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【Objective】 To explore the effect and mechanism of Fasudil in the treatment of experimental autoimmune myocarditis (EAM) in mice so as to provide a theoretical basis for the clinical use of Fasudil in treating myocarditis. 【Methods】 Balb/c male mice were used as the research objects, and the EAM mice model was constructed using MyHC-α614-629 polypeptide. Mononuclear cells were isolated and cultured to detect the number of mononuclear cells in mouse spleen. Inflammation infiltration, fibrosis and IL-6 expression in mouse myocardial tissue were detected by HE staining, Masson staining and immunohistochemistry, respectively. The protein expressions of Notch1 and IL-6 were detected by Western blotting. qRT-PCR was used to detect the expressions of pro-inflammatory factors (IL-1α, IL-1β and IL-6) as well as key genes of TLRs and NOTCH signaling pathway. 【Results】 EAM mice showed increased HW, decreased BW, increased HW/e-BW, and increased inflammatory infiltration and fibrosis in myocardial tissue. The above-mentioned symptoms or pathological features were improved in EAM mice treated with Fasudil. The analysis showed that the pro-inflammatory factors IL-1α, IL-1β and IL-6 in the myocardial tissue of EAM mice were significantly increased, but only the expression of IL-6 was statistically different after Fasudil treatment compared with the control group. In addition, TLRs signaling pathway might also play an important role in the EAM mice treated with Fasudil. The expressions of IL-6 and Notch1 were consistent, and the expressions of the key genes of NOTCH signaling pathway (Notch1, Hes1 and Jag2) were down-regulated after Fasudil treatment. 【Conclusion】 Fasudil exerts a protective effect on down-regulation of IL-6 expression by inhibiting the NOTCH signaling pathway in EAM mice.
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Objective: To explore whether curcumin can improve cardiac function in rats with experimental autoimmune myocarditis (EAM) by enhancing myocardial autophagy activity. Methods: We used 6-week-old Lewis rats to establish a model of autoimmune myocarditis by the classical method of porcine myocardium immunoglobulin. We randomly divided 21 rats into three groups: control group (Con), autoimmune myocarditis group (EAM), and curcumin-treated group (Cur), with 7 in each group. The rats were treated with curcumin for 21 days. The structure and function of rat heart was evaluated by echocardiography on day 22. Western blot was used to detect the expression of microtubule-associated protein light chain 3 (LC3). The expressions of Beclin-1 and ATG-5 in the myocardium were measured by Real-time PCR. Results: The echocardiography showed that left ventricular systolic function was abnormal in EAM rats compared with rats in Con group. After administration of curcumin, rats in Cur group had better left ventricular systolic function than EAM rats. Compared with that in Con group, LC3 expression in EAM group was significantly increased (P=0.026). After curcumin administration, the expression of LC3 increased more obviously than that in EAM group (P=0.014). Beclin-1, an upstream factor of LC3, was significantly increased in EAM group (P=0.041) and increased even higher under administration of curcumin (P=0.003). ATG-5, another LC3 upstream factor, did not significantly differ among the three groups (P=0.108). Conclusion: Curcumin can improve cardiac function in rats with autoimmune myocarditis by inducing myocardial autophagy activity, which is mediated through the promotion of Beclin-1.
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Objective To explore the expression of macrophage migration inhibitory factor(MIF)in rat hearts with experimental autoimmune myocarditis(EAM)and the possible mechanism of resveratrol's therapeutic effect.Methods Experimental myocarditis model was established by using porcine myocardial immunoglobulin. Twenty-four male 6-week-old Lewis rats were randomly divided into control group(Con),EAM model group(MC) and resveratrol treatment group(MC+ Res).All the rats were detected and compared in the cardiac function according to echocardiographic analysis,and the expression of MIF was detected by Western blot.The degree of myocardial injury was detected by HE staining and the degree of macrophage infiltration in the myocardium was detected by immunohistochemistry.Results In control group,there was no significant inflammatory infiltration or myocardial injury in the myocardium.Heavy local infiltration of macrophages,and dissolved and fractured myocardial fibers were observed in model group.Resveratrol significantly decreased macrophage density and myocardial injury in the heart(P＜ 0.05).Compared with those in control group,LVEDs were significantly increased(P＜0.01)while LVEF and LVFS were markedly decreased in model group(P＜0.01).Compared with model group,LVEDs were significantly decreased(P＜0.05)while LVEF and LVFS were markedly increased in resveratrol group(P＜0.05).The protein expression of MIF was markedly increased in rats of model group(P＜0.01),but was decreased in resveratrol group compared with model groups(P＜ 0.05).Conclusion Increased expression of MIF may be involved in the pathogenesis of EAM.The therapeutic effect of resveratrol on EAM may be associated with down-regulated MIF expression and decreased macrophage infiltration.
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Objective:To observe the expression of B7-H4 in experimental autoimmune myocarditis (EAM).Methods:BALB/c mice were randomly divided into 2 groups:the control group and the experimental group.The mice of experimental group were injected with myosin to establish EAM models , while the mice of control group were injected with complete Freund 's adjuvant and normal saline.All the mice were killed separately at the 14th,21st,30th and 45th day for lymphocyte proliferation assay ,hematoxylin-eosin staining,immunohistochemical staining and real-time PCR.Results:The inflammation infiltration of heart was most serious at the 14th and 21st day,then it was gradually relieved with time;the results of lymphocyte proliferation assay and real-time PCR were similar to that of the inflammation infiltration of heart ,which were in high level at the 14th and 21st day,and they were both higher than that of the control group ( P<0.05 );B7-H4 protein were only detected in the experimental group ,and it was constantly expressed during the whole experiment on the endothelium of heart with myocarditis.Conclusion:B7-H4 participates in the progress of EAM ,and it may be a new way of studying the mechanism of myocarditis.
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Objective To investigate the effect of CD40siRNA on the pathologic changes and T helper lymphocyte (Th)-17 cells of myocardium and IL-17,IL-23 of serum in rats with experimental autoimmune myocarditis (EAM).Methods Forty 6-8 week old healthy male Lewis rats with body weight of 185-210 g were divided into EAM group,CD40siRNA group,siRNA group,and normal control group randomly,with 10 rats in each group.The rats in EAM group,CD40siRNA group and siRNA group were induced by immunization with cardiac C protein and completed Freund adjuvant in double foot pads.The rats in normal control group were injected with PBS buffer in double foot pads.On the 8th day after immunization,the rats in CD40siRNA group were injected with CD40 siRNA expression vector,and the rats in siRNA group were injected with siRNA expression vector.The rats were sacrificed on day 21 after inoculation.The histopathologic changes were observed by light microscope and the myocardial histopathology scores were calculated.The expression of RORC mRNA of myocardium was detected by real-time quantitative polymerase chain reaction (RTPCR).Enzyme linked immunoabsorption assay was used to determine the serum level of IL-17 and IL-23.Results Compared with EAM group,the myocardial histopathology score(2.34 ±0.60 vs 3.40 ±0.35,P ＜0.05),the expression ofRORC mRNA(2.13 ±0.28 vs 2.93 ±0.36,P ＜0.05) and the serum level of IL-17 (114.38 ± 8.29 vs 148.70 ± 5.04,P ＜ 0.05) and IL-23 (107.00 ± 7.69 vs 136.98 ± 23.16,P ＜ 0.05) were significantly lower in CD40 siRNA group.Conclusions It is suggested that CD40 siRNA expression vector might reduce myocardial injury by inhibiting Th-17 activation and down-regulating the expression of IL-17 and IL-23.
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To better understand the role of macrophages in early stages of experimental autoimmune myocarditis (EAM), we compared the expression of inducible nitric oxide synthase (iNOS) and arginase-1, markers for classically activated M1 and alternatively activated M2 macrophages, respectively, in the hearts of EAM-affected and control rats. Immunohistochemical evidence revealed that both iNOS-positive and arginase 1-positive macrophages were found in EAM lesions, while some cells were co-localized with both markers. This finding suggests that the increased level of arginase-1, which is partly from M2 macrophages, contributes to the modulation of EAM, possibly through the reduction of nitric oxide in the lesion.
Subject(s)
Animals , Rats , Arginase , Heart , Macrophages , Myocarditis , Nitric Oxide , Nitric Oxide Synthase Type IIABSTRACT
ObjectiveTo assess the efficacy of herpes virus entry mediator (HVEM) gene modifled dendritic cells (DCs) in protecting against myosin induced myocarditis,and to investigate the involving mechanism.MethodsWe treated experimental autoimmune myocarditis (EAM) mice with myosin-pulsed DCs which were transfected with HVEM-expressing adenovirus (Ad-HVEM) or control vectors,then evaluated myocarditis,plasm cTn [ and autoantibody by histopathology,fluoroimmunoassay,and ELISA,respectively.ResultsWe found that DCs transfected with Ad-HVEM (DC-Ad-HVEM) could protect against EAM.Further study showed DC-Ad-HVEM could produce regulatory cytokine IL-10,and IL-10 promoted the production of a key regulatory T cell subset which is important in peripheral tolerance.The T cells mediated protection against EAM.ConclusionThis study suggest that myosin-DC-Ad-HVEM cell gene therapy is a safe and effective way for inhibiting the development of EAM,and the signal net mediated by HVEM plays different roles in different cells.