| Part one: Pathological study of human abdominal aortic aneurysm Objective:To observe the pathological characteristics of human abdominal aortic aneurysm(AAA)and the expression of SIRT1,NF-κBp65 and CD40 in AAA tissues.so as to establish a foundation for further study on the drug treatment methods and related mechanisms of early AAA.Methods:1.Three cases of surgically resected AAA specimens and three cases of normal abdominal aorta specimens were collected.HE staining was used to observe the pathological manifestations of the tissue.VG staining was used to observe the damage degree of elastic fibers in the vascular wall.2.The levels of CD31 and CD68 were detected by immunohistochemical staining to evaluate the levels of neovascularization and inflammatory cell infiltration in AAA tissues.3.Expression of SIRT1,NF-κBp65,MMP-2,MMP-9 and CD40 in AAA tissues was detected by immunohistochemical staining.Results:1.HE staining showed that the media of human AAA tissue was thinner than that of normal abdominal aorta,Significant neovascularization and inflammatory cell infiltration were observed in the media and adventitia of human AAA.VG staining of AAA showed degradation of elastic fibers in the vascular wall,interruption of fibrin continuity,and destruction of the medial membrane by a large number of inflammatory cells.2.Immunohistochemical staining showed that CD31 and CD68 were almost not expressed in the normal abdominal aorta,but they were significantly increased in the AAA wall,mainly in the media and adventitia,and the difference was significant compared with the normal abdominal aorta(P <0.05).3.The expression of CD40,NF-κB p65,MMP-2 and MMP-9 in normal abdominal aorta tissues was low,but the expression of SIRT1 was high.The expression of CD40,NF-κB p65,MMP-2 and MMP-9 in AAA tissue was significantly increased,and the expression of SIRT1 protein was decreased,which was significantly different from that in normal abdominal aorta tissues(P <0.05).Conclusion:The formation of human AAA is related to inflammatory infiltration,neovascularization,expression of MMPs,SIRT1,NF-κB and CD40,etc.Therefore,it is of certain significance to further study the internal treatment and mechanism of AAA through in vivo and in vitro experiments from these perspectives.Part two: Study on the early protective effect of phenyl ethyl caffeate on the formation of AAA in ratsObjective:To study the role of caffeic acid phenethyl ester(CAPE)in the early formation of AAA in the elastase rat model and its possible mechanisms,and to explore the potential of CAPE in the medical treatment of early AAA.Methods:1.Eighteen healthy male SD rats were randomly divided into sham operation group,model group and CAPE intervention group(6 rats in each group).Rat AAA model was established by injection of elastinase method.CAPE intervention group was intraperitoneally injected with CAPE 10 μmol/kg/d for 14 days,and the model group and sham operation group were injected with the same dose of normal saline at the same time.Fourteen days later,the rats were sacrificed and the tissue samples of AAA and normal abdominal aortic were collected.The maximum diameter of abdominal aorta was measured with a vernier caliper to determine the size of aneurysm.2.Histopathological structures were observed by HE staining and VG staining.3.α-SMA expression was detected by immunohistochemistry and apoptosis of vascular smooth muscle cells was detected by TUNEL;4.Fluorescence method was used to detect the level of ROS in abdominal aortic tissue;5.Immunohistochemical staining was used to detect the expression of CD31 in abdominal aortic tissue;6.Immunohistochemical staining was used to detect the expressions of CD68,iNOS and CD206 to study the phenotypic changes of macrophages.7.Immunohistochemical method was used to detect the expressions of MMP-2 and MMP-9 in abdominal aortic tissue;8.Western-blot was used to detect the expressions of IL-6,SIRT1,NF-κBp65,MCP-1,MMP-2,MMP-9 and CD40 in abdominal aortic tissue.Results:1.The AAA formation rate was 100% in the model group and 83% in the CAPE intervention group 14 days after surgery.There was no significant difference in the diameter of abdominal aorta before elastase perfusion in the three groups(P >0.05).Also there was no statistically significant difference between the model group and the CAPE intervention group immediately after perfusion(P>0.05),but they were significantly increased compared with the sham operation group(P <0.05).14 days after operation,the diameter of abdominal aorta in the model group and the CAPE intervention group were significantly increased compared with the sham operation group(P <0.05),but the CAPE intervention group was significantly decreased compared with the model group(P <0.05).2.HE staining showed that the arterial wall of the model group was tumor-like dilatation,with disorganized tissue structure and significant inflammatory cells infiltration of the arterial wall.After intervention with CAPE,the tumor-like expansion was not obvious,the tissue structure was relatively complete,and the inflammatory infiltration was significantly reduced compared with the model group.VG staining showed that the muscle fibers in the vascular wall of rats in the model group were significantly reduced,the collagen fibers were discontinuous and degraded significantly.But the medial membrane muscle fibers in the CAPE intervention group were increased,and the fracture and degradation of collagen fibers were decreased.3.The level of α-SMA in the model group was significantly lower than that in the sham operation group(P <0.05),while the level of α-SMA in the CAPE intervention group was significantly higher than that in the model group(P <0.05).TUNEL assay was used to detect the apoptosis level of vascular smooth muscle cells in each group.The results showed that the apoptotic cells in the sham operation group were almost invisible,but the number of apoptotic cells in the model group and the CAPE group was significantly increased(P <0.05).The number of apoptotic cells in the CAPE intervention group was significantly decreased compared with the model group(P <0.05).4.Compared with the sham operation group,ROS content in the AAA tissues of rats in the model group was significantly increased(P <0.05),while that in the CAPE intervention group was significantly decreased compared with the model group(P <0.05).5.Compared with the sham operation group,the expression of CD31 in the AAA tissues of rats in the model group was significantly increased(P <0.05),but which in the CAPE intervention group was significantly decreased compared with the model group(P <0.05).6.Compared with the sham operation group,the expression of CD68,CD206 and i NOS in the AAA tissues of rats in the model group were significantly increased(P <0.05),but the expression levels of CD68 and iNOS in the CAPE intervention group were significantly decreased compared with the model group(P <0.05),while the expression level of CD206 was increased(P <0.05).7.Compared with the sham operation group,the expression of MMP-2 and MMP-9 in the AAA tissues of rats in the model group were significantly increased(P <0.05),but which in the CAPE intervention group were significantly decreased compared with the model group(P <0.05).8.Compared with the sham operation group,the expression of CD40,IL-6,MCP-1,MMP2,MMP9 and NF-κBp65 in the AAA tissues of rats in model group were significantly increased(P <0.05),while the expression of SIRT1 was significantly decreased(P <0.05).Compared with the model group,the expression of CD40,IL-6,MCP-1,MMP9 and NF-κBp65 in the CAPE intervention group were significantly decreased(P<0.05),while the expression of SIRT1 was significantly increased(P<0.05).Although the expression of MMP-2 was decreased,there was no significant difference compared with the model group(P >0.05).Conclusion:CAPE has a protective effect on early AAA in rats,and this protective effect may be related to the inhibition of inflammatory infiltration,oxidative stress,neovascularization,apoptosis of vascular smooth muscle cells and the expression of SIRT1,NF-κB,CD40 and other proteins.Part three: Study on the mechanism of CAPE inhibiting the inflammatory response of human umbilical vein endothelial cells induced by TNF-αObjective:To investigate the inhibitory effect of CAPE on the inflammatory response of human umbilical vein endothelial cells stimulated by TNF-α and the possible molecular mechanisms related to SIRT1,NF-κB and CD40 proteins,and to provide a theoretical basis for exploring the protective effect of CAPE on early AAA.Methods:1.Human umbilical vein endothelial cells were cultured in vitro,the cells were divided into control group and TNF-α stimulated group(40μg/L,20μg/L and 10μg/L),the protein expression levels of IL-6 and MCP-1 after incubation for 12 h were detected by Western blot;2.The cells were divided into control group,TNF-α group and CAPE intervention group(80 μM,40 μM and 10 μM).The protein expression levels of IL-6 and MCP-1 in each group were detected by Western blot;3.The cells were divided into control group,TNF-α group and CAPE group,and the protein expression level of SIRT1 in each group was detected by Western blot.4.The cells were divided into control group,TNF-α group,CAPE group,CAPE+NAM group and PDTC group,the protein expression level of CD40 in each group was detected by Western blot.5.The cells were divided into control group,TNF-α group,CAPE group and CAPE+NAM group,and the protein expression level of NF-κBp65 in each group was detected by Western blot.Results:1.TNF-α affected the expression of IL-6 and MCP-1 protein in a concentration-dependent manner,and the expression levels of IL-6 and MCP-1 were significantly increased after 10μg/L TNF-α stimulation compared with the control group(P <0.05).2.CAPE at 80μM,40μM and 10μM significantly inhibited the increase of MCP-1 expression after TNF-α stimulation.CAPE treatment at 80μM and 40μM significantly inhibited the increase of IL-6 expression induced by TNF-α stimulation(P <0.05),but there was no significant difference in IL-6 expression between CAPE group treatment at 10μM and TNF-α group(P >0.05).3.SIRT1 was highly expressed in normal endothelial cells,and the expression level of SIRT1 was significantly decreased after 10μg/L TNF-α stimulation(P <0.05),while it was significantly increased after 40μM CAPE intervention compared with TNF-α group(P <0.05).4.Compared with the control group,the expression of CD40 was significantly increased after 10μg/L TNF-α stimulation(P <0.05).Compared with TNF-α group,The expression of CD40 was significantly decreased after 40μM CAPE intervention(P <0.05),but the inhibitory effect of CAPE on CD40 was significantly decreased after adding NAM(P <0.05).Compared with TNF-α group,the expression of CD40 in PDTC group was significantly decreased(P <0.05),and the effect of PDTC on CD40 was not significantly different from that of CAPE group(P >0.05).5.Compared with the control group,the expression of NF-κBp65 was significantly increased after 10μg/L TNF-α stimulation(P <0.05),40μM CAPE could significantly inhibit the increase of NF-κBp65 induced by TNF-α stimulation(P <0.05),but the inhibitory effect of CAPE on NF-κBp65 was weakened after the addition of NAM(P <0.05).Conclusion:CAPE inhibited TNF-α-induced inflammatory response in human umbilical vein endothelial cells,and the mechanism of this effect was related to the inhibition of CD40 expression in TNF-α-induced endothelial cells by SIRT1-mediated deacetylation of NF-κB p65 subunit.Therefore,we hypothesized that the mechanism of CAPE’s protective effect on early AAA might be related to SIRT1/NF-κB/CD40 pathway. |
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