Study On The Mechanism Of Ellagic Acid Ameliorates Hepatic Steatosis In Mice And The Preparation Of Urolithin A Liposomes

Object: In recent years,with changes of people's living habits and dietary structure,the incidence of nonalcoholic fatty liver disease(NAFLD)is increasing year by year and showing a trend of younger.Therefore,it is an urgent problem to explore effective methods for the prevention of NAFLD and elucidate its mechanism.Ellagic acid is a natural polyphenolic compound widely found in Punica granatum L.,Rhus chinensis Mill.,Sanguisorba officinalis L.,Phyllanthus emblica Linn.,and other Chinese herbal plant tissues,which has the effect of anti-oxidation and improving metabolic syndrome,suggesting that ellagic acid has the potential to prevent NAFLD.Ellagic acid has a low bioavailability and it is mainly metabolized as urolithins in the gastrointestinal tract of mammals.However,the role and mechanism of ellagic acid and its intestinal metabolites in the prevention of NAFLD have not been fully elucidated.Therefore,in this study,hydrodynamic transfection was used to establish hepatic steatosis model induced by AKT-overexpression,studing the the improvement and molecular mechanism of ellagic acid on AKT-overexpression induced hepatic steatosis in mice.Meanwhile,in vitro cell experiments confirmed the mechanism of regulating fatty acid metabolism by urolithin A,the main metabolite of ellagic acid.However,the short half-life of oral administration and low bioavailability limit the development and application of urolithin A.Hence,to overcome this limitation,a stable and long-circulation urolithin A liposome was prepared by modern pharmacy technology in this study.The purpose of this study was to provide a preclinical basis for the conversion of ellagic acid into urolithins to improve hepatic steatosis and the development of a new formulation of urolithin A.Methods: 1.The effect and mechanism of ellagic acid ameliorates hepatic steatosis induced by AKT-overexpression in mice 1.1 The effect of ellagic acid ameliorates hepatic steatosis induced by AKT-overexpression in mice Hydrodynamic transfection was used to establish hepatic steatosis model of wild-type FVB/N mice induced by AKT-overexpression.Three days after hydrodynamic transfection with AKT,healthy FVB/N mice were used as control group.Low and high doses(150 and 300 mg/kg)of ellagic acid were administered to the drug group for 5 weeks.After administration,liver histomorphological changes were observed in each group.H&E staining and oil red O staining were used to observe liver histopathological changes in mice.Serum alanine aminotransferase(ALT),aspartic aminotransferase(AST),triglyceride(TG),total cholesterol(T-CHO),low density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)were measured.1.2 The mechanism of ellagic acid ameliorates hepatic steatosis induced by AKT-overexpression in mice Western Blotting,real-time fluorescence quantitative PCR and immunohistochemistry were used to detect the effect of ellagic acid on the expression levels of key gene proteins or m RNAs of t-AKT,p-AKTThr308,p-RPS6,SREBP-1c,FASN and ACC in liver tissues.Meanwhile,the effects of ellagic acid on the expression levels of inflammatory factors such as NF-?B?TNF-??IL-6 and COX-2 in liver tissues of mice were detected.2.Study on the effect and mechanism of urolithin A,the main metabolite of ellagic acid,on ameliorating steatosis 2.1 Urolithin A ameliorates steatosis induced by oleic acid in hepatoma cell lines The steatosis model of Hep G2 and Huh-7 cells was induced by oleic acid(1.0 mmol/L).After intervention with different concentrations of urolithin A,oil red O staining was used to observe the lipid accumulation in cells.The TG content in each group was detected by the kit.2.2 The mechanism of urolithin A ameliorates steatosis of hepatoma cell lines First,Western Blotting was used to detect the effect of urolithin A on the protein expression levels of p-AKTThr308,SREBP-1c and FASN genes related to the de novo lipogenesis in Hep G2 and Huh-7 cells induced by oleic acid.Subsequently,hepatoma cells were transfected with AKT plasmid to simulate the animal experimental environment,confirming the regulatory effect of urolithin A on AKT mediated de novo lipogenesis.3.Preliminary study on preparation and physicochemical properties of urolithin A-long circulating liposomes Urolithin A-long circulating liposomes were prepared by the thin film dispersion method.The encapsulation efficiency and particle size were used as evaluation indexes to investigate the effects of different ultrasonic conditions,the weight ratio of urolithin A to soy phosphatidylcholine,the weight ratio of soy phosphatidylcholine to cholesterol,and the m PEG2000-DSPE dosage on the preparation of urolithin A-long circulating liposomes.The stability of urolithin A-long circulating liposomes during was investigated,and the release behavior,cellular uptake,cytotoxicity,and pharmacokinetics of free urolithin A,urolithin A liposomes and urolithin A-long circulating liposomes were also investigated.Results: 1.Five weeks of hydrodynamic transfection of AKT can induce hepatic steatosis with significant increase in liver weight,severe lipogenesis,and substantial lipid accumulation in mice.After administration of ellagic acid(150,300 mg/kg),it can significantly reduce the degree of hepatic lipidization and lipid accumulation,and ameliorate the levels of liver function(ALT,AST)and lipid levels(TG,T-CHO,LDL-C and HDL-C)in mice.2.Ellagic acid significantly reduced the expression levels of p-AKTThr308,SREBP-1c,FASN and ACC genes related to de novo lipogenesis and inflammation-related factors such as NF-?B,TNF-?,IL-6 and COX-2 in mice.3.Urolithin A significantly ameliorated oleic acid(1.0 mmol/L)induced steatosis of Hep G2 and Huh-7 cells and reduced the content of TG.4.Urolithin A significantly reduce the expression levels of key genes involved in de novo lipogenesis,such as p-AKTThr308,SREBP-1c and FASN in the models of Hep G2 and Huh-7 cells induced in oleic acid-induced and AKT transfected.5.Urolithin A-long circulating liposomes was prepared and its preparation technology was optimized.The particle size,polydispersion index,encapsulation efficiency and Zeta potential of urolith A-long circulating liposomes were 122.8 ± 7.4 nm,0.245 ± 0.16,94.6 ± 1.6%,-25.5 ± 2.3 m V,respectively.The urolithin A-long circulating liposomes was spherical and uniform in shape,with good dispersion,and could be stored stably for 30 days at 4?.The total release of free urolithin A,urolithin A liposomes and the urolithin A long circulating liposomes in vitro at 48 h were 85.2± 4.9%,57.7 ± 3.1% and 47.2 ± 5.7%,respectively.Compared with free urolithin A,urolithin A-long circulating liposomes can significantly increase the cellular uptake and cytotoxicity of hepatoma cells.Additionally,urolithin A-long circulating liposomes was able to significantly reduce the plasma clearance rate of urolithin A and prolong the half-life to improve the bioavailability of urolithin A in rats.Conclusions: Ellagic acid can ameliorate the hepatic steatosis induced by the overexpression of AKT in mice,and the mechanism may be that ellagic acid converted to urolithin A,which regulates the AKT/SREBP-1c/FASN signaling pathway and inhibits the de novo lipogenesis,reversing lipid deposition in liver cells.Urolithin A-long circulating liposomes displayed high-entrapment efficiency,small size and increased stability.Compared with free urolithin A,urolithin A-long circulating liposomes substantially increase the cellular uptake of urolithin A through the endocytosis and exhibit elevated cytotoxicity in human hepatoma cells.Moreover,urolithin A-long circulating liposomes showed the preferable sustained release effect for urolithin A in vivo.Altogether,these results suggest that urolithin A-long circulating liposomes might be a potential formulation for the clinical application of urolithin A.