Astragalus polysaccharides inhibit oxidation in high glucose-challenged or SOD2-silenced H9C2 cells

Figure 1. SOD2 protein and activity in H9C2 cells after silencing of SOD2. Notes: (A) Representative autoradiographs of Western blots. (B) Bar graphs showing SOD2 protein levels. (C) Bar graphs showing SOD2 activities. H9C2 cells were transfected with siRNASOD2 or scrambled siRNA (n=4 per group). SOD activity was measured by the extent of reduction in the appearance of NBT-diformazan utilizing the SOD Assay Kit. SOD protein levels were analyzed by Western blotting. Values are presented as mean ± SEM. Two-group analysis was performed using t-test. *P<0.05 vs ctrl. Abbreviations: NBT, nitroblue tetrazolium; SOD, superoxide dismutase; Ctrl, normal control group.

Figure 2. APS abrogated the ultrastructural abnormalities of mitochondria induced by high glucose or silencing of SOD2. Notes: (A) Confocal microscopy images of cultured cells (×200 magnification). (B) Representative ultrastructural profiles of cultured cells observed using a transmission electron microscope (×23,300 magnification). H9C2 cells cultured in high or normal concentration of glucose were transfected with siRNASOD2 or scrambled siRNA and were further cultured in the presence of APS or vehicle. Cultured cells were observed by a confocal microscope and evaluated using a transmission electron microscope to define cardiac mitochondrial structural changes. Abbreviations: APS, Astragalus polysaccharides; APS-HG, APS-treated high glucose-induced group; Ctrl, normal control group; HG, high glucose-induced group..

Figure 3. APS reduced the cell apoptosis in H9C2 cells induced by high glucose or silencing of SOD2. Notes: (A) Bar graph showing the percentage of hairpin-1-stained positive cells in cultured cells by flow cytometry quantification. (B) Immunocytochemistry microphotographs showing the hairpin-1-stained positive (red) cells (×2,000 magnification). (C) Flow cytometry analysis for the quantification of hairpin-1-stained positive cells in cultured cells. H9C2 cells cultured in high or normal concentration of glucose were transfected with siRNASOD2 or scrambled siRNA and were further cultured in the presence of APS or vehicle (n=4 per group). Hairpin oligonucleotide probes were employed for the detection of cell apoptosis. Hairpin-1-positive cells were gated and quantified by flow cytometry analysis. Each experiment was performed in triplicate and repeated three times. Values are presented as mean ± SEM. Two-group analysis was performed using t-test between HG and ctrl, APS-HG and HG, APS-siRNASOD2 and siRNASOD2, and siRNASOD2 and ctrl. *P<0.05 vs ctrl, !P<0.05 vs HG, and #P<0.05 vs siRNASOD2, corresponding to all the repetitions in the study. Abbreviations: APS, Astragalus polysaccharides;…

Figure 4. APS inhibited ROS formation and oxidative damage induced by high glucose or silencing of SOD2. Notes: (A) Fluorescence microphotographs showing DHE-stained positive (green) cells (×2,000 magnification). (B) Bar graph showing the mean DHE fluorescence intensity of nuclei in culture cells. (C) Bar graph showing the intensity of 8-OH-dG signals in culture cells. (D) Bar graph showing the intensity of nitrotyrosine signals in culture cells. H9C2 cells cultured in high or normal concentration of glucose were transfected with siRNASOD2 or scrambled siRNA and were further cultured in the presence of APS or vehicle (n=4 per group). Superoxide production in cultured cells was determined using the fluorescent dye DHE. The images were processed, and the mean DHE fluorescence intensity of nuclei was evaluated using a Bio-Rad Radiance 2100MP multiphoton microscope and ImagePro advanced software. Nitrotyrosine antibodies were employed to detect oxidative damage to cytoplasmic proteins, whereas 8-OH-dG antibodies were utilized to detect oxidative stress in the nuclei. Each experiment was performed in triplicate and repeated three times. Values are presented as mean ± SEM. Two-group analysis was…

Figure 5. APS enhanced the SOD2 protein expression and activity in H9C2 cells induced by high glucose or silencing of SOD2. Notes: (A) Representative autoradiographs of Western blots. (B) Bar graphs showing the activities of SOD1 and SOD2 in cultured cells. (C) Bar graphs showing the protein levels of SOD1 and SOD2 in cultured cells. H9C2 cells cultured in high or normal concentration of glucose were transfected with siRNASOD2 or scrambled siRNA and then exposed to APS or vehicle (n=4 per group). SOD activity was measured by the extent of reduction in the appearance of NBT-diformazan utilizing the SOD Assay Kit. SOD protein levels were analyzed by Western blotting. Each experiment was performed in triplicate and repeated three times. Values are presented as mean ± SEM. Two-group analysis was performed using t-test between HG and ctrl, APS-HG and HG, APS-siRNASOD2 and siRNASOD2, and siRNASOD2 and ctrl. *P<0.05 vs ctrl, !P<0.05 vs HG, and #P<0.05 vs siRNASOD2, corresponding to all the repetitions in the study. Abbreviations: APS, Astragalus polysaccharides; NBT, nitroblue tetrazolium; SOD, superoxide dismutase; APS-HG, APS-treated high glucose-induced group; Ctrl, normal control group; HG,…