The Effect And Mechanism Of S1P/S1P1 Signal Pathway During Post Conditioning Of Hypertrophic Cardiomyocytes

Objective:Separating and culturing original generation of rats myocardial cells and induce hypertrophy, useing the hypertrophic cardiomyoctyes to built ischemia-reperfusion modle, base on these, adding drug intervention to detect the effect of S1P/S1P1 signaling pathways during the postconditiong of hypertrophic cardiomyocytes. Methods:Isolation of cultured neonatal rat cardiomyocytes, myocardial cells were identified by immunofluorescence. Using NE to induce cardiomyocytes hypertrophy, and then detectd by measuring cell area and the expression of β-MHC to decide the appropriate NE concentration. Using three gas icubator to creat anoxia and reoxygenation enviroment to simulate ischemia-reperfusion and postconditioning, and through TUNEL and AnnexinV-FITC/PI to detect twhether the modle were successfully established and testes the expression of S1P1 through RT-PCR. Drugs was intervented into hypertrophic cardiomyoctyes postconditiong to detect the relvent singal pathways, according to the adding drugs, hypertropic cardiomyoctyes postconditioning were divde into five groups, (1) IPost group, (2) IPost+SIP group, (3) IPost+W-146 +S1P group, (4) IPost+PD98059+S1P group, (5) IPost+LY-294002+S1P group. All these group were deteced by flow cytometry for apoptosis rate, TUNEL test for myocardial cells apoptosis morphology and Western-blot for the expression of protein in the singal pathway. Results: (1) the survival cardiomyocytes was 97%, immunofluorescence showed that cardiomyocytes were approximately more than 98% purity, these myocardialcytes can be used for experiment. (2) the result of RT-PCR showed that with the increasing of the concentration of NE, the expression of β-MHC incresed, but when the NE concentration come up to 3 uM, the expression of β-MHC come to the max, and won’t increse when the concentration increased. The change of cell area is the same to the expression of β-MHC. (3) TUNEL and AnnexinV-FITC/PI found that cell apoptosis rate of I/R group and IPost group were both higher than the Sham group, while IPost group is lower than I/Rgroup. This result proved that the postconditioning modle was successfully built. RT-PCR found that the expression of S1P1 is higher in IPost group than both Sham group and I/R group. (4) After adding drugs, TUNEL found that IPost+S1P group and IPost+LY-294002+S1P group had lower apoptosis rate than other groups, Western-blot also showed these two groups had lower expression of Caspase-3 than others, while the expression of p-ERK is higher than others. There is no different of the expression of p-Akt between those groups. Conclusion:SIP can protect hypertrophic myocardialcytes during postconditioning, this function via S1P1 receptor by activating ERK1/2 signal pathway to realize.