Yupingfeng San extract ameliorates cisplatin induced renal interstitial fibrosis via IL-6/JAK/STAT3 mediated restoration of mitochondrial homeostasis

Ethnopharmacological relevance Yupingfeng San (YPFS), a classic tonic formula in Traditional Chinese Medicine (TCM), has been extensively applied clinically for centuries. Originating from Jiuyuan Fang , its earliest extant record is Danxi Xinfa . YPFS is composed of Huangqi ( Astragalus membranaceus Bunge), Baizhu ( Atractylodes rubra Dekker), and Fangfeng ( Saposhnikovia divaricata (Turcz.) Schischk). Due to its traditional efficacy of " replenishing qi and consolidating the exterior," YPFS is commonly prescribed for allergic rhinitis, nephrotic syndrome, and chronic nephritis. Recent studies have confirmed that YPFS protects renal function and alleviates renal fibrosis in mice with diabetic nephropathy, while simultaneously improving lung function and reducing pulmonary fibrosis in bleomycin-induced rats. Taken together, these results suggest YPFS possesses substantial potential for mitigating organ fibrosis. Purpose This study investigated the renoprotective efficacy and underlying mechanism of YPFS extract in a cisplatin-induced mouse model of renal interstitial fibrosis. Using an integrated in vivo and in vitro approach, we sought to validate the therapeutic potential of this traditional formula against chemotherapy-induced nephrotoxicity. Methods The chemical components of YPFS extract were analyzed using UPLC-Q-TOF -MS. For the in vivo study, male BALB/c mice received YPFS extract (1, 1.95, or 3 g/kg) by oral administration once daily for 7 days after a single injection of cisplatin (20 mg/kg, i.p.). Renal function was assessed by measuring serum creatinine, plasma BUN, fasting blood glucose, and the urine protein-to-creatinine ratio. Kidney tissues were examined using H&E and Masson's trichrome staining for histopathological analysis, and the expression of fibrosis markers (α-SMA, collagen I, fibronectin) was measured by immunohistochemistry and western blotting. For the in vitro study, MPC-5 podocytes were treated with high glucose (25 mM) for 48 h to induce injury, and then intervened with YPFS extract at concentrations of 20, 40, or 80 μg/mL, along with JAK2 inhibitor AG490 and STAT3 agonist Colivelin for mechanism verification. Cell viability was measured by MTT assay, apoptosis was assessed by Hoechst 33342 staining, the expression of α-SMA was measured by cellular immunofluorescence, and mitochondrial morphology was observed using MitoTracker Red staining and TEM. Mitochondrial function was evaluated by Na + /K + -ATPase and Complex I activities, and oxidative stress levels were determined by T-SOD, GSH, and MDA assays. RNA-seq was conducted to identify DEGs in MPC-5 cells, and Western blotting was used to verify key protein expression in both cells and tissues. Results UPLC-Q-TOF-MS analysis identified 57 compounds in YPFS extract, predominantly flavonoids (40.35%) and saponins (21.05%). In cisplatin-treated mice, the extract dose-dependently reduced serum creatinine, blood urea nitrogen, Urine protein-to-creatinine ratio, and fasting blood glucose. It also decreased collagen deposition and α-SMA overexpression in kidney tissues. In MPC-5 cells, the extract restored cell viability, reduced apoptosis and α-SMA overexpression, improved mitochondrial membrane potential and redox balance, and restored mitochondrial fusion-fission balance by increasing Mfn1/2 and decreasing Drp1 expression. Transcriptomic analysis revealed that the extract reversed the high glucose-induced activation of gene pathways related to IL-6/JAK/STAT3 signaling, oxidative phosphorylation, and apoptosis, with findings confirmed at the protein level. Mechanistic investigations using the JAK2 inhibitor AG490 and STAT3 agonist Colivelin confirmed that the cytoprotective and anti-fibrotic effects of YPFS were primarily dependent on the IL-6/JAK/STAT3 signaling pathway. Conclusion YPFS extract ameliorates cisplatin-induced renal interstitial fibrosis by suppressing IL-6/JAK/STAT3 signaling and restoring mitochondrial homeostasis. These findings provide a pharmacological basis for the traditional use of this formula and highlight its potential as an adjunct therapy for reducing chemotherapy-related kidney injury.