3.3 Autophagy may contribute to the effect of XKP ...
Figure 3(https://www.daowen.com)
(a)Cell immunofluorescence analysis determined the levels of autophagy of MIN-6 cells under five
different treatments(basic medium；basic medium with 33.3mmol/L glucose；basic medium with 33.3mmol/L glucose and 1μL/mL XKP mixture；basic medium with 33.3mmol/L glucose，1μL/mL XKP mixture and 2mmol/L 3-MA)for 36h.(b)Statistical results of cell immunofluorescence test.At least three replicates were performed.Error bars，s.d.；P＜0.01 compared to control cells by one-way ANOVA.The amount of autophagosomes per cell was quantified in at least 30 cells from three separate experiments.(c)Flow cytometry analysis determined cell apoptosis rates of MIN-6 cells under four different treatments(basic medium with 33.3mmol/L glucose and 1μL/mL XKP mixture；basic medium with 33.3mmol/L glucose，1μL/mL XKP mixture and 2mmol/L 3-MA；basic medium with 33.3mmol/L glucose and 10ng/mL rapamycin；basic medium with 33.3mmol/L glucose，10ng/mL rapamycin and 2mmol/L 3-MA)for 36h.(d)Statistical results of means±S.D.of apoptosis rate for each treatment from the three separate experiments.One way ANOVA was used to measure statistical differences in all experiments(P＜0.01).(e)Western blotting analysis.MIN-6 cells were treated with different conditions(basic medium；basic medium with 33.3mmol/L glucose；basic medium with 33.3mmol/L glucose and 1μL/mL XKP mixture；and basic medium with 33.3mmol/L glucose，1μL/mL XKP mixture and 2mmol/L 3-MA)for 36h，and total cell extracts were immunoblotted with antibodies against LC3，procaspase-3，cleaved-caspase-3 and GAPDH.
High glucose showed no effect on autophagy level ofβ-cells(Figure 3a，b).However，where XKP was added with glucose，autophagy of MIN-6 cells significantly increased and where 3-MA was included，autophagy decreased(Figure 3a，b).Our results on apoptosis rates showed that addition of XKP decreased the rate ofβ-cells under high glucose stress from 23.24%to 10.92%(Figure 1).The opposite pattern was observed between the XKPand XKP+3-MA-treated groups of cells where the addition of 3-MA potentially increased the apoptotic rate of glucosestressedβ-cells from 10.92%to 23.94%，respectively(Figure 3c，d).The expression of autophagy/apoptosis-related proteins(LC3 and Caspase 3)was consistent with these results(Figure 3e).