Protective Effect Of Schisandrin A On Diabetic Nephropath

Objective: Diabetic nephropathy(DN)is a major cause of chronic kidney disease worldwide.Its molecular mechanisms are complex and unclear,effective treatments are lacking,and there is a need to develop new drugs for the treatment of DN.Schisandra chinensis has good pharmacokinetic properties and has various pharmacological effects such as anti-inflammatory,anti-cancer,hepatoprotective,antioxidant,neuroprotective,anti-diabetic and musculoskeletal protection.This paper focuses on exploring the potential therapeutic mechanisms of SA for the treatment of DN through network pharmacological approaches,molecular docking techniques and in vivo animal experiments.Methods:Part I: Network pharmacology and molecular dockingThe Swiss Target Prediction database was used to predict SA targets,and the TTD database,Gene Cards database,OMIM database and Dis Ge NET database were used to obtain relevant targets for the treatment of DN.Venn diagrams were then constructed to obtain the intersection targets of SA and DN,and the obtained intersection targets were uploaded to the String database to construct the PPI network.Based on the DIVID database,gene ontology and Kyoto gene and genome database enrichment analyses were performed on the intersecting targets,and the results were visualised;based on this,key pathways were screened and the drug-target-pathway-disease network was constructed using Cytoscape 3.9.0 to screen the core targets.Finally,Autodock Tools 1.5.7 was used to perform molecular docking to verify the binding strength of SA to the core targets.Part II: In vivo animal experimentsA disease model of diabetic nephropathy induced by diabetes mellitus in SD rats using streptozotocin was treated by gavage of SA for four consecutive weeks.The overall condition of SD rats was recorded,glucose tolerance measurements were performed and the kidney/body weight ratio was assessed.Histopathological diagnosis was made by HE staining method and Sirius scarlet staining method,and immunohistochemistry,protein immuno immunohistochemistry,protein immunoblotting and immunofluorescence to detect the expression of core and related proteins of key pathways to investigate the mechanism of action of SA in the treatment of DN.Results:Network pharmacology and molecular docking results1 100 predicted targets of SA action and 2524 relevant targets for the treatment of DN were obtained,with a total of 43 intersecting targets of SA and DN;2 The results of GO functional enrichment analysis and KEGG signaling pathway enrichment analysis showed that there were 256 GO entries,including 167 BP entries,28 CC entries,61 MF entries and 111 KEGG signaling pathways,of which the key pathway was epidermal growth factor receptor tyrosine kinase inhibitor resistance.3 Constructed a "Schizandrin A-target-pathway-diabetic nephropathy" association network and screened three core targets,PIK3 CA,EGFR and MAPK10,with degree values of 27,25 and 20,respectively.4 The molecular docking results showed that Schizandrin A had good docking activity with PIK3 CA,EGFR and MAPK10,with binding energies of-6.73kcal/mol-1,-7.18kcal/mol-1 and-6.58kcal/mol-1,respectively;among them,the binding energy of Schizandrin A with EGFR was less than-7kcal/mol-1,suggesting that Schizandrin A had good docking activity with EGFR.This indicates that Schizandrin A has strong binding activity with EGFR,suggesting that EGFR is likely to be a key target for SA in the treatment of DN.Results of in vivo animal experiments1 The moulding results showed that SD rats all showed signs of depression,chorionic villus clumping and reduced self-cleaning activity after moulding,and there was significant excessive drinking,polyphagia,polyuria,weight loss and a significant increase in the total kidney weight/body weight ratio,which decreased after SA treatment,and the glucose tolerance situation improved.2 HE staining showed a small amount of tubular dilatation and narrowing and vacuolisation of the tubular epithelium in the DN group.The glomerular capillaries were clear in the visual field,with granulocyte infiltration and some glomerular capillaries were congested.After SA treatment,the granulocyte infiltrate was relatively reduced and the situation improved.3 The results of Sirius scarlet staining showed that the collagen fibres of the glomeruli and tubules of the rats in the DN group showed a deep and distinct red staining.After SA treatment,the red staining of the glomeruli and tubules subsided significantly,the collagen was evenly distributed and the fibrotic nodules were significantly reduced.4 Immunohistochemical methods,protein immunoblotting method and immunofluorescence method detected that the phosphorylation of EGFR was increased in the kidney of rats in the DN group after moulding,while the expression of PTRF was also increased,the phosphorylation of Akt was decreased and the expression of Bcl-2 was also decreased,while the phosphorylation of GSK-3β was increased,all of which were changed after SA treatment.Conclusion:1 SA can retard the development of DN;2 SA may have implications for improving diabetic nephropathy by inhibiting EGFR phosphorylation and regulating PTRF expression.