Salidroside Regulates Phenotype Of Osteoclast Based On Osteoblast-osteoclast Interaction Via HIF-1α Pathway

ObjectMicroenvironmental hypoxia is a common feature of osteolytic diseases such as osteoporosis,rheumatoid arthritis,primary bone tumors and bone metastatic carcinoma,and is closely associated with disease progression and poor prognosis.Hypoxia-inducible factor-1(HIF-1)is a core transcription factor that regulates oxygen homeostasis and consists of two subunits,α and β.HIF-1α is an oxygen regulatory protein that regulated by oxygen partial pressure strictly.Under normoxic conditions,HIF-1α is hydroxylated by PHDs,recognized by p VHL and then degraded by protein ubiquitination hydrolysis system.When cells under hypoxic conditions or p VHL is functionally deficient,HIF-1α accumulates and translocates to the nucleus to bind with HIF-1β to form the HIF-1 complex,which acts as a transcription factor and binds to the hypoxia response element(HRE).In turn,it activates transcription of downstream genes,which triggers a series of oxygen-tolerant adaptive responses in tissue cells.HIF-1α is expressed in both osteoblasts and osteoclasts,and hypoxia promotes osteoclasts differentiation into multinucleated cells and increases their bone resorption activity.It has been shown that VEGF and ANGPTL4 are downstream target genes of HIF-1α,while our group has demonstrated that IL-6 and RANKL are also new downstream target genes of HIF-1α throughluciferase assay and Ch IP experiments,which are involved in the regulation of bone formation and bone resorption activity jointly.The Tibetan medicine Rhodiola rosea has various pharmacological effects such as anti-hypoxia,pro-angiogenesis and anti-osteoporosis.Our previous studies have demonstrated that its main active ingredient,rhodiogenin(SAL),can increase bone density and promote fracture healing in OVX rats by upregulating the angiogenic bone formation coupling through the HIF-1α/VEGF pathway in osteoblasts.In this study,we investigated whether SAL regulates the phenotypic expression of co-cultured osteoblasts via the hypoxic osteoblast HIF-1α pathway.Subsequent work by the group showed that SAL inhibited lipopolysaccharide(LPS)-induced bone loss in vivo,and promoted the differentiation and maturation and bone resorption activity of osteoblastic precursor cells by upregulating the HIF-1α/VEGF,IL-6 and ANGPTL4 pathways under hypoxic conditions in vitro.This study laid the foundation for a comprehensive and in-depth elucidation of the mechanism of SAL against bone loss,and provided a scientific basis for the future clinical application of SAL in the prevention and treatment of bone loss.Methods1.MC3T3-E1 and MOB were used as experimental objects,and MOB was extracted by combined trypsin-collagenase digestion and identified by alkaline phosphatase staining.The effects of different concentrations of SAL(1 n M,10 n M,100 n M,1000 n M,10000 n M)on the viability of osteoblasts were detected by MTT,and the drug concentrations for subsequent experiments were determined.The effects of SAL on HIF-1α,VEGF,IL-6,ANGPTL4,RANKL gene and protein expression in the HIF-1α signaling pathway under hypoxia were measured by qRT-PCR,Western blot,ELISA.2.RAW264.7 and BMMs were differentiate into multinucleated osteoclasts by RANKL and M-CSF,and osteoblasts(MC3T3-E1 cells and MOB)were pretreated with SAL.The culture supernatant was collected as conditioned medium mixed with osteoclasts medium at a ratio of 1:3 for osteoclasts.The osteoclasts were tested by MTT and flow cytometry for viability and apoptosis;qRT-PCR for osteoclasts differentiation and functional marker gene expression;TRAP staining for osteoclasts differentiation;and toluidine blue staining for osteoclasts bone resorption activity.Aim to detect the effects of SAL on osteoclasts viability,apoptosis rate,differentiation and bone resorption function.Results1.Effects of SAL on HIF-1α pathway in hypoxic osteoblastThe cranial MOB of C57/BL6 suckling mice were extracted by trypsin-collagenase digestion and identified as osteoblasts by alkaline phosphatase staining.MTT results showed that five concentrations(1 n M,10 n M,100 n M,1000 n M,10000 n M)of SAL promoted the viability of MC3T3-E1 cells and MOB cells compared with the control group.The qRT-PCR results showed that hypoxia promoted the gene expression of HIF-1α,VEGF,IL-6,ANGPTL4 and RANKL in the HIF-1α signaling pathway compared with the control group.Compared with the hypoxic group,SAL upregulated the mRNA expression levels of VEGF and downregulated the mRNA expression levels of HIF-1α,IL-6,ANGPTL4 and RANKL.Western blot results showed that hypoxia promoted the protein expression of HIF-1αcompared with the control group,and SAL down-regulated the protein expression of HIF-1αcompared with the hypoxic group.ELISA results showed that hypoxia promoted the protein secretion of VEGF,IL-6,ANGPTL4 and RANKL compared with the control group,and SAL up-regulated the protein levels of VEGF and down-regulated the protein levels of IL-6,ANGPTL4 and RANKL compared with the hypoxic group.2.Effects of SAL on osteoclast phenotype via HIF-1 α /VEGF,IL-6,ANGPTL4 and RANKL pathways in osteoblastMC3T3-E1 and MOB culture supernatant were collected as conditioned medium(CM)acting on RAW264.7 cells and BMMs.MTT results showed that after the addition of SAL-activated osteoblasts culture supernatant,osteoclasts viability was reduced in the hypoxic CM+SAL group compared with the hypoxic CM group.The flow results showed that after the addition of SAL-activated osteoblasts culture supernatant,the apoptosis rate of osteoclasts was higher in the hypoxic CM+SAL group compared with the hypoxic CM group.qRT-PCR results showed that after the addition of SAL-activated osteoblasts culture supernatant,the osteoclast-specific differentiation marker gene RANK,TPRA,CTR and functional marker gene MMP-9,Cat K were downregulated in the hypoxic CM+SAL group compared with the hypoxic CM group.The effect was diminished after the addition of blocker YC-1 and neutralizing antibodies Avastin,Anti-IL-6,MBL,Denosumab-activated osteoblasts culture supernatant.TRAP staining showed that compared with the hypoxic CM group,the number of TRAP-positive multinucleated cells was decreased,the number of cell nucleus was decreased and the differentiation of osteoclasts was inhibited in hypoxic CM+SAL group of RAW264.7.The effect was diminished after the addition of blocker YC-1 and neutralizing antibodies Avastin,Anti-IL-6,MBL,Denosumab-activated osteoblasts culture supernatant.Toluidine blue staining showed that the bone resorption traps area of RAW264.7 in the hypoxic CM+SAL group were reduced compared with the hypoxic CM group,and the bone resorption function of osteoclasts was inhibited.The effect was diminished after the addition of blocker YC-1 and neutralizing antibodies Avastin,Anti-IL-6,MBL,Denosumab-activated osteoblasts culture supernatant.Conclusion1.SAL downregulated the expression of HIF-1α,IL-6,ANGPTL4,RANKL and upregulated expression of VEGF in osteoblasts under hypoxia.2.SAL inhibited the cell viability,marker gene expression,differentiation and bone resorption activity of co-cultured osteoclasts and promoted apoptosis of osteoclasts through regulation of HIF-1α/VEGF,IL-6,ANGPTL4 and RANKL pathways in hypoxic osteoblasts.