Effect Of Neuregulin-1 On Senescence Of Mouse Bone Marrow Mesenchymal Stem Cells Induced By Hydrogen Peroxide

Objective:The aim of this study was to explore the effect of neuregulin-1（Neuregulin-1,NRG-1）on the senescence of mouse bone marrow mesenchymal stem cells（Bone marrow mesenchymal stem cells,BMSCs）induced by hydrogen peroxide.Methods:The senescent model of mouse bone marrow mesenchymal stem cells was induced by H₂O₂.BMSCs were divided into three groups:control group,H₂O₂group and H₂O₂+NRG-1 group.The survival rate of BSMCs treated with different concentrations of H₂O₂was detected by CCK8 kit.The concentration gradient of H₂O₂0,15,30,60,90,180,360,480μmol/L were used to detect the cell survival rate,and the appropriate concentration of cell senescence induction was selected.The control group received no special treatment,and the H₂O₂+NRG-1 group was pretreated with 50μmol NRG-1 for1 h before senescence induction.Then,H₂O₂group and H₂O₂+NRG-1 group were treated with complete medium containing 250μmol H₂O₂for 2 hours,then the liquid was changed,and then the complete medium was changed and cultured for 24 hours,and the related indexes were detected.Flow cytometry was used to detect apoptosis,Senescence-associated-β-galactosidase（SA-β-gal）staining was used to detect cell senescence.Flow cytometry was used to detect the proportion of BMSCs cell cycle in control group,H₂O₂group and H₂O₂+NRG-1 group,that is,G0/G1 phase,S phase and G2/M phase.Quantitative polymerase chain reaction（Quantitative polymerase chain reaction,q PCR）was used to detect the expression of aging-related genes NRG-1,P53 and P21 m RNA.Western blot was used to detect the expression of Erb B2（Phospho-Tyr877）and aging-related proteins acetylated p53 and P21,the migration of cells was detected by transwell test and scratch test.Results:According to the results of CCK8 detection,the cell survival rate of BMSCs was calculated,and H₂O₂with a concentration of 250μL was selected as exogenous oxidant to induce BMSCs senescence.Apoptosis results showed that:compared with the control group,there was no difference in apoptosis in the H₂O₂group（P>0.05）;compared with the H₂O₂group,the apoptosis in the H₂O₂+NRG-1 group was slightly decreased（P<0.05）,indicating that the concentration of H₂O₂does not significantly cause apoptosis of BMSCs and can be used to induce senescence.The results ofβ-galactosidase staining showed that the percentage of SA-β-gal positive cells in H₂O₂group was higher than that in control group（P<0.01）,and the percentage of SA-β-gal positive cells in H₂O₂+NRG-1 group was lower than that in H₂O₂group（P<0.01）.The results of cell cycle experiment detected by flow cytometry:compared with the control group,the proportion of cells in G0/G1 phase increased（P<0.05）,the proportion of S phase decreased and the proportion of G2/M phase decreased in H₂O₂group（P<0.05）,suggesting that the proliferation of BMSCs was significantly inhibited.Compared with H₂O₂group,the proportion of G0/G1 phase in H₂O₂+NRG-1group decreased（P<0.05）,the proportion of S phase increased,and the proportion of G2/M phase increased（P<0.05）,suggesting that the inhibition of proliferation in H₂O₂+NRG-1 group was significantly improved.The results of q RT-PCR assay showed that the expression of NRG-1 m RNA in H₂O₂group was down-regulated（P<0.05）,and p53 and P21 m RNA in BMSCs in H₂O₂group was higher than that in control group,while the expression of NRG-1m RNA in H₂O₂+NRG-1group was up-regulated（P<0.05）,and p53 and P21m RNA in H₂O₂+NRG-1 group was down-regulated compared with H₂O₂group（P<0.05）.The results of Western blot showed that compared with H₂O₂group,the expression level of p-Erb B2 was no significant different（P>0.05）and acetylated p53 and P21 protein increased significantly in H₂O₂+NRG-1 group（P<0.05）,while the protein phosphorylation level of Erb B2 increased in H₂O₂+NRG-1 group compared with H₂O₂group P<0.05).The results of scratch test showed that the cell migration ability of H₂O₂group was significantly lower than that of control group（P<0.01）,and that of H₂O₂+NRG-1 group was significantly higher than that of H₂O₂group（P<0.05）.The results of transwell assay showed that the cell migration ability of H₂O₂group was significantly lower than that of H₂O₂group,and the cell migration ability of H₂O₂+NRG-1group was significantly higher than that of H₂O₂+NRG-1 group（P<0.05）.Conclusion:（1）NRG-1 can improve the senescence of BMSCs induced by hydrogen peroxide and protect the proliferation and migration of BMSCs.（2）NRG-1 can attenuate the senescence of BMSCs by regulating the senescence-related gene p53 and P21,and regulating the phosphorylation of downstream protein Erb B2 and the acetylated p53 and P21 of senescence-related proteins.