Effect Of SAL On Regulating Phenotypic Expression Of Osteoblast Through HIF-1Signaling Passway

Objectives: Studies have showed that SAL has significant effect onanti-hypoxia and angiogenesis. It has been proved that SAL can promoteproliferation activity and differentiation function of primarily culturedosteoblast. But the regulation function of SAL on phenotypic expression andits related signaling passway in normoxia and hypoxia environment have notbeen established. Here we choose human osteoblast-like cell line MG-63andrat osteoblastic cell as the research models to study the effect of SAL onhypoxia-inducible factor-1alpha(HIF-1α) signaling pathway and phenotypicexpression of osteoblast, and also explore possible molecular mechanism ofSAL.Methods:1The proliferation activity assay with MTT method.2Apoptosis of osteoblast in hypoxia environment was detected byAnnexin V/PI double staining and flow cytometry.3Alkaline phosphatase (ALP) activity assay with colorimetric methodwas performed to detect the effect of salidroside on early differentiation ofosteoblast.4The mRNA expression levels of HIF-1α,vescular endothelial growthfactor(VEGF), osteocalcin(OC), von hippel-lindau protein(pVHL), Osterix andRunx2were detected by reverse transcription polymerase chain reaction(RT-PCR).5Secretion capacity of VEGF, OC and collagen Ⅰ synthesis wereexamined by enzyme-linked immnosorbent assay(ELISA).6The protein expression levels of HIF-1α, pVHL, Osterix, Runx2wereinvestigated by Western Blot.7Immunofluorescence confocal microscope technology was used to explore the regulation function of SAL on nuclear translocation of HIF-1α.8Regulation effect of SAL on mineralized node of ROB stained by ARS.Results:1The effect of SAL on HIF-1signaling passway of osteoblastIn normoxic state, SAL could significantly promote protein HIF-1αandVEGF expression and inhibit pVHL expression in MG-63cells. In hypoxicenvironment， it could elevate protein HIF-1α and VEGF levels anddown-regulate pVHL protein expression level. But compared with it，pretreatment with SAL might markedly decrease HIF-1αexpression level andpromote expression of VEGF and pVHL.Under normoxic conditions，little HIF-1αexpressed in MG-63cells. Butin hypoxic environment，HIF-1αcould be translocated into nuclear andexpressed a lot. Compared with hypoxia control group，pretreatment with SALmight prevent HIF-1α into nuclear.2Effect of SAL on regulating phenotypic expression of osteoblastSAL could markedly promote MG-63cell proliferation and inhibithypoxia-induced apoptosis in MG-63cells. SAL stimulated cell differentiationthrough promoting activities of ALP and secretion capacity of OC andcollagenⅠof MG-63cell in normoxic and hypoxic environment. Regulationeffect of SAL on mineralized node of ROB was also remarkable. In normoxicenvironment，SAL could promote ROB to form more typical mineralizednodes compared with normal control group. However， the outline ofmineralized nodes became unclear and non-typical under hypoxia. Pretreatedwith SAL，mineralized nodes became much better and quantity increased.SAL could also up-regulate expression levels of Osterix and Runx2，andthen promote growth and development of osteoblast.Conclusions:1SAL could stimulate osteoblast proliferation, differentiation andmineralization in normoxia and hypoxia environment. Meanwhile, SAL couldalso inhibit osteoblast apoptosis which was induced by hypoxia. 2SAL could protect osteoblast from hypoxia damage and promotephenotypic expression of osteoblast through HIF-1αsignaling pathway.