Study On Pharmacodynamic Substances And Mechanism Of Swertia Mileensis In Improving Nonalcoholic Fatty Liver Disease

Objective: Swertia mileensis is a dry whole grass of Swertia in Gentianaceae.It has the effects of clearing liver and biliary,clearing heat and reducing humidity,and is often used for the treatment of liver diseases.It has been reported in the literature that the active components of Swertia mileensis,such as iridoid glycosides and xanthones,have the effect of improving nonalcoholic fatty liver disease(NAFLD).However,few studies have been conducted on the improvement of NAFLD by Swertia mileensis.Therefore,this paper aims at the regulation of Swertia mileensis on lipid metabolism in NAFLD mice induced by high fat diet,especially the regulation of triglyceride metabolism,so as to clarify the pharmacodynamic substances and mechanism of action of Swertia mileensis,and provide theoretical and experimental basis for the clinical application of Swertia mileensis.Methods:(1)The whole dried grass of Swertia mileensis in Mile city of Yunnan Province was collected,and the extract of Swertia mileensis(SME)was obtained by 70% ethanol heating reflux.The content of gentiopicroside(GPS)and demethylbellidifolin(DMB)in the SME was detected by HPLC based on the literature reports and the previous laboratory testing conditions.(2)NAFLD model of C57BL/6 mice induced by high fat diet.The total extract of Swertia mileensis was given by oral administration at doses of 100 mg/kg,200 mg/kg and 400 mg/kg for 3 months.After the administration,biochemical indexes in serum and liver of mice were detected.The efficacy of the total extract of Swertia mileensis was determined by hematoxylin-eosin staining(H&E)and oil red staining on liver tissues.Quantitative real-time reverse transcription PCR(q-RT-PCR)and western blot(WB)were used to detect the transcription and protein expression of genes related to lipid metabolism in the liver,and the potential mechanism of improving NAFLD by Swertia mileensis was preliminarily determined.(3)A vitro model of lipid accumulation was established by inducing Hep G2 cells with sodium oleate.The cells were given total extract of Swertia mileensis and gentiopicroside for 24 h to detect triglyceride(TG)and total cholesterol(TC)for evaluating the lipid-lowering effect of Swertia mileensis and gentiopicroside in vitro.Furthermore,WB was used to detect the transcription and protein expression levels of gentiopicroside on genes related to lipid metabolism,and to clarify the mechanism of action Swertia mileensis on NAFLD.Results:(1)The content of gentiopicroside in the total extract of Swertia mileensis in Mile City,Yunnan Province was 4.28 mg/g and the content of DMB was 9.79 mg/g by using HPLC.(2)After the detection of serum and liver biochemical indexes,it was found that,compared with the normal group,TG and TC in the liver of the model group were significantly increased,and TC,alanine amino transferase(ALT),aspartate amino transferase(AST)and low density lipoprotein cholesterol(LDL-C)in the serum were significantly increased.TG in Serum increased,but there was no statistically significant difference,and high density lipoprotein cholesterol(HDL-C)in serum did not significantly change.Compared with the model group,the drug administration group with the total extract of Swertia mileensis significantly reduced the contents of TG and TC in the liver of the mice,decreased the contents of TG,ALT,AST and LDL-C in the serum,and increased the contents of HDL-C in the serum.The liver staining results of H&E and oil red O showed that the total extract of Swertia mileensis could significantly reduce the lipid accumulation in the liver of NAFLD mice,improve the hypertrophy of hepatocytes,and alleviate liver injury.It has been shown initially that total extract of Swertia mileensis could improve lipid accumulation and liver damage and protect the liver.Further researches were conducted on the mechanism of action of Swertia mileensis.The q-RT-PCR results showed that the total extract of Swertia mileensis could significantly increase the gene transcription levels of carnitine palmitoyl transferase 1(CPT-1),hormone sensitive lipase(HSL)and adipose triglyceride lipase(ATGL)in mice liver tissue,and decrease the gene transcription levels of sterol regulatory element binding protein 1c(SREBP-1c)and fatty acid synthase(FAS).WB results showed that the total extract of Swertia mileensis significantly up-regulated the protein expression of silent information regulator 1(SIRT-1),adenosine monophosphate activated protein kinase(AMPK),p-AMPK,phosphorylated acetyl-Co A carboxylase(p-ACC)/ACC,CPT-1,HSL,p-HSL and ATGL and down-regulated the protein expression of SREBP-1c,FAS and stearyl coenzyme A desaturase 1(SCD-1).(3)By detecting the TG and TC in the lipid accumulation model of Hep G2 cells after drug administration,it was found that the contents of TG and TC in the model group cells significantly increased compared with those in the normal group.Compared with the model group,total extract of Swertia mileensis and gentiopicroside could significantly reduce the contents of TG and TC in Hep G2 cells induced by sodium oleate.The results of oil red O staining showed that the total extract of Swertia mileensis and gentiopicroside could significantly improve the accumulation of lipid droplets in Hep G2 cells induced by sodium oleate.WB results showed that gentiopicroside significantly up-regulated the protein expression of SIRT-1,AMPK,p-AMPK,p-ACC /ACC,p-HSL /HSL and CPT-1,and down-regulated the protein expression of SREBP-1c,FAS and SCD-1.Conclusions: The total extract of Swertia mileensis can significantly regulate lipid metabolism in NAFLD mouse model,improve NAFLD and protect the liver.Its mechanism of action may be to reduce fat synthesis,promote fat oxidation and hydrolysis,and improve NAFLD by activating AMPK/SIRT-1/SREBP-1c,AMPK/ACC/CPT-1 and AMPK/ATGL pathways-related gene transcription and protein expression.This study provides a scientific basis for the application of Swertia mileensis in the development of new drugs and rational clinical use for NAFLD.