Figure 4

Puerarin inhibited oxidative stress in murine podocytes cultured in high glucose. (A,B) Immortalized mouse podocytes were cultured in either normal glucose (NG: 5 mM glucose + 25 mM mannitol) or high glucose (HG: 30 mM glucose) treated with DMSO vehicle or 10 μM puerarin for 24 hours. Superoxide production was determined by incubation of cells with dichlorofluorescin diacetate (DCFDA) fluorogenic dye and by detection of oxidized 2′, 7′-dichlorofluorescein (DCF). Puerarin reduced superoxide generation in podocytes cultured in high glucose media. Representative image is shown in (A) and ratio of DCF+ cells/total (DAPI+) cells per field are shown in (B) (***p < 0.001, n = 3, 7 fields per group). (C) Real-time PCR analysis of NOX4 mRNA levels in podocytes cultured with NG or HG treated with vehicle or 10 μM puerarin for 4 hours. GAPDH mRNA was used as an internal control. ***p < 0.001, n = 3. (D) Representative western blot analyses of NOX4 expression in podocytes cultured in NG or HG conditions for 24 hours. The representative blots of three individual experiments are shown here. (E) Quantification of NOX4 protein expression normalized to GAPDH is shown. **p < 0.01 and ***p < 0.001, n = 3.