The Mitochondrial Mechanism In Protective Effects Of S1P Against The Injury Of Cardiomyocytes During Hypoxia/Reoxygenation

Objective: To observe the correlation between various receptor subtypes in S1 P and S1 P resists the injury of cardiomycytes during hypoxia/reoxygenation,and to investigate how S1 P regulates the mitochondrial function in rats after hypoxia/reoxygenation thus play a role in resisting the injury of cardiomycytes during hypoxia/reoxygenation.Methods: 1.Effects of S1 P and its receptor antagonist on myocardial injury in rat cardiomyocytes during hypoxia and reoxygenation.(1)Model of cells exposed hypoxia reoxygenation injury: H9c2 cells were placed with DMEM medium containing 10% FBS and cultured for 24 hours,then subjected to 16 hours hypoxia followed by 4 hours reoxygenation.To simulate hypoxia,the culture medium was replaced by hypoxia buffer,and then incubated for 16 h in a 95% N2 and 5% CO2 gas mixture.For reoxygenation,cells were exposed to normal conditions for 4 hours and different drugs were appied simultaneously.(2)Groups: The H9c2 rat myocardial cells were randomly divided into 13 groups: normal control group(C group);hypoxia/reoxygenation group(H/R group);S1P group;W146 + S1 P group;W146 group;CAY10444 + S1 P group;CAY10444 group;JTE013 + S1 P group;JTE013 group;VPC23019 + S1 P group;VPC23019 group;PTX + S1 P group;PTX group.(1)control(C)group: cells were normally cultured for 24 hours,then cells were placed with the only serum-free DMEM for 4 h;(2)hypoxia/reoxygenation(H/R)group: cells were subjected to hypoxia for 16 h and reoxygenation for 4 h;(3)S1P group: 4 μM S1 P was added when reoxygenation for 4 h;(4)W146 + S1 P group(W146 + S1P): cells were pretreated with 10 μM W146(inhibitor of S1P1)30 min before reoxygenation,then cotreated with W146 and S1 P during reoxygenation for 4 h;(5)W146 group: cells were pretreated with 10 μM W146 30 min before reoxygenation,then reoxygenation for 4 hours;(6)CAY10444 + S1 P group(CAY10444 + S1P): cells were pretreated with 10 μM CAY10444(inhibitor of S1P3)30 min before reoxygenation,then treated with the mixture of CAY10444 and S1 P during reoxygenation for 4 h;(7)CAY10444 group: cells were pretreated with 10 μM CAY10444 30 min before reoxygenation,then reoxygenation for 4 hours;(8)JTE013 + S1 P group(JTE013 + S1P): cells were pretreated with 10 μM JTE013(inhibitor of S1P2)30 min before reoxygenation,then treated with the mixture of JTE013 and S1 P during reoxygenation for 4 h;(9)JTE013 group: cells were pretreated with 10 μM JTE013 30 min before reoxygenation,then reoxygenation for 4 hours;(10)VPC23019 + S1 P group(VPC23019 + S1P): cells were pretreated with 5 μM VPC23019(inhibitor of S1P1、3)30 min before reoxygenation,then treated with the mixture of VPC23019 and S1 P during reoxygenation for 4 h;(11)VPC23019 group: cells were pretreated with 5 μM VPC23019 30 min before reoxygenation,then reoxygenation for 4 hours;(12)PTX + S1 P group(PTX + S1P): cells were pretreated with 1 μg/m L PTX(inhibitor of G protein-coupled receptor)30 min before reoxygenation,then treated with the mixture of CAY10444 and S1 P during reoxygenation for 4 h;(13)PTX group: cells were pretreated with 1 μg/m L PTX 30 min before reoxygenation,then reoxygenation for 4 hours.(3)Experimental assays:(1)Cell viability was determined by MTT assay.(2)Cell apoptosis was measured by flow cytometry.(3)Detection of LDH content in cell culture medium.2.The mitochondrial mechanism in protective effects of S1 P against the injury of cardiomyocytes during hypoxia/reoxygenation.(1)Groups:Cells were randomly divided into 5 groups: normal control group(C group);hypoxia/reoxygenation group(H/R group);S1P group;STATTIC + S1 P group;STATTIC group.(1)control(C)group: cells were normally cultured for 24 hours,then cells were placed with the only serum-free DMEM for 4 h;(2)hypoxia/reoxygenation(H/R)group: cells were subjected to hypoxia for 16 h and reoxygenation for 4 h;(3)S1P group: 4 μM S1 P was added when reoxygenation for 4 h;(4)STATTIC + S1 P group(STATTIC + S1P): cells were pretreated with 1 μM stattic(inhibitor of STAT3)30 min before reoxygenation,then treated with the mixture of stattic and S1 P during reoxygenation for 4 h;(5)STATTIC group: cells were pretreated with 1 μM STATTIC 30 min before reoxygenation,then reoxygenation for 4 hours.(2)Experimental assays:(1)Cell viability was measured by MTT assay.(2)Cell apoptosis was measured by flow cytometry.(3)Detection of lactate dehydrogenase(LDH)content in cell culture medium.(4)Mitochondrial membrane potential,mitochondrial calcium ion concentration,mitochondrial permeability transition pore and mitochondrial ROS were detected by laser confocal microscopy.(5)The content of cytochrome C was measured by immunofluorescence assay.(6)Protein expression level of Bcl-2 and Bax,Pro-caspase3 and Cleaved caspase3 were detected by Western blot.Results: 1.Effects of S1 P and its receptor antagonist on myocardial injury in rat cardiomyocytes during hypoxia and reoxygenation.Compared with group C,the survival rate of H/R group was significantly decreased,the apoptosis rate and LDH content were significantly increased;Compared with the hypoxia / reoxygenation group(H/R group),S1 P group significantly increased H9c2 cell viability after the treatment of H/R(P<0.01),and the apoptosis rate(P<0.01)and LDH content were significantly decreased(P<0.01).Compared with S1 P group,PTX + S1 P,W146 + S1 P,CAY10444 + S1 P and VPC23019 + S1 P group all significantly reduced the survival rate of H9c2 cells(P<0.01),increased the apoptosis rate(P<0.01)and cell culture medium LDH content(P<0.01).S1 P receptor antagonists which include: PTX,W146,CAY10444,VPC23019 can significantly block the role of S1 P above,in addition to JTE013 that is S1P2 receptor antagonist(P<0.01).2.The mitochondrial mechanism in protective effects of S1 P against the injury of cardiomyocytes during hypoxia/reoxygenation.Compared with group C,the survival rate of H/R group was significantly decreased(P<0.01),the apoptosis rate and LDH content increased significantly(P<0.01);the mitochondrial membrane potential decreased significantly(P<0.01),the calcium level and the reactive oxygen species increased significantly(P<0.01),the degree mitochondrial permeability transition pore opening significantly increased(P<0.01),the release of cytochrome C significantly increased,not only the relative expression level of protein Bax/Bcl-2 but also cleaved caspase3/Pro-casepase3 was increased.(P<0.01);Compared with the hypoxia / reoxygenation group(H/R group),the survival rate of H9c2 cells after hypoxia / reoxygenation was significantly increased in S1 P group(P<0.01),and the apoptotic rate and the LDH content was significantly decreased(P<0.01),the mitochondrial membrane potential increased significantly(P<0.01),the calcium level and the reactive oxygen species decreased significantly(P<0.01),the degree mitochondrial permeability transition pore opening significantly decreased(P<0.01),the release of cytochrome C significantly decreased,not only the relative expression level of protein Bax/Bcl-2 but also cleaved caspase3/Pro-casepase3 was decreased(P<0.01);Compared with S1 P group,the cell viability of Stattic + S1 P group was significantly lower(P<0.01),and the apoptosis rate,LDH content increased significantly(P<0.01),the mitochondrial membrane potential decreased significantly(P<0.01),the calcium level and the reactive oxygen species increased significantly(P<0.01),the degree mitochondrial permeability transition pore opening significantly increased(P<0.01),the release of cytochrome C significantly increased,not only the relative expression level of protein Bax/Bcl-2 but also cleaved caspase3/Pro-casepase3 was increased(P<0.01).Conclusion: 1.The effect of S1 P resists the injury of cardiomyocytes during hypoxia/reoxygenation was related to the S1P1、3 receptors of the cell membrane activated.2.S1 P can decrease the intracellular calcium concentration and ROS level;decrease mitochondrial membrane potential;inhibite mitochondrial permeability transition opening;inhibite mitochondrial cytochrome C releasing thus inhibiting cytosolic Caspase3-independent signaling pathway activated.Eventually,inhibite apoptosis induced from related mitochondrial pathway.Moreover,it can reverse the damage of mitochondria and improve the function of mitochondria,in order to play a role in resisting the injury of cardiomyocytes during hypoxia and reoxygenation.