A Preliminary Experimental Study On Mitochondrial Lesion And Its Mechanisms In Kawasaki Disease

Part I: Murine model of Kawasaki disease induced by Candida albicans water-soluble fractionObjective:Repeated intraperitoneal injection with Candida albicans water soluble fraction(CAWS) in mice to induce immune vasculitis, and to verify its similarity with vascular disease in Kawasaki disease, in order to provide an experimental model for the study on Kawasaki disease.Methods:Sixty C57BL/6 male mice(4-6 weeks old) were randomly divided into the experimental group and control group, 30 mice in each group. Experimental and control groups were injected intraperitoneally with 0.1 ml CAWS or saline for 5 days. After the last injection, blood samples of 10 mice each in the both groups were collected on day 3, 17 and 24, and then mice were sacrificed and heart specimens were collected for histopathological examination, while the coronary arteries, the aorta and abdominal aorta were collected for transmission electronmicroscope(TEM) observation; plasma tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) levels in mice were detected using sandwich enzyme-linked immunosorbent assay(ELISA) method.Results:1. Histopathological changes: the incidence of coronary immune vasculitis: 100% in the experimental group and 0% in the control group.(1) HE staining: Day 3(acute phase): all the mice in the experimental group had infiltration of aortic root and part coronary by mainly lymphocytes, monocytes and few neutrophil. Degeneration of endothelial cells, discontinuous endothelium, and varying degrees of myocarditis, endocarditis and percarditis were also observed in the experimental group. Day 24(recovery phase): coronary artery ectasia, infiltration by lymphocytes and macrophage and proliferation of fibroblast were observed in the experimental group.(2) TEM results: day 3(acute phase): in the experimental group, vascular endothelial cells showed extensive vacuolar degeneration, necrosis and detachment. Basement membrane loose, damages on mitochondria and endoplasmic reticulum expanding were also observed in the experimental group.2. Immunological parameters detections: Day 3 after the last injection with CAWS(acute phase), plasma TNF-α, IL-6 concentrations the experimental group were significantly increased in comparison with the control group, the difference was statistically significant(P <0.01): Day 24(recovery phase): plasma TNF-α, IL-6 concentrations in experimental group were obviously decreases than those in the acute phase, the difference was statistically significant(P <0.01):Conclusion:This study demonstrated that CAWS could induce immune vasculitis in C57BL/6 mice, which feature is similar to the immune vasculitis changes in Kawasaki disease in humans. Therefore this can be used as an experimental model of Kawasaki disease. Part II: Preliminary experimental study on the correlation between mitochondrial lesion and NFAT expression in Kawasaki diseaseObjective:Based on the murine model of Kawasaki disease, this study was to observe the pathological changes in endothelial cells mitochondria and the expression of nuclear factor of activated T cell(NFAT) in different inflammatory periods, to preliminarily study the correlation between mitochondrial pathological changes and NFAT expression.Methods:Thirty-six C57BL/ 6 male mice(4-6 weeks old) were randomly divided into experimental group and control group, 18 mice in each group. Experimental and control groups were injected intraperitoneally with 0.1 ml Candida albicans water soluble fraction(CAWS) or saline consecutively for 5 days. After the last injection, 6 mice each in the both groups were sacrificed on day 3, 17 and 24. Heart specimens were collected, and the coronary arteries, the aorta, abdominal aorta were collected for transmission electronmicroscope(TEM) observation; m RNA expression levels of NFAT1, NFAT2 in peripheral blood mononuclear cells were measured using Real-time PCR analysis.Result:1. TEM results: acute phase(3d): in the experimental group, endothelial cells mitochondria in coronary arteries, the aorta and abdominal aorta showed severe swelling, vacuolar degeneration, matrix loose and disappeared internal structure; sub-acute phase(17d): alleviated mitochondrial swelling and vacuolar degeneration, reduced matrix loose, and unclear internal structure were observed in the experimental study; recovery phase(24d): reduced the swelling of mitochondria with visible cristae and normal structure of some mitochondria were detected in the experimental group.2. acute phase(3d): NFAT1 and NFAT2 m RNA expression levels were significantly higher than those in the control group(P <0.01); recovery phase(24d): NFAT1 and NFAT2 m RNA expression levels were significantly decreased compared with acute phase, the difference was statistically significant(P < 0.01); recovery phase(24d): NFAT1, NFAT2 m RNA expression levels were increased compared with control group, however, the difference was not statistically significant(P > 0.05).Conclusion:1. Vascular endothelial cell mitochondria lesion of Kawasaki disease model is most severe in the acute phase(3d), less severe in recovery phase(24d) with some recovered mitochondria; 2. NFAT1 and NFAT2 m RNA expression levels of Kawasaki disease were significantly increased in the acute phase(3d), and markedly decreased in recovery phase(24d) than those in acute phase; 3. These results suggest that NFAT and vascular endothelial cell mitochondria lesion in Kawasaki disease may be relevant, but further experiments remain to identify the mechanism.