Research On The Mechanism Of SAL On Regulating Phenotypic Expression Of Rat Calvarial Osteoblasts

Objectives The neonatal rat calvarial osteoblasts(ROB) obtained by the repeatedenzymatic digestion method. Under the normoxic condition, we observed the regulationof the HIF-1alpha and VEGF and the affect of proliferation, differentiation andmineralization by different concentrations of salidroside acting on research model-ROB.We want to investigate the possible mechanism of salidroside that effected on ROB ofphenotypic expression through HIF-1signaling passway under normoxic condition.Methods1The rat calvarial osteoblasts(ROB) obtained by enzymatic digestion methodand purified by the method of differential adhesion. We identified the ROB by themethod of cell morphology, Gomori method staining for ALP and mineralized nodulesstaining by ARS. The third generation of the purification up to90%were used for study.2The ROB cell proliferative activity was detected by MTS assay.3Using alkalinephosphatase kit to determine the effect of SAL on ROB cells ALP activity-a earlydifferentiation marker gene-by benzene disodium phosphate method.4The mRNAexpression levels of the HIF-1α, VEGF, Osterix (Osx), Runx2and osteocalcin weredetected by reverse transcription polymerase chain reaction.5The OC,VEGF and Col-1expression levels were detected by enzyme-linked immnosorbent assay.6Effect on ROBmineralization with SAL were detected by ARS and quantitative analysis.Results1Gomori method staining for ALP in rat calvarial osteoblasts shows blackdeposits in cytoplasm. ROB were not subcultured formed mineralized nodules after13days, which can appear some black opaque area under an inverted microscope and somered calcificated extracellular matrix by ARS staining.2SAL can significantly promoteROB proliferation.3SAL can significantly promote the content of ALP, Col-1and OC inROB.4SAL can promote ROB to form mineralized nodules.5SAL can significantlyincrease the mRNA expression levels of osteoblast specific transcription factor-Oterixand Runx2.6SAL can promote the expression of the gene of HIF-1α and VEGF underthe normoxic condition.Conclusions SAL can promote ROB proliferation, differentiation and mineralization;The mechanism of SAL promote ROB proliferation, differentiation and mineralizationmay up-regulate osteoblast specific transcription factor-Runx2and Osx and activate the gene of HIF-1α and VEGF.