Research Paper Volume 13, Issue 4 pp 5342—5357

Coumestrol mitigates retinal cell inflammation, apoptosis, and oxidative stress in a rat model of diabetic retinopathy via activation of SIRT1

CMS suppressed HG-induced oxidative stress and inflammation in hRMECs through activating SIRT1. hRMECs were treated with sh-NC, sh-SIRT1, sh-SIRT1 + DMSO and sh-SIRT1 + CMS, respectively. (A) Expression pattern of iNOS as determined by RT-qPCR in hRMECs, normalized to β-actin. (B) Representative Western blots of iNOS protein and its quantitation in hRMECs, normalized to β-actin. (C), Expression pattern of NO as determined by nitrite test in hRMECs. (D–F), Expression patterns of IL-6 (D), TNF-α (E), and CRP (F) as measured by ELISA in the cell supernatant. *p **p ***p #p ##p ###p post hoc test. The cell experiments were repeated three times independently. NC, negative control; CMS, coumestrol, HG, high glucose; hRMECs, human retinal microvascular endothelial cells; SIRT1, sirtuin 1; DMSO, dimethyl sulfoxide; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; IL-6, interleukin-6; TNF-α, tumor necrosis factor α; CRP, C-reactive protein; ELISA, Enzyme linked immunosorbent assay; ANOVA, analysis of variance.

Figure 6. CMS suppressed HG-induced oxidative stress and inflammation in hRMECs through activating SIRT1. hRMECs were treated with sh-NC, sh-SIRT1, sh-SIRT1 + DMSO and sh-SIRT1 + CMS, respectively. (A) Expression pattern of iNOS as determined by RT-qPCR in hRMECs, normalized to β-actin. (B) Representative Western blots of iNOS protein and its quantitation in hRMECs, normalized to β-actin. (C), Expression pattern of NO as determined by nitrite test in hRMECs. (DF), Expression patterns of IL-6 (D), TNF-α (E), and CRP (F) as measured by ELISA in the cell supernatant. *p < 0.05, **p < 0.01, ***p < 0.001, compared to sh-NC-treated cells, and #p < 0.05, ##p < 0.01, ###p < 0.001, compared to cells stimulated with sh-SIRT1 and treated with DMSO. The results were measurement data and expressed as mean ± standard deviation. Comparisons between multiple groups were analyzed by one-way ANOVA with Tukey’s post hoc test. The cell experiments were repeated three times independently. NC, negative control; CMS, coumestrol, HG, high glucose; hRMECs, human retinal microvascular endothelial cells; SIRT1, sirtuin 1; DMSO, dimethyl sulfoxide; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; IL-6, interleukin-6; TNF-α, tumor necrosis factor α; CRP, C-reactive protein; ELISA, Enzyme linked immunosorbent assay; ANOVA, analysis of variance.