The Precautive Role Of Valproic Acid In Glucocorticoid-induced Osteonecrosis Of Femoral Head In Rat

Valproic acid is a widely used antiepileptic and anticonvulsant drug.In 2001,Gottlicher et al.found that valproic acid is a histone deacetylases inhibitor（HDACi）,influencing gene transcription by regulating acetylation of histone.An increasing amount of evidence show that HDACi promote can promote osteogenic differentiation of several cell types,such as bone marrow-derived mesenchymal stem cell and adipose-derived mesenchymal stem cell.Accordingly,valproic acid,as a commonly clinical medicine and an HDAC,can be used to promote osteoblast maturation and bone healing.Glucocorticoid-induced osteonecrosis of femoral head（ONFH）and bone defect after trauma or osteotomy often cause trouble to orthopedists.The mechanism of glucocorticoid（GC）-induced ONFH is associated inhibition of osteogenic differentiation of BMSCs.Besides,the scaffold bone defect is filled with scaffold whose osteoinduction is limited.Consequently,valproic acid is promising in precaution of ONFH and in scaffold establishment with adulteration of valproic acid to promote osteogenesis.OBJECTIVE:The current research aimed to study the effect of VPA on glucocorticoid treated hBMSCs,HMEC-1 and to study the effect of VPA on glucocorticoid-induced ONFH in rat.Method:（1）the harvest,culture and identification of hBMSCs with bone marrow from patients.（2）we treated hBMSCs with gradient concentrations of VPA to see its effect on proliferation and apoptosis:the CCK-8 assay and Annexin V-FITC/PI double staining with FCM are performed to evaluate the proliferation and apoptosis.（3）the effect of VPA on hBMSCs osteogenic differentiation is evaluated by measurements of ALP activity,osteogenic-associated proteins and genes expression,e.g.Runx2,ALP,OCN,Col I,calcium nodule by alizarin red.（4）the effect of VPA on hBMSCs osteogenic differentiation on condition of DEX is evaluated by measurements of ALP activity,osteogenic-associated proteins and expression,e.g.Runx2,ALP,OCN,Col I,calcium nodule by alizarin red.（5）the effect of VPA on hBMSCs migration is evaluated in a manner of Transwell chamber assay,and the associated SDF-1α/CXCR4 signaling pathway is also measured in terms of protein and gene expression.（6）the effect of VPA on angiogenesis is evaluated with tube formation assay after culture of HMEC-1 in condition of DEX with or with VPA for 2 days.（7）the rat model of ONFH is established in a manner that MP was intramuscularly injected once in 3 consecutive days for 3 weeks and the evaluation method included micro-CT,micro-fill and HE staining.（8）during the period of model establishment,we intraperitoneally injected 300mg/kg once in 3 weeks and the preventive role of VPA was evaluated in a manner similar to the 6^th part.RESULTS:（1）hBMSCs were successfully harvested with positive in CD29（95±3.7%）,CD44（91±2.1%）,CD73（98±1.4%）,CD90（98±2.3%）,CD105（76±6.2%）and could differentiate into osteo-lineage and adipo-lineage after specific induction.（2）VPA in lower concentration（0.5²mM）did not significantly influence the proliferation of hBMSCs while higher concentration（5mM）significantly inhibited the proliferation of hBMSCs,however,the apoptosis is not influenced.（3）VPA upregulated the osteogenic-associated proteins and genes expression,promoted ALP activity and calcium nodule deposition.（4）VPA attenuated the inhibitive role of DEX on the proliferation and osteogenic differentiation of hBMSCs by improvement in ALP activity,calcium nodule deposition and expression of osteogenic-associated proteins and genes.（5）the hBMSCs from patients with ONFH manifested inhibited migration ability with lower CXCR4 and SDF-1αexpression both in proteins and genes.With addition of VPA,the lowered CXCR4and SDF-1αexpression were reversed and migration ability of hBMSCs was improved with more cells transiting into the down chamber.（6）VPA improved angiogenesis of HMEC-1 in condition of DEX and upregulated the associated genes expression.（7）the successful rat model of ONFH was characterized with thinner and sectioned trabecula with empty lacuna,degeneration of epiphyseal plate,bone marrow necrosis（8）VPA lowered the occurrence of rat ONFH.CONCLUSION:VPA could attenuate the inhibitive effect of DEX on the proliferation,osteogenic differentiation and migration ability of hBMSCs,on the angiogenesis of HMEC-1,and lead to the precaution of ONFH in rats.