlipidemia and obesity.Methods: In this study,8-week-old Wistar rats were selected and randomly divided into normal diet(ND)group(maintenance diet,n=8)and high-fat diet group(high-fat diet,n=32).After six weeks of feeding,the rats in the high-fat diet group were randomly divided into four groups: high-fat diet group(HFD)(n=8),low-dose ferulic acid group(HFD＿LFA)(n=8),medium-dose ferulic acid group(HFD＿MFA)(n=8)and high-dose ferulic acid group(HFD＿HFA)(n=8).The ferulic acid intervention group was gavaged with the appropriate dose of ferulic acid solution,and the normal diet and high-fat diet groups were gavaged with saline.After four weeks,fresh feces,blood and liver tissue were obtained by autopsy.Liver was homogenized and diluted to the appropriate concentration and then the antioxidant enzyme concentration was measured.Plasma was taken from fresh blood after heparin anticoagulation to determine the antioxidant enzyme concentration.Liver function and lipid parameters were determined using a fully automated analyzer.The structure and abundance of rat fecal gut microbiota were analyzed by 16 S r RNA method.Differential metabolites in rat feces,liver and serum were determined by metabolomic method.The mRNA transcript levels in liver and blood were analyzed by transcriptomic approach.Results: Ferulic acid slowed down the weight gain of rats induced by high-fat diet,upregulated the expression of mRNAs related to fat degradation pathway,PPAR signaling pathway and adipocytokine signaling pathway,and improved hyperlipidemia and liver function of rats fed with high-fat diet in a dose-dependent manner.Ferulic acid exerted its antioxidant effects in three ways: firstly,ferulic acid increased the expression of antioxidant pathway-related proteins in the liver.Secondly,ferulic acid up-regulated the expression of antioxidant pathway-related mRNAs.Finally,ferulic acid reversed the reduction of antioxidant enzyme concentrations in the liver and plasma induced by high-fat diet.In addition,ferulic acid has a moderating effect on the structure and abundance of gut microbiota,and could inhibit the increase in the abundance of pathogenic bacteria such as Deferribacterota,Fibrobacterota,MBNT15,Proteobacteria,Actinobacteriota and the decrease in the abundance of beneficial bacteria such as Faecalibacterium and Prevotellaceae induced by high-fat diet.It had an ameliorating effect on the high-fat diet-induced increase in the ratio of Firmicutes to Bacteroidetes(F/B value)in rats.The correlation analysis between gut microbiota and antioxidant indexes revealed that Prevotellaceae＿NK3B31＿group,NK4A214＿group,Prevotellaceae＿UCG＿001 were significantly and positively correlated with Catalase(CAT)and Glutathione(GSH)(P<0.05),and negatively correlated with malondialdehyde(MDA)in liver(P<0.05).Alistipes showed significant negative correlation with NO and SOD in liver(P<0.05).Alloprevotella showed significant positive correlation with SOD(P<0.05),CAT(P<0.01),GSH(P<0.01)and NO(P<0.05),and significant negative correlation with MDA(P<0.01)in liver.Prevotellaceae＿NK3B31＿group,NK4A214＿group,Prevotellaceae＿UCG＿001 showed significant positive correlation with blood glutathione peroxidase(GSH-Px),glutathione S-transferase(GST)and total antioxidant capacity(T-AOC)(P<0.05),and significant negative correlation with blood MDA(P<0.05).Alistipes showed a significant negative correlation with blood CAT and SOD(P<0.05).Alloprevotella showed a significant positive correlation with blood GSH-Px(P<0.01),CAT(P<0.05),SOD(P<0.05),GST(P<0.01)and T-AOC(P<0.01),and a significant negative correlation with blood MDA(P<0.01).Ferulic acid regulated metabolic pathways such as protein digestion and absorption,cholesterol metabolism,glutathione metabolism,biosynthesis of unsaturated fatty acids,and lipolysis.The correlation analysis between metabolites and antioxidant indexes revealed that Linoleic acid was significantly and positively correlated with SOD and NO(P<0.01),Uridine showed a significant negative correlation with CAT and GSH(P<0.01),and a significant positive correlation with MDA(P<0.01),Allantoin showed a significant positive correlation with CAT and GSH(P<0.01),and a significant negative correlation with MDA(P<0.01).Docosahexaenoic Acid,Acetylcarnitine and Thymidine were significantly and positively correlated with SOD and NO(P<0.05).Sedoheptulose 1,7-bisphosphate showed significant negative correlation with CAT and GSH(P<0.05),and significant positive correlation with MDA(P<0.05)in liver.LPC 22:5 in blood was significantly and negatively correlated with GSH-Px(P<0.01),CAT(P<0.05),SOD(P<0.05),GST(P<0.01)and T-AOC(P<0.01),and significantly positively correlated with MDA(P<0.01).(±)13-HODE and Orotic Acid was significantly and positively correlated with MDA(P<0.05),and negatively correlated with GSH-Px,GST and T-AOC(P<0.05).Vitamin B2 showed a significant positive correlation with CAT and SOD(P<0.05)in blood.Conclusion: Ferulic acid not only had lipid-lowering and weight-reducing effects,but also significantly improved liver function in rats.Ferulic acid could also improve the ability of the liver to combat oxidative stress by regulating the Keap1/Nrf2/ARE antioxidant pathway,upregulating the expression of antioxidant pathway-related mRNAs,and enhancing antioxidant enzyme activity.In addition,ferulic acid improved the dysbiosis of gut microbiota in rats induced by high-fat diet,and the gut microbiota.Prevotellaceae＿NK3B31＿group,NK4A214＿group,Prevotellaceae＿UCG＿001,and Alloprevotella were significantly and positively correlated with antioxidant capacities of liver and blood.Alistipes was significantly and negatively correlated with antioxidant capacities of liver and blood.Ferulic acid could regulate the metabolic pathways such as glutathione metabolism and lipolysis,and the metabolites Linoleic acid,Allantoin,Docosahexaenoic Acid,Acetylcarnitine and Thymidine showed significant positive correlation with hepatic antioxidant capacity.Uridine and Sedoheptulose 1,7-bisphosphate showed significant negative correlation with hepatic antioxidant capacity,the metabolite Vitamin B2 was significantly and positively correlated with blood antioxidant capacity,LPC 22:5,(±)13-HODE,and Orotic Acid were significantly and negatively correlated with blood antioxidant capacity.