Research Paper Volume 13, Issue 10 pp 14355—14371

κ-opioid receptor stimulation alleviates rat vascular smooth muscle cell calcification via PFKFB3-lactate signaling

Effects of κ-OR stimulation on VSMC calcification induced by β-GP. Control: VSMCs were incubated in the absence of β-GP. β-GP: VSMCs were incubated in 10% FBS-DMEM containing β-GP (10 mmol/L) for ten days. U50,488H (10 - 80 mmol/L) was added before β-GP treatment to investigate the effect of U50,488H on VSMC calcification. (A) Cell lysates were collected and analyzed for RUNX2 using western blotting. (B) Quantitative analysis of RUNX2 expression. (C) Calcium nodules were stained with Alizarin red. Red nodules indicate calcium deposition. Scale bar = 50 μm. (D) Quantification of mineralization. (E, F) The contents of intracellular calcium and ALP were detected using calcium assay kits and ALP activity kits. U, U50,488H; β-GP, β-Glycerophosphate disodium salt pentahydrate; N, nor-BNI; Data obtained from quantitative densitometry were presented as means ± SEM. n=4 in each group. *P **P #P ##P $P

Figure 1. Effects of κ-OR stimulation on VSMC calcification induced by β-GP. Control: VSMCs were incubated in the absence of β-GP. β-GP: VSMCs were incubated in 10% FBS-DMEM containing β-GP (10 mmol/L) for ten days. U50,488H (10 - 80 mmol/L) was added before β-GP treatment to investigate the effect of U50,488H on VSMC calcification. (A) Cell lysates were collected and analyzed for RUNX2 using western blotting. (B) Quantitative analysis of RUNX2 expression. (C) Calcium nodules were stained with Alizarin red. Red nodules indicate calcium deposition. Scale bar = 50 μm. (D) Quantification of mineralization. (E, F) The contents of intracellular calcium and ALP were detected using calcium assay kits and ALP activity kits. U, U50,488H; β-GP, β-Glycerophosphate disodium salt pentahydrate; N, nor-BNI; Data obtained from quantitative densitometry were presented as means ± SEM. n=4 in each group. *P < 0.05 versus the control group, **P < 0.01 versus the control group, #P < 0.05 versus the β-GP group, ##P < 0.01 versus the β-GP group, $P < 0.05 versus the β-GP+U group.