| BackgroundViral myocarditis is lymphocytic myocarditis caused by viral infection.Current treatment methods are not satisfactory.As an innate anti-inflammatory and immunomodulator,IL-37 played an important role in immune diseases such as Systemic Lupus Erythematosus(SLE),Ankylosing Spondylitis(AS)and Rheumatoid Arthritis(RA).Studies have also confirmed that IL-37 played an important role in the occurrence and development of cardiovascular diseases such as atherosclerosis(AS),Acute Coronary Syndrome(ACS)and Heart Failure(HF).IL-37 can reduce inflammatory cells infiltration,regulate immune imbalance,and inhibit cell apoptosis through the p38 Mitogen-actived Protein Kinase(p38 MAPK)signal pathway.The pathogenesis of viral myocarditis is related to cardiac lymphocyte infiltration and cardiomyocyte apoptosis.Whether IL-37 can inhibit the apoptosis of viral myocarditis by inhibiting the p38 MAPK pathway in myocarditis has not been demonstrated.Objective1.Acute Viral Myocarditis(VMC)mice models were established by intraperitoneal injection of Coxsackie virus group B type 3(CVB3).Recombinant IL-37 was used to intervene VMC mice.The pathological damage of myocardial tissue in mice induced by CVB3 and IL-37 were observed;2.The regulatory effects of IL-37 on inflammatory cells and myocardial function in acute viral myocarditis were studied through the weight,mortality,general manifestation,serum inflammatory cytokine expression,myocardial histopathological examination,cardiac ultrasound myocardial function test,molecular biology and immunopathological expression of apoptosis related proteins of mice in each group;3.Objective to investigate the expression of p38 MAPK pathway in acute VMC and the effects of IL-37 on the expression of p38 MAPK pathway.Methods1.Model construction and experimental groupingForty normal 4-6 weeks old BALB/c mice were randomly divided into four groups,each group contained 10 mice,namely the control group(normal mice.Control),the drug-only group(normal mice+IL-37,Control+IL-37),the experimental group(virus mice,Virus),and drug-treatment group(virus mice+IL-37,Virus+IL-37).① The Virus group were intraperitoneal injection of 25μl CVB3(108TCID50)diluted with 100μl PBS and 200μl normal saline;②The mice in the Control group were intraperitoneally injected with 100μl PBS and 200μl normal saline on day 0;③ The mice in the Control+IL-37 group were intraperitoneally injected with 100μl PBS and 200μl normal saline on day 0,100μl PBS and IL-37(2μg)dissolved in 200μl normal saline on days 5.8,and 11;④The mice in the Virus+IL-37 group were intraperitoneally injected with 25μl CVB3(108TCID50)diluted with 100μl PBS and 200μl normal saline on day0,IL-37(2μg)dissolved in 200μl normal saline and 100μl PBS on day 5,8,and 11;2.General manifestations,weight change and survival curve of miceWe observed the changes in the weight,death,and general manifestions of each group of mice,such as mobility and hair color.We eatablished the survival curve and body weight curve.3.Cardiac ultrasoundOn the 14th day,all mice were subjected to cardiac ultrasound to evaluate the cardiac function of mice in each group.The cardiac hemodynamic parameters of four groups of mice were measured by small animal high resolution ultrasound imaging system(Vevo 2100,Fuji Visual Sonics).Parasternal Short-axis View(PSAX),parasternal Long-axis View(PLAX)and M-mode ultrasound images were collected from long axis and short axis views of sternum to determine left ventricular activity and function.IVS-s,IVS-d,LVID-s,LVID-d,LVPW-s,LVPW-d and LVEF were measured by 12 MHz probe.The data above were collected to evaluate the cardiac function of mice.After anesthesia,the mice were sacrificed to extract cardiac tissue.4.Pathological examination of myocardiumAfter 14 days of model construction,pentobarbital sodium(concentration:2%,dosage:70mg/kg)was injected intraperitoneally to anesthetize the mice,opened the chest,taked the mouse myocardial tissue,photoed.and taked the general specimen picture.Blood was collected from the apex of the heart and fixed in paraformaldehyde overnight,then embedded in dehydrated paraffin.Paraffin sections with 5 μm thickness were prepared for pathological examination.Hematoxylin&Eosin(HE)staining was used to observe the myocardial inflammatory cell infiltration,Masson Trichrome staining was used to observe myocardial fibrosis and quantitative methods were evaluated.5.ELISAELISA methods were used to detect serum TNF-α,IL-1,and C-TnI expression of each group to evaluated the inflammatory cytokines and myocardial necrosis markers each group.6.Western BlotThe total protein of myocardial tissue were extracted,Western Blot measured the expression of apoptosis proteins and p38 MAPK pathway related proteins.7.Immunohistochemical staining(IHC)Immunohistochemical staining was used to determine the expression of specific lymphocytes and apoptosis-related proteins.Optical microscope were used to observe,photograph.Quantitative analysis were used to analysis.8.TUNELThe TUNEL methods were used to determine the apoptosis of cardiomyocytes of each group.Optical microscope was used to observe,photograph.Quantitative analysis were used to analysis.Results1.General manifestations,weight change of each groupWe observed that the mice in the Control group mice had a good diet,active and lively,thick hair,with bright hair color,soft and smooth.when we grasped the mice,they performed obvious struggle actions and when we gived stimulation,they performed obvious resistance;Compared to the normal mice,the manifestations of viral myocarditis in the Virus group were obvious,including reduced diet,reduced activity,sparse hair,with dark hair color,rough touch.when we grasped the mice,they performed not obvious struggle actions and when we gived stimulation,they performed not obvious resistance.However,mice treated with recombinant IL-37 showed significant improvement.From the first day of model construction,we weighed and recorded the body weight of mice every day.The results showed that:the weight of mice in the Control group and the Control+IL-37 group had been increasing.From the day1 to the day4,compared to the normal mice,the weight of mice in the Virus group decreased significantly(p<0.05),and the rate of weight loss in the Virus+IL-37 group also decreased significantly(p<0.05).From the day 4,compared with the normal mice,the weight loss in Virus group was more significant(day6(p<0.01),day8(p<0.01),day10(p<0.001),day12(p<0.001),day14(p<0.001)).The weight loss in the Virus+IL-37 group was significantly reduced(day6(p<0.05),day8(p<0.05),day 10(p<0.01).day 12(p<0.01),day 14(p<0.01).2.Survival curve analysisNo mice died in the Control group and Control+IL-37 group(survival rate:100%),The survival rate was 60%in the Virus group and 90%in the Virus+IL-37 group.Compared with the normal mice,the survival rate of the Virus group was significantly lower(p=0.0208);Compared with the Virus group,the survival rate of the Virus+IL-37 group was also significantly statistical different(p=0.0238).3.IL-37 attenuated myocardial injury and cardiac remodeling in viral myocarditisOn the 14th day,the hearts of the Control group and the Control+IL-37 group were ruddy,without edema,smooth myocardial surface,and no necrotic tissue deposition.Compared with the normal mice,the hearts of mice in Virus group showed obvious pale edema,large white necrotic tissue on the surface of myocardium,the blood vessels on the surface of myocardium could not be observed clearly.Compared with the Virus group,the myocardiums of the Virus+IL-37 group were slightly white,the edema of the myocardial tissue was significantly reduced,and the areas of white necrotic tissue on the surface of the heart were significantly reduced.In the HE staining,the structures of myocardial fibers in the Control group and the Control+IL-37group were clear,regular,normal in size,and no abnormal cells infiltration were found in the myocardial interstitium.Compared with the normal mice,the structures of myocardial fibers in the Virus group were disordered,the myocardial cells were edema,abnormal in shape and size,some myocardial cells were dissolved and necrotic,and a large number of inflammatory cells were infiltrated in the myocardial interstitiums.In the Virus+IL-37 group,the disorder of myocardial fibers structures were significantly improved,the myocardial cells were still edematous,abnormal in shape and size,and inflammatory cells infiltration were still seen in the myocardial interstitium,but they were lighter than that in the Virus group.Compared with the normal mice,the average pathological scores of myocarditis in the Virus group were significantly higher(p<0.001),and the average pathological scores of myocarditis in the Virus+IL-37 group were significantly lower than that in the Virus group(p<0.01).Masson staining showed that there were no obvious collagen fibers deposition in the Control group and Control+IL-37 group.Compared with the normal mice,there were obvious collagen fibers deposition in the myocardial interstitiums of the Virus group(p<0.001).Compared with the Virus group,the deposition of collagen fibers and the areas of myocardial fibrosis in the Virus+IL-37 group were significantly reduced(p<0.01).We also observed a phenomenon worthy exploring,the epicardium of mice in Control+IL-37 group showed inflammatory cells infiltration and myocardial fiber necrosis.Mice in the Virus+IL-37 group also showed the same phenomenon.The epicardiums of the control group was normal.4.IL-37 improved myocardial function in mice with viral myocarditisCompared with the normal mice,the heterogenous and diffuse hyperechoic shadow of cardiac motion were observed in the Parasternal Long-axis view(PLAX)and Parasternal Short-axis view(PSAX)of mice in the Virus group.Meanwhile,compared with the normal mice,the M-mode ultrasound images showed that the ventricles of mice in the Virus group were significantly enlarged and the ability of ventricular wall motion was significantly weakened.In the Virus+IL-37 group,there were still heterogeneous hyperechoic shadows in PLAX and PSAX,but the areas were significantly reduced,the ventricles were still enlarged,and the ventricular wall activity was still weakened,but it was significantly reduced compared with the Virus group.In addition,compared with the normal mice,the LVEF(p<0.05)was significantly decreased,IVS-s(p<0.05),IVS-d(p<0.05)were significantly thinner,LVID-s(p<0.05),LVID-d(p<0.05)were significantly reduced,LVPW-s(p<0.01),LVPW-d(p<0.01)were significantly decreased.Compared with the Virus group,the LVEF(p<0.05)of Virus+IL-37 treatment group was significantly increased,IVS-s(p<0.05),IVS-d(p<0.05)were significantly thickened,LVID-s(p<0.05),LVID-d(p<0.05)and 1VPW-s(p<0.05)were significantly increased,1VPW-d(p<0.05)was significantly increased.5.IL-37 intervention can reduce serum inflammatory cytokines and myocardial injury markers in viral myocarditisTNF-α,C-TNI,IL-1 were closely related to the occurrence and development of viral myocarditis.TNF-α and IL-1 were two important factors of inflammation and C-TNI was an important index of myocardial necrosis.Compared with Control group,the expression of TNF-α(p<0.01),C-TnI(p<0.05),IL-1(p<0.001)in Virus group were significantly increased.Compared with the Virus group,the Virus+IL-37 group reduced the expression of TNF-α(p<0.05),C-TnI(p<0.05),IL-1(p<0.05).6.IL-37 reduced lymphocytes and macrophages infiltration in viral myocarditisThe expression of CD3+ T cells,CD4+T cells,CD8+T cells and CD68+macrophages in four groups of mice were detected by immunohistochemistry to assess the inflammatory infiltration.In the Control group,the myocardial fibers were dense and evenly arranged,the size and staining of myocardial cells were normal,and there were not obvious lymphocytic cells infiltration in the myocardial interstitiums.Compared with the normal mice,the myocardial fibers of the Viral group were swollen and uneven in size.A large number of CD3+T cells,CD4+T cells,CD8+T cells and CD68+macrophages were seen in the myocardial interstitiums.The positive staining areas increased significantly under the optical microscope.The inflammatory cells infiltration were also seen in the Virus+IL-37 group,but positive staining areas were]ess than that in the Virus group.Compared with the normal mice,there were significant differences in CD3+T cells(p<0.001),CD4+T cells(p<0.001),CD8+T cells(p<0.001)and CD68+macrophages(p<0.001)in the Virus group.Compared with the Virus group,there were significant differences in myocardial CD3+T cells(p<0.01),CD4+T cells(p<0.01),CD8+T cells(p<0.05)and CD68+macrophages(p<0.01)in the Virus+IL-37 group.7.IL-37 inhibited cardiomyocyte apoptosis in viral myocarditisIHC method was used to label apoptosis related proteins to evaluate the myocardial apoptosis of viral myocarditis.Compared with the normal mice,myocardial fibrosis and necrosis and a large number of stained brown abnormal nucleis could be seen in the Virus group.The degree of myocardial necrosis in the Virus+IL-37 group was lighter than that in the Virus group.Compared with the normal mice,the IHC staining positive rates of apoptosis related proteins such as Cleved-caspase3(p<0.001),Caspase3(p<0.01),Cleved-caspase8(p<0.01),Caspase8(p<0.01)and Bax(p<0.0001)in the Virus group were increased.Compared with the Virus group,the IHC positive rates of Cleaved-caspase3(p<0.05),Cleaved-caspase,(p<0.05),Cleaved-caspase8(p<0.05),Caspase8(p<0.05)and Bax(p<0.01)protein in the Virus+IL-37 group were lower.On the contrary,compared with the normal mice,the expression of Bcl-2 in Virus group decreased(p<0.01),but increased in Virus+IL-37 group(p<0.05).Western blot was used to evaluate the expression of apoptosis related proteins.Compared with the normal mice,the expression of proteins in the Virus group were significantly higher,and the expression of Cleved-caspase3(p<0.05),Caspase-3(p<0.01),Cleved-caspase8(p<0.01),Caspase-8(p<0.01),and Bax(p<0.01)were significantly higher in the Virus group;Compared with the Virus group,the expression of Cleved-caspase3(p<0.05),Caspase-3(p<0.05),Cleved-caspase8(p<0.05),Caspase8(p<0.05),Bax(p<0.05)were significantly reduced in Virus+IL-37 group.In addition,the ratio of Cleved-caspase 3/Caspase-3(p<0.05),and the ratio of Cleved-caspase8/Caspase8(p<0.05)in the Virus+IL-37 group was lower than that of Virus group.The expression of Bcl-2(p<0.05)and the ratio of Bcl-2/Bax(p<0.001)in the Virus group were lower than that in the Control group,while the relative expression of Bcl-2(p<0.05)and Bcl-2/Bax(p<0.05)increased in the Virus+IL-37 group.We used the TUNEL method to detect the myocardial apoptotic cells.Compared with the normal mice,a large number of cells in the Virus group had nuclear membrane rupture,decomposition,deep staining,aging and shrinkage.TUNEL positive staining and nuclear morphological changes of myocardial cells in Virus+IL-37 were less than those in Virus group.The results of statistical analysis were as follows:Compared with the normal mice,the mumbers of myocardial apoptotic cells in the Virus group were increased obviously(p<0.001),compared with the Virus group,the numbers of myocardial apoptotic cells in the Virus+IL-37 was significantly decreased(p<0.01).8.IL-37 may alleviate cardiomyocyte apoptosis in viral myocarditis by inhibiting p38 MAPK signal pathwayWestern blot was used to detect the expression of p38 MAPK and p-p38 MAPK protein in cardiomyocytes of four groups.There was no obvious difference of the total p38 MAPK protein expression in each group.Compared with the normal mice,the expression of p-p38MAPK protein in the Virus group was obviously increased(p<0.05).On the contrary,compared to the Virus group,the expression of p-p38MAPK protein in the Virus+IL-37 group was significantly decreased(p<0.05).In addition,compared with the normal mice,the ratio of p-p38 MAPK/p38 MAPK(p<0.01)in the Virus group was increased,while compared with the Virus group,the ratio of p-p38 MAPK/p38 MAPK(p<0.05)in the Virus+IL-37 group was decreased.Conclusion1.IL-37 can reduce the myocardial pathological injury in acute VMC mice;2.IL-37 can reduce the mortality,improve cardiac function,reduce the expression of inflammatory factors,the infiltration of inflammatory cells,and the expression of apoptosis related proteins.3.The expression of p38 MAPK signal pathway in acute VMC myocardium was increased,and IL-37 could reduce the expression of p38 MAPK signal pathway. |
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