Fig 2: GN combination ameliorated H2O2-induced apoptosis in HUVECs. (A) The concentration of GSH in cell supernatants. (B) The concentration of SOD in cell supernatants. (C) The concentration of MDA in cell supernatants. (D) Representative images of cellular apoptosis detected by flow cytometry. (E) ROS generation induced by H2O2 in HUVECs on fluorescence microscopy (scale bar: 1,000 μm, 100×). (F) The expression of NOX2, p22phox and GAPDH in HUVECs was determined by WB. (G) The relative protein levels of NOX2 and p22phox were normalized to GAPDH. (H) The expression of Bax, Bcl2, caspase-3 and GAPDH in HUVECs was determined by WB. (I) The relative protein levels of caspase-3, Bax and Bcl2 were normalized to GAPDH. All the data are presented as the mean ± SD, n = 3. *p < 0.05 vs. control group, **p < 0.01 vs. control group, # p < 0.05 vs. model group, ## p < 0.01 vs. model group.

Fig 3: (A) Western blot analysis of the protein expression levels of p-PI3K, p-AKT, p-mTOR, NOX2 and Nrf2 in the rat kidney. Aldosterone (0.75 µg/h subcutaneous infusion) upregulated the expression levels of (B) p-PI3K, (C) p-AKT, (D) p-mTOR and (E) NOX2 and downregulated the expression of (F) Nrf2, which was attenuated by spironolactone (100 mg/kg/day). *P<0.05 vs. con group, **P<0.05 vs. UUO group, ***P<0.05 vs. UUO + ald group. Data are presented as the mean ± standard deviation (n=3). p-PI3K, phosphorylated phosphatidylinositol 3-kinase; p-AKT, phosphorylated AKT serine/threonine kinase; p-mTOR; phosphorylated mammalian target of rapamycin; UUO, unilateral ureteric obstruction; NOX2, NADPH oxidase 2; Nrf2, nuclear transcription factor erythroid-related factor 2; con, control; ald, aldosterone; spir, spironolactone.

Fig 4: GN combination inhibited apoptosis in HFD-fed ApoE−/− mice. Serum level of GSH. (A), MDA (B) and SOD. (C) (n = 6). (D) The expression of NOX2, p22phox and GAPDH was determined by WB. (E) The relative protein levels of NOX2 and p22phox were normalized to GAPDH (n = 3). (F) The expression of caspase-3, Bax, Bcl2, and GAPDH was determined by WB. (G) The relative protein levels of caspase-3, Bax, and Bcl2 were normalized to GAPDH (n = 3). All data are shown as the mean ± SD. *p < 0.05 vs. control group, **p < 0.01 vs. control group, # p < 0.05 vs. model group, ## p < 0.01 vs. model group.

Fig 5: Effect of aldosterone on the protein expression levels of (A) AIF-1, p-PI3K, p-AKT, p-mTOR, NOX2 and Nrf2 in normal rats using western blotting. Aldosterone (0.75 µg/h subcutaneous infusion) increased the expression of (B) AIF-1, (C) p-PI3K, (D) p-AKT, (E) p-mTOR and (F) NOX2 and decreased the expression of (G) Nrf2 in normal rats, which was attenuated by spironolactone (100 mg/kg/day). *P<0.05 vs. con group, **P<0.05 vs. ald group, ***P<0.05 vs. ald + spir group. The data are presented as the mean ± standard deviation (n=3). AIF-1, allograft inflammatory factor-1; p-PI3K, phosphorylated phosphatidylinositol 3-kinase; p-AKT, phosphorylated AKT serine/threonine kinase; p-mTOR, phosphorylated mammalian target of rapamycin; NOX2, NADPH oxidase 2; Nrf2, nuclear transcription factor erythroid-related factor 2; con, control; ald, aldosterone; spir, spironolactone.