| Objectives: Sepsis is a life-threatening organ dysfunction caused by abnormal host response to infection.Persistent sepsis can lead to myocardial dysfunction,that is,the intrinsic systolic and/or diastolic dysfunction of myocardium caused by sepsis,which is manifested in ventricular dilatation,decreased myocardial compliance,and decreased responsiveness to fluid resuscitation and catecholamine stimulation.More than 40% of patients with sepsis have cardiac dysfunction,which is a common complication of severe sepsis.The mortality of patients with cardiac dysfunction increases to 70%-90%,which can be used as an independent risk factor for the mortality of patients with septic shock.It is urgent to find a positive and effective way to protect myocardium.Endostatin(ES)is a potent vascular endothelial growth factor inhibitor.Many animal experiments have found that endostatin has therapeutic effect on many acute and chronic inflammatory diseases,and can reduce the levels of TNF-α,IL-1β and IL-6 in serum of septic animals.Therefore,we speculate that exogenous endostatin can protect cardiomyocyte function by reducing apoptosis after sepsis.Sphingosine-1-phosphate(S1P)is an intracellular second messenger,which is involved in a variety of pathological reactions of the heart.It is a potential target for the treatment of heart failure and myocardial ischemia-reperfusion injury.The aim of this study was to investigate the relationship between exogenous endostatin and cardiomyocyte apoptosis after lipopolysaccharide induced sepsis in adolescent rats,and whether endostatin affects cardiomyocyte apoptosis through S1 P signaling pathway.Methods: Part one:(1)112 healthy male Sprague Dawley(SD)rats(21 days old,weighing 45g-60g)were randomly divided into control group,model group and endostatin group.The model group was divided into 7 time points: 2h,4h,6h,12 h,24h,48 h and 72 h.The endostatin group was divided into 6h and 12 h time points,and each time point was further divided into 4 groups.There were 8 rats in each group.Sepsis model was established by intraperitoneal injection of lipopolysaccharide(LPS,5mg/kg,10ml/kg).Endostatin(4μg/kg,10ml/kg)was injected 30 min later,on different time points(2h,4h,6h,12 h,24h,48 h,72h),Luminex and enzyme linked immunosorbent assay(ELISA)and biochemical analyzer were used to detect serum endostatin,inflammatory factors(TNF-α,IL-1β,IL-6)and myocardial enzyme spectrum to observe the changes of endostatin in septic animals and whether exogenous endostatin was protective.HE staining and TUNEL staining were used to observe the apoptosis of myocardium.RT-PCR and Western blot were used to detect the apoptosis of myocardium.The expression of apoptosis related proteins Bcl-2,Bax and cleavedcaspase-3 were detected by blot.(2)Rat cardiomyocytes(H9C2 cells)were randomly divided into H9C2 group,H9C2+ES group,H9C2+LPS group and H9C2+LPS+ES group.In H9C2+ES group,endostatin(4μg/ml)was added;in H9C2+LPS group,LPS(10μg/ml)was added;in H9C2+LPS+ES group,LPS(10μg/ml)was administrated firstly,and endostatin(4μg/ml)was injected after 0.5h in incubator.TUNEL immunofluorescence(IF)double staining and flow cytometry were used to count the proportion of apoptosis in each group.RT-PCR and Western blot were used to detect the expression of Bcl-2,Bax and cleaved-Caspase3.Part two: Groups and processing methods are same as part one,PCR,western blot and immunofluorescence were used to determine whether rh ES could affect the expression of S1 P and its receptors(S1PR1,S1PR2 and S1PR3)in the myocardial tissue and H9C2 cell line in the acute stage of sepsis.Part three:(1)Animal experiments: 48 healthy male 21-day old SD rats were randomly divided into four groups: control group,NC group,model group and endostatin group.The model group and endostatin group were further randomly divided into two groups: 6h group and 12 h group.There were 8 rats in each group.The rats in NC group were intraperitoneally injected with JTE-013(10mg/kg,10ml/kg),and the rats in model group and endostatin group were intraperitoneally injected with LPS(5mg/kg,10ml/kg)half an hour after JTE-013.After another half an hour,the rats in endostatin group were intraperitoneally injected with endostatin(5mg/kg,10ml/kg).(2)Cell experiments: H9C2 cells were transfected with S1PR2-shRNA to establish stable cell line.The cells were randomly divided into four groups: NC group,shRNA group,LPS group and LPS + ES group.NC group and shRNA group were used as normal medium;LPS group was treated with stable S1PR2-shRNA strain added with LPS(10μg/ml);LPS+ES group was treated with stable S1PR2-shRNA strain added with LPS(10μg/ml),cultured in incubator for 30 min,then added with endostatin(4μg/ml).TUNEL,RT-PCR,Western blot and flow cytometry were used to detect cardiomyocyte apoptosis after S1PR2 and H9C2 cells were silenced.(3)CCK-8 was adapted to evaluate the proliferation of all cell groups.(4)Cell cycle was tested by flow cytometer to show cell distribution in different stages.Results: Part one:(1)In the acute stage of sepsis(4h),the expression of endostatin in serum and myocardial tissue was significantly decreased(P<0.01),although the expression of endostatin slowly increased with time,it was still significantly lower than that of the control group(P<0.01)until 72h;(2)at each time point of sepsis,the level of inflammatory factors was significantly higher than that of the control group(P<0.05),in which the expression of endostatin at 6h and 1h was significantly higher than that of the control group(P<0.05)The serum concentrations of inflammatory factors TNF-α,IL-1β and IL-6 reached the peak at 2h(P<0.01).Compared with the model group,endostatin could significantly reduce the serum levels of TNF-α,IL-1β and IL-6(P<0.01),and the appropriate concentration of endostatin was 4μg/kg.Exogenous endostatin can also significantly reduce the serum CK,CKMB and LDH levels(P<0.01).(3)in the animal experiment,compared with the control group,the expression of Bcl-2 protein and mRNA in the model group decreased significantly(P<0.01),and the expression of Bax protein and mRNA increased significantly(P<0.01),Compared with the model group,the expression of Bcl-2 protein and mRNA in the endostatin group increased(P<0.01),and the expression of Bax protein and mRNA and the expression of cleaved-Caspase3 protein decreased(P<0.01).TUNEL results showed that the proportion of apoptotic cells in the model group was significantly higher than that in the control group(P<0.01),and the expression of Bax protein and mRNA in the endostatin group was significantly lower than that in the model group(P<0.01)The percentage of apoptotic cells decreased significantly(P<0.01).(4)Compared with H9C2 group,the expression of apoptosis related proteins in H9C2+ES group had no significant difference,while the expression of Bcl-2 protein and mRNA in H9C2+LPS group decreased significantly(P<0.01),and the expression of Bax protein and mRNA increased significantly(P<0.01),Compared with H9C2+LPS group,the expression of Bcl-2 protein and mRNA in H9C2+LPS+ES group increased(P<0.01),the expression of Bax protein and mRNA decreased significantly(P<0.01),and the expression of cleaved-Caspase3 protein decreased(P<0.05).The results of TUNEL-IF and flow cytometry showed that compared with H9C2 group,the proportion of apoptotic cells in H9C2+ES group had no significant difference,the apoptosis in H9C2+LPS group was significantly increased(P<0.01),and the apoptotic cells in H9C2+LPS+ES group were significantly decreased(P<0.01).Part two:(1)In the animal experiment,the expression of S1 P protein and mRNA decreased in the model group and the control group(P<0.01),while the expression of S1 P protein and mRNA increased in the endostatin group compared with the model group(P<0.01,except 12 hRT-PCR,P<0.05).Compared with the control group,the protein expression of S1Pr1 in the 12 h model group had no significant change,but the mRNA expression was decreased(P<0.05).The protein and mRNA expressions of S1PR1,S1PR2 and S1PR3 in the other model groups were decreased(P<0.01).Compared with the model group,the protein and mRNA expression of S1PR1 in endostatin group had no difference at 6h,but decreased at 12h(P<0.05),while the mRNA expression had no difference;the protein and mRNA expression of S1PR2 in endostatin group increased significantly(P<0.01);the protein expression of S1PR3 had no change at 6h,but decreased at 12h(P<0.01),and decreased at 12h(P<0.05).The results of immunofluorescence showed that the expression of S1PR2 in model group was significantly decreased(P<0.01).(2)In cell experiment,compared with H9C2 group,the expression of S1 P protein and mRNA in H9C2+ES group had no significant difference.The expression of S1 P protein and mRNA in H9C2+LPS group was significantly decreased(P<0.01).Compared with H9C2+LPS group,the expression of S1 P protein and mRNA in H9C2+LPS+ES group was increased(P<0.05)and increased(P<0.01).Part three:(1)In animal experiments,after administration of S1PR2 inhibitor JTE-013,although the trend of Bcl-2 decreased(P<0.01)and Bax increased(P<0.01)in the model group did not change,compared with the model group,there was no significant difference in the expression of Bcl-2 and Bax protein and mRNA,and there was no difference in the expression of cleaved-Caspase3 protein.TUNEL results showed that the apoptotic cells in the model group were more than those in the control group(P<0.01),but there was no difference between the endostatin group and the model group.(2)Compared with NC group,the expressions of Bcl-2,Bax and cleavedCaspase3 protein and mRNA in shRNA group had no difference,while the expressions of Bcl-2 protein and mRNA in shRNA+LPS group were decreased(P<0.01),the expressions of Bax protein and mRNA had no difference,but cleaved-Caspase3 protein expression was increased(P<0.01).TUNEL and flow cytometry analysis showed that the apoptosis rate of shRNA+LPS+ES group was higher than that of shRNA+LPS group(P<0.01).(3)CCK-8 showed no difference in cell proliferation between H9C2 group and H9C2+ES group,while that of H9C2+LPS group decreased significantly(P<0.01),while that of H9C2+LPS+ES group increased(P<0.01);after S1PR2 was blocked,there was no difference in proliferation between LPS+ES group and LPS group.(4)Cell cycle tested by flow cytometry: the cell proportion of each phase was not different between H9C2 and H9C2+ES groups;the ratio of G1 phase of H9C2+LPS group decreased(P<0.01),the proportion of G2 cells increased(P<0.01);compared with H9C2+LPS group,the ratio of G1 phase of H9C2+LPS+ES group increased(P<0.01),the proportion of G2 cells decreased(P<0.01);after S1PR2 was blocked,the proportion of cell cycle in LPS+ES group was no difference Different.Conclusions: 1.In the early stage of sepsis(4h),serum endostatin is decreased;2.Exogenous endostatin can protect myocardial block by down regulating the expression of inflammatory factors in serum and reducing cardiomyocyte apoptosis;3.Endostatin can up regulate the expression of S1P/S1PR2 in sepsis model,which may be a potential target for endostatin to regulate cardiomyocyte apoptosis;4.Provide a theoretical basis for endostatin in the clinical treatment of sepsis. |
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