| Background:Previous study showed that sustained damage to myocardial tissue by virus infection may be the important reason in virus myocarditis(VMC).The mainly mechanism of VMC are myocardial cell degeneration and necrosis,cardiac fibers and perivascular connective tissues inflammatory cell infiltration,and interstitial fibrosis leads to the occurrence of molecular events such as myocardial remodeling and eventually leads to left ventricular dilatation and cardiac dysfunction,and then a continuous pathological process of virus myocarditis-dilated cardiomyopathy-heart failure formed.As a whole pathological process,the mechanism of myocardial death in the initial stage has not been fully elucidated,and the necrosis of myocardial cells is the core power to promote the progress of the pathological process.Therefore,it is of great important to explore the exact mechanism of myocardial cell necrosis and to find a new target to prevent myocardial cell injury effectively.Necroptosis can be regulated by activation of the death receptor(DRs)and ligand and the activation of RIPs proteins.RIPs,as an important signaling molecule,initiates and regulates cell stress responses,involves in and decides the fate of cells toward to apoptosis,necrosis,or survival.Two RIPs proteins,namely RIP1/RIP3,play a key role in the regulation of programmed necrosis pathways.Necroptosis has the typical necrosis feature that like morphological changes,which can cause significant inflammatory reaction,a large number of inflammatory cells are infiltrating and activating and are accompanied by autophagy during cell necrosis,the process can be inhibited by Necrostatin-1(Nec-1)but specifically without being blocked by zVAD-fin and 3-MA,the specific inhibitors of apoptosis and autophagy.As a new form of necroptosis,it plays an important role in the pathological process of many diseases,especially acute and severe diseases,such as acute inflammation,ischemic injury and virus infection.The necrosis of myocardial cells are the primary pathological manifestations of virus myocarditis.Whether a myocardial cell live or die is very importment in the progress of VMC,but the research of the relationship between necroptosis and myocardial injury of virus myocarditis is still in the blank stage at home and abroad,therefore,this paper aims to establish an animal model of acute severe virus myocarditis induced by coxsackievirus B3,molecular biology and Immunology techniques are used to investigate the role of RIP1/RIP3 necrosis mediating myocardial cell programmed necrosis in acute severe virus myocarditis.The main modes and mechanisms of myocardial cell injury in virus myocarditis are discussed from a new perspective,so as to provide a new theory evidence and therapeutic target for its prevention and treatment.Part 1 Objective An acute severe virus myocarditis model was constructed by CVB3 infection in BALB/c mice.Methods according to the method of GayR.T.et al,we determined that the inoculation amount of virus was 1000 TCID50 0.1ml in the formal experiment,4 week old male BALB/c mice were taken,the model group received intraperitoneal sterile inoculation with 0.1ml 1000 TCID50 dose of CVB3 to make the acute sever.The mice were treated with intraperitoneal injection for 7 consecutive days,and the infectious TCID50 per 1g was calculated by Karder method.Results The survival rate of mice in normal group was 100%,and the physiological indexes were normal.CVB3 infected virus myocarditis group:death began to appear on the 4th day,the mortality rate was 50%from the 4th day to 7th day,consistent with the survival rate performance of acute severe virus myocarditis;on the 7th day of the virus infection,the levels of serum CK,CK-MB and cTnI in CVB3 group mice were significantly higher than those in normal group mice(P<0.01),which indicated that the myocardial cells were seriously damaged;light microscopy results showed that myocardial cells necrosis,disintegrate,nuclei and cell contours disappear were occurred in CVB3 infected mice,and a large number of inflammatory cells infiltrating around;electronic microscope results showed that mitochondria swelling and many vacuoles forming in CVB3 infection group.Conclusion We successfully constructed an acute severe virus myocarditis model in CVB3 infected BALB/c mice,with rapidly progression of illness,high mortality,and heavy inflammatory reactions.Therefore,the scope of this study belonged to acute severe virus myocarditis.Part 2 Objective To explore the expression and significant role of RIP 1/RIP3 in CVB3-induced acute severe virus myocarditis in mice.Methods 60 male BALB/c mice were divided into normal control group(10),CVB3 infection group(20),drug treatment group(20),and blank control group(10).Normal control group mice were injected with 0.9%saline intraperitoneally,0.1ml/mice;mice in CVB3 infection group were given 0.1%DMSO at different doses according to different doses of the drug except for intraperitoneal injection of CVB3 0.1 ml per mice.In drug treatment group,each mice was given an intraperitoneal injection of CVB3 0.1ml,after 4 hours,different drugs were injected into the caudal vein(Z-VAD-fmk,lmg/kg;3-MA,15mg/kg;Nec-1 0.6mg/kg;dissolve the drug with 0.1%DMSO).Mice in blank control group received no treatment.The mice above were treated with intraperitoneal injection for 7 consecutive days.The expression of necroptosis protein RIP1/RIP3,MLKL、PGAM5L,apoptosis protein caspase-3,and autophagy related protein light chain 3(LC3-II)were detected by Western blot and immunohistochemistry.Results Compared with the normal control group,Western blot results showed that the expression of RIP1/RIP3、MLKL、PGAM5L in myocardial tissue of CVB3 group was significantly increased(P<0.01),when used Nec-1,the expression of MLKL and PGAM5L in myocardial tissue of CVB3 group was significantly decreased(P<0.01);In CVB3 infection group,the expression of LC3-II,caspase-3 and RIP1/RIP3 in the myocardium were increased in the early(the 4th day)(P<0.05).However,the 7th day after infection,only LC3-II and RIP1/RIP3 was significantly increased(P<0.01);the expression of LC3-II protein was significantly decreased after use Nec-1(P<0.01);Immunohistochemistry results showed that the expression of RIP1/RIP3 in myocardial cells of CVB3 infected group was significantly increased and presented as diffuse distribution.Conclusion The necroptosis mediated by RIP1/RIP3 plays an important role in CVB3-induced acute severe virus myocarditis in mice.Its downstream signaling may be achieved via RIP1-RIP3-MLKL-PGAM5L pathway.Nec-1 leads to a decrease in the expression of LC3-II,indicating that myocardial autophagy may be the necroptosis downstream reaction mediated by RIP1/RIP3,the formation of autophagic tissue in VMC myocardium may be caused by necroptosis,necroptosis is the major mode of programmed cell death.Part 3 Objective To explore the protective effect of Nec-1 on CVB3-induced myocardial injury in severe acute virus myocarditis.Methods 60 male BALB/c mice of 4 weeks old were divided into normal control group(10),CVB3 infection group(20),CVB3 + Nec-1 group(20)and blank control group(10).Model group preparation:Intraperitoneal aseptic inoculation of 0.1ml 1000 TCID50 dose CVB3 to make acute severe virus myocarditis animal model.The mice in the normal control group were injected intraperitoneally with 0.9%saline,0.1ml/mice.The mice in CVB3 infection group,in addition to intraperitoneal injection of CVB3 0.1ml,and then were injected an equal dose of 0.1%DMSO according to different pairings.The mice in CVB3+ Nec-1 group were treated with intraperitoneal injection of CVB3,0.1ml,and 4 hours later,and then received drug injections at the caudal vein(Nec-l 0.6mg/kg,the above drugs were dissolved in 0.1%DMSO).Mice in blank control group received no treatment.The mice above were treated with intraperitoneal injection for 7 consecutive days.The weight loss rate,survival rate,heart index,serum CK.CK-MB and cTnI levels and HE staining in heart tissue were measured on the 7th day.The pathological changes of myocardial tissue were observed by electron microscope.The cardiac function of the left ventricle was measured by echocardiography to evaluate the induction extent of virus myocarditis.Results Compared with the CVB3 infection group,the survival rate of the CVB3+Nec-1 group was increased from the 5th day,reached to 90%and remained stable;the body weight index rose from the second day and remained stable.The CK,CK-MB and cTnI of CVB3+Nec-1 group was significantly decreased(P<0.01);CVB3+Nec-1 group could significantly reduce the levels of RIP1 and RIP3 in virus myocarditis group(P<0.01).Immunohistochemistry results showed that the CVB3+Nec-1 group could significantly reduce the positive staining of RIP1 and RIP3 in the virus myocarditis group.The histopathological results showed that CVB3+Nec-1 group could obviously improve the myocardial tissue lesion degree in CVB3 infected mice,and had significant myocardial protection effect.Electron microscopy results showed that the myocardial lesions in the CVB3+Nec-1 group were alleviated,the myofibrils were arranged neatly,no obvious lytic necrosis,Z line was clearer,mitochondria swelling and vacuoles were decreased.The result of echocardiography showed that the cardiac EF value and LVDESD were improved significantly after Nec-1(P<0.01).Conclusion Nec-1 has obvious protective effect on myocardial cells of CVB3 induced acute severe virus myocarditis in mice,which may be achieved by inhibiting RIP1/RIP3 necrotic bodies.In summary,this research shows that three types of programmed cell death are involved in the death process of myocardial cell injury in acute severe virus myocarditis,however,necroptosis is the major mode of programmed cell death.It also shows that the necrosis of myocardial cells is a procedural and controllable process in acute severe virus myocarditis.The specific inhibitor Nec-1 has a significant protective effect on the myocardial cells of CVB3 induced acute severe virus myocarditis in mice,which may have great clinical significance on the treatment of acute severe virus myocarditis. |
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