The effect of CGRP on autophagy regulation through ALKBH5/m6A in hypoxia/reoxygenation-induced cardiomyocyte proliferation and apoptosis
Objective To explore the effect of CGRP on autophagy regulation through ALKBH5/m6A in hypoxia/reoxygenation-induced cardiomyocyte proliferation and apoptosis.Methods H9c2 cardiomyocytes were randomly divided into Control group(H9c2 cells cultured in normal culture medium),H/R group(preparation of hypoxia/reoxygenation-induced H9c2 cell injury model),CGRP group(1 × 10-1 mol/L CGRP added before hypoxia/reoxygenation for 20 min),and CGRP+sh-ALKBH5 group(sh-ALKBH5 transfected into H9c2 cells,cultured for 24 h,followed by the addition of 1×10-1 mol/L CGRP for 20 min,and then preparation of hypoxia/reoxygenation model).CCK-8 and EdU staining were used to detect cell viability of H9c2 cells;flow cytometry was used to measure cell apoptosis rate;lactate dehydrogenase(LDH)activity was measured in cardiomyocytes;Dot blot was performed to measure m6A methylation levels;protein immunoblotting and RT-PCR were performed to measure the expression of ALKBH5,Beclin-1,p62,LC3 Ⅱ,LC3I proteins,and mRNA in cells.Results Compared with the Control group,the H/R group showed significantly decreased cell survival rate(46.27％±4.09％),EdU positive cell rate(3.89％± 0.87％),and increased cell apoptosis rate(13.62％±1.27％),LDH concentration(276.32％±25.14％),and m6A relative expression level(2.46％±0.18％)(P＜0.05).Compared with the H/R group,the CGRP group exhibited significantly increased cell survival rate(87.31％±6.24％),EdU positive cell rate(40.71％±2.53％),and decreased cell apoptosis rate(3.17％±0.82％),LDH concentration(78.24％±7.19％),and m6A relative expression level(1.42％±0.13％)(P＜0.05).The cell survival rate(61.04％±5.48％)and EdU positive cell rate(8.76％±1.24％)in the CGRP+sh-ALKBH5 group were significantly lower than those in the CGRP group,while the cell apoptosis rate(10.19±0.95％),LDH concentration(229.06±22.43％),and m6A relative expression level(2.09±0.15％)were significantly higher than those in the CGRP group(P＜0.05).Compared with the Control group,the ALKBH5 protein(0.34±0.03),p62 protein(0.32±0.04),and mRNA expression(0.21±0.04)in the H/R group were significantly reduced,while Beclin-1 protein(0.97±0.13),mRNA expression(2.14±0.17),and LC3 Ⅱ/LC3I ratio(0.64±0.08)were significantly increased(P＜0.05).Compared with the H/R group,the CGRP group showed significantly increased ALKBH5 protein(0.78±0.09),p62 protein(0.71±0.08),and mRNA expression(0.87± 0.08),while Beclin-1 protein(0.41±0.06),mRNA expression(1.26±0.12),and LC3 Ⅱ/LC3I ratio(0.23±0.03)were significantly decreased(P＜0.05).Compared with the CGRP group,the CGRP+sh-ALKBH5 group exhibited significantly decreased ALKBH5 protein(0.50±0.07),p62 protein(0.40±0.05),and mRNA expression(0.30± 0.05),while Beclin-1 protein(0.86±0.10),mRNA expression(1.95±0.14),and LC3 Ⅱ/LC3I ratio(0.58±0.06)were significantly increased(P＜0.05).Conclusion CGRP can inhibit hypoxia/reoxygenation-induced autophagy and apoptosis in myocardial cells,increase cell survival rate,and its mechanism of action may be related to the promotion of m6A demethylase ALKBH5 expression.
CardiomyocytesCalcitonin gene related peptide(CGRP)Alkylation repair homolog pro-tein 5/N6-Methyladenosine(ALKBH5/m6A)Autophagyhypoxia/reoxygenation-inducedProliferationApoptosis
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