Study On The Material Basis And Mechanisms Of Taohe-chengqi Decoction Against Renal Fibrosis

Objective:Taohe-Chengqi Decoction,a classic formula recorded in“Treatise on Febrile Diseases”,has been widely prescribed for different chronic kidney diseases with definite curative effect.At present,the research on Taohe-Chengqi Decoction is mostly focused on the clinical efficacy,but the researches on material basis and mechanisms are scarce.Based on the clinical effect,we integrated the interdisciplinary research methods of evidence-based medicine,traditional Chinese medicine chemistry,network pharmacology,traditional Chinese medicine pharmacology,molecular biology and analytical chemistry.Then,the meta-analysis of Taohe-Chengqi Decoction in the treatment of chronic renal failure was carried out,the effective substances of Taohe-Chengqi Decoction against renal fibrosis were selected,and the main components of the effective part were analyzed and identified.In addition,the pharmacodynamics and mechanisms of the effective components were studied in vitro and in vivo based on network pharmacology analysis.Finally,the mechanism of action,material basis and clinical efficacy evaluation of Taohe-Chengqi Decoction were expounded systematically,which will provide scientific basis for its further development and utilization.Methods:1 Meta-analysis of Taohe-Chengqi Decoction on the treatment of chronic renal failureDatabases such as CNKI,VIP,WanFang,CBMdisc,PubMed,EMbase and Medline were retrieved from the inception to January 2020.The randomized controlled trials?RCTs?of Taohe-Chengqi Decoction in the treatment of chronic renal failure were conducted.The quality of the study was evaluated according to Cochrane system evaluation method.Two evaluators independently selected the literature,data and evaluated the bias risk of the study according to the inclusion and exclusion criteria.RevMan 5.3 software was used for statistical processing of the extracted data.2 Extraction and separation of different polar parts of Taohe Chengqi DecoctionFour of Chinese herbal medicines?Persicae Semen,Radix et Rhizoma Rhei,Cinnamomi Ramulus,and Glycyrrhizae Radix et Rhizoma?in Taohe-Chengqi were homogenized and extracted with eightfold of 70%ethanol for 2 h and 1.5 h respectively.The ethanol extracts were concentrated to dryness under vacuum,diluted with water and then partitioned by petroleum ether,chloroform,ethyl acetate,and n-butanol successively.Natrii Sulfas?sodium sulfate?was directly treated as an extracted part.The entire solvent was evaporated under reduced pressure to obtain petroleum ether extract,chloroform extract,ethyl acetate extract,n-butanol extract.3 Study of screening the effective parts of Taohe-Chengqi Decoction against renal fibrosisThe HK-2 cell fibrosis model induced by TGF-?1 was used to intervene the cells in different parts of Taohe-Chengqi decoction with different concentrations.ELISA kit assay was used to detect Col-I?1 and FN in supernatant to screen the main active parts.CCK-8 method was used to determine the best concentration of intervention site of bioactive components.Western blot analysis was used to detect the expression level of Col-I,Col-III,MMP2,TIMP2,CTGF.Immunofluorescence assay was used to detect the expression of?–SMA.Real time PCR analysis was used to detect the expression of PAI-1 mRNA.4 Chemical composition analysis of the effective n-butanol extract in Taohe-Chengqi DecoctionUPLC-Q/TOF-MS/MS analysis was conducted using an analytical Welch Ultimate TM UPLC XB-C18 column?100 mm×2.1 mm,1.8?m?.The mobile phase A consisted of 0.1%formic acid in H₂O,and mobile phase B consisted of acetonitrile;the solvent gradient varied from 5%B in the first 25 min to 95%B up to 40 min at a flow rate of 0.3 mL/min;the column oven temperature was 40°C.The detection was performed at 210nm.The acquisition of high-resolution mass spectra was conducted in the positive and negative ion modes at the dry gas flow rate of 800 L/h and the gas temperature of 200°C.The capillary voltage was 30kV;the analysis was carried out using a scan from m/z 50 to 1500.5 Mechanism of n-butanol extract of Taohe-Chengqi Decoction?NE-THCQ?on anti-renal fibrosis based on network pharmacologyTCMSP was retrieved to obtain the OB and DL values of the 26compounds in NE-THCQ.OB?30%and DL?0.18 as thresholds to screen the potential active ingredients.The potential targets of NE-THCQ were predicted by SEA and SIB databases,and the diseases targets were retrieved from DisGeNet and NCBI database.Target protein-protein interaction?PPI?network topology parameters was constructed using String Version 10.5 database.Enrichment analysis of KEGG pathway was analyzed by using Cluego version 2.5.4 plug of Cytoscape software.The key targets were analyzed by the gene ontology?GO?enrichment using David 6.8 software.Cytoscape Version 3.6.0 software was used to construct the PPI network?active ingredient-key target network and the ingredient-target-signal pathway network.6 Study on the mechanisms of n-butanol extract in Taohe Chengqi Decoction?NE-THCQ?on anti-renal fibrosis?1?In vitro study:TGF-?1 induced HK-2 cells was constructed as renal fibrosis model in vitro.CCK8 assay was used to detect the viability of HK-2cells with the treat of NE-THCQ.Western blot analysis was used to detect the expression leves of?-SMA,E-cadherin,FN,and PI3K/AKT/mTOR and HIF-1?/VEGF signal pathway.Immunofluorescence was used to detect the expression of?-SMA,E-cadherin,FN and TNF–?.F-actin microfilament protein was observed by FITC-Phalloidin staining.?2?In vivo study:UUO rats was established by unilateral ureteral ligation as renal fibrosis model.36 SPF male Wistar rats were randomly divided into three groups:sham group,UUO group and NE-THCQ group.The rats were killed separately on 7th and 14th day after operation.Blood was collected to prepare serum samples,and kidneys were removed.Serum creatinine and urea nitrogen levels were detected.Renal pathological changes were observed by H&E staining,Masson staining and transmission electron microscopy.The contents of IL-6 and IL-1?in serum were detected by ELISA kit.Immunohistochemistry was used to detected the expressions of CoL-I,CoL-?,?-SMA and E-cadherin.The expression levels of PI3K/AKT/mTOR and HIF-1?/VEGF signaling pathways were detected by Western blot.Result:1 Meta-analysis of Taohe-Chengqi Decoction on the treatment of chronic renal failureA total of 630 patients were included in 8 articles,including 321 in the treatment group and 309 in the control group.Meta-analysis showed that compared with the control group,Taohe-Chengqi Decoction could improve the total effective rate of CRF patients'symptoms[OR=4.89,95%CI[3.28,7.29]],inhibit serum BUN[SMD=-0.62,95%CI[-0.81,-0.43]]and Scr[SMD=-3.12,95%CI[-4.72,-1.52]],improve Ccr rate[SMD=-0.56,95%CI[-0.34,-077]],eGFR[SMD=0.62,95%CI[0.30,0.94]]and Hb[SMD=-0.71,95%CI[0.39,1.03]].2 Study of screening the effective parts of Taohe-Chengqi Decoction against renal fibrosisEthyl acetate extraction site,n-butanol extraction site and chloroform extraction site could significantly reduce the Col-I?1 and FN content under the concentration of 200 and 400?g/ml.CCK8 method selected the best intervention concentration as 200?g/ml.Compared with the model group,ethyl acetate extract,n-butanol extract and chloroform extract all decreased the expression of Col-I,Col-III,TIMP2 and CTGF in HK-2 cells induced by TGF-?1,and increased the expression of MMP2.The effect of n-butanol extract was the most significant?P<0.01?.The results of immunofluorescence showed that ethyl acetate extract,n-butanol extract and chloroform extract could inhibit the expression of?-SMA to some extent,and n-butanol extract had the most significant effect?P<0.01?.The results of real-time fluorescence quantitative analysis showed that PAI-1mRNA expression was inhibited by all the three parts,and the most significant was n-butanol extract?P<0.01?.3 Study on the chemical constituents of n-butanol extract of Taohe Chengqi Decoction?NE-THCQ?In NE-THCQ,26 possible compounds of were identified.Among them,2 were from Persicae Semen,1 from Cinnamon,8 from Rhubarb and15 from Licorice.They are mainly saponins,flavonoids and terpenes.4 The network pharmacology study of n-butanol extract of Taohe Chengqi Decoction?NE-THCQ?against renal fibrosisAmong 26 compounds of NE-THCQ,7 potential active compounds were screened out,484 targets and 118 potential therapeutic targets were obtained.Through the active component target network,7,2',4'-trihydroxy-5-methoxy-3-arylcoumarin exhibited the highest correlation with renal fibrosis targets,and the rest were licorice glycoside E,hederagenin,beta-sitosterol,liquiritin,gallic acid-3-O-?6'-O-galloyl?-glucoside,and 18?-hydroxyglycyrrhetic acid.The first 16 key targets in PPI network are ALB,IL6,AKT1,VEGFA,MAPK3,EGFR,TNF,CASP3,SRC,PTGS2,MAPK8,FN1,MMP9,JUN,KDR,IL1B.The Go enrichment analysis showed that NE-THCQ mainly affected cell proliferation,apoptosis and intracellular signaling cascade.In addition,MAPK,TNF,RAS,HIF-1,NOD-like receptor,Toll-like receptor,VEGF and mTOR signaling pathways are directly or indirectly involved in the process of renal fibrosis.5 Study on the mechanisms of n-butanol extract in Taohe Chengqi Decoction?NE-THCQ?on anti-renal fibrosis?1?In vitro study:CCK8 assay demonstrated that with incubation of200?g/ml NE-THCQ for 12 h,24 h and 48 h,had no effect on the viability of cells.NE-THCQ can significantly reduce the expression levels of?-SMA and FN,and up-regulate the expression of E-cadherin,the effect of NE-THCQ on these genes was more significant at 48 h?P<0.01?.NE-THCQ significantly inhibited the expression of TNF-??P<0.01?and reversed the rearrangement of F-actin protein in HK-2 cells induced by TGF-?1.NE-THCQ inhibited the expression of phosphorylated protein in PI3K/Akt/mTOR signaling pathway in HK-2 cells induced by TGF-?1,and significantly down-regulated the expression of p-PI3K/PI3K?P<0.01?,p-mTOR/mTOR and p-AKT/AKT?P<0.05?.The expression of HIF-1??P<0.01?and VEGF?P<0.05?in HK-2 cells induced by TGF-?1 were significantly inhibited.?2?In vivo study:NE-THCQ alleviated the renal morphological and pathological changes of UUO rats,reduced the levels of SCr and BUN?P<0.01?.NE-THCQ significantly reduced the contents of serum IL-6 and IL-?1?P<0.01?.NE-THCQ significantly inhibited the expression of CoL-I,CoL-?and?-SMA,up-regulated the expression of E-cadherin?P<0.05 or P<0.01?.NE-THCQ inhibited the phosphorylation protein of PI3K/AKT/mTOR signaling pathway,and significantly down-regulated the expression of p-PI3K/PI3K?P<0.05?,p-mTOR/mTOR and p-AKT in the kidney of UUO rats?P<0.01?.The expression of HIF-1?and VEGF in the kidney of UUO rats was significantly inhibited?P<0.01?.Conclusion:1 Taohe-Chengqi Decoction can improve clinical efficacy,renal function and survival status of patients,compared with simple western medicine treatment on CRF.But the quantity and quality of the literature are limited,and the relevant conclusions need to be further verified.2 The effective fractions of Taohe-Chengqi Decoction were chloroform extract,ethyl acetate extract and n-butanol extract.Among them,the n-butanol extract showed most active.3 In the n-butanol extract of Taohe-Chengqi Decoction,26 potential compounds were identified and 7 bioactive components were screened out,which laid a foundation of the further study on its pharmacological mechanisms.4 Through the study of network pharmacology,it is speculated that the mechanisms of n-butanol extract of Taohe-Chengqi Decoction against renal fibrosis may be related to its regulation of PI3K/AKT,mTOR,HIF,VEGF and other signaling pathways to inhibit the expressions of inflammatory factors?such as TNF,IL-6,IL-1?,etc.?,improve the biological activity of ECM degrading enzymes?such as MMP1,MMP2,MMP3,etc.?and related activators on inflammation,hypoxia,angiogenesis,ECM synthesis and degradation.5 In vivo and in vitro experiments confirmed that PI3K/AKT/mTOR and HIF-1?/VEGF signaling pathways mediated renal fibrosis.The mechanisms of NE-THCQ on anti-renal fibrosis may be related to the regulation of PI3K/AKT/mTOR and HIF-1?/VEGF signaling pathways to inhibit cytoskeleton rearrangement,reverse epithelial mesenchymal transition?EMT?,inhibit extracellular matrix?ECM?synthesis and secretion,inhibit inflammatory response,improve renal function and adapt to hypoxia injury.