Study On The Mechanism Of Dahuang Huangqi Decoction Inhibiting Renal Interstitial Fibrosis Based On Network Pharmacology

Background: Chronic kidney disease(CKD)has become one of the important diseases endangering the world’s health.Renal interstitial fibrosis(RIF)is a pathological process leading to the destruction of renal tissue structure and loss of function in chronic kidney disease.Dahuang-Huangqi are widely used in the treatment of CKD in clinic.RIF is a common pathological pathway in the end stage of CKD.At present,western medicine has no specific drugs for the treatment of RIF,so it is very important to study and explore anti-RIF drugs.According to the in-depth mining and analysis of the medication experience data of more than 70 old traditional Chinese medicine,the two herbs with the highest frequency are:Dahuang and Huangqi.Zheng Xin,a master of Chinese medicine,also used Dahuang-Huangqi as an important component of Shen-shuai-ling prescription,which has achieved good therapeutic effect in the clinical treatment of CKD.Therefore,the research group focuses on whether the action mechanism of Dahuang-Huangqi on the treatment of CKD is related to its anti-RIF.The aim of this study are:(1)to analyze the network pharmacology of multi-component,multi-target and multi-channel therapeutic effect of DHHQD on renal interstitial fibrosis of chronic kidney disease through various modern databases,and to construct the network of "herbs-chemical components-targets-disease".(2)Through animal experiments,the mechanism of DHHQD on rats with renal interstitial fibrosis was clarified.(3)Through in vivo and in vitro experiments,the mechanism of rhein improving RIF by inhibiting SHH signaling pathway was revealed.SECTION ONE THE NETWORK PHARMACOLOGICAL ANALYSIS OF DAHUANG HUANGQI DECOCTION ON CHRONIC KIDNEY DISEASEObjectives To evaluate the target of Dahuang-Huangqi in the treatment of CKD-RIF through network pharmacological analysis.Methods In this study,the chemical components of Dahuang-Huangqi were analyzed by Ultra-high-performance Liquid Chromatography/Tandem Mass Spectrometry(UHPLC-MS/ MS),and the chemical components and action targets of Dahuang-Huangqi were analyzed by TCMSP,literature review and so on and mass spectrometry.Combine OMIM and Gene Card databases to obtain the disease target of "chronic kidney disease-renal interstitial fibrosis".Using R software to obtain the herbs and disease targets in Dahuang-Huangqi and CKD-RIF,find the intersection target genes and draw Venny map.Using Cytoscape 3 5.1 the software constructs the visual network of active components and targets.PPI network were constructed by string database.Go enrichment analysis of biological processes,cell components and molecular functions and KEGG enrichment analysis of signal pathways were carried out through David and KEGG databases.Results: Combined with mass spectrometry analysis,a total of 631 potential pathophysiological targets were collected through online pharmacology databases,including frequently studied genes,such as MAPK,NOS,MMP,STAT,PPAR,SOD,EGFR and IL6.After intersecting with the disease target,139 common target genes were finally obtained.The drug chemical component target disease target network was established.There were 2615 correlations among the targets of protein-protein interaction network(PPI).Most of the targets identified in the analysis were also closely related to inflammation such as mapk3 / 8,IL-6 and Jun and apoptosis such as Akt,VEGFA,EGFR and EGF.The oxidative stress enriched in target genes was observed through go analysis(go: 0006979)The cellular response to oxidative stress(go: 0034599)and reactive oxygen species(go: 0034614)are closely related to inflammation.In addition,the response to mechanical stimulation(go: 0009612),the regulation of apoptosis signal pathway(go: 2001233),the response to radiation(go:0009314)and the cellular response to external stimulation(go: 0071496)are positively related to fibrosis.In KEGG analysis,131 targets were located in 123 KEGG pathways,including Akt / PI3 K signaling pathway,IL-17 signaling pathway,TNF signaling pathway,apoptosis,HIF-1signaling pathway,EGFR tyrosine kinase inhibitor resistance and toll like receptor signaling pathway.Through the analysis of data and related biological processes,20 go and KEGG signal pathways with the highest significance were selected.Conclusions By integrating the mass spectrum and network pharmacological analysis of Dahuang-Huangqi,it is found that Dahuang-Huangqi can treat CKD-RIF through multi-components,multi-targets and multi-channels.This study provides a theoretical basis for the mechanism of Dahuang-Huangqi in the treatment of RIF.SECTION TWO MECHANISM OF DAHUANG-HUANGQI INHIBITING RENAL INTERSTITIAL FIBROSIS THROUGH PI3 K / AKT SIGNALING PATHWAYObjectives To investigate the mechanism of Dahuang-Huangqi decoction(DHHQD)on RIF in UUO model rats.Methods UUO renal interstitial fibrosis model rats were established and randomly divided into sham operation(sham),UUO group,high-dose DHHQD group(DHHQD-H,20 ml/kg/D)and low-dose DHHQD group(DHHQD-L,10 ml/kg/D).Except the sham operation group,the rats in the other 3 groups were modeled,the left ureter was exposed and dissected directly,and the abdominal cavity of DHHQD group was perfused with different concentrations of traditional Chinese medicine decoction for 14 days.At the end of the experiment,the serum of rats in each group was collected for renal function detection,and the renal tissue samples were collected for HE staining,Masson staining,PAS staining and transmission electron microscope observation to evaluate the pathological injury of rat renal tissue.The expression of related proteins and genes was detected by immunohistochemistry,ELISA,Western blotting and q PCR.Results The levels of serum creatinine and blood urea nitrogen decreased,renal tubular atrophy and necrosis,interstitial fibrosis,extracellular matrix proliferation and abnormal deposition,and the microstructure of mesangial matrix and glomerular basement membrane were changed in rats treated with Dahuang Huangqi decoction.Through immunohistochemical detection,DHHQD treatment significantly reduced the transforming growth factor-β 1(TGF-β 1)And α Smooth muscle actin(α-SMA)of UUO rats,which may be one of the keys to improve renal function and renal fibrosis.Combined with network pharmacology,DHHQD was detected by ELISA to reduce the expression of e NOS,IL-6,EGFR and VEGF proteins in UUO rats,and reduce the core targets involved in inflammatory pathways,such as PI3 K,Akt,TLR4 and NF-κExpression of B protein and RNA.DHHQD treatment potentially regulates PI3 K / Akt and TLR4 / NF-κ B signaling pathway to improve the severity of RIF,which may be the key to its inhibition of renal fibrosis.Conclusions DHHQD regulates PI3 K / Akt and TLR4 / NF-κ B signal activity,further inhibit the degree of renal interstitial fibrosis and protect renal function.SECTION THREE MECHANISM OF RHEIN INHIBITING RENAL INTERSTITIAL FIBROSIS THROUGH SHH SIGNALING PATHWAYObjectives To investigate the mechanism of rhein on RIF in UUO model rats.Methods UUO renal interstitial fibrosis model rats were established and randomly divided into sham operation(sham),UUO group,rhein group.Except the sham operation group,the rats in the other 2 groups were modeled,the left ureter was exposed and dissected directly,the Rhein group rats were administered an intragastric gavage of rhein for 14 d.Kidney function-related indicators were monitored in these rats,while indexes of pathologic aspects were determined histologically.The expression of α-SMA,TGF-β1 was evaluate by Immunohistochemical staining.The expression of SHH,Gli1,and Snail was quantified using real-time polymerase chain reaction and western blotting.The NRK-49F cells were incubated with and without SHH for 48 hours.The SHH activated NRK-49 F cells were incubated with cyclopamine or rhein.The Gli1 and Snail m RNA and protein level was detected.Results In vivo experiment,rhein could improve the renal dysfunction and renal pathological damage caused by UUO.However,these changes could be significantly reversed by the administration of rhein.Compared with the untreated UUO group,the Rhein group showed reduced kidney tubular atrophy and necrosis,interstitial fibrosis,hyperplasia,and abnormal deposition of extracellular matrix.Rhein reduced the m RNA and protein expression of SHH,Gli1 and Snail in UUO rats.In vitro experiment,CNP or rhein treatment decreased the expression of Gli1 and Snail on m RNA and protein levels in SHH-induced NRK-49 F cells,suggesting that CNP or rhein suppresses SHH-induced NRK-49 F activation.Taken together,these results demonstrated that rhein suppresses SHH-Gli1-Snail signal pathway activation in NRK-49 F cells induced by SHH.Conclusions Treatment with rhein remarkably ameliorated renal interstitial fibrosis in UUO rats by regulating the SHH-Gli1-Snail signal pathway.