FIGURE LEGENDS
FIGURE 1 SIRT1 was down-regulated in STZ induced DR rats. A, Retinal tissues observed by HE staining (× 400). B, Ultrastructure of retinas of rats observed by TEM (× 10000). C, The expression of SIRT1 measured by RT-qPCR. D, The expression of SIRT1 measured by Western blot analysis. * p < 0.05 compared to the sham-operated rats, and # p < 0.05 compared to the rats injected with STZ. The results were measurement data and expressed as mean ± standard deviation. Comparisons between multiple groups should be analyzed by one-way ANOVA with Tukey’s post hoc test. (n = 15). DR, diabetic retinopathy; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SIRT1, Sirtuin 1; TEM, transmission electron microscopy; ANOVA, analysis of variance; n, number.
FIGURE 2 CMS reduced oxidative stress, inflammation and apoptosis in the retina of DR rats. Rats were treated with sham, STZ, CMS + STZ, respectively. A, The expression of iNOS in rats determined by RT-qPCR. B, The protein level of iNOS in rats measured by Western blot analysis. C, The expression of NO in rats measured by nitrite test. D - F, The expression of IL-6, TNF-α, and CPR in rats measured by ELISA. G, Cell apoptosis in rats detected by TUNEL (× 400). H, The expression of apoptotic factors (Cyt-C and Caspase-3) in rats determined by Western blot analysis. * p < 0.05 compared to the sham-operated rats, and # p < 0.05 compared to the rats injected with STZ. The results were measurement data, which were expressed as mean ± standard deviation. Comparisons between multiple groups should be analyzed by one-way ANOVA with Tukey’s post hoc test (n = 15). iNOS, inducible nitric oxide synthase; CMS, coumestrol, NO, nitric oxide.
FIGURE 3 CMS activated the expression of SIRT1. ARPE-19 cells were transfected with sh-SIRT1-1, sh-SIRT1-2, and sh-SIRT1-3, respectively and treated with HG-FFA, 0.5-CMS, 1-CMS, and 2-CMS, respectively. A, The expression of SIRT1 in ARPE-19 cells detected by RT-qPCR. B, Protein levels of SIRT1 in ARPE-19 cells detected by Western blot analysis. C - D, The SIRT1 activity and nuclear accumulation in ARPE-19 cells detected by immunofluorescence staining (× 400). E, The expression of SIRT1 in ARPE-19 cells detected by RT-qPCR.* p < 0.05 compared to the controls, and # p < 0.05 compared to cells stimulated with NC. The results were measurement data and expressed as mean ± standard deviation. Comparisons between multiple groups should be analyzed by one-way ANOVA with Tukey’s post hoc test. (n = 15). NC, negative control.
FIGURE 4 CMS suppressed oxidative stress and inflammation of retinal cells induced by DR through activating SIRT1. A, The expression of iNOS in ARPE-19 cells determined by RT-qPCR. B, Protein level of iNOS in ARPE-19 cells determined by Western blot analysis. C, The expression of NO in ARPE-19 cells measured by nitrite test. D - F, The expression of IL-6, TNF-α, and CPR in ARPE-19 cells measured by ELISA.* p < 0.05 compared to the controls, and # p < 0.05 compared to cells stimulated with sh-SIRT1. The results were measurement data and expressed as mean ± standard deviation. Comparisons between multiple groups should be analyzed by one-way ANOVA with Tukey’s post hoc test. (n = 15)
FIGURE 5 CMS suppressed apoptosis of retinal cells of DR. A - B, Cell cycle and apoptosis of retinal cells detected by flow cytometry analysis. C, Protein levels of Cyt-C and Caspase-3 in ARPE-19 cells detected by Western blot analysis. * p< 0.05 compared to the controls, and #p < 0.05 compared to cells stimulated with sh-SIRT1. The results were measurement data and expressed as mean ± standard deviation. Comparisons between multiple groups should be analyzed by one-way ANOVA with Tukey’s post hoc test. (n = 15)