| Objective: Based on the network pharmacology to predict the mechanism of Yu Ping Feng granules for the treatment of Ig A nephropathy(Ig AN),and validate the mechanism in Ig AN model mice with Th1/Th2 imbalance as the entry point based on the network pharmacology results.Methods: The active ingredients and action targets of Yu Ping Feng granules were retrieved from TCMSP database;Ig AN targets were obtained from Gene Cards,OMIM and TTD databases,and intersection targets were obtained using Venny 2.1.0 and Cytoscape 3.7.2software to construct Yu Ping Feng granules-main ingredients-targets-Ig AN relationship network;Using String database to construct PPI network,and using DAVID database for GO and KEGG enrichment analysis;perform molecular docking by Auto Dock Vina software,and visualize the results by Pymol software.Sixty SPF KM mice,10 in the normal group and 50 in the model group,Ig AN model mice using BSA(oral)+ LPS(tail vein injection)+ CCl4(subcutaneous injection).After successful modeling,the model group was divided into model group,Yu Ping Feng granules high,medium and low groups,western medicine group according to the random number table method,and administered for 8 weeks.Observation indexes: general condition of mice;biochemical method to detect renal function and liver function;HE staining and PAS staining to observe renal histopathological changes;immunofluorescence method to detect Ig A deposition in renal tissue;ELISA method to detect the expression of Ig A and Th1/Th2 related factors in serum;ELISA method,IHC method,and WB method to detect the expression of TGF-β1 in serum and kidney tissues.Results:(1)34 active ingredients of Yu Ping Feng granules were screened,222 drug targets;1436 disease targets;102 intersection targets;the main compounds were quercetin,kaempferol,etc.;the key targets were TNF,IL-6,IL-4,IL-2,etc.;the molecular docking results showed that quercetin had the good binding activity with TNF,IL-6,IL-4,IL-2;GO enrichment analysis yielded 689 biological processes(BP),54 cellular components(CC),115 molecular functions(MF).KEGG enrichment analysis yielded 157 signaling pathways,such as IL-17 and NF-κB signaling pathways,etc.(2)Body weight of mice in each group: the body weight of mice in the model Group was significantly lower than that of the normal Group(P <0.01),and the control group and the high group of Yu Ping Feng granules was significantly higher than the model group(P < 0.01).(3)Kidney function of mice in each group: compared with the normal group,24h-UTP,BUN and Scr were significantly higher in the model group(P < 0.01);the control group and the high,medium and low dose groups of Yu Ping Feng granules were lower compared with the model group(P < 0.01,P <0.05).(4)Histopathology of mice in each group: HE staining showed the model group’s glomerular swelling,proliferation of thylakoid cells and endothelial cells increased extracellular matrix and expansion of thylakoid area;compared with the model group,the histopathological damage of kidney in each treatment group was reduced,among which the glomeruli in the high group of Yu Ping Feng granules tended to be normal and improved more obviously;PAS staining showed the model group’s proliferation of thylakoid cells and increase in extracellular matrix,and after drug treatment,the degree of renal histopathology in each treatment group changed from moderately severe to moderately mild,among the effect of Yu Ping Feng granules high group was better.(5)Ig A immunofluorescence in kidney tissues: compared with the normal group,strong green Ig A deposition was seen in the thylakoid region of mice in the model group;All treatment groups showed a decrease in the intensity of Ig A deposition compared to the model group,while the decrease was more pronounced in the Yu Ping Feng granules high group,and the reduction was more significant in the high group of Yu Ping Feng granules.(6)The expression of Ig A and Th1/Th2 in the serum of mice in each group: ELISA showed that in comparison with normal group,the expression of Ig A in the serum of mice in the model group was significantly higher,and compared with the model group,the expression of Ig A in the serum of mice in the high,medium and low groups of Yu Ping Feng granules and the western group was reduced(P < 0.01,P < 0.05),and between the groups of Yu Ping Feng granules in comparison,the high group was better than the middle and low group(P < 0.01).Compared with the normal group,the Th1/Th2 ratio in the model group mice was significantly decreased(P < 0.01),and the Th1/Th2 ratio in the serum of mice in each treatment group was adjusted upward(P < 0.01);there was no major differences between the different intervention drug groups and the Yu Ping Feng granules group(P > 0.05).(7)The expression of TGF-β1 in serum and kidney tissues of mice in each group: the expression level of TGF-β1 in serum of mice in the model group was significantly increased(P < 0.01);the serum levels of TGF-β1 were significantly decreased in each treatment group of mice compared with the model group(P<0.01);immunohistochemical showed the expression level of TGF-β1 in the basement membrane and thylakoid zone of glomeruli of mice in the model group was significantly increased(P < 0.01);after drug treatment,the expression of TGF-β1in the kidney tissues of mice in each group was significantly reduced,and the effect was more significant in the high group of Yu Ping Feng granules.WB showed TGF-β1 protein expression in the kidney tissues of all treatment groups was reduced,and the effect was more obvious in the high group of Yu Ping Feng granules than in the western group.Conclusion:Network pharmacology studies have shown that the mechanism of Yu Ping Feng granules for the treatment of Ig A nephropathy may be through the targets of TNF、IL-6、IL-4、IL-2,ect.Animal experiments have shown that Th1/Th2 imbalance is involved in the development of Ig AN;Yu Ping Feng granules have better therapeutic effects on Ig AN,and the mechanism may be achieved by correcting Th1/Th2 imbalance through TGF-β1,reducing the immune inflammatory response in vivo,improving renal function and renal histopathology,and delaying renal fibrosis. |
PDF Full Text Request