Research Paper Volume 12, Issue 3 pp 2084—2100

Insulin impedes osteogenesis of BMSCs by inhibiting autophagy and promoting premature senescence via the TGF-β1 pathway

Figure 4. TGF-β1 promotes senescence, inhibits autophagy and osteogenic differentiation of DM-BMSCs. DM-BMSCs were incubated under hyperglycemic conditions, and H-BMSCS were incubated under normoglycemic conditions. The expression of the TGF-β1 signaling pathway was measured by western blot (A). DM-BMSCs were incubated under hyperglycemic conditions with or without insulin addition of the SB435142 or hrTGF-β1 for 3 days. The expression of LC3 and P62 were detected by western blot after serum deprivation for 6 h (B). Protein bands were quantified and analyzed by densitometric analysis (C, D). Cellular senescence was detected by SA-β-Gal staining after incubation in the corresponding condition for 3 days (E). The number of positive cells was calculated (F). DM-BMSCs were cultured in osteogenic medium for 7 days with or without insulin under hyperglycemic conditions stimulated with SB431542 or hrTGF-β1. mRNA level expression of Alp (G), Runx2 (H), Ocn (I), and Opn (J) was detected by real-time PCR. NG, normoglycemic condition, HG, hyperglycemic condition. Data are presented as the mean ± standard deviation, n=3. *p<0.05, #p>0.05. Scale bar = 100 μm.