The Role Of NRF2-ARE Signaling Pathway In Osteogenic Differentiation Injury Of MC3T3-E1 Cells Induced By Hydrogen Peroxide And The Intervention Of Resveratrol

Objective:To investigate the role of nuclear factor E2-related factor 2（NRF2）-antioxidant response element（ARE）signaling pathway in the differentiation and injury of mouse osteogenic precursor cells（MC3T3-E1）induced by hydrogen peroxide（H₂O₂）.To observe the effects of resveratrol（Res）on H₂O₂-induced osteoblast differentiation injury,and to explore the function of NRF2-ARE signaling pathway in the process.Methods:1.CCK-8 method was used to gauge the effects of different concentrations of H₂O₂ on the survival rate of cells.The cells were separated into normal control（NC）group,200μM H₂O₂（200H）group,400μM H₂O₂（400H）group.The m RNAs and proteins were extracted after 24h H₂O₂intervention,Real-time quantitative PCR（RT-PCR）and Western blot（WB）was used to measure type 1 collagen（COL1）,Runt-associated transcription factor 2（RUNX2）,NRF2,heme oxygenase-1（HO-1）,catalase（CAT）,quinone oxidoreductase 1（NQO1）m RNA and protein expressions in each group.After drug intervention,osteogenic induction fluid was replaced to induce osteogenic differentiation,Alkaline phosphatase（ALP）staining was performed on day 7 to evaluate the ability of early osteogenic differentiation,and alizarin red staining was performed on day 21 to evaluate the degree of cell mineralization.2.To observe the effects of inhibiting NRF2-ARE signaling pathway on H₂O₂-induced osteogenic differentiation injury:The cells were divided into NC group,400H group,NRF2 specific inhibitor（5μM ML385,M）group and 400μM H₂O₂+5μM ML385（400H+M）group,Cells were pretreated by ML385 for 12h before H₂O₂ intervention,Reactive oxygen species（ROS）levels were detected by flow cytometry or fluorescence microscopy after H₂O₂treatment for 6h.NRF2-ARE signaling pathway and osteogenic differentiation and mineralization markers were measured by the same methods as above.3.To observe the effect of H₂O₂ on osteogenic differentiation and NRF2-ARE signaling pathway under Res intervention.CCK-8 method was used to gauge the effects of different concentrations of Res on the survival rate of cells.The cells were separated into NC group,400H group,400μM H₂O₂+0.1μM Res（400H+0.1R）group,400μM H₂O₂+1μM Res（400H+1R）group,400μM H₂O₂+5μM Res（400H+5R）group,400μM H₂O₂+5μM NAC（400H+5N）group.Res or NAC pretreated cells for 12h before H₂O₂ intervention.ROS levels,m RNA or protein expression levels of COL1、RUNX2、ALP、osteocalcin（OCN）NRF2、HO-1、NQO1、CAT,and early osteogenic differentiation ability and mineralization degree of cells in each group were detected by the same methods as above.4.Effect of inhibition of NRF2-ARE signaling pathway on Res resistance to H₂O₂ induced osteogenic differentiation injury:The cells was divided into control group,400H group,400H+1R group,400μM H₂O₂+1μM Res+5μM ML385（400H+1R+M）group.Prior to Res intervention,cells were pretreated with ML385 for 12h,H₂O₂ was then used for intervention.The ROS level,NRF2-ARE signaling pathway and osteogenic differentiation and mineralization markers of cells in each group were detected by the same methods as above.Results:1.Compared with NC group,the mRNA and protein expression levels of COL1,RUNX2,NRF2,HO-1,CAT and NQO1,and the ALP staining and mineralization in H₂O₂ group were significantly decreased,and the m RNA and protein expression levels of NRF2,HO-1,CAT and NQO1 were significantly up-regulated,and the parameters all above were more obvious in 400H group than 200H group（all P<0.05）.2.Compared with NC group,ROS level in 400H group increased significantly;Compared with400H+M group,the m RNA and protein expression levels of NRF2,HO-1,NQO1,CAT,COL1 and RUNX2 in 400H+M group were significantly down-regulated,and the ALP staining and mineralization in 400H+M group were significantly decreased,and the ROS level in 400H+M group was significantly up-regulated（all P<0.05）.There was no significant difference in the above parameters between the M group and the NC group.3.Compared with 400H group,the mRNA expression levels of Col1a1 and Runx2,protein expression levels of RUNX2,COL1,ALP and OCN,the ALP staining and mineralization in Res or NAC groups were significantly up-regulated,and the ROS level of cells in all Res or NAC groups was significantly down-regulated（all P<0.05）.There was no significant difference in the above indexes between all Res or NAC groups.Compared with 400H group,the m RNA and protein expression levels of NRF2,HO-1,CAT and NQO1 in all Res groups were significantly up-regulated（P<0.05）,which were significantly down-regulated（P<0.05）in 400H+5N group,and there was no statistical significance among all Res groups.4.Compared with 400H+1R group,the protein or m RNA expression levels of NRF2,HO-1,NQO1,CAT,RUNX2,COL1,ALP and OCN in 400H+1R+M group were significantly down-regulated,the ALP staining and mineralization in 400H+1R+M group was also significantly down-regulated,and the ROS level was significantly up-regulated（all P<0.05）.Conclusion:1.The process of osteogenic differentiation and injury of MC3T3-E1 cells induced by H₂O₂was accompanied by the activation of NRF2-ARE signaling pathway,which to some extent alleviated the oxidative damage of osteogenic differentiation of MC3T3-E1 cells,and the mechanism may be related to the antioxidant effect of NRF2-ARE signaling pathway.2.Resveratrol can alleviate H₂O₂-induced osteogenic differentiation injury of MC3T3-E1 cells,and the mechanism may be related to resveratrol further activating NRF2-ARE signaling pathway and inhibiting H₂O₂-induced ROS level increase.