Mechanism of Action of Ursolic Acid in Ameliorating Mitochondrial Dysfunction in Podocytes Induced by High Glucose

Objective: This study aimed to investigate the protective effects of ursolic acid on mitochondrial dysfunction in podocytes induced by high glucose and to explore the underlying mechanism. Methods: The CCK-8 method was used to determine the experimental conditions and the experiments were divided into five groups: control (CN), high glucose (200 mmol/L, HG), low-dose ursolic acid (200 mmol/L glucose + 0.703 μmol/L ursolic acid), medium-dose ursolic acid (200 mmol/L glucose + 1.406 μmol/L ursolic acid) and high-dose ursolic acid (200 mmol/L glucose + 2.813 μmol/L ursolic acid). The immunofluorescence method was used to observe mitochondrial morphology and detect the reactive oxygen species (ROS) level, the degree of mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (MMP). Flow cytometry was used to detect the ROS level and the rate of apoptosis of podocytes. Western Blot was used to detect the expression of cyclophilin D (Cyp D), voltage-dependent anion channel (VDAC), Bcl-2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2) and cytochrome c (Cyt c) proteins. Results: Compared with the CN group, the proliferation rate of podocytes was significantly reduced by intervention of 200 mmol/L glucose (P < 0.01), which was therefore used for modeling. Compared with the HG group, all doses of ursolic acid significantly increased the proliferation rate of podocytes (P < 0.01). Immunofluorescence assays indicated that they restored mitochondrial morphology, inhibited ROS overproduction, repressed the abnormal opening of mPTP and increased MMP. The results of flow cytometry showed that ursolic acid at all three doses reduced the apoptosis rate of podocytes compared with the HG group (P < 0.01). The Western blot results showed that compared with the HG group, they significantly down-regulated the expression of Cyp D, VDAC, Bax and Cyt c proteins but up-regulated the expression of Bcl-2 protein. Conclusion: Ursolic acid may exert its effect against diabetic nephropathy by inhibiting mitochondrial dysfunction and apoptosis caused by HG-induced oxidative stress in podocytes.