Mechanism Of Inhibitory Effects Of Vinpocetine On The Osteoblastic Differentiation Of Vascular Smooth Muscle Cells Through The ERK/Akt Pathway

Objective: Vascular calcification is the calcification of tunica media,which is similar to osteoblastic differentiation of vascular smooth muscle cells;however, its exact mechanism remains unclear. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the proliferation of VSMCs; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of VSMCs. So the experiment,s objectives are:(1)To determine the effect of vinpocetine on the osteoblastic differentiation of vascular smooth muscle cells;(2) To determine whether vinpocetine stimulates the ERK/Akt signaling in vascular smooth muscle cells;(3) To determine whether vinpocetine affects the osteoblastic differentiation of vascular smooth muscle cells via it,s receptor TSPO.Methods: 10 mM of beta-glycerophosphate(β-GP) was used to induce osteoblastic differentiation of mouse vascular smooth muscle cells(VSMCs),vinpocetine was treated on the β-GP-stimulated VSMCs. The protein expression of Run related transcription factor 2(Runx2), bone morphology protein-2(BMP-2), ERK, p38, JNK, Akt and the phosphorylation of them were determined by Western Blot. The mRNA expression of Run related transcription factor 2(Runx2), bone morphology protein-2(BMP-2) were determined by qPCR. Alkaline phosphatase(ALP) assay kit was used todetermine the ALP activity. The formation of mineralized nodules was determined by Alizarin Red S staining.VSMCs were treated with 15μM vinpocetine at 0, 5, 10, 15, 30, 60 min.ERK, c-jun N-terminal kinases(JNK), p38 MAP kinases(p38), Akt and their phosphorylated isoforms were determined by Western Blot. The activities of ERK/Akt were inhibited by their specific inhibitor PD98059/LY294002. After48 h incubation, cells were harvested and levels of phosphorylated and total ERK/Akt protein were determined by Western Blot, and then the ALP activities were determined by ALP assay kit.VSMCs were treated with 5-15μM vinpocetine, after 7d incubation, cells were harvested and levels of TSPO mRNA determined by qPCR. VSMCs were treated with 15μM vinpocetine, after 3, 6, 9, 12 d incubation, cells were harvested and levels of TSPO mRNA determined by qPCR. VSMCs were treated with siRNA, after incubation, cells were harvested and levels of TSPO and ERK/Akt protein were determined by Western Blot, and then the ALP activities were determined by ALP assay kit.Results:(1) Vinpocetine significantly decreased ALP activity, inhibited the expression of Runx2 and BMP-2, and attenuated the formation of mineralized nodule.(2) The phosphorylation of ERK and Akt were increased5 min after incubation with 15μM vinpocetine and the peaked effect was at15 min. The phosphorylation of p38 and JNK were not determined.(3)Expression of TSPO mRNA was significantly induced by vinpocetine.Expression of TSPO protein was significantly reduced by siRNA. Silence of TSPO inhibited the phosphorylation of ERK/Akt and abolished the decreased ALP activity by vinpocetine.Conclusion: Vinpocetine could stinulate its receptor TSPO to inhibit the osteoblastic differentiation of VSMCs via ERK/Akt signaling pathway.