*3.2. E*ff*ect of Curcumin on Apoptosis of MC3T3 Cells in High-Glucose Conditions*

Flow cytometry showed that curcumin treatment had an effect on glucose-induced apoptosis in MC3T3 cells (Figure 2A). The first-quadrant cells were necrotic and consisted of late apoptotic cells, which could be simultaneously stained with Annexin V-FITC and PI. The second-quadrant cells were PI-negative. In the third quadrant, normal living cells were not stained with Annexin V-FITC or PI. The fourth-quadrant cells were only stained with Annexin V-FITC, indicating early apoptotic cells. The early apoptosis rates of the control and glucose groups were 9.03% ± 0.67%, 14.70% ± 0.26%, and 18.70% ± 0.61%, and the curcumin-treated groups were 13.30% ± 0.10%, 12.00% ± 0.85%, 16.80% ± 0.20%, and 13.77% ± 0.21%. Glucose significantly increased the rate of apoptosis (*p* < 0.05), whereas treatment with both low and high doses of curcumin significantly decreased the apoptosis rate for cells under different concentrations of glucose (*p* < 0.05). In the high glucose group, the apoptosis rate in the high concentration of the curcumin group was significantly lower than that in the low concentration of the curcumin group (*p* < 0.05; Figure 2B), whereas there were no differences in the medium glucose group. It can be concluded that treatment with curcumin significantly protected cells from hyperglycemia-triggered apoptosis of MC3T3 cells under high-glucose conditions. This was shown in

a dose-dependent manner under a high concentration of glucose, but not under a low concentration of glucose, under which a low concentration of curcumin can achieve an optimal protective effect on cells. This expands on previous observations that curcumin mitigates hyperglycemia-related clinical symptoms [29,30].

**Figure 2.** Effects of curcumin on apoptosis of MC3T3 cells in high-glucose conditions. Cell apoptosis was assessed by staining the cells with Annexin V-FITC/PI double staining kit and subsequent analysis by flow cytometry. (**A**) Detection of the early apoptosis rate of MC3T3 cells using flow cytometry. (**B**) The bar chart shows the mean of early apoptosis rate (*n* = 3). \**p* < 0.05 vs the control group; #*p* < 0.05 vs the medium glucose group; *p* < 0.05 vs the high glucose group; and &*p* < 0.05 vs the high glucose/low curcumin group.
