Mechanisms Of Qi-Dan-Dihuang Decoction In Ameliorating Renal Fibrosis In Diabetic Kidney Disease Based On Network Pharmacology

Diabetic kidney disease,one of the most common complications of diabetes,is threatening public health with increasing incidence.Our previous studies had found that Qi-dan-Dihuang Decotion(QDD)is effective in decreasing urinary protein,protecting renal function and alleviating renal fibrosis of DKD,but the mechanism is unclear.[Objective]To predict the renal protective mechanisms through network pharmacology,verify the mechanism through in vivo and in vitro experiments,and clarify the specific mechanism of QDD on diabetic renal fibrosis.[Methods]1.Active compounds and corresponded targets QDD were screened through TCMSP and TCM Database@Taiwan,targets of DKD renal fibrosis were mined through Genecards database and the herb-disease-interaction targets were screened out.The PPI network of herb-disease-interaction targets were obtained from STRING database and the topological properties of the network were analyzed to dig out key targets.Potential biological processes and signaling pathways were found through GO functionally and KEGG pathway enrichment analysis.2.Mice in control(db/m mice)and model(db/db mice)groups were given normal diet,and Dap,QDD-L,QDD-H groups were fed with chow supplemented with 50mg/lg dapagliflozin,2%or 4%QDD.The body weight,blood glucose,oral glucose tolerance were measure during the experiment.The albumin and creatinine from 24 hours urine were measured after 12 weeks of treatment.When treatment finished,lipids and renal function were measured from serum and kidney histology changes were observed by HE,PAS or Masson stained sections.The protein levels of Fibronectin,E-Cadherin,ZO-1,α-SMA,N-Cadherin,Vimentin,NF-κB,p-NF-κB,IL-1β,IL-18,p38MAPK,p-p38MAPK,AKT,p-AKT,mTOR and p-mTOR were detected by western blot.3.After 48 hours administration of 30mmol/L glucose and 10、20、40 mg/L lyophilized QDD powder in renal tubular epithelial cell lines HK-2 and NRK-52E,cell viability was detected by MTT assay and cell migrative ability were detected by wound healing test.Western blot was performed to detect the protein level of E-Cadherin,ZO-1,α-SMA,N-Cadherin,Vimentin,NF-κB,p-NF-κB,IL-1β,IL-18,p38MAPK,p-p38MAPK,AKT,p-AKT,mTOR and p-mTOR.[Results]1.According to TCMSP,TCM Database@Taiwan and GeneCards databases,143 active ingredients and 161 herb-disease interaction targets were identified in QDD.30 key targets were identified by network analysis.GO functionally and KEGG pathway enrichment analysis show that herb-disease interaction targets are significantly enriched in processes and pathways that are closely related to DKD,such as glucose metabolism,lipid metabolism,inflammatory response,EMT,positive regulation of cell migration,PI3K-Akt pathway,VEGF pathway,MAPK pathway,mTOR pathway,NF-κB pathway,TGF-β pathway,etc..2.Compared to model group,mice in model group showed impaired glucose tolerance,increased fast blood glucose,24hU-Pro,UACR,Scr,TG,CHO,HDL and kidney fibrosis.Compared to model group mice,24hU-Pro,UACR,Scr,CHO,HDL-C,kidney pathological changes including collagen and glycogen deposits were significantly ameliorated after 12 weeks of QDD administration.The western blot showed that the protein level of E-Cadherin and ZO-1 were significantly decreased in db/db mice compared to model group,and Fibronectin,α-SMA,N-Cadherin,Vimentin,NF-κB,IL-1β,IL-18,p-p38MAPK,p-AKT and p-mTOR were increased.QDD significantly increased the protein level of E-Cadherin and ZO-1,decreased the expression of Fibronectin,N-Cadherin,α-SMA,Vimentin,IL-1β,IL-18,p-p38MAPK,p-AKT and p-mTOR.3.Compared to normal group,migration capacity of HK-2 and NRK-52E cells was significantly elevated in high glucose group,and protein levels of E-Cadherin,ZO-lwere decreased,protein levels of N-Cadherin,α-SMA,Vimentin were increased.QDD significantly decreased migration capacity of HK-2 and NRK-52E cells,increased the protein levels of E-Cadherin and ZO-1,decreased the protein levels of N-Cadherin,a-SMA,and Vimentin compared to high glucose group.Compared to normal group,protein levels of p-p38MAPK,p-AKT,p-mTOR,p-NF-κB,IL-1β and IL-18 protein in HK-2 and NRK-52E cells were increased in high glucose group.QDD significantly reduced the protein expression of p-p38,p-AKT,p-mTOR,p-NF-κB,IL-1β and IL-18 compared to high glucose group.[Conclusion]QDD protects renal function,ameliorates urinary albumin and delays renal fibrosis in diabetic mice,whose mechanisms are related to the regulation of inflammatory response and EMT in epithelial cells through p38MAPK andAKT/mTOR signaling pathways.