Effects Of Hydrogen Peroxide And Retinoic Acid On The Expression Of P-p38, MKP-1 And HDAC-1,-2,-3 In A549 Cells

Part 1 Effects of different concentrations of hydrogen peroxide on the expression of p-p38,MKP-1 and HDAC-1,-2,-3 in A549 cellsObjective The effects of hydrogen peroxide?H₂O₂?on the expression of p-p38,MKP-1 and HDAC-1,-2,-3 in A549 cells were investigated by establishing the experimental models of A549 cells with different concentrations of H₂O₂ intervention.Methods We established the experimental models of A549 cells by interventing of different concentrations of H₂O₂ for 12 hours.The apoptosis of A549 cells was detected by MTT,CCK8 and LDH methods,the expressions of p-p38,MKP-1 and HDAC-1,-2,-3 were detected by Western blot.Results1.The survival rate of A549 cells decreased in a concentration and time dependent manner with the increase of the concentration of H₂O₂.Combined with the results of CCK8,MTT and LDH,we selected the 12 hours as the intervention time in the follow-up study,which could not only have a relatively obvious impact on A549 cells,but also not cause too much damage to A549 cells to affect the subsequent experimental results.2.When different concentration of H₂O₂ interfered A549 cells for 12 hours,compared with the control group?the concentration of H₂O₂ is 0 umol/L?,the expression levels of p-p38 and MKP-1 were gradually increasing with the increase of concentration of H₂O₂.And when the concentration of H₂O₂ increased to 400 umol/L,the expression levels of p-p38 and MKP-1 were significantly different from the control group?p<0.01?.The expression levels of HDAC-1,-2,-3 were gradually reducing with the increase of concentration of H₂O₂,however,HDAC-1,-2,-3 have different sensitivity to the concentration of H₂O₂.When the concentration of H₂O₂ reached 100 umol/L,the expression of HDAC-1 was specific statistically differences compared with the control group?p<0.05?;when the concentration of H₂O₂ increased to 200 umol/L,the expression of HDAC-3 was specific statistically differences compared with the control group?p<0.05?;when the concentration of H₂O₂ increased to 400 umol/L,the expression of HDAC-2 was specific statistically differences compared with the control group?p<0.05?.Conclusion We successfully established animal and cells in vitro models of oxidative stress lung injury,and confirmed that MKP-1,p38 MAPK signaling pathway and HDACs epigenetic mechanism were involved in the oxidative stress injury process of lung cells.Part II Effects of hydrogen peroxide and retinoic acid on the expression of p-p38,MKP-1 and HDAC-1,-2,-3 in A549 cellsObjective The effects of H₂O₂ and retinoic acid?RA?on the expression of p-p38,MKP-1 and HDAC-1,-2,-3 in A549 cells were investigated by establishing the experimental models of A549 cells with H₂O₂ and RA intervention.Methods The A549 cells in logarithmic phase were randomly divided into four groups?control group,H₂O₂ group,RA group,and H₂O₂ +RA group?for intervention8?12?24 hours,the expressions of p-p38,MKP-1 and HDAC-1,-2,-3 were detected by Western blot,and the apoptosis of A549 cells was detected by flow cytometry.Results1.Compared with the control group,the expression levels of p-p38 protein in the H₂O₂ group increased after 8 hours in A549 cells?p<0.05?,RA could partly decrease the expression level of p-p38 after the intervention of H₂O₂,but there was no significant statistical difference compared with H₂O₂ group?p>0.05?.RA and H₂O₂ could increase the expression of MKP-1?p<0.01?,and the intervention of both RA and H₂O₂ could further increase the expression of MKP-1,the difference was statistically significant compared with H₂O₂ group?p<0.01?.The expression levels of HDAC-1,-2,-3 in the H₂O₂ group significantly decreased to different degrees?p<0.05?,RA could increase the expression level of HDAC-1,-2 after the intervention of H₂O₂,and the difference was statistically significant compared with H₂O₂ group?p<0.05?.However,there was no statistically significant difference in the pro-expression effect of HDAC-3?p>0.05?.Compared with the control group,the expression levels of p-p38 protein in the H₂O₂ group increased after 12 hours and 24 hours in A549 cells?p<0.05?,RA could partly decrease the expression level of p-p38 after the intervention of H₂O₂?p<0.05?.The expression trend of MKP-1 was basically consistent with that of 8 hours.The expression levels of HDAC-1,-2,-3 in the H₂O₂ group significantly decreased to different degrees?p<0.01?,RA could increase the expression level of HDAC-1,-2,-3 after the intervention of H₂O₂,and the difference was statistically significant compared with H₂O₂ group?p<0.05?.2.At the intervention time of 8 hours,H₂O₂ significantly increased the number of early apoptotic cells compared with the control group?p<0.01?,but it has no obvious effect on late apoptosis and dead cells?p>0.05?,and RA had no significant protective effect on H₂O₂ induced cell damage?p>0.05?.When the intervention time was extended to 12 hours and 24 hours,both early apoptotic cells and late apoptotic cells were significantly increased by H₂O₂?p<0.001?,RA could partially reverse the apoptosis and necrosis of A549 cells induced by H₂O₂,and the difference was statistically significant compared with H₂O₂ group?p<0.001?.Conclusion It has been preliminarily confirmed that RA can regulate the expression of HDACs and MKP-1 to adjust the activity of p38 MAPKs signaling pathway,and play a protective role in oxidative stress lung injury.