The Effect Of Intravenous Immmunoglobulins And Vitamin C On The Progression Of Experimental Autoimmune Myocarditis In Mouse

Background and objectiveViral myocarditis is a common disease that badly intimidates the health of children. Although the pathogenesis of viral myocarditis remains unknown, more and more researches have confirmed that both cellular and humoral components of immune system which are induced by virus infection participate in heart injury. Experimental Autoimmune Myocarditis (EAM) is induced by immunization against porcine cardiac myosin which is free of microorganism and represents immunization-mediated disorder that progresses towards dilated cardiomyopathy similar to human equivalent. The onset of EAM is about day 15 after immunization and the disorder is transferable by T cells.IVIG is increasingly used in patients with immuno-mediated diseases. The mechanisms mediating the protective effects are not precisely defined but probably include multiple pathways such as inhibition of T-cell- and autoanti- body-mediated damage, as well as modulation of the cytokine milieu in a manner that favors an anti-inflammatory pattern. The administration of IVIG was reported to be of clinicalvalue against some virus infections, and this effect was due to the capacity of immunoglobulin to neutralize the viruses. Previous study demonstrated that immunoglobulin therapy suppresses coxsackievirus B3 myocarditis by transferring the neutralizing antibody into the host in the acute stage, because human immunoglobulin contains the antibody against coxsackievirus B3, which is most common in humans. It is possible that immunoglobulin administration may alter immune responses, thus leading to a decrease in cardiac inflammation. IVIG may bind to Fc receptors on macrophages and prevent internalization of the antigens. Some reports showed that immunoglobulin therapy could suppress virus myocarditis not by virus neutralizing activity but by the reduction of plasma catecholamines, IFN-y, and sICAM-1. IVIG may be beneficial in selected autoimmune diseases by modulating the function of the idiotypic network. Several lines of evidence suggest that IVIG contain antiidiotypes against a variety of autoantibodies from patients with autoimmune diseases and against natural autoantibodies from normal individuals.Vitamin C, a potent water-soluble antioxidant, has been used to many diseases. Some evidences have demonstrated that pathogenesis of VMC is closely related to the disequilibrium of the oxidizing and anti-oxidizing reaction. It can protect myocytes by antioxidation and cleaning the radicals. There is report that Oral Vitamin C can extenuate the inflammation of the heart in VMC.Intravenous Immunoglobulin(IVIG) and which is demonstrated to have immunomodulatory effects and Vitamin C were reported to be used in clinic and animal researches, but there are few reports about these two drugs on EAM . The aim of our study is to investigate the effects of IVIG in mice with autoimmune myocarditis and the possible mechanisms that could mediate these beneficial effects. We are also to observe if two dose of Vitamin C and combined IVIG and Vitamin C can eliminate the heart damage in mice with EAM and the related mechanisms.Materials and Methods1. Experiment procedure: Balb/c mice were randomized into six groups: mice without any treatment(blank group, I );mice immunized with porcine cardiac myosin and simultaneously injected with normal saline intraperitoneally(control group, II);mice immunized with porcine myosin and simultaneously injected with Vitamin C (150mg/kg/d) intraperitoneally(small dosage VC group, III1);mice immunized with porcine cardiac myosin and simultaneously injected with Vitamin C(300mg/kg/d) intraperitoneally(large dosage VC group, III2);mice immunized with porcin myosin and simultaneously injected with IVIG(lg/kg/d) intraperitoneally (IVIG group, IV);mice immunized with porcine cardiac myosin and simultaneously injected with IVIG(lg/kg/d) and Vitamin C(150mg/kg/d) intraperitoneally (IVIG+small VC group, V). All mice were sacrificed 14 and 21 days later. The level of Tumor Necrosis Factor a (TNF- a ) was detected with enzyme linked immunoabsorbent assay(Elisa);The heart, spleen and kidney of the mice were gained to calculated the rates of heart to body weight(CR), spleen to body weight(SR), kidney to body weigh (RR);The hearts were removed to observe the hisopathological change.2. Statistical analysis: Results are expressed as means ± s. Differences between groups were compared employing a one-way ANOVA test. Differences were considered significant at P < 0.05. The difference between two independent groups were compared employing t-test;The difference of the morbility in groups were compared employing x 2 test.Result1. Pathology result:I group: there were no inflammatory cells infiltrating in the myocardium and the myocardium is normal;II group: Different pathological changes happened in the heart and a lot of inflammatory cells infiltrated in the myocardium. The damage andnecrosis areas were near the epicardium. Calcification was found approaching pericardium. But all hearts had no collagen hyperplasia;III 1 group: There were inflammatory cells such as lymphocytes and plasma cell infiltrating in the myocardium interstitium. Calcification was less than II group;III2 group: Calcification was not found and the inflammatory change was similar to IIIl group;IV group: Inflammatory cells were occasionally found and there was no calcification;V group: The change was almost the same as IV group.2. Statistical result of morbidity: P<0.05.There was significant difference.3. The result of SR had significant difference, P<0.001;The result of CR had significant difference, P<0.05;The result of RR had no significant difference, P>0.05. The result of 14 day and 21day had no significant difference.4.The levels of TNF- a in the groups had significant difference, P<0.001;After compared every two group, we found that:?The level of II, IIIl, III2 group was much higher than that of I group and had significance (P<0.05);?The level of IV, V group was higher than that of I group, but there was no significance (P>0.05);(3)The level of IIIl and III2 group was lower than that of II group, but there was no significance (P>0.05);?The level of IV, V group was much lower than that of II group and had significance (P<0.001);?The level of IV, V group was much lower than that of IIIl, III2 group and had significance (P<0.05);But there is no significant difference between IV and V group;?The level of III2 group was lower than that of IIIl group, but there was no significance (P>0.05);?The level of V group was lower than that of IV group, but there was nosignificance (P>0.05);5. Sporadic fluorescence area was found occasionally in I group ?, wide fluorescence stripe was found in II group along extracellular matrix surrounding the damaged cardiomyocytes which coincided with the pathological change;Both density and intensity of fluorescence in III1, III2 group were lower that of II group;There were much wider fluorescence stripe in IV, V group and the intensity strengthened.6. The myofilaments in I group were well-arranged and sarcomere had no breakage and all organelles were normal;The myofilaments in II group were in wild disorder and sarcomere had severe breakage. Chondriosome hypertrophy and vacuolar degeneration were found;The damage lessened in IIIK III2 group;Both myofilaments and sarcomeres in IV, V group were almost normal and the Chondriosome was normal.Conclusion1. Two dose of VitC can both attenunate the pathological damage of myocardium. They have some effect on cellular immunity and humoral immunity and may decrease the damage in EAM by suppressing the damage of immune system.2. IVIG at doses twice the dose applied for human disease and IVIG combined with small dose VitC are effective in ameliorating the progression of EAM by decreasing the level of TNF-a in serum. The mechanisms of IVIG on humoral immunity depend on being studied further.3. IVIG and Vitamin C have some protective and theraputic effect on the progression of EAM. Especially the administration with IVIG combined with Vitamin C is more effective. IVIG and Vitamin C may be found clinically feasible for autoimmune myocarditis.