Network Pharmacology, Molecular Docking, and Experimental Verification to Reveal the Mitophagy-Associated Mechanism of Tangshen Formula in the Treatment of Diabetic Nephropathy

Yinfeng Chen,* Xiaying Wang,* Jie Min, Jie Zheng, Xuanli Tang, Xiaoling Zhu, Dongrong Yu, De Jin Department of Nephrology, Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University, Hangzhou, Zhejiang Province, 310007, People’s Republic of China*These authors contributed equally to this workCorrespondence: Dongrong Yu; De Jin, Department of Nephrology, Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University, Hangzhou, Zhejiang Province, 310007, People’s Republic of China, Email yudr68@163.com; 826901274@qq.comPurpose: This study investigated the mechanism of TSF in treating DN through network pharmacology, molecular docking, and experimental validation.Methods: To identify critical active ingredients, targets, and DN genes in TSF, multiple databases were utilized for screening purposes. The drug-compound-target network was constructed using Cytoscape 3.9.1 software for network topological analysis. The protein interaction relationship was analyzed using the String database platform. Metascape database conducted enrichment analysis on the key targets using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes. The renoprotective effect was evaluated using a mouse model of diabetic nephropathy (db/db mice) that occurred spontaneously. Validation of the associated targets and pathways was performed using Western Blot (WB), Polymerase Chain Reaction (PCR), and Immunohistochemical methods (IHC).Results: The network analysis showed that the TSF pathway network targeted 24 important targets and 149 significant pathways. TSF might have an impact by focusing on essential objectives such as TP53, PTEN, AKT1, BCL2, BCL2L1, PINK-1, PARKIN, LC3B, and NFE2L2, along with various growth-inducing routes. Our findings demonstrated that TSF effectively repaired the structure of mitochondria in db/db mice. TSF greatly enhanced the mRNA levels of PINK-1. WB and IHC findings indicated that TSF had a notable impact on activating the PINK-1/PARKIN signaling pathway in db/db mice, significantly increasing LC3 and NRF2 expression.Conclusion: Our results indicate that TSF effectively addresses DN by activating the PINK-1/PARKIN signaling pathway and enhancing Mitochondrion structure in experimental diabetic nephropathy.Keywords: tangshen formula, diabetes nephropathy, network pharmacology, mitophagy, PINK1/Parkin pathway