| Acute myocardial infarction(AMI)is a kind of heart disease with acute occlusion of coronary artery and complete obstruction of blood flow,which leads to continuous myocardial ischemia and hypoxia and necrosis.For AMI patients,timely reperfusion therapy can not prevent myocardial fibrosis and ventricular remodeling in some patients.After myocardial infarction,local hypoxia leads to myocardial cell necrosis and apoptosis,and then immune cells are recruited to remove necrotic cells and tissue debris,produce inflammatory response,and then start reparative fibrosis.However,on the other hand,over activation or over inhibition of immune inflammatory response can also lead to excessive myocardial damage or fibrosis,resulting in adverse ventricular remodeling,It causes heart failure.After myocardial ischemia and necrosis,multiple inflammatory pathways are activated,and the immune system activation plays an important role in the process of myocardial fibrosis after myocardial infarction.Previous studies have shown that innate immune system such as neutrophils,monocytes macrophages and T lymphocytes in adaptive immunity play an important role in the process of persistent inflammation and fibrosis.B lymphocytes,or B cells for short,are important adaptive immune cells that activate T cells by secreting antibodies,cytokines and presenting antigens.Studies have suggested that B cells are closely related to ventricular remodeling in cardiomyopathy.After depletion of B cells with anti-CD22 antibody,collagen deposition in local myocardial interstitium and cardiomyocyte apoptosis were significantly reduced.Some studies have also found that a large number of B cells can be seen in the local ischemic myocardial tissue in the mouse model of AMI,which aggravates the myocardial injury and leads to poor ventricular remodeling.These results suggest that B cells may be involved in myocardial fibrosis.However,what is the role and mechanism of B cells in myocardial fibrosis after AMI?So far,there is no relevant report.Therefore,in order to explore the role and mechanism of B cells in myocardial fibrosis after acute myocardial infarction and provide a new target for the prevention and treatment of left ventricular remodeling after AMI.The research contents include:(1)the effect of B cells on myocardial fibrosis after AMI in mice;(2)effect of B cells on collagen metabolism of fibroblasts in vitro;(3)changes and clinical significance of peripheral blood B cells and their subsets in patients with acute myocardial infarction.Part 1 Effect of B Cells on Myocardial Fibrosis in Mice with Acute Myocardial InfarctionStudy One The changes of B cells and related cytokines in different stages of AMI in a mouseObjective: To observe the changes of myocardial pathology,B cells and cytokines in different stages of AMI in mice.Methods: 88 male C57BL/6 mice aged 12 weeks were randomly divided into Sham operation group(n=24)and acute myocardial infarction group AMI group(n= 64).The two groups were divided into 4 subgroups: 3-day,5-day,1-week and 2-week.There were 6 mice in each subgroup of sham group and 16 mice in each subgroup of AMI group.In AMI group,the left anterior descending branch(LAD)was ligated permanently according to conventional thoracotomy to make AMI mouse model.In Sham group,the heart of the mice was opened routinely,and the thread was threaded under LAD without ligation of blood vessels.The peripheral blood,spleen and heart were collected after the mice were killed at corresponding time points.The pathological changes of myocardium were observed by hematoxylin & eosin(HE)staining,and the degree of myocardial fibrosis was detected by Masson staining.The expressions of TNF-α,IL-1β,IL-6,TGF-β1 mRNA were detected by RT-PCR.The concentrations of TNF-α,IL-1β,IL-6,TGF-β1 in peripheral blood were detected by ELISA.Results:(1)The infiltration of inflammatory cells was the most obvious at the 5-day,and a few inflammatory cells and obvious fibrosis were observed at the 2-week.(2)In myocardial tissue,the proportion of activated B cells in AMI group were significantly higher than that in Sham group at 3-day,5-day and 1-week(P<0.05),but there was no significant difference between AMI group and Sham group at 2-week(P>0.05),and it was the highest at 5-day(P<0.05).In spleen and peripheral blood,the proportion of activated B cells in AMI group was significantly higher than that in sham group at 3-day and 5-day(P<0.05),and the highest at 5-day(P<0.05),and there was no significant difference between AMI group and sham group at 1-week and 2-week(P>0.05).(3)In myocardial tissue,the mRNA levels of TNF-α,IL-1β,IL-6,TGF-β1 in AMI group were significantly higher than those in sham group at 3-day,5-day,1-week and 2-week(P<0.05),3-day and 5-day were the highest(P<0.05).Although 5-day was higher than 3-day,there was no statistical significance(P>0.05).(4)In peripheral blood,the concentrations of TNF-α,IL-1β,IL-6 in AMI group were significantly higher than those in Sham group at 3-day,5-day,1-week and 2-week(P<0.05).Among the 4 time points,the highest values were found at 3-day and 5-day(P<0.05).The level of TGF-β1 was significantly higher than that in Sham group at four time points,with the highest level in 5-day group(P<0.05),and higher than that in 3-day group at 1-week(P<0.05).Conclusion: The models of C57BL/6 mice with AMI were established successfully.On the 5th day of AMI,the infiltration of inflammatory cells in myocardium were the most obvious.The ratio of activated B cells were the highest in myocardium,spleen and peripheral blood.The mRNA expression and blood concentration of TNF-α,IL-1β,IL-6 and TGF-β1 were also the highest.It is suggested that B cells were activated in acute stage of AMI,and the cytokines secreted by B cells were increased significantly,reaching the peak on the 5th day.Study Two The effect of B cells on myocardial collagen metabolism after acute myocardial infarction(AMI)in mice and its mechanismObjective: To investigate the effect of B cells on myocardial collagen metabolism after acute myocardial infarction(AMI)in mice and its possible mechanism.Methods: Wild type C57BL/6 mice were divided into sham group(n=12)and AMI group(n=18),and BKO group(n=18).The mRNA expression levels of TNF-α,IL-1β,IL-6 and TGF-β1 in myocardium were detected by RT-PCR.The serum levels of TNF-α,IL-1β,IL-6 and TGF-β1 were measured by ELISA.The left ventricular size and EF value were evaluated by transthoracic echocardiography(TTE)in 2-week subgroups,and then the mice were killed to take the myocardial tissue.The myocardial pathological changes were observed by HE staining,and the degree of myocardial fibrosis was detected by Masson staining.RT-PCR was used to detect the mRNA expression levels of type I collagen(COL1-A1),type III collagen(COL3-A1),tissue inhibitor of matrix metalloproteinase-1(TIMP-1)and matrix metalloproteinase-9(MMP9).Result:(1)On the 5th day of AMI,the mRNA levels of TNF-α,IL-1β,IL-6 and TGF-β1 in myocardium of BKO group were significantly lower than those of WT group(P < 0.05)and higher than those of Sham group(P < 0.05);the concentrations of TNF-α,IL-1β,IL-6 and TGF-β1 in peripheral blood of BKO group were significantly lower than those of WT group(P<0.05)and higher than those of Sham group(P<0.05).(2)At 2 weeks after AMI,the mRNA levels of COL1-A1,COL3-A1,MMP9 and TIMP in BKO group were significantly lower than those in WT group(P<0.05),but higher than those in Sham group(P <0.05).(3)At 2 weeks after AMI,the left ventricular diastolic and end systolic diameters in BKO group were significantly smaller than those in WT group,and LVEF was significantly higher than that in WT group(P<0.05).Conclusion:In AMI mice,B cell deletion could reduce myocardial collagen synthesis and myocardial fibrosis,reduce left ventricular remodeling and protect left ventricular function.This may be related to the down-regulation of myocardial mRNA expression and blood concentration of TNF-α,IL-1β,IL-6 and TGF-β1.Part 1 conclusion: B cells were activated in acute stage of AMI,and the cytokines secreted by B cells were increased significantly,reaching the peak on the 5th day.In AMI mice,myocardial B cells may promote collagen synthesis and myocardial fibrosis by secreting TNF-α,IL-1β,IL-6 and TGF-β1,thus aggravating left ventricular remodeling and impairing left ventricular function.Part 2 Effect of B Cells on Collagen Metabolism of Fibroblasts in VitroObjective: To observe the level of cytokines secreted by B cells of AMI mice in vitro and to verify the effect of B cells of AMI mice on fibroblasts in vitro.Methods: Wild type C57BL/6 mice were used to make AMI model(AMI group)and Sham operation group(Sham group)as control group.The mice were killed on the 5th day of AMI.The spleen was aseptically taken to make single cell suspension of spleen.B cells were sorted according to mouse CD19 microbads magnetic beads,and the purity of B cells was detected by flow cytometry.After 2 days of stimulation with LPS(10μg/ml)and anti-CD40(2.5μg/ml),B cells and their supernatants were collected.The TNF-α,IL-6 and TGF-β1 mRNA expression levels of B cells were detected by RT-PCR.The concentrations of TNF-α,IL-6 and TGF-β1 in the supernatant of B cells were determined by ELISA.The supernatant of B cell culture in AMI group and Sham group was collected.Ten C57BL/6 suckling mice(male or female)of 5-day old were selected.The hearts were taken out aseptically,cut into pieces,digested into single cell suspension by several times.The primary cells of cardiac fibroblasts were collected by differential adherent method,cultured and passaged.After identification,the second-fourth generation cells were used for follow-up experiments.The obtained fibroblasts(FB)were divided into three groups,Fibroblast group(FB group),Fibroblasts+supernatant of Sham group B cells culture group(FB+Sham group)and Fibroblast+AMI group B cell supernatant culture group(FB+AMI group).After 48 hours,the fibroblasts and supernatant were collected.The mRNA expression levels of COL1-A1,COL3-A1 and Ki67 were detected by RT-PCR.The protein levels of COL1-A1 and COL3-A1 in supernatant were detected by ELISA.Results:(1)The mRNA expression levels of TNF-α,IL-6 and TGF-β1 in B cells of AMI group were significantly higher than those of Sham group(P<0.05);(2)The concentrations of TNF-α,IL-6 and TGF-β1 in B cell culture supernatant of AMI group were higher than those of Sham group(P<0.05);(3)The mRNA expression levels of COL1-A1,COL3-A1 and Ki67 in FB + AMI group were significantly higher than those in FB + Sham group and FB group(P<0.05)(4)the protein levels of COL1-A1 and COL3-A1 in FB+AMI group were significantly higher than those in FB+Sham group and FB group(P<0.05).Part two conclusion: In vitro,B cells of AMI mice could promote collagen synthesis of cardiac fibroblasts by secreting TNF-α,IL-6 and TGF-β1.Part 3 Changes and Clinical Significance of B Cells and Their Subsets in Patients with Acute Myocardial InfarctionObjective:To investigate the changes and clinical significance of B cells and their subsets in patients with AMI.Methods: Fifty nine patients with acute anterior myocardial infarction(AMI group),who underwent emergency PCI in the Department of Cardiology of our hospital,32 patients with unstable angina pectoris(UA group)and 20 healthy controls(CON group)were selected.B cells and their subsets: naive B cells,preswitch memory B cells and post-switch memory B cells in peripheral blood were detected by flow cytometry.The serum levels of TNF–α,IL-6,TGF-β1 and BAFF were measured by ELISA.The clinical test results of hs CRP,hs Tn T and NTpro-BNP were collected.Left ventricular end diastolic volume(LVEDV)and left ventricular ejection fraction(LVEF)were collected at admission and 6 months after AMI.According to the criteria,AMI patients were divided into LVR group(n=15)and non LVR group(n=44).The differences of B cells and their subsets at admission were compared between the two groups.Results:(1)The levels of B cells(25.36±6.13 vs 14.20 ±3.21 vs13.47±3.36)×104/ml,naive B cells(17.88±4.36 vs 10.15±2.35 vs9.73±2.46)×104/ml,pre-switch memory B cells(1.07 ±0.31 vs 0.63 ±0.17 vs 0.60 ±0.20)×104/ml and post-switch memory B cells(1.07±0.31 vs 0.63±0.17 vs0.60±0.20)×104/ml in AMI group were significantly higher than those in UA group and CON group,(P<0.001)(2)The levels of serum hs CRP,TNF-α,IL-6,TGF-β1 and BAFF in LVR group were significantly higher than those in UA group and CON group at admission(P<0.05);(3)The levels of B cells and their subsets in LVR group were significantly higher than those in non LVR group at admission(31.23±2.80 vs 23.36±5.66)×104/ml,naive B cells were significantly higher than those in non LVR group(21.97±2.03vs16.48± 4.06)×104/ml,pre-memory B cells(1.33±0.19vs0.98±0.29)×104/ml,post-memory B cells(4.10±0.68vs3.14±0.82)×104/ml(P<0.001);Conclusion: The proportion of blood B cells and their subsets in patients with AMI were significantly increased.B cells were associated with left ventricular remodeling 6 months after AMI.The results of this study suggest that:(1)B cells were activated in the acute phase of AMI,and the cytokines secreted by B cells increased significantly,reaching the peak on the 5th day.In AMI mice,myocardial B cells may promote collagen synthesis and myocardial fibrosis by secreting TNF-α,IL-1β,IL-6 and TGF-β1,thus aggravating left ventricular remodeling and impairing left ventricular function.(2)In vitro,B cells of AMI mice could promote collagen synthesis of cardiac fibroblasts by secreting TNF-α,IL-6 and TGF-β1.(3)The proportion of blood B cells and their subsets in patients with AMI increased significantly,indicating that B cells were related to left ventricular remodeling 6 months after AMI. |
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