| Objective:It is discovered in recent years that Sphingosine-1-phosphate(S1P) is a kind of Membrane phospholipid metabolites and has important physiological function.S1P can act as an intracellular second messenger and also act on specific cell surface receptors to play an important biological functions.S1P is closely related to a variety of cell acttivities, including cell proliferation, differentiation, apoptosis and migration.In recent years,substantial studies have shown that S1P has a role in myocardial protection, reducing ischemia/reperfusion injury.But its mechanism is not yet fully clarified.In this study,by means of the primary culture of neonatal rat cardiomyocytes and building hypoxia-reoxygenation injury model,we want to explore the protective effect and mechanisms of Sphingosine-1-phosphate on hypoxia/r- eoxygenation injury.Methods:1. The primary culture of neonatal rat cardiomyocytesCut neonatal rat ventricular, born within three days,into pieces.Digest the pieces with 0.125% trypsin.Purify ventricular myocytes by means of differential adhesion.Add 20% DMEM medium,and then sent to 37℃,95% O2 +5% CO2 carbon dioxide Incubator to culture. Replace the cell culture medium every 48h.Experiment when the single-layer myocardial cells cover the bottom more than 80%.2. Building and identification the hypoxia/reoxygenation injury model in cultured neonatal rat ventricular cardiomyocytes.Replace the cell culture medium when the single-layer Myocardial cells cover bottom more than 80% and culture 12h to synchronize the cells.Cover the surface of the culture medium with liquid paraffin sterilized by high-temperature, and then curture 6h.Remove the liquid paraffin,replace the cell culture medium,and then curture 12h in the same culture conditions.Measure cell mortality and apoptosis in different exprimental groups by means of trypan blue and PI staining and identification the hypoxia/ reoxygenation injury model in cultured neonatal rat ventricular cardiomyocytes.3. The protective effect and mechanisms of S1P in curtured rat neonatal cardiomyocytes during simulated hypoxia/reoxygenation.Observe the the impact of S1P on cell mortality and apoptosis in curtured rat neonatal cardiomyocytes during simulated hypoxia/reoxygenation by means of trypan blue and PI staining.Observe the the impact of S1P on p-Akt1 in curtured rat neonatal cardiomyocytes during simulated hypoxia/reoxygenation by means of western blot.Observe the the impact of S1P on mitochondrial membrane potential in curtured rat neonatal cardiomyocytes during simulated hypoxia/reoxygenation by means of Rhodamine 123 staining.Results:1. The primary culture of neonatal rat cardiomyocytesThe myocardial cells are round or oval before adhesion.The cells begin to adhere and proliferate after 4h,and the cells'shapes change into spindle or polygonal. The living cells adhere completely after 24h. The vast majority of cells are polygonal or rod-shaped and assemble into concentric radial cell clusters.On the third day,the cells'number reach a peak,the single-layer myocardial cells cover the bottom more than 80%.Cells are in contact with each other,and in the occurrence of contact inhibition.Cells in the same cluster beat synchronously with steady frequency,rhythm and strength,about 80-120 times per minute.2. Building and identification the hypoxia/reoxygenation injury model in cultured neonatal rat ventricular cardiomyocytes.The result of trypan blue staining:the cell mortality in normal curtured rat neonatal cardiomyocytes is (7.52±2.13)%,and rise to (41.25±3.96)%(P<0.005)after 6h hypoxia.The cell mortality in curtured rat neonatal cardiomyocytes during simulated hypoxia/reoxygenation is (93.14±2.66)%,it is higher than H group (P<0.001).The result of PI staining:On the DNA histogram,there is a small number of apoptosis in Control group,Sub-G1 is not obvious, apoptosis in Control group is only (8.47±0.75)%;there is a low Sub-G1 in H group, apoptosis rise to (26.87±3.17)%(P<0.01);Sub-G1 in H/R group is prominent,apoptosis reach (58.83±3.55)%,it is higher than H group(P<0.005).3. The protective effect and mechanisms of S1P in curtured rat neonatal cardiomyocytes during simulated hypoxia/reoxygenation.The result of trypan blue staining:S1P can obviously reduce cell mortality from (93.14±2.66)% to (47.42±2.72)%(P<0.002) in curtured rat neonatal cardiomyocytes during simulated hypoxia/reoxygenation;but wortmannin can inhibit this effect of S1P partially and raise cell mortality back to (46.97±2.27)%(P<0.005),but it is still lower than H/R group(P<0.01).The result of PI staining:S1P can obviously reduce apoptosis from(58.83±3.55)% to (32.87±3.06)%(P < 0.005) in curtured rat neonatal cardiomyocytes during simulated hypoxia/reox- ygenation;but wortmannin can inhibit this effect of S1P partially and raise apoptosis back to (46.97±2.27)% (P<0.01),but it is still lower than H/R group(P<0.02).The result of western blot:the value of R in Control and H/R group is very low, (0.1838±0.0031) and (0.1957±0.0033) respectively.S1P can obviously rise the level of p-Akt1 in curtured rat neonatal cardiomyocytes during simulated hypoxia/reoxygenation, the value of R in H/R+S1P group is as high as (0.6908±0.0018),it is greatly higher than H/R group(P<0.001);but wortmannin can inhibit this effect of S1P partially,the value of R in H/R+S1P+W group drop to (0.3623±0.0022)(P<0.001),but it is still higher than H/R group(P<0.001). The result of Rhodamine 123 staining:the value of mean fluorescence intensity(MFI) in Control group is (437.33±22.19).In H/R group,the value of MFI drop to 265.67±23.18 prominently(P<0.01).S1P can obviously raise the value of MFI to (370.33±22.03 )in H/R+S1P group,it is higher than H/R group(P<0.05);but wortmannin can inhibit this effect of S1P partially, the value of MFI in H/R+S1P+W group drop to (310.33±20.13)(P<0.05),but it is still higher than H/R group(P<0.05).Conclusion:1. S1P can obviously mitigate the hypoxia/reoxygenation injure and reduce cell mortality and maintain the integrity of myocardial cell membrane in curtured rat neonatal cardi- omyocytes during simulated hypoxia/reoxygenation.Wortmannin can inhibit this effect of S1P partially2. S1P can obviously restrain apoptosis in curtured rat neonatal cardiomyocytes during simulated hypoxia/reoxygenation.Wortmannin can inhibit this effect of S1P partially.3. S1P can activate PI3K-Akt1 signaling pathway,raise the level of p-Akt1. Wortmannin can inhibit this effect of S1P partially. This result prompts the existence of non-PI3K signaling pathway by which S1P can also activate Akt1.4. S1P can stabilize mitochondrial membrane potential in curtured rat neonatal cardiomyocytes during simulated hypoxia/reoxygenation. Wortmannin can inhibit this effect of S1P partially.Innovation:1. In this study, for the first time we observe that Wortmannin can partly inhibit activation of Akt1 by S1P in cultured neonatal rat cardiomyocytes during simulated hypoxia/reo- xygenation.2. In this study,for the first time we observe that S1P can stabilize mitochondrial membrane potential in curtured rat neonatal cardiomyocytes during simulated hypoxia/reo- xygenation and wortmannin can inhibit this effect of S1P partially.This result prompts that S1P can affect the mitochondrial membrane permeability through PI3K-Akt signaling pathway and affect the mitochondrial membrane potential in turn.
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