The Effect And Mechanism Of Alginate Oligosaccharide On Autophagy In Hydrogen Peroxide-induced Senescent Cardiomyocytes

Objective:To investigate the protective effects of Alginate oligosaccharide?AOS?on senescent cardiomyocytes induced by hydrogen peroxide?H₂O₂?and its relationship with autophagy,and to explore the potential mechanisms.Methods:This experiment used H9c2 rat cardiomyocyte line.The cells were divided into six groups:Control group,Model group?H₂O₂?,Alginate oligosaccharide low-dose?H₂O₂+AOS?L??group,Alginate oligosaccharide middle-dose?H₂O₂+AOS?M??group,Alginate oligosaccharide high-dose?H₂O₂+AOS?H??group and AMPK inhibitor Compound C?H₂O₂+AOS?H?+Cpd C?group.The cells in Control group were given synchronous replacement of medium during the experiment,and the cells in other groups were exposed to H₂O₂?100?mol/L?for 4 h to induce the senescence model of H9c2cardiomyocytes.After that,the H₂O₂+AOS?L?group,H₂O₂+AOS?M?group and H₂O₂+AOS?H?group cells were treated with different concentrations of AOS?10?g/mL,50?g/mL,100?g/mL?,and the H₂O₂+AOS?H?+Cpd C group cells were pretreated with Compound C,AMPK inhibitor,for 2 h,before treated with AOS?100?g/mL?.After AOS treatment for 48 h,the senescent cardiomyocytes were detected by Senescence-associated?-galactosidase?SA-?-gal?Staining.Cell cycle was detected by flow cytometry.The expressions of autophagy related proteins LC3-?,beclin1,AMPK,p-AMPK,mTOR and p-mTOR were detected by Western blot.The expressions of LC3-?and p62 mRNA were detected by RT-PCR.Results:1.SA-?-gal Staining showed that,compared with the Control group,the number of SA-?-gal Staining positive cells was obviously increased in the H₂O₂ group,and the difference was statistically significant?p<0.05?.Compared with the H₂O₂ group,AOS reduced the number of SA-?-gal Staining positive cells in a dose-dependent manner,and the difference was statistically significant?p<0.05?.AMPK inhibitor Compound C pretreated cells for 2 h,inhibited the protective effect of AOS on senescent cardiomyocytes,and the difference was statistically significant compared with the H₂O₂+AOS?H?group?p<0.05?.2.Cell cycle detection results showed that compared with the Control group,cell cycle arrest was observed in the H₂O₂ group,with increased cells in the G1 phase and decreased cells in the G2 phase,and the difference was significant?p<0.05?.Compared with the H₂O₂group,AOS alleviated cell cycle arrest induced by H₂O₂ in a dose-dependent manner,and decreased cells in the G1 phase and obviously increased cells in the G2 phase,the difference was significant?p<0.05?.Compound C pretreated cells for 2 h,inhibited the effects of AOS?p<0.05?.There was no significant difference in the proportion of S phase cells in each group?p>0.05?.3.Western blot showed that compared with the Control group,the expressions of autophagy marker proteins LC3-?and beclin1 were decreased significantly,the difference was statistically significant?p<0.05?.Compared with the H₂O₂ group,AOS increased the protein expressions of LC3-?and beclin1?p<0.05?.Compound C pretreated cells for 2 h,inhibited the activation effect of AOS on autophagy?p<0.05?.4.Western blot showed that compared with the Control group,the protein expression of p-AMPK was significantly decreased and the protein expression of p-mTOR was significantly increased in the H₂O₂ group?p<0.05?.Compared with the H₂O₂ group,AOS significantly increased the expression of p-AMPK and decreased the expression of p-mTOR,the difference was significant?p<0.05?.Compound C pretreated cells for 2 h,significantly inhibited the activation effect of AOS on AMPK/mTOR signaling pathway?p<0.05?.5.RT-PCR results showed that compared with the Control group,the mRNA expression of LC3-?was decreased,the mRNA expression of p62 was increased in the H₂O₂ group,the difference was significant?p<0.05?.Compared with the H₂O₂ group,AOS increased the mRNA expression of LC3-?,and reduced the mRNA expression of p62,the difference was significant?p<0.05?.Compound C pretreated cells for 2 h,inhibited the effects of AOS?p<0.05?.Conclusions:AOS delay the H9c2 cardiomyocytes senescence induced by H₂O₂through up-regulating autophagy,and its mechanism may be related to the activation of AMPK/mTOR signaling pathway.