Combination Relationship Research Of The Effect Of Supplemental Kidney Qi Pill(Decoction)on Rats With Kidney-yang Deficiency And Edema Model

Objective1.By means of network pharmacology,the target and signal pathway of Supplemented Kidney Qi Pill in the treatment of renal edema were studied to explore the pharmacodynamic mechanism of Supplemented Kidney Qi Pill in treating renal edema.2.Observing and studying the effects of Supplemented Kidney Qi Pill and its recipes in the model of kidney-yang deficiency edema,so as to provide scientific basis for the theory of“warm yang and promote urination”in traditional Chinese medicine.Method1.TCMSP databases were used to screen the potential active ingredients of Supplemented Kidney Qi Pill;Swiss Target Prediction database and TCMSP database was used to predict and screen the potential active components of Supplemented Kidney Qi Pill;Based on the Gene Cards,OMIM,Drug Bank and Pubmed databases to predict and screen the active components of kidney kidney-yang deficiency edema and cross-compare the potential targets;The Cytoscape software was used to construct the“component-target”Network,and Network Analyzer plug-in was used for parameter analysis;The protein-protein interactions(PPI)network of antidepressant targets was established through the String database and Cytoscape software;The targets were analyzed by GO enrichment analysis and KEGG pathway analysis through DAVID database.2.70 SPF healthy male SD rats were randomly divided into control group,model group,original formula group,warm yang and promote urination group,supplement kidney and promote urination group,warm and supplement kidney yang group,and promote urination group,with 10 rats in each group.The rat model of kidney-yang deficiency edema was duplicated by hydrocortisone and doxorubicin hydrochloride,After modeling,each group was given the drug by gavage for 14 days.Observe the general status of rats:anal temperature,body mass,and urine volume recorded for 24 hours;the biochemical indicators were measured by an automatic biochemical analyzer;Kidney tissues were collected to make pathological section by htoxylin eosin(HE)staining;kidney yang deficiency indicators and inflammation indicators were measured by enzyme-linked immunosorbent assay(ELISA);expression levels of IL-17 in kidney were measured by immunohistochemistry;AQP2,V2R and MAPK signaling pathway related proteins were carried out by Western blot.Result1.The theoretical study:Through network pharmacology,111 potential active ingredients were screened from Supplemented Kidney Qi Pill,involving 120 potential targets for treating renal edema.17 94 targets of renal edema were retrieved.By cross comparison,94 common targets were obtained for the combined action of Supplemented Kidney Qi Pill.and renal edema.93 nodes targets were selected based on the PPI interaction database,Through GO and KEGG signaling pathway enrichment analysis,active components participated in inflammatory response,proteolysis,redox reaction,immune response and other biological processes,involving mitogen-activated protein kinase(MAPK)signaling pathway,interleukin-17(IL-17)signaling pathway,Tumor Necrosis Factor(TNF)signaling pathway,nuclear factor kappa-B(NF-κB)signaling pathway and so on.Based on relevant literature,IL-17/MAPK signaling pathway could be important step.2.The experimental study:(1)Effects on the general state of rats:Each dose group showed a regulatory effect on general state and anal temperature;urinary volume in 24 h and body mass in each period increased whereas urinary protein content for 24 h decreased in the kidney-yang deficiency edema model rats.Day 14 of administration,the body mass of original formula group was significantly higher than that of promote urination group,whereas urinary protein content of original formula group for 24 h was significantly lower than that of promote urination group and warm and supplement kidney yang group(2)HE staining results:Each dose group showed a regulatory effect on renal pathological damage of kidney-yang deficiency edema model rats.(3)Results of biochemical criterion:Each dose group decreased the levels of Crea and BUN,increased the levels of TP and ALB of kidney-yang deficiency edema model rats(4)Results of kidney-yang deficiency criterion:Each dose group except promote urination group had markedly increased levels of cAMP and cAMP/cGMP,but markedly decreased levels of cGMP,the levels of CRH,ATCH,Cor were markedly increased,in the kidney-yang deficiency edema model rats(5)Results of ELISA criterion:Each dose group had markedly decreased levels of AVP,IL-1β,IL-6,TGF-β1 in the kidney-yang deficiency edema model rats.The IL-1β,IL-6 and TGF-β1 serum level of original formula group,warm yang and promote urination group were significantly lower than that of promote urination group The IL-1β,IL-6 serum level of original formula group were significantly lower than that of warm and supplement kidney yang group,The TGF-β1 serum level of original formula group,warm yang and promote urination group was significantly lower than that of warm and supplement kidney yang group(6)Results of immunohistochemical experiments:Original formula group,warm yang and promote urination group,supplement kidney and promote urination group can inhibited the high expression of IL-17 in renal tissue of rats in the kidney-yang deficiency edema model rats.(7)Results of Western blot experiments:①Original formula group,warm yang and promote urination group,supplement kidney and promote urination group can inhibited higher expression of AQP2 and V2R in renal tissue in the kidney-yang deficiency edema model rats.The expression of AQP2 in renal tissue of original formula group was significantly lower than that of warm yang and promote urination group,supplement kidney and promote urination group.The expression of V2R in renal tissue of original formula group was significantly lower than that of supplement kidney and promote urination group②Warm yang and promote urination group,supplement kidney and promote urination group could inhibit expression of p-p3 8 and p-ERK1/2 in the kidney-yang deficiency edema model rats but did not impact the expressions of p38 and ERK1/2,compared with the model group.The expression of p-ERK 1/2 in renal tissue of original formula group was significantly lower than that of warm yang and promote urination group,supplement kidney and promote urination group.The expression of p-p3 8 in renal tissue of original formula group was significantly lower than that of supplement kidney and promote urination groupConclusion1.Supplemented Kidney Qi Pill and its separated groups had certain effects in treating kidney Yang deficiency edema,which could significantly improve the general state,urinary protein and renal function of rats.Supplemented Kidney Qi Pill were more effective than separated groups.Under the condition of this experiment,the formula reflects the correct guiding significance of the compatibility rule of " warming Yang and benefiting water”2.Supplemented Kidney Qi Pill play the role of warming Yang and benefiting water through multiple components,multiple targets and multiple pathways,its plays the role of "warm yang and promote urination" may be:reduce the expression level of IL-17,block the MAPK signaling pathway,and protective role in kidney;reduce the expression level of AVP,decrease the expression level of AQP2 protein,improve the body fluid metabolism disorders.