| Background and objective:Sepsis is a dysregulated host response to infection.The incidence is growing at a rate of 1.5%-9%annually.Although with the level of medical progression,sepsis mortality is still as high as 20%-40%.With a complex pathogenesis,inflammatory disorder is the pathophysiological basis of sepsis.Patients with sepsis prone to multiple organ dysfunction,but the heart is the easiest organ involved.Recent studies have shown that patients with sepsis had myocardial depression at a rate of nearly 40%-50%,7%of them got heart failure.So there are practical value to make clear the sepsis-induced cardiac dysfunction causes and mechanisms and then for effective prevention and treatment are also very valuable.SIP is a important lipid mediator and interacts with five GPCRs named S1P1-5 five subtypes.The GPCRs transduce intracellular signals to regulate cellular behavior.Because of its receptors are widely distributed in the different systems of the body,previous studies have shown that,SIP has a great relationship with the pathophysiology of sepsis,coronary heart disease,stroke and other diseases.FTY720 is a structure analogue of SIP and could combine with four receptors namely S1P1,S1P3,S1P4,S1P5 receptor except for S1P2 receptor.It is used for autoimmune diseases,inflammation,organ transplant rejection response and the treatment for the other diseases,but its impact on the autocrine of myocardial cells in sepsis is not clearly.Therefore,in this research,with different doses of FTY720 intervention,by determine the inflammatory cytokines and protein expression of cardiomyocytes treated with LPS,we seeks to elucidate the protective effects of S1P on septic myocardial cells and the possible mechanism.Methods:1?Cell culture:H9C2 cells were cultured in high glucose DMEM supplemented with 10%fetal bovine serum and fostered in a humidified atmosphere consisting of 5%C02 at 37?,the cells are adherent cells.2?The effect of inflammatory cytokine secretion of cardiomyocytes after LPS stimulation:The cardiomyocytes were treated with LPS with different concentrations of 0,1,2,5,10?g/ml for 8,12,24,36 and 48h.The supernatant was collected.After centrifugation,the IL-6 and IL-10 of supernatant were measured by ELISA.3?The determination of inflammation reaction pathway:Using the same concentration(5?g/ml)of LPS to stimulate of cardiac myocytes Oh,8h,12h,24h,36h,48h,Then measures the expressions of P-I?B? in cells by Western blot analysis.4?Effects of LPS on cardiac myocyte apoptosis and proliferation:with the same method of step 3.Measureing the expressions of cleaved caspase-3?AKT in cells by Western blot analysis.5?Changes in cytokine secretion of different concentrations of FTY720 Intervention:The cells were divided into 6 groups.Control group:cells were untreated;LPS group:with only 5?g/ml LPS stimulation;FTY720(10nM)+LPS group:cells were treated with 10nM FTY720 and 5?g/ml LPS stimulation;FTY720(50nM)+LPS group:cells were treated with 50nM FTY720 and 5?g/ml LPS stimulation;FTY720(100nM)+LPS group:cells were treated with 100nM FTY720 and 5?g/ml LPS stimulation;FTY720(200nM)+LPS group:cells were treated with 200nM FTY720 and 5?g/ml LPS stimulation.cell supernatants were collected after the intervention 24h.The supernatant was collected and the concentrations of IL-6 and IL-10 were measured by ELISA.6?Effect of the intervention of FTY720 on the inflammatory response pathway and apoptosis proteins:Western blot was used to test the expression of P-I?B??cleaved caspase-3 and AKT.7?The effects of selective inhibitors of receptor S1P1,3 VPC23019 and AKT kinases inhibitor wortmanin on AKT protein expression:The cells were divided into two groups.LPS group:with only 5?g/ml LPS stimulation;FTY720(100nM)+LPS group:cells were treated with 100nM FTY720 and 5?g/ml LPS stimulation;FTY720(100nM)+LPS+VPC23019 group:cells were treated with 100nM FTY720,5?g/ml LPS and VPC23019 stimulation;VPC23019+LPS group:cells were treated with 5pg/ml LPS and VPC23019 stimulation;LPS group:with only 5?g/ml LPS stimulation;FTY720(100nM)+LPS group:cells were treated with 100nM FTY720 and 5?g/ml LPS stimulation;FTY720(100nM)+LPS+wortmanin group:cells were treated with 100nM FTY720?5?g/ml LPS and wortmanin stimulation;wortmanin+LPS group:cells were treated with 5?g/ml LPS and wortmanin stimulation.Western blot was used to test the expression of AKT.Results:1?LPS affect the secretion of IL-6 and IL-10 in myocardial cells:LPS can stimulate cardiac cells to secrete inflammatory cytokines IL-6 and IL-10,and secretion of IL-6 express the concentration-dependent of LPS.The secretion of IL-6 peaked in 36h.The secretion of IL-10 had no significant relationship between the time gradient.2?The determination of inflammation reaction pathway and apoptosis protein:Western Blot showed,LPS(5?g/ml)stimulation of cardiac myocytes can increase the expression levels of P-I?B? in cytoplasm,which reached a peak within 24 hours,compared with the control group with significant difference(P<0.05).3?LPS could affect the expression of cleaved caspase-3,which arrived at the highest level at the time of 12h at the stimulation of LPS(5?g/ml),compared with control group(P<0.05)and the expression of AKT arrived at the lowest level at the 8h,recovered at 48h.4?The changes of cytokine after FTY720 intervention:FTY720 could affect the secretion of the inflammatory cytokine IL-6.The levels of IL-6 in the LPS group?FTY720(50nM)+LPS group?FTY720(100nM)+LPS group and FTY720(200nM)+LPS group were 146±13.7 pg/ml,96.5±7.7 pg/ml,71.1±8.3 pg/ml and 56.1±7.2 pg/ml.The levels in the FTY720(50nM)+LPS group?FTY720(100nM)+LPS group and FTY720(200nM)+LPS group were lower than that in the LPS group.The levels of IL-10 were 21.7±2.8 pg/ml,20.9±1.9 pg/ml,19.5±1.8 pg/ml,21.2±2.2 pg/ml,18.9±2.4 pg/ml respectively.It were observed that the FTY720 did not significantly affect the secretion of IL-10 in the septic myocardial cells.5?FTY720 could affect the phosphorylation of I?B? in the cytoplasm of myocardial cell:The expressions of P-I?B? of FTY720(50nM)+LPS group?FTY720(100nM)+LPS group and FTY720(200nM)+LPS group were respectively reduced by 62%,98%,189%,compared with LPS group.6?FTY720 could affect LPS-induced myocardial cell apoptosis and cell survival:50nM,100nM,200nM concentrations of FTY720 can make the cleaved caspase3 expression reduced by 95%,123%,66%,FTY720 intervention at a concentration of 100nM,200nW,compared with LPS group,the expression of AKT was increased by 100%and 167%.7?S1P1,3 receptor selective inhibitors VPC23019 and AKT kinases inhibitor wortmanin could restrain the AKT protein expression:VPC23019 and wortmanin can make AKT expression decreased,FTY720 through S1P1,3pathway activated AKT pathway.Conclusion:1?LPS could stimulate the secretion of TNF-? and IL-6 and in H9C2 cardiomyocytes by the pathway of NF-?B,and its secretion in a time and concentration-dependent manner.LPS can increase the secretion of IL-10,but only with concentration-dependent,but without the time-dependent.The expression of cleaved caspase-3 were higher after LPS stimulation,while the expression of AKT was lower.2?FTY720 could reduce the secretion of the inflammatory cytokine like IL-6 and inhibit the phosphorylation of IicBa,reduce the expression of cleaved caspase-3 and increase the expression of AKT.Moreover,FTY720 activated AKT through S1P1,3 receptor protein-dependent pathway.FTY720 had protective effect through anti-inflammatory,resistance to apoptosis,promoting survival in septic cardiomyocytes. |
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