The Effect Of Notch1Signal Pathway On H9c2Cardiomyocytes Apoptosis In Ischemic Postconditioning

Objective:To investigate the mechanism of Notch1signaling pathway mediating cardiopro-tection after ischemic postconditioning (IPost) by reducing cardiomyocyte apopto-sis and offer a new idea for clinical of ischemia reperfusion injury.Methods:Rat heart-derived H9c2cells were randomly divided into six groups as following:Control group, Hypoxia/reoxygenation (H/R) group, N1ICD+H/R group, H-postgroup, miRNA+H-post group, Mock group. Flow cytometry were employed todetect apoptosis rate Bcl-2/Bax mRNA and Bcl-2/Bax protein levels wererespectively determined by quantitative reverse transcriptase-polymerase chainreaction (qRT-PCR) and Western-blottingting analysis. Caspase-9and caspase-3activity were measured using the colorimetric activity assay.Results:1. Apoptosis rate was examined using flow cytometry: the results revealed thatthe apoptosis rate of the H/R group was higher than the Control group(6.21±0.98%VS0.80±0.12%, P<0.05), the H-post group was lower than the H/R group(1.98±0.18%VS6.21±0.98%, P<0.05). When Notch1signaling was activated usingtransfected with pcDNA3.1-Myc-His plasmid, the apoptosis rate wasreduced(3.10±0.17%VS6.21±0.98%;N1ICD+H/R VS H/R, P<0.05).Meanwhile,inthe miRNA+H-post group, the apoptosis rate was increased (6.30±026%VS1.98±0.18%;miRNA+H-post VS H-post, P<0.01).These results demonstrated thatthe Notch1signaling could inhibit cardiomyocyte apoptosis in IPost.2. The expression of Bcl-2mRNA was decreased in the H/R group (H/R VSControl, P<0.05) and elevated in the H-post group (H-post VS H/R, P<0.05). Aftertransfected with Notch1miRNA, the expression of Bcl-2mRNA was significantlylower in the miRNA+H-post group (miRNA+H-post VS H-post, P<0.01),whentransfected with pcDNA3.1-Myc-His plasmid,the expression of Bcl-2mRNAelevated(N1ICD+H/R VS H/R, P<0.05,).Meanwhile, the level of Bax miRNA was significantly increased in the H/R group (H/R VS Control, P<0.05), and the level ofBax was significantly decreased in H-post group (H-post VS H/R, P<0.05). Aftertransfected with Notch1miRNA, the expression of Bax mRNA was significantlyincreased in the miRNA+H-post group (miRNA+H-post VS H-post, P<0.01), butdecreased in the N1ICD+H/R group (N1ICD+H/R VS H/R, P<0.05); the Bcl-2protein and Bax protein expression were consistent with the gene expression3. To test whether activation of caspase pathway was involved in the apoptosisof H9c2cell. Caspase-9and caspase-3activities were measured. The results revealedsignificant increase of both caspase-9and caspase-3activities were detected in theH/R group compared with the Control group (P<0.05). The N1ICD+H/R group hadsignificantly decreased caspase-9and caspase-3activities (N1ICD+H/R VS H/R,P<0.05). In the H-post group, both caspase-9and caspase-3activities were attenuatedsignificantly (H-post VS H/R, P<0.05); Notch1miRNA counteracted the effects ofdown-regulation of increased caspase-9and caspase-3activities due to the H-post(miRNA+H-post VS H-post, P<0.05).Conclusion:Notch1signaling inhibits cardiomyocyte apoptosis in heart ischemicpostconditioning,which may cast new lights on myocardial IPost protection.