Mechanism of Naringenin in amelioraing glucocorticoid-induced osteonecrosis of the femoral head by regulating HO-1/HIF-1α/VEGF axis

ZHANG Xinwei; SONG Ming; ZHU Hongxun; DAI Shouping; ZHANG Yusong; WEI Biaofang

doi:10.13431/j.cnki.immunol.j.20250106

Abstract：Objective To investigate the mechanism of Naringenin(NGN)in the treatment of steroid(glucosteroid)-induced osteonecrosis of the femoral head(SONFH).Methods A SONFH rat model was established using Methylprednisolone(MPS)treatment,followed by intervention with NGN and zinc protoporphyrin(ZnPP).Micro-CT was used to analyze the morphological changes in femoral head tissues,and the levels of osteocalcin(OCN)in rat serum as well as heme oxygenase-1(HO-1)and hypoxia-inducible factor-1α(HIF-1α)in femoral bone tissue were measured.A cellular model was constructed by treating MC3T3-E1 cells with Dexamethasone(DEX),followed by NGN intervention.Bioinformatics analysis combined with molecular docking technology was used to predict the target of NGN,and the Pulldown experiment was performed for validation.The expression of HO-1 was knocked down through cell transfection,to analyze the viability,proliferation,apoptosis,and migration of MC3T3-E1 cells,and angiogenesis assays were conducted to evaluate the angiogenic potential of human umbilical vein endothelial cells(HUVECs).Results Micro-CT analysis revealed that,compared with the control group,the trabecular thickness and trabecular number were significantly reduced in the MPS group,while the bone surface area/bone volume ratio and trabecular separation were significantly increased(P<0.001).In vitro experimental results indicated that DEX inhibited the proliferation of MC3T3-E1 cells,promoted cell apoptosis,and increased reactive oxygen species generation(P<0.01),and that DEX suppressed the formation of mineralized nodules,a key indicator of osteogenic differentiation,and downregulated the expression of osteogenesis-related genes(Runt-related transcription factor 2,osteopontin,osteocalcin)(P<0.01).However,NGN treatment partially reversed these effects.DEX significantly inhibited the migration of HUVECs,angiogenesis,and the expression of angiogenesis-related markers(platelet endothelial cell adhesion molecule-1,vascular endothelial growth factor,and von Willebrand factor)(P<0.01).In contrast,NGN treatment did not significantly affect the aforementioned effects,but the treatment with NGN conditioned medium[CM(NGN)]partially reversed these effects(P<0.01).Bioinformatics analysis combined with Pulldown assay results indicated that HO-1 was the target of NGN.DEX treatment significantly downregulated the expression of HO-1,while NGN intervention partially counteracted the inhibitory effect induced by DEX(P<0.01);knockdown of HO-1 negated the therapeutic effects of NGN(P<0.01).Compared with MPS administration alone,the combined administration of NGN and MPS upregulated the expression of HO-1 and HIF-1α in rat femoral head tissues.However,the HO-1 inhibitor ZnPP further upregulated the expression of HO-1 but downregulated the protein level of hypoxia-inducible factor-α(HIF-1α)(P<0.01).Conclusion NGN exerts its therapeutic effects on SONFH by activating the expression and activity of HO-1,which regulates the HIF-1α/VEGF pathway to promote osteoblast differentiation,bone formation,and angiogenesis.