| Chapter 1 The establishment of an isolated rat myocardial perfusion model with coronary endothelial dysfunctionObjective:To establish an isolated rat myocardial perfusion model with coronary endothelial dysfunction in order to investigate the impact of Triton X-100 on cardiac function and incidence of arrhythmia.Triton X-100 was used,based on the Langendorff perfusion modelMethods:SD rats were randomly divided into two groups(n=6 rats/group):CK group and ED group.The isolated rat hearts were perfused with K-H buffer for 20 min after the heart rate reached standard levels.CK group was perfused with saline for 10 min;ED group was perfused with 0.01%Triton X-100 for 10 min,and then perfused with K-H buffer for 140 min.CPP(%)was evaluated at T0 and T1.Hemodynamic indexes(CPP,LVSP,LVEDP,HR,+dp/dtmax,-dp/dtmax,LVDP)were recorded at T0,T1,T2,T3,T4 and T5,Arrhythmia index was calculated.Myocardial injury markers were collected.Myocardial infarction areas were detected by TTC method.Afterwards,the coronary artery was dissected and the morphological changes of endothelial cells were observed by scanning electron microscope.Results:The changes in CPP:In the case of histamine injection,results showed that CPP in the ED group was significantly lower than that of the CK group at T1.There were no significant differences between the two groups in CPP after sodium nitroprusside injection.There were no significant differences between the two groups in hemodynamic changes,arrhythmia index,myocardial injury markers,and infarction size at each time point.Regarding the morphological changes of coronary endothelium observed with scanning electron microscope:the coronary endothelium was imbricate,intact,continuous and smooth in the CK group.However,the coronary endothelium was rough,disordered and partially damaged in the ED group as evidenced by the exposure of subendothelial structures.Conclusion:Triton X-100 can cause coronary endothelial dysfunction in isolated rat hearts without affecting the function of cardiomyocytes.The model of isolated rat hearts with coronary endothelial dysfunction was successfully established.Chapter 2 The effect of oxytocin pretreatment on myocardial ischemia/reperfusion injury in rats with coronary endothelial dysfunctionObjective:Based on the experiments described in Chapter 1,we further determined the effect of oxytocin pretreatment on myocardial ischemia/reperfusion injury in rats with coronary endothelial dysfunction.Again,the changes in hemodynamics,myocardial injury markers and infarct sizes were recorded.A scanning microscope was used to determine the morphological changes in coronary artery endothelium.We further explored whether oxytocin pretreatment can alleviate ischemia/reperfusion injury in isolated rat hearts with endothelial dysfunction.Methods:SD rats were randomly divided into 6 groups(n=10 rats/group):CK group,I/R group,OT-I/R group,ED group,ED-I/R group,OT-ED-I/R group.The isolated rat hearts were perfused with K-H buffer for 20 min after the heart rate reached standard levels.The CK group was injected with normal saline for 10 min;ED group was injected with 0.01%Triton X-100 for 10 min;OT group was injected with oxytocin(0.01?mol/L)for 40 min;I/R group had 30 minutes of ischemia followed by 60 min of reperfusion.CPP(%)was evaluated at T1 and T5.hemodynamic changes(LVSP,LVEDP,HR,+dp/dtmax,-dp/dtmax,LVDP,RPP,SI)were recorded at T0,T1,T2,T3,T4 and T5,and arrhythmia index was calculated.Myocardial injury markers were collected and infarct sizes were calculated.Afterwards the coronary artery was dissected and the morphological changes in endothelial cells were observed by scanning electron microscope.Results:The changes in CPP:In the case of histamine injection,results showed that CPP of CK group T5 point was significantly higher than that of ED-I/R group and ED-OT-I/R group at T1 point,ED group,ED-I/R group and ED-OT-I/R group at T1 and T5 time point;OT-I/R group was significantly higher than that of I/R group at T5 time point;I/R group at T5 point was significantly higher than that of ED group,ED-I/R group and ED-OT-I/R group at T5 time point.There were no significant differences in CPP among the six groups after sodium nitroprusside injection.There were no significant changes among six groups in HR at T0-T5.Compared to the CK group,the hemodynamic indexes of the ED group had no statistical difference.compared to CK group,the LVDP,ądp/dtmax and RPP of I/R group,OT-I/R group,ED-I/R group and ED-OT-I/R group were decreased at each time point after reperfusion.But the arrhythmia index,hs-cTnT,SI and infarct sizes were increased.compared to I/R group.the LVDP,ądp/dtmax and RPP of OT-I/R group were increased at each time point after reperfusion,but the arrhythmia index,hs-cTnT,SI and infarct sizes were decreased.compared to the ED-I/R group;the LVDP,ądp/dtmax and RPP of ED-OT-I/R group were increased at each time point after reperfusion,but the arrhythmia index,hs-cTnT,SI and infarct area were decreased.There was no statistical difference between the OT-I/R group and the OT-ED-I/R group for each index.The scanning electron microscope results indicated that the endothelial cells were basically intact,smooth,and imbricate in CK group.There were no damaged endothelial cells found in CK group.In the I/R group,the integrity of endothelial cells was destroyed.In the OT-I/R group,the severity of endothelial injury was reduced.There were slight interruptions and holes in the endothelial cells.However,in ED group,ED-I/R group,and ED-OT-I/R group,the endothelial cells were irregularly arranged and were abnormally attached.The endothelia were almost completely stripped,which were more severe than those in I/R group.Conclusions:Oxytocin pretreatment can reduce ischemia/reperfusion injury,improve cardiac systolic and diastolic functions,and reduce the incidence and severity of arrhythmias.Oxytocin pretreatment can also increase the rate of pressure production,and reduce shock index.The expressions of myocardial injury markers and infarct sizes were reduced by this pretreatment.This therapeutic effect was present in isolated rat hearts with healthy and dysfunctional coronary endothelium,and There was no difference in therapeutic effect.oxytocin pretreatment can reduce the ischemia/reperfusion injury of coronary artery endothelia.Chapter 3 The therapeutic mechanism of oxytocin pretreatment on hypoxia/reoxygenation injury of rat coronary endothelial cells co-cultured with H9C2 cellsObjective:To establish the co-culture model of the H9C2 cells and the coronary endothelial cells isolated from rats.Transwell culture model was used to observe the effects of oxytocin pretreatment on H9C2 cells and rat coronary endothelial cells.And,we aimed to explore the mechanism of oxytocin in alleviating hypoxia/reoxygenation injury of cardiomyocytes and endothelial cells at cellular level and molecular biological level.Methods:Rat coronary endothelial cells and H9C2 cells were cultured separately and co-cultured in Transwells.They were divided into 8 groups:H group,C group,H-H/R group,C-H/R group,C-H-H/R group,OT-C-H-H/R group,OT-C-H-H/R group and OT-C-H-H/R group.Group H:the H9C2 cells were cultured normally for 8 h;C:the rat coronary endothelial cells were cultured normally for 8 h;H/R treatment:the cells had hypoxia for 4 h and reoxygenation for 4 h;OT treatment:the cells had oxytocin(0.01 ?mol/L)pretreatment for 40 min.Then the cells were analyzed for cell morphology under scanning microscope.The cell viabilities were detected by CCK8 method.The cTnT and eNOS of these cells were detected by ELISA.Cellular NO production was detected by the Griess method.Results:The observations in cell morphology under scanning microscope demonstrated that the cells grew normally in H group and C group.The cells were flat and long spindle shaped,with similar morphology and even distribution.In the H-H/R group,the H9C2 cells were damaged and deformed;the adherence rate of the C-H/R cells was decreased,showing a round dot shape;the morphology of H9C2 cells in the C-H-H/R group was similar to that in the H-H/R group;the morphology of H9C2 cells in the oxytocin treatment group was improved.Compared to the first group,after the H/R treatment,the H-H/R group,OT-H-H/R group,C-H-H/R group and OT-C-H-H/R group were decreased in cell activities and increased in cTnT expressions,compared to the H group.Compared to the second group,the C-H/R group was decreased in cell activities,compared to the C group.Compared to the third group,the OT-H-H/R group and OT-C-H-H/R group were increased in cell activities and decreased in cTnT expressions,compared to the H-H/R group.Compared to the fourth group,the OT-C-H-H/R group was increased in cell activities and decreased in cTnT expressions,compared to the C-H-H/R group.Compared to the fifth group,the OT-C-H/R group was increased in cell activities,compared to the C-H/R group.And lastly,there were no significant differences in the cell activities and the expressions of cTnT between the OT-H-H/R and OT-C-H-H/R groups.Regarding the expressions of eNOS and NO productions,they were decreased in the H-H/R group and OT-H-H/R group,compared to the H group.Second,the expressions of eNOS and the NO productions were decreased in the C-H/R group and were increased in the OT-C-H/R group and OT-C-H-H/R group compared to the C group.Third,the expressions of eNOS and the NO productions were increased significantly in the OT-C-H-H/R group,compared to the OT-H-H/R group.Fourth,there were no significant differences in the expressions of eNOS and the NO productions between the OT-H-H/R group and the H-H/R group.And lastly,there were no significant differences in eNOS expressions and the NO productions between the OT-C-H-H/R group and OT-C-H/R group.Conclusions:Oxytocin pretreatment can reduce the hypoxia/reoxygenation injury in H9C2 cells.The protective effect of oxytocin pretreatment on hypoxia/reoxygenation injury of H9c2 cells was not different between H9c2 cells cultured alone and co-cultured with rat coronary endothelial cells.The therapeutic effects of oxytocin on the hypoxia/reoxygenation injured H9C2 cells did not correlate with rat coronary endothelial cells.Oxytocin pretreatment can increase the expressions of eNOS and the NO productions,and can reduce the hypoxia/reoxygenation injury of rat coronary endothelial cells.Chapter 4 The mechanism of ERK1/2 pathway on oxytocin pretreatment in alleviating hypoxia/reoxygenation injury of rat coronary endothelial cells co-cultured with the H9C2 cellsObjective:Based on the Transwell culture model,the co-culture model of the H9C2 cells with rat coronary endothelial cells was established.The mechanism of ERK1/2 pathway on oxytocin pretreatment in alleviating hypoxia/reoxygenation injury of rat coronary endothelial cells co-cultured with the H9C2 cells was identified by using ERK1/2 phosphorylation inhibitor U0126.Methods:Rat coronary endothelial cells and H9C2 cells were cultured separately and co-cultured in Transwells.They were divided into 6 groups:H group,H-H/R group,OT-H-H/R group,OT-H-H/R-U0126 group,OT-C-H-H/R group,and OT-C-H-H/R-U0126 group.Group H:the H9C2 cells were cultured normally for 8 h;H/R treatment:the cells were cultured under hypoxic conditions for 4 h/reoxygenation for 4 h;OT treatment:cells were pretreated with oxytocin for 40 min.OT-U0126 treatment:cells were pretreated with oxytocin and U0126 for 40 min.Cell morphology was observed under microscope.Cell viabilities were detected by CCK8 method.The cTnT and eNOS were detected by ELISA.NO productions were detected by Griess method.And the expressions of ERK1/2 and p-ERK1/2 were detected by western blot(WB).Results:The observations of cell morphology under microscope showed that the H group cells were healthy,with long spindle shape and normal adherent rate.The H-H/R cells were deformed and disrupted.And some cells in this group floated on the culture medium.The OT-H-H/R group cells were less damaged than H-H/R group cells,with less abnormality in cell morphology.The OT-H-H/R-U0126 cells were similar to H-H/R group cells in morphology and damage.The OT-C-H-H/R group:the levels of cell damage in this group were reduced.The status of cells were significantly improved.The morphological changes in H9C2 cells were similar to that of OT-H-H/R group.In the OT-H-H/R-U0126 group,the cell shapes of H9C2 cells were similar to those in OT-H-H/R-U0126 group,and the cell shapes of endothelial cells were similar to those in OT-C-H-H/R group.Regarding the cell viabilities and cTnT expressions:(1)The cell viabilities were decreased significantly,whereas the cTnT expressions were increased in the H-H/R group,OT-H-H/R group,OT-H-H/R-U0126 group,OT-C-H-H/R group and OT-C-H-H/R-U0126 group,compared to the H group.(2)The cell viabilities were increased,whereas the cTnT expressions were decreased in the OT-H-H/R group and OT-C-H-H/R group,compared to the H-H/R group.(3)The cell viabilities were decreased,whereas the cTnT expressions were increased in the OT-H-H/R-U0126 group,compared to the OT-H-H/R group.(4)The cell viabilities were decreased,whereas the cTnT expressions were increased in the OT-C-H-H/R-U0126 group,compared to the OT-C-H-H/R group.(1)The expressions of eNOS and the NO productions were decreased in the H-H/R group,OT-H-H/R group and OT-H-H/R-U0126 group,compared to the H group,(2)The expressions of eNOS and the NO productions were increased in the OT-C-H-H/R group and OT-C-H-H/R-U0126 group,compared to the H group.(3)The expressions of eNOS and the NO productions had no statistical differences between the OT-H-H/R-U0126 group and the OT-H-H/R group.(1)The phosphorylation rate of ERK1/2 in the H-H/R group was decreased,compared to the H/R group.(2)The phosphorylation rate of ERK1/2 in the OT-H-H/R group was increased,compared to the H-H/R group.(3)The phosphorylation rate of ERK1/2 in the OT-H-H/R-U0126 group was decreased,compared to the OT-H-H/R group.Conclusions:Oxytocin pretreatment can reduce the hypoxia/reoxygenation injury of the H9C2 cells by increasing ERK1/2 phosphorylation.U0126 can block ERK1/2 phosphorylation,thus dramatically reducing the therapeutic effects of oxytocin.U0126 did not affect the expression of eNOS and the NO productions.ERK1/2 phosphorylation did not affect the therapeutic effects of oxytocin pretreatment on the hypoxia/reoxygenation injury of the coronary endothelium. |
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