强骨康疏方含药血清调控HIF-1α/Wnt/β-catenin信号通路抑制铁死亡从而促进低氧处理的MC3T3-E1细胞成骨分化OA

AIM:To investigate the mechanism by which Qianggu-Kangshu formula-containing serum regu-lates the hypoxia inducible factor-1α(HIF-1α)/Wnt/β-catenin pathway to inhibit ferroptosis and promote osteogenic dif-ferentiation of hypoxic MC3T3-E1 cells.METHODS:(1)The MC3T3-E1 cells were exposed to 1%O₂ for 24,48 and 72 h.Cell viability was assessed by CCK-8 assay,and the protein expression of HIF-1α,β-catenin,glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11)and Runt-related transcription factor 2(RUNX2)was detected by Western blot to determine the optimal hypoxic modeling duration.The optimal intervention concentration of Qianggu-Kangshu formula-medicated serum at low,medium and high doses was screened by CCK-8 assay.(2)The cells were di-vided into control,hypoxia model and hypoxia+medicated serum(low,medium and high doses)groups.Osteogenic dif-ferentiation was evaluated by alizarin red S staining and alkaline phosphatase(ALP)activity assay.Oxidative stress and ferroptosis were assessed by detecting malondialdehyde(MDA),glutathione(GSH),reactive oxygen species(ROS)and ferrous ion levels,combined with transmission electron microscopy.The protein and mRNA expression of RUNX2,osteo-protegerin(OPG),GPX4 and SLC7A11 were detected by Western blot and RT-qPCR.(3)The cells were divided into control,hypoxia and hypoxia+medicated serum groups.The protein and mRNA expression of HIF-1α,Wnt3a,β-catenin,T-cell factor 4(TCF4)and phosphorylated glycogen synthase kinase-3β at Ser9(p-GSK-3β Ser9)were detected by Western blot and RT-qPCR.For reverse validation,cells were divided into control,hypoxia model,hypoxia+1,4-DPCA,and hypoxia+1,4-DPCA+high-dose medicated serum groups.RESULTS:(1)Compared with normoxia control group,72 h of hypoxia significantly inhibited cell viability(P<0.05),increased HIF-1α protein expression(P<0.01),and de-creased the protein expression of RUNX2,β-catenin,GPX4,and SLC7A11(P<0.05).Among different concentrations of the medicated serum,5%yielded the best cell viability(P<0.05),and was therefore selected as the uniform interven-tion concentration.(2)Compared with hypoxia model group,treatment with Qianggu-Kangshu formula-containing serum significantly attenuated mitochondrial damage,reduced the levels of ROS,MDA and ferrous ion,and increased the levels of GSH and ALP(P<0.05).It also down-regulated HIF-1α protein and mRNA levels(P<0.05),and up-regulated the protein levels of GPX4,SLC7A11,RUNX2,OPG,Wnt3a,β-catenin,TCF4 and p-GSK-3β Ser9,as well as the mRNA levels of RUNX2 and GPX4(P<0.05).(3)Treatment with 1,4-DPCA(4 μmol/L)significantly increased HIF-1α pro-tein levels and inhibited the Wnt pathway(P<0.01).Compared with 1,4-DPCA alone group,co-intervention with high-dose medicated serum significantly decreased HIF-1α protein expression and up-regulated the protein expression of Wnt3a,β-catenin and TCF4(P<0.01).CONCLUSION:Qianggu-Kangshu formula-medicated serum inhibits hypoxia-induced aberrant activation of HIF-1α,restores Wnt/β-catenin signaling pathway function,attenuates mitochondrial damage,and reduces oxidative stress,thereby suppressing ferroptosis in osteoblasts and promoting osteogenic differentia-tion and mineralization.