Effects Of Notch Signal Pathway On H9c2Cardiomyocytes Apoptosis Induced By Hypoxia/reoxygenation Via Rock2

Background:Ischemic heart disease(IHD) is a disease characterized by ischaemia (reducedblood supply) or necrosis of the heart muscle due to coronary artery disease(atherosclerosis of the coronary arteries), which is also a serious threat to humanhealth. So, it is very important to explore the new strategies of prevention andtreatment of IHD in molecular level. Notch signaling pathways are very expedient interms of the protection and recovery of myocardial when the heart muscle beingimpaired. It is also known that ROCKs are closely related to the apoptosis ofcardiomyocytes. We demonstrated that there are interactions between Notch signalingpathways and ROCKs on cell apoptosis in H9c2cardiomyocytes model of H/R?Objective:1.To investigate the effects of Notch signaling pathway on hypoxia/reoxyg-enation (H/R)-induced apoptosis of H9c2cardiomyocytes in rat.2.To reveal the mechanisms of Notch signaling pathway regulatingcardiomyocytes apoptosis and recovery after H/R by studying the interaction betweenROCK2and NICD on H/R.Methods:1. The total length of cDNA fragment encoded with NICD was obtained fromH9c2cardiomyocytes from rat by reversing transcription polymerase chain reaction(RT-PCR), then the cDNA fragment was inserted into pCMV-Tag2B vector. Therecombinant plasmid was confirmed by restricition endonudease (EcoRâ… and Salâ… )digestion and DNA sequencing. H9c2cardiomyocytes were transfected bypCMV-Tag2B-NICD and the expression of NICD was detected by Western blot.2. All of the cultured H9c2cardiomyocytes were randomly divided into sixgroups: Control group; H/R group; NICD group; NICD+H/R group; DAPT group;DAPT+H/R group. Apoptosis of each group was analyzed by flow cytometry(FCM), and Western blot was applied to assess the expression of ROCK2and NICD proteins.Results:1. The recombinant plasmid pCMV-Tag2B-NICD was successfully constructed.After pCMV-Tag2B-NICD was transfected into H9c2cardiomyocytes, Western blotanalysis was done to showed a higher expression of NICD in H9c2cardiomyocytes.2. The overexpression of NICD results in a rise of protein expression of NICD,and reduces the protein expression of ROCK2and apoptosis of H9c2cardiomyocytes;on the contrary, the supression of NICD leads to a reduction of protein expression ofNICD, and increases the the protein expression of ROCK2and apoptosis of H9c2cardiomyocytes.Conclusion:1. The recombinant plasmid pCMV-Tag2B-NICD was successfully constructed.2. After hypoxia/reoxygenation, the protein expression of NICD wassignificantly increased, and the protein expression of ROCK2was decreased,compared to control group.3. Notch signaling pathway reduced the H/R-induced apoptosis of rat H9c2cardiomyocytes via supression of ROCK2from the rat.