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)