Study On The Improvement Of Polygonum Multiflorum Thunb.on Non-alcoholic Fatty Liver Disease

With the increase of people’s standard of living,the incidence of Non-alcoholic fatty liver disease(NAFLD)is increasing rapidly.Recently,the prevalence of NAFLD is as high as 26%-45% in China,ranking first in all chronic liver diseases.There is little selection of drugs for NAFLD,so it is urgent to find more safe and effective drugs for NAFLD treatment.Traditional Chinese medicine(TCM)is the treasure of Chinese medicine,and plays a crucial role in the prevention and treatment of a variety of chronic diseases.Previous studies have found that TCM has a huge advantage in the treatment of NAFLD.Polygonum Multiiflori Radix(PMR)is the dry root of Polygonum multiflorum Thunb..PMR has the effect of detoxification,relieving carbuncle,intercepting malaria and relaxing bowel.The processed product of PMR is Polygoni Multiflori Radix Praeparata(PMRP),which has many effects including nourishing liver and kidney,boosting essence blood,darkening hair and beard,strengthening muscles and bones,and lowering turbidity and lipid.This study aims to investigate the improvement of Polygonum multiflorum Thunb.on NAFLD.Firstly,the water and ethanol extracts of both PMR and PMRP were prepared.The contents of 2,3,5,4′-tetrahydroxy-stilbene-2-O-β-D-glucoside(TSG),emodin and physcione in those above four extracts were detected by using high performance liquid chromatography(HPLC).It was found that TSG,emodin and physcion were the main chemical constituents,and the content of TSG was higher than the content of emodin and physcione in those above four extracts.Secondly,the amelioration of PMR and PMRP on NAFLD was investigated in vivo.The improvement of those above four extracts of PMR and PMRP on NAFLD in rats or mice induced by high-fat diet(HFD)or methionine-choline-deficent diet(MCD).The results demonstrated that the amount of liver triglyceride(TG)and total cholesterol(TC)were increased and hepatic lipid accumulation appeared inHFD-induced NAFLD in rats.But,the water and ethanol extracts of both PMR and PMRP improved all those above phenomena.In MCD-induced NAFLD in mice,serum alanine/aspartate aminotransferase(ALT/AST)activity,hepatic TG and TC amount were increased obviously,but the water and ethanol extracts of both PMR and PMRP reduced all these elevations.Results of liver histological evaluation and oil red O staining showed liver steatosis in MCD-induced NAFLD in mice,but the water and ethanol extracts of both PMR and PMRP alleviated those above pathological alternations.All those results suggest that the water and ethanol extracts of both PMR and PMRP alleviated the hepatic lipid accumulation induced by HFD in rats and MCD in mice,and they also improved liver injury induced by MCD in mice.Next,it was investigated whether there were differences between the improvement of Polygoni Multiflori Radix water extract(PMRWE)and Polygoni Multiflori Radix Praeparata water extract(PMRPWE)on NAFLD.It was found that PMRPWE had a better activity than PMRWE on ameliorating cellular lipid accumulation induced by 0.5 m M oleic acid/palmitic acid(OA/PA)(2:1)in human normal liver L-02 cells.Serum ALT/AST activity,liver reactive oxygen species(ROS)level,hepatic content of TG,TC and non-esterified fatty acids(NEFA)were all increased in HFD-induced NAFLD in rats.PMRWE(70,140,280 mg/kg)and PMRPWE(70,140,280 mg/kg)reduced the elevated serum ALT/AST activity,hepatic TG and TC amounts,and liver ROS level in HFD-induced NAFLD in rats.Moreover,PMRPWE showed a better improvement on reducing hepatic ROS formation in rats than PMRWE.PMRWE(280 mg/kg)and PMRPWE(70,140,280mg/kg)reduced the elevated hepatic NEFA content in HFD-induced NAFLD in rats.Results of liver histological evaluation and oil red O staining showed that PMRWE and PMRPWE alleviated liver steatosis in HFD-induced NAFLD in rats.All these results revealed that both PMRPWE and PMRWE reduced the elevated liver steatosis and oxidative stress injury in HFD-induced NAFLD in rats,and the improvement of PMRPWE was better than PMRWE.Serum ALT/AST activity,liver contents of TG and NEFA,and liver ROS level were all enhanced in MCD-induced NAFLD in mice.PMRWE(100,200 mg/kg)and PMRPWE(50,100,200 mg/kg)decreased the elevated serum ALT/AST activity,the enhanced hepatic contents of TG and NEFA,and hepatic formation of ROS in MCD-induced NAFLD in mice.But PMRWE(50mg/kg)had no effect.Results of liver histological evaluation and oil red O staining showed that PMRWE and PMRPWE alleviated liver steatosis in MCD-induced NAFLD in mice.In summary,both PMRWE and PMRPWE had protection against MCD-induced NAFLD in mice,and the effect of PMRPWE was better than PMRWE.Further results of Real-time PCR analysis showed that PMRWE and PMRPWE reversed the reduced expression of genes related with the β-oxidation of fatty acids in both HFD-induced NAFLD in rats and MCD-induced NAFLD in mice.These results suggest that the β-oxidation of fatty acids in the mitochondria might be involved in the protection of PMRWE and PMRPWE against NAFLD.Fianlly,0.5 m M OA/PA(2:1)was used to induce cellular lipid accumulation in human normal liver L-02 hepatocytes.The inhibition of cellular lipid accumulation provided by various compounds in PMRWE and PMRPWE was further detected in vitro.It was found that TSG,resveratrol,emodin and physcion all reduced cellular lipid accumulation induced by OA/PA(2:1)in hepatocytes.In conclusion,the water or ethanol extract of PMR and PMRP all improved NAFLD induced by HFD in rats or MCD in mice.The results of experiments in vivo and in vitro suggest that PMRPWE had better protective effects against NAFLD than PMRWE.The improvement of PMRWE and PMRPWE on NAFLD may be due to the improvement of β-oxidation of fatty acids in mitochondria.The experiment in vitro implies that TSG,resveratrol,emodin and physcion may be the main active ingredients in PMRWE and PMRPWE for alleviating NAFLD.