The Effect Of Hydrogen Peroxide On Autophagy Of Osteoblasts And The Intervention Of Resveratrol

Objective:To observe the changes of autophagic flow during oxidative damage of osteogenic precursor cells MC3T3-E1 cells in mouse caused by hydrogen peroxide?H₂O₂?,and The intervention of resveratrol?Res?on the oxidative damage of H₂O₂ to osteoblasts,and to explore the protective effect of Res on oxidation injury osteoblasts and the role of autophagy in it.Method:1.the mouse osteogenesis precursor cell line MC3T3-E1 cells were incubated in incubator with saturated humidity,5%CO₂ and 37?at constant temperature.2.P3-P4 osteoblasts with good growth state were obtained.By detecting the effects of different concentrations of H₂O₂?0.1²00?M?on the activity of osteoblasts to screen the appropriate damage concentration of H₂O₂and The effect of H₂O₂ on the time change of autophagy marker protein in osteoblasts was used to screen the suitable injury time.The oxidative stress injury models of osteoblasts were constructed.The intervention of different concentrations of Res?0.1-50?M?on the degradation of osteoblast was detected to screen the concentration of Res intervention was preliminarily.And the interference effect of Res?1,5,10?M?on the oxidative stress level and apoptosis of H₂O₂ induced osteoblasts was detected to screen the appropriate concentration of Res.The autophagy inducer,rapamycin?Rapamycin,Rap?and the inhibitor 6-amino-3-methyl purine?3-Methyladenine,abbreviated as 3-MA?intervened autophagy of osteoblasts,and the relationship between the interference of Res on H₂O₂ oxidative damage to osteoblasts and autophagy was observed.3.the well growing P3-P4generation osteoblasts were randomly divided into the control group?control?,H₂O₂ group?H₂O₂100?M?,Rap group?rapamycin 100nM?,Rap+H₂O₂ group?rapamycin 100nM+H₂O₂100?M?,Resgroup?resveratrol,10?M?,Res+H₂O₂group?resveratrol10?M+H₂O₂100?M?,Res+3MA?res veratrol10?M+3-MA5mM?,Res+H₂O₂+3-MAgroup?resveratrol10?M+H₂O₂100?M+3-MA 5mM?and group H₂O₂+3-MA?H₂O₂100?M+3-MA5mM?;4.flow cytometry was used to detect the cell apoptosis rate and the active oxygen?ROS?level;.5.detect the level of MDA,SOD,and T-AOC in each cell culture fluid.;6.Western Blot was used to detect changes in the expression of autophagy marker protein LC3II/LC3-I,Beclin-1,P62 and apoptotic protein Caspase3 and cleaved Caspase3.Results:1.The effects of H₂O₂ on the viability and autophagy of osteoblasts showed that low concentration?0.1,1?M?of H₂O₂ showed no significant difference in cell viability,10?M concentration of H₂O₂ can significantly promote cell proliferation?p<0.05?,In the range of 25²00?M,the inhibition of H₂O₂ on cell vitality gradually increased.In the short time of H₂O₂ action cells,As computing time extension,the ratio of autophagic marker protein LC3-II/LC3-I and the expression of Beclin-1 in osteoblasts gradually increased,and the expression of P62 decreased gradually.Compared with the control group,the expression of LC3-II/LC3-I ratio and Beclin-1 and P62 expression in 1h were significantly different?P<0.05?.2.The effect of Res on H₂O₂ injury osteoblasts showed that Compared with H₂O₂ group,the cell viability of H₂O₂+Res?1-50?M?was increased by different degrees?p<0.05?,and compared with H₂O₂+Res?1-50?M?,H₂O₂+Res10?M was the most significant increase in cell vitality?p<0.05?.Compared with the control group,the apoptosis rate of bone cells,the ratio of cleaved-Caspase3/Caspase3 to apoptotic protein and the level of ROS increased significantly?P<0.05?,and Res?1,5,10?M?+H₂O₂ groups were all lower than the H₂O₂ group?P<0.05?,and the effect of Res on the oxidative damage induced by H₂O₂induced osteoblasts was in a concentration dependent range.Compared with the control group,the LC3-II/LC3-I ratio and the expression of Beclin-1 increased in group Res and H₂O₂,and the expression of P62 decreased?P<0.05?.Res could promote the effect of H₂O₂ on autophagy and showed a concentration dependence.3.after regulating autophagy,the experimental results of autophagy showed that compared with the control group,the LC3-II/LC3-I ratio and Beclin-1expression increased?P<0.05?and P62 expression decreased?P<0.05?in group Rap,Res group and H₂O₂ group,and the LC3-II/LC3-I ratio and the amount of Beclin-1 in H₂O₂+Res and H₂O₂+Rap groups were further increased.Step reduction?P<0.05?.3-MA significantly inhibited the effect of Res.Compared with the H₂O₂+Res group,the LC3-II/LC3-I ratio and Beclin-1 expression in the H₂O₂+Res+3-MA group decreased significantly?P<0.05?,and the expression of P62 increased significantly?P<0.05?.Res and Rap could improve the effect of H₂O₂ on the oxidative damage of osteoblasts.Compared with the H₂O₂ group,the apoptosis rate,the cleaved-Caspase3/Caspase3ratio of apoptotic protein,the ROS level and the MDA content of H₂O₂+Res and H₂O₂+Rap significantly decreased?P<0.05?.The effect of osteoblasts on oxidative damage?P<0.05?.Conclusion:H₂O₂ can increase the level of autophagy while promoting the oxidative damage of osteoblasts.Res has antioxidant protection against oxidative damaged osteoblasts and induces autophagy.and 3-MA can partially block the effect of Res.The mechanism may be that Res can protect the osteoblasts from H₂O₂ induced oxidative damage by reducing oxidative stress via autophagy upregulation.