Study On The Correlation And Mechanism Of Inflammatory Factors,Retinol-binding Protein 4 And Aortic Dissection

Part One The serum concentration levels of inflammatory factors,MMP-2 and RBP4 in rat model of aortic dissectionObjective:In this study,we first established an animal model of aortic dissection(AD)in rats,and then explore the pathology and morphological changes of aortic wall and changes of serum inflammatory factors.Method:Thirty male SD rats(3 weeks)were randomly(random number)divided into control group(10 rats)and model group(20 rats).model group rats were induced for AD using 0.25%?-aminopropionitrile(BAPN)treatment for 6 weeks,and were subsequently divided into model ?(with AD)and model ?(no AD).Aorta was separated and observed,while HE staining and Victoria blue staining observed pathology and morphological changes of aortic tissues.Enzyme linked immunosorbent assay(ELISA)explored the serum concentration levels of interleukin-6(IL-6),C-reactive protein(CRP),tumor necrosis factor-a(TNF-a),matrix metalloproteinase-2(MMP-2)and retinol binding protein 4(RBP4).The thickness of aortic media was measured by computer image analytical method.Result:1.A total of 16 rats had AD(80%formation rates).A total of 10 rats died due to AD during experiment,with average dead time 30 ± 6 days after BAPN treatment.The weight gain of the model group was lower than that of the control group.HE staining showed dilated aorta cavity in model rats,with aneurysm-like morphology.Victoria blue staining showed regular arrangement of elastic fibers in rat aorta tissues without rupture.After AD formation in model rats,disorganization or discontinuous segments of elastic fibers were visible,and elastic fiber gap was increased,which contains abundant erythrocytes to form a pseud-cavity,indicating blood inside AD cavity.Model?I group had significantly larger aorta diameter and aortic media thickness compared to model ? or control group(p<0.05).2.Compared with the control group,the serum concentrations of IL-6,CRP,TNF-?,MMP-2 and RBP4 in the model I group were significantly higher.Except for TNF-?,the concentrations of IL-6,CRP,MMP-2 and RBP4 in the model ? group were also significantly higher than those in the control group.Conclusion:In the rat model of aortic dissection,inflammatory factors,MMP-2 and RBP4 release increased,the body is in inflammatory stress state.Part Two Expression and significance of inflammatory factors and BRP4 in aortic dissectionObjective:To investigate the expression and clinical significance of IL-6,Interferon-y(IFN-?),MMP-2,smooth muscle 22 alpha(SM22a)and RBP4 in rat aortic dissection and their correlations,and explore theirs roles and possible mechanism in aortic dissection.Method:Aortic specimens were obtained from part one.Immunohistochemistry(IHC)staining was performed to evaluate the presence of IL-6,IFN-? and MMP-2 in rat aorta tissues.TUNEL staining was used to observe apoptosis of aortic vascular smooth muscle cells.Western-blot Analysis was used to measure the exact protein content of IL-6,IFN-?,MMP-2,SM22a and RBP4.Then the correlation was analyzed by spearman correlation.Result:1.Cellular expression of IL-6,IFN-y and MMP-2 were shown as yellow-brown.IHC staining results showed elevated positive expression of IL-6,IFN-? and MMP-2 in model groups compared with the control group.2.Model ? group had significantly elevated apoptosis of vascular smooth muscle cells compared to model ? group or control group(p<0.05).No statistical significance existed between model rats without AD formation and control group(p>0.05).3.The protein expression of IL-6,IFN-?,:MMP-2 and RBP4 in model ? group and model ? group was significantly higher than that in control group,and the difference was statistically significant(p<0.05).The expression of SM22a protein was opposite to that of the control group(p<0.05).4.The correlation analysis showed that the expression levels of IL-6 versus MMP-2(r=0.421,p<0.05),IL-6 versus RBP4(r=0.514,p<0.05),IFN-y versus MMP-2(r=0.568s,p<0.05),IFN-? versus RBP4(r=0.675,p<0.05)were significantly positively correlated in model rats with AD.The expression levels of IFN-y versus SM22a(r=-0.603,p<0.05),RBP4 versus SM22a(r=-0.728,p<0.05)were significantly negatively correlated in model rats with AD.5.The protein levels of IL-6(r=0.506,P<0.05),IFN-y(r=0.532,P<0.05)and RBP4(r=0.651,P<0.05)were significantly positive correlation with apoptosis of aorta vascular smooth muscle cells in model rats with AD.6.The protein levels of IL-6(r=0.657,P<0.001),IFN-?(r=0.787,P<0.001),MMP-2(r=0.752,P<0.001),and RBP4(r=0.6836,P<0.001)were significantly positive correlation with the process of aortic dissection.The protein levels of SM22a were significantly negatively correlation with the process of aortic dissection(r=-0.573,P<0.001).Conclusion:1.Inflammatory factors,MMP-2 and BRP4 expressed highly in aortic dissection.The apoptosis and phenotype transformation of vascular smooth muscle cells is likewise correlated with AD formation.2.IL-6 and IFN-y expressed highly in aortic dissection.IL-6 may increase the expression of MMP-2 which accelerate the degradation of aortic media matrix,and cause the apoptosis of vascular smooth muscle cells.IFN-y may increase the expression of MMP-2 which accelerate the degradation of aortic media matrix,and cause the apoptosis and phenotype transformation of vascular smooth muscle cells.3.BRP4 expressed highly in aortic dissection.BRP4 may cause the apoptosis and phenotype transformation of vascular smooth muscle cells in aortic dissection.4.There was a positive correlation between the protein levels inflammatory factors and RBP4 and the process of aortic dissection.5.The protein levels of inflammatory factors(IL-6,IFN-?)and RBP4 were significantly positive correlation in aortic dissection.BRP4 may cause the inflammatory reaction in aortic dissection.However,the exact mechanism needs further study.Part Three The regulation mechanism of retinol binding protein 4 in aortic dissectionObjective:To investigate the possible regulation mechanism of RBP4 in the pathogenesis of aortic dissection at the cellular level.Method:The primary cultured aortic smooth muscle cells were co-incubated with RBP4,the cell proliferation was detected by scratch assay,and the expression of apoptosis related protein was detected by Western-blot Analysis.Result:1.Using RBP4(4ul/ml)for co-incubation,can see the proliferation of aortic smooth muscle was significantly lower than the control group,the two groups were significantly statistically significant differences(p<0.05).2.The expression of caspase3,caspase7 and caspase9 in the aortic smooth muscle cells was induced by RBP4,and the dose-dependent characteristics were observed.Conclusion:Increased expression of RBP4,can reduce the proliferation of aorta VSMCs and enhance its apoptosis,thus affecting the reconstruction of the aortic wall,may be an important factor in promoting the incidence of aortic dissection.