Study Of The Role And Mechanism Of Sphingosine 1-phosphate Receptor 3 In Sepsis-induced Cardiomyocyte Apoptosis

Objective:To investigate the role and mechanism of sphingosine 1-phosphate receptor 3 in sepsis-induced cardiomyocyte.Methods:Part I experiments:To investigate the myocardial injury,apoptosis and the relationship with S1PR3 in sepsis.C57/B6 mice were divided into Sham Operation Group（Sham group,n=6）and sepsis group（CLP group,n=6）.The myocardial tissues of two groups of mice were taken for corresponding experiments.1.WB method was used to detect the expression level of S1PR3 protein and determine the time point of the highest expression level of S1PR3protein firstly.2.H&E method was used to observe the pathological changes of myocardial injury,q PCR and WB method were used to detect the expression levels of myocardial injury markers BNP and c Tn I.The relative optical density of CD45 on the surface of leukocytes and CD68 on the surface of macrophages was measured by IHC.and the expression of inflammatory factors was detected by q PCR and WB methods.3.The expression of S1PR3was detected by q PCR,WB and IF methods.4.The level of cardiomyocyte apoptosis was detected by WB and TUNEL methods.Part II experiments:To verify the correlation between S1PR3 and cardiomyocyte apoptosis in sepsis.The mice were divided into WT Sham group,WT CLP Group,S1PR3^-/-Sham group and S1PR3^-/-CLP group with 6 samples in each group.The myocardial tissues of four groups of mice were taken for corresponding experiments.1.H&E method was used to observe the pathological changes of myocardial injury,q PCR and WB method were used to detect the expression levels of myocardial injury markers BNP and c Tn I,The relative optical density of CD45 on the surface of leukocytes and CD68 on the surface of macrophages was measured by IHC,and the expression of inflammatory factors was detected by q PCR and WB methods.2.The level of cardiomyocyte apoptosis was detected by WB and TUNEL methods.Part III experiments:S1PR3-m TOR pathway was explored.For the previous grouping,the phosphorylation expression level of m TOR was detected by WB method.Injection of m TOR inhibitor rapamycin（Rapa）and rapamycin solvent medium（Veh）.The mice were divided into S1PR3^-/-Sham group,S1PR3^-/-CLP group,S1PR3^-/-Sham+Veh group,S1PR3^-/-CLP+Veh group,S1PR3^-/-Sham+Rapa group,S1PR3^-/-CLP+Rapa group,with 6 samples in each group.The myocardial tissues of six groups of mice were taken for corresponding experiments.The WB method was used to detect the level of cardiomyocyte apoptosis.Results:Part I:1.The relative expression levels of S1PR3 protein in myocardial tissue of mice in the Sham group and the CLP method post-modeling at 0h,6h,12h,18h,24h,36h,and 48h sepsis groups were 0.50±0.19,0.69±0.15,0.77±0.05,0.78±0.04,0.78±0.06,1.13±0.02,1.11±0.03,and 1.06±0.06,respectively,Compared with the Sham group,S1PR3 protein expression was significantly increased at 24h（P=0.0019,n=6）,36h（P=0.0023 n=6）and 48h（P=0.0049,n=6）after CLP modeling.And it reached a peak at 24h.There was no statistical difference between the remaining time groups and the Sham group（P>0.05,n=6）.Therefore,the highest peak of S1PR3 protein expression at 24h after CLP modeling was selected for subsequent experiments.2.Compared with the Sham group,mice in the CLP group showed disturbed myocardial fiber arrangement,marked edema and thickening of myofibers,extensive transverse fractures within myofibers,massive inflammatory cell infiltration in the interstitial space,degeneration of some cells,and a significant increase in myocardial histopathological score（P<0.0001,n=6）;a significant increase in the expression of BNP m RNA,a marker of myocardial injury（P=0.0006,n=6）and c Tn I protein expression（P=0.0018,n=6）;increased inflammatory cell infiltration,increased relative optical density of leukocyte surface marker CD45（P=0.0413,n=6）and macrophage surface marker CD68（P=0.0016,n=6）;decreased m RNA expression of the anti-inflammatory factor IL-10（P=0.0201,n=6）.decreased（P=0.0219,n=6）,pro-inflammatory factor IL-6 m RNA expression increased significantly（P<0.0001,n=6）,TNF-αm RNA expression increased significantly（P<0.0009,n=6）;inflammatory factor IL-10 protein expression decreased significantly（P=0.0036,n=6）,pro-inflammatory factor IL-6 protein expression increased significantly（P=0.0077,n=6）,and TNF-αprotein expression（P=0.0123,n=6）.3.Compared with the Sham group,myocardial tissue S1PR3 m RNA expression was increased（P=0.0150,n=6）,protein expression was significantly increased（P=0.0056,n=6）,and mean fluorescence intensity was increased（P=0.0014,n=6）in the CLP group mice.4.Compared with the Sham group,the CLP group showed increased cardiomyocyte apoptosis,decreased expression of anti-apoptotic Bcl2 protein（P=0.0289,n=6）,significantly increased expression of pro-apoptotic Bax protein（P=0.0077,n=6）,significantly increased expression of Cleaved-Caspase3/Pro-Caspase3 protein（P=0.0021,n=6）,and a significant increase in apoptosis index in TUNEL-positive cells（P=0.0009,n=6）.Part II:1.Compared with the WT CLP group,the S1PR3^-/-CLP group showed significantly lower myocardial histopathology score（P<0.0001,n=6）;significantly lower BNP m RNA expression（P=0.0011,n=6）;significantly lower c Tn I protein expression（P<0.0001,n=6）;significantly lower relative optical density of leukocyte surface marker CD45（P<0.0001,n=6）;a significant decrease in the relative optical density of macrophage surface marker CD68（P<0.0001,n=6）;an increase in IL-10 m RNA expression（P=0.0246,n=6）,a significant decrease in IL-6 m RNA expression（P<0.0001,n=6）and TNF-αm RNA expression（P<0.0001,n=6）,n=6)were significantly decreased;IL-10 protein expression was significantly increased（P<0.0001,n=6）and IL-6 protein expression（P<0.0001,n=6）and TNF-αprotein expression（P<0.0001,n=6）were significantly decreased.2.Compared with WT CLP group,myocardial tissue of S1PR3^-/-CLP mice showed significantly increased expression of anti-apoptotic Bcl2 protein（P<0.0001,n=6）,significantly decreased expression of pro-apoptotic Bax protein（P<0.0001,n=6）and Cleaved-Caspase3 protein（P=0.0002,n=6）.TUNEL-positive apoptosis index was significantly reduced（P<0.0001,n=6）.Part III:1.Myocardial tissue p-m TOR protein expression was significantly decreased in mice in the CLP group compared with the Sham group（P=0.0003,n=6）.2.Myocardial tissue p-m TOR protein expression was significantly increased in S1PR3^-/-CLP group mice compared with WT CLP group（P<0.0001,n=6）.3.Compared with the S1PR3^-/-Sham group,there was no difference in the expression of anti-apoptotic Bcl2 protein（P=0.9372,n=6）,pro-apoptotic Bax protein（P>0.9999,n=6）and Cleaved-Caspase3 protein（P>0.9999,n=6）in the myocardial tissue of S1PR3^-/-CLP group.Compared with the S1PR3^-/-CLP group,there was no difference in the expression of anti-apoptotic Bcl2 protein（P=0.6311,n=6）,pro-apoptotic Bax protein（P=0.9998,n=6）and Cleaved-Caspase3 protein in the myocardial tissue of S1PR3^-/-CLP+Veh mice（P=0.9998,n=6）.Compared with the S1PR3^-/-CLP group,the S1PR3^-/-CLP+Rapa group mice showed decreased expression of anti-apoptotic Bcl2 protein（P=0.0185,n=6）,significantly increased expression of pro-apoptotic Bax protein（P<0.0001,n=6）,and increased expression of Cleaved-Caspase3 protein in myocardial tissue（P=0.0241,n=6）.Conclusion:1.Knockout of S1PR3 gene can improve the degree of myocardial injury,inflammatory cell infiltration and the level of pro-inflammatory factors in sepsis.2.S1PR3 promotes apoptosis in septic cardiomyocytes by inhibiting phosphorylation of m TOR.