Experimental Study On The Mechanism Of NF-κB Signaling Pathway In Coronary Artery Injury In Kawasaki Disease Model Mice

Objective: Intraperitoneal injection of candida albicans water soluble fraction(CAWS)induced a model of immune vasculitis in mice similar to human Kawasaki disease vasculitis damage,and observed changes in the expression levels of common inflammatory factors,vasoactive substances and NF-κB and their correlation to explore the mechanism of NF-κB signaling pathway in coronary artery injury in rats with Kawasaki disease.Methods: A total of 80 C57 BL / 6 mice aged 4-6 weeks were randomly divided into experimental and control groups of 40 mice each.At the same time every day,mice in the experimental group were injected intraperitoneally with candida albicans water soluble fraction(CAWS)(80mg/ml,8mg / head),and mice in the control group were injected intraperitoneally with saline(0.1ml / head).Mice were treated for 5 consecutive days.Observe the general changes in mice.In the experimental group and the control group,10 mice were randomly selected at different time points(3d,17 d,24d)after the last injection.Peripheral blood was removed from the eyeballs,placed in an EDTA tube for anticoagulation,and then the mice were sacrificed to obtain heart and coronary specimens.Peripheral blood of mice was separated to obtain plasma,and the levels of TNF-α,IL-6,IL-1β,and ET-1 in plasma were measured by enzyme-linked immunosorbent assay(ELISA)at 3,17,and 24 days after the last injection.The nitrate reductase method was used to determine the plasma NO content of mice at different times after the last injection.Heart specimens were pathologically sectioned to observe changes in the aorta and coronary arteries.Immunohistochemical staining was used to observe the site of vessel wall staining,and the expression levels of NF-κB p65 and p-NF-κB p65 at different time points on the vessel wall were detected.Image pro-plus 6.0 was used for semi-quantitative detection.Results: 1.By observing the coronary arteries and aorta of mice,the Kawasaki disease model induced by candida albicans water soluble fraction(CAWS)was successfully determined.2.Except before injection,the body weight of mice in the experimental group was lower than that in the control group at other time points,and the differences were statistically significant(P<0.01).The weight of the mice in the experimental group began to decrease after the first injection of CAWS,and decreased significantly at 1d and 3d after the last injection.There was no significant difference between the two(P> 0.05).Then the weight of mice in the experimental group gradually increased,Compared with the weight of the group at 3d,17 d and 24 d after the last injection,the differences were statistically significant(P <0.01).A total of 5 mice died in the experimental group,and no mice died in the control group.3.The levels of plasma inflammatory factors TNF-α,IL-6,IL-1β,and vascular endothelial active substances ET-1 and NO in the experimental group were significantly higher than the corresponding control at 3d(acute phase)and 17d(subacute phase)after the last injection,and the difference was statistically significant(P <0.01).Compared with the control group,the level measured at 24d(recovery period)after the last injection was not statistically significant(P> 0.05).The levels of TNF-α,IL-6,IL-1β,ET-1 and NO in the experimental group of mice gradually decreased with time at different time points,and content comparison in groups at different time points,and the differences were statistically significant(P <0.01).4.The positive expression sites of NF-κB p65 and p-NF-κB p65 on the vascular wall were vascular endothelial cells and vascular smooth muscle cells.The former was expressed in the cytoplasm and nucleus of the two groups without significant differences between the groups,and the latter was mainly expressed in the nucleus.The expression of p-NF-κB p65 in experimental group was more significant than that in control group.5.There was no statistically significant difference in the total optical density of total NF-κB p65 in the blood vessel wall of the experimental group and the control group at different time points(P> 0.05).The positive expression level of p-NF-κB p65 and the ratio of p-NF-κB p65 / NF-κB p65 in the blood vessel wall of the experimental group were significantly higher than those of the corresponding control group at 3d(acute phase)and 17d(subacute phase)after the last injection,and the difference was statistically significant(P <0.01).Compared with the control group,the levels measured at 24 days(recovery period)after the last injection were not statistically significant(P> 0.05);The positive expression of p-NF-κB p65 and the ratio of p-NF-κB p65 / NF-κB p65 in the blood vessel wall of the experimental group gradually decreased with time at three time points.The levels were compared at different time points,and the differences were statistically significant(P <0.01).6.The positive expression of p-NF-κB p65 in vascular endothelium has a significant positive correlation with the levels of plasma inflammatory factors TNF-α,IL-1β,IL-6and vascular endothelial active factors ET-1 and NO(r = 0.899,r = 0.832,r = 0.879,r = 0.831,r = 0.841,P <0.01).Conclusion: 1.Candida albicans water soluble fraction(CAWS)can successfully induce a mouse model of Kawasaki disease;2.Inflammatory factors TNF-α,IL-6,IL-1β and vascular endothelial active substances ET-1 and NO are involved in the development of immune coronary arteritis in mice;3.The NF-κB signaling pathway may be involved in the occurrence of coronary artery injury in Kawasaki disease by mediating the expression of inflammatory factors and vasoactive substances.