Kunkui Baoshen Granule attenuates diabetic kidney disease via the SIRT3/FOXO3a signaling pathway: An integrated multi-omics analysis

Ethnopharmacological relevance: Kunkui Baoshen Granule (KBG) is a traditional Chinese Medicine (TCM) formula derived from clinical experience and is composed of four medicinal herbs. It has been used for many years at the Jiangsu Province Hospital of Chinese Medicine. It has obtained both a patent and an in-hospital preparation approval from the Jiangsu Provincial Medical Products Administration. KBG has been consistently used to treat diabetic kidney disease (DKD) and has demonstrated significant therapeutic efficacy.

Aim of study: DKD is one of the most prevalent microvascular complications of diabetes mellitus and a leading cause of end-stage Renal Disease. Mitochondrial dysfunction is closely associated with the development and progression of DKD. KBG has been demonstrated to reduce proteinuria and impede DKD progression. However, further research is required to elucidate the mechanisms by which KBG treats DKD. This study explored the therapeutic effects of KBG in DKD and the underlying molecular mechanisms.

Methods: Blood-circulating compounds from KBG were identified using serum pharmacochemistry. In the in vivo experiments, db/db mice were treated with different dosages of KBG. Biochemical parameters, renal histopathological changes, apoptotic indices, inflammatory factors, and oxidative stress levels were systematically evaluated. Potential targets and mechanisms of action were investigated using proteomic analysis. Molecular docking and molecular dynamics simulations were used to preliminary validate potential therapeutic targets. Untargeted metabolomics was used to analyze the serum metabolic profiles of mice. The expression levels of key proteins were assessed using Western blot, ELISA, immunohistochemistry and immunofluorescence. In vitro experiments, HK-2 cells treated with high glucose and palmitic acid as a cellular model. Inhibitors or lentivirus-mediated knockdown of key targets were employed to further validate the underlying mechanisms.

Results: A total of 11 compounds in KBG were identified as prototype components that could enter the bloodstream. In vivo experiments demonstrated that KBG improved renal function, alleviated pathological damage, and mitigated epithelial-mesenchymal transition, inflammation, and oxidative stress in db/db mice. Through proteomics, mitochondrial function and Mitophagy were identified as potential mechanisms underlying the therapeutic effects of KBG. Molecular docking and molecular dynamics simulations confirmed good binding stability between the blood-circulating compounds and the key target SIRT3. KBG inhibited NLRP3 inflammasome activation by regulating Mitophagy through the SIRT3/FOXO3a signaling pathway. Untargeted metabolomics analysis revealed that KBG primarily remodeled metabolic homeostasis by regulating tryptophan metabolism, TCA cycle, and pyruvate metabolism. Furthermore, in vitro cell experiments demonstrated that KBG improved mitochondrial function and prevented NLRP3 inflammasome activation. Notably, the combination with 3-methyladenine or SIRT3 knockdown abolished the protective effects of KBG in HK-2 cells.

Conclusion: KBG regulated mitochondrial homeostasis and Mitophagy through the SIRT3/FOXO3a signaling pathway, thereby inhibiting NLRP3 inflammasome activation. KBG modulated various metabolic pathways and mitigated metabolic dysregulation. These findings indicate that KBG is a potential multi-component TCM formulation for the effective treatment of DKD.