Study On The Protective Effect Of Astragaloside IV And Lumbrokinase On NRK-52E Cells Under Oxidative Stress State Based On SITR1 Pathway

Purpose:To investigate the regulatory effect of astragaloside IV and lumbrokinase on SIRT1 pathway-related factors and antioxidant activity in NRK-52E cells under oxidative stress.Methods:NRK-52E cells were resuscitated and passaged.The cells were randomly divided into normal group?Norm?,model group?H₂O₂?,resveratrol group,astragaloside IV group?AS-IV?and lumbrokinase group?Lumb?.Each group of cells were inoculated 24h,and then were given different drug before 24h when H₂O₂were given,including:Res group with 25?mol/L resveratrol broth,AS-IV group with 25mmol/L Astragaloside IV culture medium,Lumb group with 150U/mL Lumbrokinase broth,Norm group and H₂O₂ group were not treated.After 24 hours,the cells were treated with 300?mol/L H₂O₂for 30 minutes except the normal group.After that,The cells were collected and observed by inverted phase contrast microscope;the expression of SIRT1,Nrf2,SOD2,FOXO3?and PGC-1?in SIRT1pathway was detected by Western blot;the lipid oxidation level of each group was detected by MDA kit;the cell viability was measured by CCK8 reagent;the fluorescent OD value of ROS in each group was detected by fluorescence microplate reader;the expression of ROS in each group was detected by flow cytometry;the fluorescence intensity of ROS in each group was observed by fluorescence inverted microscope.The experimental data were analysed by SPSS software for statistical analysis and P<0.05 was considered statistically significant.Results:1.Cell morphology changes:When compared with the normal group,the cells in the model group showed increased cell permeability to the backlight,broadened the cell gap and irregular vacuolar changes in the intercellular junctions;when compared with model group,resveratrol group,astragaloside IV group and lumbrokinase group were improved;2.CCK8:?1?H₂O₂ and drug stimulation 24h respectively:the cell viability of the model group decreased significantly?P<0.05?,and decreased with the increase of H₂O₂ concentration;the low doses of resveratrol group,astragaloside IV group and lumbrokinase group had no difference in cell viability when compared with the normal group?without H₂O₂ in the drug groups?,and the high doses of resveratrol group and astragaloside IV group increased cell viability?P<0.05?,while the high doses of lumbrokinase group decreased cell viability?P<0.05?;?2?H₂O₂ stimulation for 30 min:within 1h after stimulation,there was no significant change in the activity of each group of cells compared with the normal group?with the drug group added H₂O₂?;while the viability of the cells in model group were decreased?P<0.05?after 1.5h stimulation end;3.The OD value of ROS probe:?1?Compared with the model group,the fluorescence OD value of each group were lower?P<0.05?;?2?Compared with the normal group,the fluorescence OD value of lumbrokinase group were higher?P<0.05?;4.MDA:Compared with the model group,MDA in each group decreased?P<0.01?;?2?Compared with the normal group,MDA in resveratrol group and lumbrokinase group increased?P<0.05?;5.Western blot:?1?SOD2:Compared with the model group,the expression of SOD2 in normal group,resveratrol group and astragaloside IV group were all increased?P<0.01?;compared with normal group,the expression of SOD2 in resveratrol group,astragaloside IV group and lumbrokinase group were lower?P<0.01?;?2?n-NRF2:Compared with the model group,the expression of n-NRF2 in resveratrol group and astragaloside IV group were increased?P<0.05?,while the n-NRF2expression in normal group and lumbrokinase group decreased?P<0.01?;compared with normal group,the expression of n-NRF2 in resveratrol group and astragaloside IV group were increased?P<0.05?;?3?PGC-1?:Compared with model group,the expression of PGC-1?in normal group,resveratrol group,astragaloside IV group and lumbrokinase group were all increased?P<0.01?;compared with normal group,the expression of PGC-1?in resveratrol group and astragaloside IV group increased?P<0.01?,and decreased in lumbrokinase group?P<0.05?.?4?SIRT1:Compared with the model group,the expression of SIRT1 in normal group,resveratrol group,astragaloside IV group and lumbrokinase group were all increased?P<0.01?;compared with normal group,the expression of SIRT1 in resveratrol group and astragaloside IV group increased?P<0.01?.?5?FOXO3?:Compared with the model group,the expression of FOXO3?in normal group,resveratrol group,astragaloside IV group and lumbrokinase group were all increased?P<0.01?;compared with the normal group,the expression of FOXO3?in resveratrol group,astragaloside IV group and lumbrokinase group increased?P<0.01?.6.Flow-detected ROS probe:compared with the model group,the positive rate of fluorescent cells in resveratrol group,astragaloside IV group and lumbrokinase group decreased?P<0.05?;compared with the normal group,the positive rate of fluorescent cells in model group,resveratrol group,astragaloside IV group and lumbrokinase group were all increased?P<0.01?;7.Fluorescence inverted phase contrast microscope:the cell or fluorescence background can not be seen in the normal group 1?without fluorescence probe?;the background of green impurities can be seen in the normal group 2?with fluorescent probe?;the probe of model group had the strongest fluorescence intensity,and lumbrokinase group weaker,and resveratrol group and astragaloside IV group weakest.Conclusion:Astragaloside IV and lumbrokinase enhance the antioxidant capacity of NRK-52E cells and reduce the damage of H₂O₂-induced oxidative stress to cells.Among them,astragaloside IV had a significant up-regulation effect on SIRT1 pathway and with similar effect from resveratrol.Lumbrokinase could not up-regulate SIRT1,while it could protect SIRT1pathway from being inhibited,meanwhile,FOXO3?was activated directly or indirectly to protect cells against oxidative stress by lumbrokinase.