茵陈蒿汤主成分治疗代谢相关脂肪性肝病的配伍剂量优化及作用机制实验研究OA

Objective:To investigate the optimal dose ratio and mechanisms of the primary active components in Yinchenhao decoction(geniposide,chlorogenic acid,and rhubarb polysaccharides)for ameliorating metabolic associated fatty liver disease(MAFLD).Methods:C57BL/6 mice were randomly divided into the normal control group,model control group,uniform design groups 1-6,and Yinchenhao decoction group;except for the normal control group,mice in all other groups were fed Western diets to establish MAFLD models,and after eight weeks of modeling,mice in the uniform design groups 1-6 and Yinchenhao decoction group were given the corresponding drugs by gavage.At 12 weeks,all mice were sacrificed:their body weight and liver weight were measured,hematoxylin-eosin staining was used to observe the histopathological changes of liver tissues,the plasma levels of alanine transaminase(ALT)and aspartate transaminase(AST)were measured,and the levels of total cholesterol(TC)and triglycerides(TG)in plasma and liver were measured.Based on these results,the optimal uniform design group was identified;subsequently,with plasma AST,plasma TG,and liver TC levels as screening indicators,the optimal dose ratio was obtained via a regression equation,which was further verified from functional indicators and tissue morphology.Meanwhile,glucose tolerance test and insulin tolerance test were conducted to evaluate glucose metabolic homeostasis and insulin sensitivity in mice,periodic acid-Schiff staining was used to observe glycogen accumulation,quantitative reverse transcription-polymerase chain reaction was employed to detect the Mrna expression of genes related to glycolipid metabolism and bile acid metabolism,Western blotting was performed to measure the protein expression of molecules involved in bile acid metabolism,and commercial kits were used to determine the plasma levels of total bilirubin(TBIL),direct bilirubin(DBIL),and total bile acid(TBA).Results:Combinations of geniposide,chlorogenic acid,and rhubarb polysaccharide all reduced the liver-to-body weight ratio,alleviated liver injury,and decreased lipid accumulation,among which the uniform design group 6(200 mg/kg geniposide+160 mg/kg chlorogenic acid+340 mg/kg rhubarb polysac-charide)exhibited the optimal efficacy.Meanwhile,regression analysis indicated that the dosage ratio of uniform design group 6 was the optimal one for MAFLD intervention.Validation experiments showed that,compared with single-drug intervention,the optimal dosage ratio resulted in significantly lower body weight,as well as lower plasma levels of ALT,AST and TC in mice(all P<0.05),along with a more pronounced reduction in the area of hepatic lipid accumulation.Mechanistic investigation experiments demonstrated that intervention with the optimal dosage ratio significantly improved glucose tolerance and insulin sensitivity in mice(all P<0.05),reduced hepatic glycogen accumulation,and downregulated the Mrna expression of glycolipid metabolism-related genes such as Gsk3,G6pc,Pck1,Fbp1,Fasn,Srebp-1c,Scd1,Slc27a2,and Slc27a5(all P<0.05);it also decreased plasma levels of TBIL,DBIL,and TBA(all P<0.05),restored the dysregulated protein expression of bile salt export pump(BSEP),farnesoid X receptor(FXR),and cytochrome P450 family 7 subfamily A member 1(CYP7A1)in the liver(all P<0.05),and reversed the abnormal Mrna expression of bile acid metabolism-related genes including Nr1h4,Cyp7a1,Cyp27a1,Slc10a1,and Slco1a1(all P<0.05).Conclusion:The combination of geniposide(200 mg/kg),chlorogenic acid(160 mg/kg),and rhubarb polysaccharide(340 mg/kg)exerts the optimal ameliorative effect on MAFLD in mice.This superior efficacy is presumably achieved by synergistically regulating the key nodes of glucose,lipid and bile acid metabolism.