The Study On The Genetic Influence Of Hujin Dcoction On AMPK/mTOR Pathway In MAFLD Mice

Objectivemetabolic(dysfunction)associated fatty liver disease(MAFLD)is a metabolic stress liver injury closely related to insulin resistance and genetic susceptibility.It is a clinical pathological syndrome characterized by diffuse hepatic bullae fatty degeneration with or without inflammation.Hujin dcoction has been used in clinical practice for many years and has definite curative effect on MAFLD.It is beneficial to detoxify dampness and invigorate the body by removing blood stasis,as well as to improve the treatment of endogenous phlegm-dampness,fatty liver caused by qi stagnation and blood stasis,hepatitis and cholecystitis.Preliminary basic studies have shown that this method can improve the blood lipid and liver function of MAFLD model mice,as well as the pathological conditions of liver tissues and the ultrastructure of liver cells.In this paper,through the investigation of different wavelength,temperature,chromatographic column and mobile phase,the HPLC fingerprint of Hujin dcoction was established,and the content of its active components was determined.The aim is to establish an effective standard for the quality control model of Hujinfang,and to evaluate its quality stability and uniformity.Hujin can reduce obesity in MAFLD mice,improve liver injury,and reduce blood cholesterol and triglyceride levels in the liver.In addition,the Sirtl/PGC-1/PPARpathway is used to increase the rate-limiting enzyme activity related to fatty acid oxidation,so as to increase fatty acid oxidation and alleviate the fatty acid deposition in the liver.On this basis,we continued to investigate the genetic influence of Tigojin prescription on AMPKmTOR pathway in MAFLD mice,and further elaborated the reversal effect of Tigojin prescription on MAFLD.Methods1.Establishment of fingerprint and determination of active components of Hujin dcoction.Ultra pure water was used to extract Tigris square in a remote proportion,and the mixture was heated and refluxed for two times.The mixture was decompressed and placed in a vacuum drying oven to obtain Tigris square water extract powder.After quantitative treatment with methanol,test samples were obtained.The chromatographic conditions were determined by investigating the wavelength,mobile phase,flow gradient,temperature and chromatographic column.Chromatographic analysis was carried out by using the similarity evaluation software of TRADITIONAL Chinese medicine chromatographic fingerprint,and THE HPLC fingerprint of the gold formula samples of the water-extracted tiger was established.The contents of Polygonin,resveratrol,Alisthenol A,sanqi saponin R1,Ganoderic acid B,hawthorn acid and curcumin in Hujin dcoction were determined by HPLC.2.The genetic effects of Hujin dcoction on AMPK/mTOR pathway in MAFLD mice.In this section,50 SPF mice C57BL/6 mice were randomly assigned after adaptive feeding with high-fat diet.Mice in the control group were fed with low-fat diet,and mice in the model group and the low,medium and high dose Tigris formula groups were fed with high-fat diet for 8 weeks.After sampling,fluorescence quantitative PCR analysis of ACC,FAS,mTOR,S6K1,SCD1,and LXRa were performed.Results1.HPLC fingerprint of Hujin dcoction was established:through similarity evaluation system(2004A version),the optimal fingerprint conditions were:Welch Ultimate XB C18250×4.6mm,5 m;DAD detector with detection wavelength of 214nm;The velocity of 1 ml/min;The column temperature was 30℃ and the injection volume was 5 L.Mobile phase A is 0.2%phosphoric acid and mobile phase B is methanol.2.HPLC-DAD method was used to determine the content of 7 components in Hujin dcoction,with good separation degree and good linear relationship(R>The RSD of repeatability,stability and precision were all lower than 3.0%,the average recovery rate was 96.27%-102.33%,and the RSD was 0.33%-2.84%.3.The gene expression levels of fatty acid synthase(FAS),rapamycin target protein(mTOR),ribosome S6 kinase 1(S6K1),stearyl coenzyme A dessaturated enzyme 1(SCD1)and liver X receptor(LXR)were significantly higher in the model group and the group with low,medium and high doses of Tigricin formula than in the normal group.The gene expression level of Acetyl-Coa carboxylase(ACC)was significantly increased in the normal group and the model group,as well as in the low,medium and high dose groups.ConclusionIt is possible that Hujin dcoction can alleviate lipid deposition and improve lipid metabolism by regulating ACC.,mTOR,FAS,S6K1,SCD1,LXRa and other genes in AMPK/mTOR pathway.