The Studies On Chemical Constitiutions And The Action Process In Vivo Of Traditional Chinese Medicine Tangshen Formula

Diabetic kidney disease(DKD)is one of the main and serious microvascular complications of diabetes(Diabetes mellitus,DM).Effective treatment of DKD has become a major problem in the world’s public health.The traditional Chinese medicine is a combination medicine,which has the characteristics of multiple components,multiple targets,multiple pathways and comprehensive curative effects.It is a potential new measure for effective treatment of DKD.Preliminary clinical trials have shown that the Tangshen formula can effectively treat DKD;multiple basic studies have also confirmed that Tangshen formula has the effects of anti-inflammatory,anti-fibrosis,lowering blood lipids,protecting podocytes,and lowering urine protein.It also passes AMPK-PPARa,NF-κB,TGF-β/Smad3,AMPK/SIRT1,TRPC6/Talinl and other pathways.However,the medicinal components of Tangshen formula and their relevent mechanisms of action are not clearly,and the in vivo action is still unclear too.Aims:To investigate effect study and compare with two water extraction processes of Tangshen formula in the treatment of DKD,and further to reveal its medicinal ingredients,related mechanism of action and their in vivo action process.Methods:1 Eight-week-old male KKAy mice were randomly divided into 3 groups,namely model group(KKAy)(n=7),KKAyTSFl group(combined secondary water extraction process,n=7),KKAyTSF2 group(combined three water extraction process,n=7),eight-week-old male C57BL/6J mice(n=7)were used as the normal control group.The normal control group mice were fed with regular ordinary diet,and all KKAy mice were fed with exclusive KK high-fat diet.The two different water-extraction process of Tangshen formula were started to intervene KKAy mice for 16 weeks.And the body weight,fasting blood glucose(FBG),urine albumin and urine creatinine(UACR)levels were examined respectively;The automatic biochemical analyzer was used to detect serum urea(BU)and blood creatinine(SCr);The PAS and HE staining were used to observe renal pathology.2 The UHPLC-Q-Orbitrap HRMS technology was used to screen the chemical compositions in Tangshen formula,and UHPLC-MS/MS technology was used to determine the content of active ingredients in two different water-extraction process of Tangshen formula.3 UHPLC-MS/MS technology was used to screen the blood components of Tangshen formula;based on blood components,network pharmacology is used to construct a drug-target-pathway network to obtain the effective components and related pathways of Tangshen formula in the treatment of DKD.And based on the three topological parameters of Betweenness Centrality,Closeness Centrality and Degree,it screens important medicinal ingredients and action pathways for Tangshen formula in the treatment of DKD.4 UHPLC-Q-Orbitrap HRMS technologyas used to detect the serum components in KKAy model group,KKAyTSF1 group and C57BL/6J mice,screen out the potential biomarkers of DKD through metabolomics technology,and combine network information and literatures to explore the relevance pathways of Tangshen formula in the treatment of DKD.5 Male SD rats were randomly divided into the experimental group(n=6)and the blank group(n=3).They were given 4 g/kg Tangshen formula e combined with the secondary water extraction process for intragastric administration.The blood was collected from the tail vein in 5min,10 min,20 min,30 min,40 min,50 min,1 h,2 h,4 h,6 h,8 h,10 h,12 h,24 h time points.UHPLC-MS/MS technology was used to determine the contents of the 20 pharmacokinetic components of Tangshen formula in plasma,and the pharmacokinetic parameters of Tangshen formula’s pharmacokinetics were calculated.Results:1 Compared with the three-time water extraction process,the Tangshen formula of the second water extraction process significantly reduced the UACR level and improved the pathological conditions of the renal tubule interstitium more significantly in of KKAy mice.2 It was successfully screened and identified 85 chemical components;And in the Tangshen formula of the second water extraction process,the contents of sweroside,calycosin-7-glucoside,verbascoside,neoeriocitrin,hyperoside,hesperidin,hesperetin,neohesperidin,ginsenoside Rg1,daidzein,poncirin,calycosin,rhein,naringenin,genistein,astragaloside Ⅳ,formononetin,aloe-emodin,and emodin were higher than those in combined three water extraction processes.3 Based on blood component and network pharmacology research,Tangshen formula has 20 medicinal ingredients in the treatment of DKD,including 7 important medicinal ingredients such as naringin,daidzein,genistein,formononetin,aloe-emodin,hesperetin and ginsenoside Rg1.And there are 30 overall mechanisms of action of Tangshen formula in the treatment of DKD,including 7 important signaling pathways,such as HIF-1 signaling pathway,TNF signaling pathway,AMPK signaling pathway,PI3K-Akt signaling pathway,Toll-like Receptor signaling pathway,NOD-like receptor signaling pathway,and insulin signaling pathway.4 Based on metabolomics,Tangshen formula may act on the metabolic pathways of unsaturated fatty acids,amino acids,and tryptophan signaling pathway.5 AUC0-t of effective components in Tangshen formula were ranged from 38 to 684 ng/mL*h and its T1/2 were ranged from 1 to 9 h.Conclusions:The combined secondary water extraction process is more significant than the combined three water extraction process for Tangshen formula in improving the kidney damage in KKAy mice.It is likely to be related to the content of sweroside,calycosin-7-glucoside,verbascoside,neoeriocitrin,hyperoside,hesperidin,hesperetin,neohesperidin,ginsenoside Rg1,daidzein,poncirin,calycosin,rhein,naringenin,genistein,astragaloside Ⅳ,formononetin,aloe-emodin,and emodin.The ginsenoside Rg1,naringin,daidzein,genistein,formononetin,aloe-emodin,aloe-emodin,and orange peel are the key active compounds among the 20 medicinal ingredients of Tangshen formula in the treatment of DKD.Among the 30 action pathways of Tangshen formula in the treatment of DKD,HIF-1 signaling pathway,TNF signaling pathway,AMPK signaling pathway,PI3K-Akt signaling pathway,Toll-like receptor signaling pathway,NOD-like Receptor signaling pathways and insulin signaling pathways are important pathways;In addition,Tangshen formula may act on the metabolic pathways in unsaturated fatty acids,amino acids,and tryptophan;And AUC0-t of effective components in Tangshen formula were ranged from 38 to 684 ng/mL*h and its T1/2 were ranged from 1 to 9 h,which is likely to provide reference for clinical reasonable dosing plan.