| Part ?GABAAergic drugs influenced ventricular remodeling and surrogate clinical outcomes in mice after myocardial infarctionObjectives:To observe the influence of GABAAergic drugs on ventricular remodeling and surrogate clinical outcomes in mice after myocardial infarction(MI)Methods:We induced MI in C57BL/6 mice(male,weighing 20-25 g,8 to 10 weeks old)by ligating the left anterior descending(LAD)artery.MI-operated mice were randomly injected intraperitoneally with PBS buffer,GABAA receptor specific agonist(topiramate)and antagonist(bicuculline),once a day consecutively until 14 days after MI.Mice in sham group were subjected to identical operation without ligating coronary artery,and were injected intraperitoneally with PBS buffer once a day.Survival rates were observed and recorded from the first day after MI operation,and dead mice were dissected to clear the cause.The cardiac function was examined by echocardiography and hemodynamics on the day of being euthanized,and hearts were stained via 2,3,5-triphenyltetrazolium chloride(TTC)staining and Masson's trichrome staining to accumulate the infarct size.In monocytes/macrophages(Mo/M?)depletion experiments,clodronate liposomes were given to mice 1 day before and 3,and 6 days after MI.The MI-operated mice were given intraperitoneally to corresponding drugs according to the divided group(sham,control,Cl2MDP-lipo+PBS,Cl2MDP-lipo+Topiramate,Cl2MDP-lipo+Bicucullie)survival rates and ventricular rupture rates were recorded,echocardiography,hemodynamics,TTC and Masson's trichrome staining were performed on day 7 post-MI.Results:Echocardiography was performed on day 1 after MI-operation to verify the MI-operated mice being successfully induced and there were no differences between groups due to the operation.On day 14 post-MI echocardiography and hemodynamics showed that compared with PBS-treated group(controls),cardiac function of topiramate-treated MI mice was improved significantly,while bicuculline-treated MI mice had more deteriorated cardiac function than controls.TTC and Masson's trichrome staining showed that compared with controls,the infarct size was smaller in topiramate-treated MI mice,whereas larger in bicuculline-treated MI mice.Masson's trichrome staining further indicated that compared with control group,collagen density in the infarcted area of topiramate-treated MI mice increased,whereas which in bicuculline-treated MI mice decreased.Meanwhile,there were no significant differences of fibrosis in the remote zone among all of the groups.In addition,on day 28 post-MI,the observation of the surrogate clinical outcomes showed that compared with controls,topiramate-treated MI mice had higher survival rates,lower ventricular rupture rates,while bicuculline-treated MI mice have reversed result.Moreover,in Mo/M? depletion experiments,on day 7 post-MI,no significant differences were observed about cardiac function,infarct size,collagen density in the infarcted area,survival rates and survival rates among Cl2MDP-lipo+PBS-treated group,C12MDP-lipo+Topiramate-treated group and Cl2MDP-lipo+Bicucullie-treated group.Conclusion:GABAAergic drugs had influence on cardiac function,infarct size,and collagen density in the infarcted region,survival rates and ventricular rupture rates.The experiment of selective depletion of Mo/M? further indicated the target might be Mo/M?.Part IIGABAAergic drugs affected monocytes/macrophages polarization in mice after myocardial infarctionObjectives:To clarify the influence of GABAAergic drugs on monocytes/macrophages(Mo/M?)polarization in myocardial infarction(MI)miceMethods:We first sought to detect expression of the constituent ?1-and ?3-subunits of the GABAA receptor(which are bicuculline and topiramate bind site)on related immune cells,cardiomyocytes,vascular endothelial cells and fibroblasts at mRNA and protein levels.In vivo,we induced MI in C57BL/6 mice(male,weighing 20-25 g,8 to 10 weeks old)by ligating the left anterior descending(LAD)artery.MI-operated mice were randomly injected intraperitoneally to PBS,the GABAA receptor specific agonist(topiramate)and antagonist(bicuculline),once a day consecutively until mice were euthanized.Mice in sham group were subjected to identical operation without ligating coronary artery,and were injected intraperitoneally to PBS once a day.The number and porportion of Ly6Chigh/Ly6Clow monocytes in bone marrow,spleen,peripheral blood and infarcted region,the number and porportion of Ml or Ly6Chigh macrophages/M2 or Ly6Clow macrophages and fetal derived macrophages in infarcted region,numbers of peripheral blood and cardiac neutrophils,splenic Th1/Th2 were detected by flow cytometric analysis on days 1,3,7 post-MI.The levels of IL-1? in peripheral blood were detected by ELISA,and the expression of IL-1? in bone marrow,spleen and infarcted region were detected by qRT-PCR respectively on days 1,3,7 post-MI.In vitro,cultured RAW264.7 macrophages were devided into several groups which were associated with M1 macrophages,including no stimulus group,IFN-y-treated group,IFN-?+ topiramate-treated group,'IFN-y+bicuculline-treated group,and groups which were associated with M2 macrophages,including no stimulus group,IL-4-treated group,IL-4+ topiramate-treated group,IL-4+ bicuculline-treated group.The porportion of Ml or M2 macrophages of different groups was detected by Immunofluorescence co-staining and flow cytometric analysis.Results:The ?3-subunits of GABAA receptor expressed in macrophages and neutrophils,but were absent in monocytes,Thl,Th2,and also didn't expressed in cardiomyocytes,fibroblasts and vascular endothelial cells.The ?1-subunits of GABAA receptor expressed in monocytes,macrophages,Thl,Th2 and neutrophils,but not in cardiomyocytes,fibroblasts and vascular endothelial cells.In vivo,in topiramate-treated MI mice,flow cytometric analysis showed that,compared with controls,the number and proportion of splenic,peripheral blood and cardiac Ly6Chigh monocytes reduced,while those of Ly6Clow monocytes increased.Moreover,the number and proportion of M1 or Ly6Chigh macrophages decreased in the infarcted myocardium,while M2 or Ly6Clow macrophages increased,and numbers of cardiac fetal derived macrophages,splenic Th2 increased on days 3,7 post-MI.On days 1,3,7 post-MI,flow cytometric analysis showed there was no significant changes in the number and proportion of bone marrow Ly6Chigh and Ly6Clow monocytes between the controls and GABAergic drug treatment.Meanwhile,no significant difference was observed about numbers of peripheral blood,cardiac neutrophils and splenic Thl on days 1,3,7 post-MI.Immunohistochemistry and flow cytometric analysis showed there was no significant change in the total macrophage numbers between the controls and GABAergic drug treatment on days 1,3,7 post-MI.ELISA showed the levels of IL-1? in peripheral blood of topiramate-treated MI mice were lower than controls on 3,7 days post-MI,while bicucullie-treated MI mice showed opposite results.In addition,the same results was found about IL-1? mRNA level in bone marrow,spleen and infarct region.In vitro,immunofluorescence co-staining and flow cytometric analysis showed that there was no significant difference on the percentage of M1 macrophages among IFN-y-treated group,IFN-?+ topiramate-treated group and IFN-?y+ bicuculline-treated group,and there was also no significant difference on the percentage of M2 macrophages among IL-4-treated group,IL-4+ topiramate-treated group,IL-4+ bicuculline-treated group.Conclusion:First,topiramate exert influence on macrophages by binding on the P3-subunits of the GABAA receptor.In addition,topiramate reduced peripheral Ly6Chigh monocyte numbers by decreasing the expression or level of IL-1? in bone marrow,spleen,peripheral blood and the infracted myocardium.Bicuculline exert influence on monocyte/macrophages by binding on the ?1-subunits of the GABAA receptor.In vivo,GABAAergic drugs regulated the polarization of Ly6Chigh/Ly6Clow monocytes,M1/M2 macrophages,Ly6Chigh/Ly6Clow macrophages and Thl/Th2 after MI.Topiramate reduced pro-inflammatory Ly6Chigh monocytes,M1 macrophages or Ly6Chigh macrophages and increased anti-inflammatory Ly6Clow monocytes,M2 or Ly6Clow macrophages,fetal derived macrophages and Th2.In vitro,neither topiramate nor bicuculline had directly influence on the polarization of macrophages.Furthermore,the experiment of selective depletion of Mo/M? demonstrated GABAAergic drugs modulates the inflammatory response mainly by acting on monocytes/macrophages after MI.Part ?GABAAergic drugs influenced the balance of myocardial injury and repair in mice after myocardial infarction by regulating the activity of macrophagesObjectives:To investigate the influence of GABAAergic drugs on the balance of myocardial injury and repair in mice after myocardial infarction(MI)by regulating the activity of macrophagesMethods:M1 or Ly6Chigh macrophage-producing or-related cytokines in the infarcted region including TNF-?,IL-6 and matrix metalloproteinase(MMP)-9 were examined by qRT-PCR,western blotting and gelatin zymography on days 1,3,7,14 post-MI,so were M2 or Ly6Clow macrophage-producing or-related cytokines including IL-10,vascular endothelial growth factor(VEGF)and TGF-?1.Levels of neovascularization and myofibroblast differentiation were detected by immunofluorescence co-staining on days 7 and 14 post-MI.Collagen I deposition in infarct area was examined by immunohistochemistry and picrosirius red staining on days 7 and 14 post-MI.Results:Compared with controls,Ml or Ly6Chigh macrophage-producing or-related cytokines including TNF-?,IL-6,MMP-9 decreased,but M2 or Ly6Clow macrophage-producing or-related cytokines including IL-10,VEGF and TGF-?1 increased in topiramate-treated MI mice by qRT-PCR,westrern blotting and gelatin zymography on 1,3,7,14 days post-MI.While bicucullie-treated MI mice showed reversed results.Immunofluorescence staining showed that compared with controls,levels of neovascularization and the myofibroblast differentiation increased in topiramate-treated MI mice on days 7,14 post-MI.While bicucullie-treated MI mice showed reversed results.Immunohistochemistry and picrosirius red staining showed that compared with controls,collagen I deposition in infarct area increased in topiramate-treated MI mice on 7,14 days post-MI.,while bicucullie-treated MI mice showed reversed results.Conclusion:Topiramate down-regulated the expression of M1 or Ly6Chigh macrophage-producing or-related cytokines including TNF-a,IL-6 and MMP-9,whereas up-regulated the expression of M2 or Ly6Clow macrophage-producing or-related cytokines including IL-10,VEGF and TGF-?1,which consequently contributed to less pro-inflammation,destroying of extracellular matrix,more neovascularization,myofibroblast differentiation and increasing collagen I deposition in infarct area.Ultimately,topiramate could relieve the injury in myocardium,and facilitate myocardial scar healing. |
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