| PartⅠ Role of Kv1.3 potassium channel in ventricular remodeling and experimental clinical events after myocardial infarctionObjective: Myocardial infarction(MI)is the most common fatal cardiovascular disease in the world,and heart failure after MI is the leading adverse outcome of disability and mortality from coronary heart disease.Kv1.3 potassium channels exist on the surface of various immunoinflammatory cells and play a key role in their activation and function.The purpose of this study was to investigate the effects of murine anti-Kv1.3-E314 peptide monoclonal antibody(Anti-Kv1.3 m Ab)prepared by our team on ventricular remodeling and experimental clinical events after MI.Methods: The MI model and sham operation model(Sham group)were established in 8 to 10 weeks old male C57BL/6 mice.The MI models were randomly divided into Control group and Anti-Kv1.3 group.PBS buffer or Anti-Kv1.3 m Ab was injected into tail vein 6h,48h and 5 days after MI surgery,respectively.Mice in each group were sacrificed after 21 days of echocardiography after MI operation.Cardiac specimens were collected and Masson staining was performed to detect the infarct size and degree of fibrosis.Survival was recorded for each of the 21 days to calculate the survival rate,and dissection of the dead mice was performed to record the heart rupture rate.Results: Masson staining showed that compared with the Control group,the Anti-Kv1.3 group has the significantly reduced infarct area,the significantly increased thickness of the infarct ventricular wall,the significantly increased collagen deposition in the infarct area,and the significantly reduced fibrosis at the infarct junction 21 days after MI.21 days after MI,compared with the Control group,the results of echocardiography showed that EF and FS values in the Anti-Kv1.3 group were notably higher,and LVEDD,LVESD,LVEDV and LVESV values were markedly lower.The survival rate and heart rupture rate of each group during 21 days after MI were recorded.Compared with the Control group,the survival rate of the Anti-Kv1.3 group was significantly increased,and the heart rupture rate was significantly reduced.Conclusion: In mouse MI model,Anti-Kv1.3 m Ab can reduce infarct area,increase ventricular wall thickness,increase collagen deposition in infarct area,reduce fibrosis in infarct junction,improve cardiac function,improve survival rate,and reduce the rate of heart rupture.PartⅡ Role of Kv1.3 potassium channel in T lymphocytes after myocardial infarction in miceObjective: To study the regulatory effect of Anti-Kv1.3 m Ab on T cells after MI in mice.Methods: Male C57BL/6 mice aged from 8 to 10 weeks were selected to establish MI model and Sham operation model(Sham group).MI models were randomly divided into Control group and Anti-Kv1.3 group.PBS buffer or Anti-Kv1.3 m Ab was injected into tail vein 6 h,48 h and 5 d after MI,respectively.MI in mice after 1,3,7 days,the proportions of Th1,γδ T,Th2,Treg,IL-10+ CD4+ T in spleen,lymph nodes and infarction region were detected by flow cytometry.The relative expression levels of T cell cytokines genes such as IFN-γ,IL-17 A,IL-4,IL-10 and Foxp3 in spleen and infarction region were detected by q RT-PCR.Results: Flow cytometry results showed that 3 and 7 days after MI,Th1 andγδ T cells proportions in the spleen,lymph nodes and heart infarction region of Anti-Kv1.3 group were notably lower than those in the Control group.At 1,3 and 7 days after MI,the proportions of Th2 cells and Treg cells in spleen,lymph node and heart infarction region of Anti-Kv1.3 group were not prominently different from those of Control group.At 3 and 7 days after MI,compared with the Control group,the proportions of IL-10+ CD4+ T cells in spleen,lymph nodes and heart infarction region in Anti-Kv1.3 group were markedly higher.q RT-PCR results showed that the relative expression levels of IFN-γ and IL-17 A in spleen and heart infarction region of Anti-Kv1.3 group were significantly lower than those of Control group at 3 and 7 days after MI.At 1,3 and 7 days after MI,the relative expression levels of IL-4 and Foxp3 in spleen and heart infarction region of Anti-Kv1.3 group were not markedly different from those of Control group.The relative expression levels of IL-10 in spleen and heart infarction region of Anti-Kv1.3 group were higher than those of Control group at 3 and 7 days after MI.Conclusion: In mouse MI model,Anti-Kv1.3 m Ab can reduce the pro-inflammatory Th1 and γδ T cells,increase the anti-inflammatory IL-10+ CD4+ T cells,without affecting the Th2 and Treg cells.That is,the pro-inflammatory/anti-inflammatory balance can be regulated by regulating the activation and differentiation of T cell subsets after MI by Anti-Kv1.3 m Ab.Part Ⅲ Role of Kv1.3 potassium channel in monocyte/macrophage polarization and myocardial damage/repair balance after myocardial infarction in miceObjective: To study the effect of Anti-Kv1.3 m Ab on the regulation of monocytes/macrophages polarization after MI in mice,and the effect of Anti-Kv1.3 m Ab on the repair of post-infarction injury.Methods: Male C57BL/6 mice aged from 8 to 10 weeks were selected to establish MI model and Sham operation model(Sham group).MI models were randomly divided into Control group and Anti-Kv1.3 group.PBS buffer or Anti-Kv1.3 m Ab was injected into tail vein 6 h,48 h and 5 d after MI,respectively.Mice in each group were sacrificed at 1,3 and 7 days after MI operation,and the number of neutrophils in peripheral blood and heart infarction region,the number of Ly6Chigh/Ly6 Clow monocytes in bone marrow,spleen,heart infarction region and peripheral blood,the proportion and number of M1 / M2 macrophages,the proportion and number of M2a/M2 c macrophages were measured by flow cytometry.MI postoperative 1,3,7 days,through the q RT-PCR and ELISA test mice spleen and heart infarction region respectively CCL2 and IL-1β m RNA expression quantity relatively,serum CCL2,CCL7,IL-1β levels.At 1,3 and 7 days after MI,the m RNA relative expression levels of TNF-α,IL-6,MMP-9 secreted by M1 macrophages and TGF-β,VEGF,IL-10 secreted by M2 macrophages in heart infarction region were detected by q RT-PCR.At 21 days after MI,capillary angiogenesis and the expression of myofibroblasts in the heart infarction region were detected by immunofluorescence,and type I collagen deposition in the heart infarction region was detected by immunohistochemistry and Sirius red picric acid staining.Results: Flow cytometry results showed that there were no statistical differences in the number of neutrophils in the peripheral blood and heart infarction area between the Control group and the Anti-Kv1.3 group at 1,3 and 7 days after MI.At 3 and 7 days after MI,compared with the Control group,the number of Ly6 Chigh monocytes in the bone marrow of the Anti-Kv1.3 group was significantly increased,while the number of Ly6 Chigh monocytes in the spleen,peripheral blood and heart infarction area was significantly decreased and the number of Ly6 Clow monocytes was significantly increased in the Anti-Kv1.3 group.At 3 and 7 days after MI,compared with the Control group,the proportion and number of M1 macrophages in the heart infarction region in the Anti-Kv1.3 group were significantly decreased,the proportion and number of M2 macrophages were significantly increased,and the proportion and number of M2 c macrophages were significantly increased.q RT-PCR and ELISA results showed that,compared with the Control group,the levels of CCL2,CCL7 and IL-1β in the spleen,heart infarction region and blood in the Anti-Kv1.3 group were notably decreased.At 1,3 and 7 days after MI,compared with the Control group,q RT-PCR results showed that the relative expression levels of TNF-α,IL-6 and MMP-9 in heart infarction region of Anti-Kv1.3 group were lower,while the relative expression levels of VEGF and IL-10 were higher.Immunofluorescence,histochemistry,and Sirius red staining results at 21 days after MI showed that the number of new capillaries,myofibroblasts,and type I collagen deposition in the infarcted area were significantly increased in the Anti-Kv1.3 group compared with the Control group.Conclusion: In mouse MI model,Anti-Kv1.3 m Ab inhibits the mobilization and generation of Ly6 Chigh monocytes,as well as their migration and invasion,regulates the polarization of monocytes to M2 macrophages(mainly M2c),down-regulates the pro-inflammatory factors and up-regulates the anti-inflammatory factors.Thus,the inflammation is inhibited,type I collagen deposition and new capillaries are increased,which significantly alleviate the tissue damage after MI and improve the quality of myocardial repair. |
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