Figure 3.

ED-71 promotes BMAL1 expression to ameliorate circadian rhythm disturbances of osteoblasts induced by HG in vivo and in vitro. (A), the mRNA expression of BMAL1 and CLOCK in bone tissues were detected by RT-qPCR in WT, T2DM, and T2DM + ED-71groups (n = 5). (B–D), Western blot detection and statistical analysis of BMAL1 protein expression in bone tissues (n = 5). (C and E), representative images of osteogenic gene RUNX2 (green) and rhythmic gene BMAL1 (red) in tibia were observed by IF double staining (n = 5), bar = 75μm. Red arrows indicated the representatives of double positive cells. (F and G), Western blot detection of BMAL1 protein expression in MC3T3-E1 cells after 48 hours of high glucose stimulation and its statistical analysis (n=3). (H and I), BMAL1 protein expression in MC3T3-E1 cells detected by IF after 48 hours of high glucose stimulation and its statistical analysis (n = 5), bar = 75μm. (J), RT-qPCR to detect changes in BMAL1 mRNA expression within the circadian time for 24 hours after HG stimulation (n = 5). Data are expressed as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001.