The Role And Molecular Mechanism Of MCPIP1 In Abdominal Aortic Aneurysms

Abdominal aortic aneurysm(AAAs)is a dilated aortic disease due to weak aging of the arterial wall and poor compliance.With the intensification of aging in China,the incidence of AAA is increasing,and the process of concealment is slow,but once broken,the mortality rate can reach 80%,which places a great burden on individuals and society.Although the death rate of AAA has decreased with the improvement of inspection methods and treatment methods,no effective drugs have been found to effectively prevent and treat AAA.At the same time,effective molecular indicators for monitoring or predicting AAA are also lacking.Therefore,in-depth exploration of the molecular mechanism of AAA occurrence and development,finding new effective targets for AAA,and developing effective drugs to block and treat AAA are of great significance for the early diagnosis,prevention and treatment of AAA.The current research found that the pathogenesis of abdominal aortic aneurysms is very complicated,which is the result of the fusion and interaction of various pathophysiology mechanisms in the process.However,the specific mechanism for the formation and development of AAA is still difficult to determine.The pathophysiological characteristics of AAA mainly include extracellular matrix(ECM)degradation,immune response,inflammation and oxidative stress response,apoptosis,and vascular remodeling.Monocyte chemotactic protein-1 induced protein(MCPIP1)is a recently discovered zinc finger protein encoded by the zc3h12a gene with transcriptional activity.With the deepening of the research on MCPIP1,the study found that MCPIP1 can participate in a variety of pathological processes in cells through transcriptional activation and/or RNase activity.And it has been considered to be a multifunctional protein related to cardiovascular disease,inflammation,autoimmunity,etc.At the same time,MCPIP1 also plays an important role in inducing differentiation,autophagy and apoptosis.However,the mechanism of MCPIP1 in AAA is rarely reported.Therefore,exploring the role of MCPIP1 in AAA and its mechanism is of great significance for the diagnosis and treatment of AAA.In this study,blood and tissue samples from patients with abdominal aortic aneurysms and controls were collected(N=10).Firstly,the clinical correlation significance of MCPIP1 and inflammatory factors was determined by detecting changes in serum MCP-1,IL-1 3,and NF-?B concentrations;Further,we explored the effects of MCPIP1 on angiotensin-induced smooth muscle cell proliferation,migration,and inflammation by constructing interference MCPIP1 cells and AAA mouse models and clarify the function of MCPIP1 in the pathogenesis of abdominal aortic aneurysms;Finally,the role of MCPIP1 in the pathogenesis of abdominal aortic aneurysms was revealed through detecting the autophagy-associated protein expression,the proportion of autophagosomes,and cell apoptosis in autophagy activators RAPA induced smooth muscle cell.Part I Collection of clinical samples of abdominal aortic aneurysms and analysis of correlation between MCPIP1 and AAAObjective:Blood and tissue samples of patients with abdominal aortic aneurysms and healthy controls(N=10)were collected,and the changes of serum MCP-1,IL-1 3,and NF-?B concentrations were first detected by ELISA;The expression of CD31 and MCPIP1 in abdominal aortic aneurysm tissue samples was analyzed to determine the clinical relevance of MCPIP1 and inflammatory factors.Methods:1.Blood samples were collected from patients with abdominal aortic aneurysms and normal blood samples without abdominal vascular lesions in 10 cases each and serum was separated;the concentrations of MCP-1,IL-1?,NF-?B in serum were detected by ELISA,and the relevance of MCPIP1 with inflammatory factors were analyzed.2.Collect arterial tissue from abdominal aortic aneurysm patients and normal tissue samples without abdominal vascular lesions were collected(10 cases).qRT-PCR and western blot were used to detect the expression of MCPIP1 in the tissues;3.All values are expressed as mean ± standard deviation(SD);statistical analysis was performed with SPSS 17.0(SPSS,Inc.,Chicago,IL,USA);T-test was used to assess differences between 2 groups groups and one-way ANOVA analysis of was used to evaluate the variance more than 2 groups;P<0.05 was considered statistically significant.Results:1.ELISA detection of serum levels of MCP-1,IL-1? and NF-?B in patients with abdominal aortic aneurysms and healthy people found that_compared with healthy controls,MCP1,IL-1? and NF-The ?B level was significantly increased;Further analysis of the correlation between MCP-1 and IL-1? and NF-?B in the serum of AAA patients found that the content of MCP1 in AAA patients was positively correlated with the content of NF-?B and IL-1?;2.qPCR and western blot detecting on the mRNA and protein levels of MCPIP1 in abdominal aortic aneurysms and healthy populations of AAA patients found that compared with healthy controls the expression of MCPIP1 in mRNA and protein levels in AAA tissues significantly increased.Conclusion:Changes in the contents of inflammatory factors IL-1? and NF-?B in abdominal aortic aneurysms indicate a significant inflammatory infiltration in abdominal aortic aneurysms;The changes of MCP-1 in serum and the expression of MCPIP1 in tissues of abdominal aortic aneurysms indicate that the increase of inflammatory factors may be related to the increase of MCPIP1 in AAA.Part II Functional analysis of MCPIP1 in the pathogenesis of abdominal aortic aneurysmsObjective:To confirm the function of MCPIP1 in the pathogenesis of abdominal aortic aneurysms.hrough the identification of aortic elastic fibers and collagen fibers and the apoptosis rates of smooth muscle cells,the expression analysis of MCPIP1 and inflammatory factors,and the effects of MCPIP1 on angiotensin in AAA mouse models The effects of cell proliferation,apoptosis,and inflammation in the cells and AAA mice model with interfering MCPIP1,Methods:1.Establish an experimental mouse abdominal aortic aneurysm model using angiotensin II(Ang-II)micropump release method.Ultrasound measurement of abdominal aorta diameter was performed on days 0,7,14,and 28 and the ultrasound measurement results were retained abdominal aorta was measured to calculate the tumor formation rate of each group.The experiment was divided into sham and model groups.2.EVG staining,Sirius red staining,immunohistochemistry and tunel were used to evaluate the aortic elastic and collagen fiber morphology and the apoptosis rate of smooth muscle cells in the AAA mouse model;3.The expression levels of MCPIP1,CD68 and CD31 in tissues were detected by HE and immunohistochemistry.4.qPCR,western blot were used to detect the MCPIP1 expression in tissues;Western blot was used to detect the expression of MCP-1,IL-1? and NF-?B in tissues;5.Abdominal aortic vascular smooth muscle cells of rat were isolated for expansion culture;And the experimental groups as follows:VSMC,VSMC+Ang-?,VSMC+Ang-?+si MCPIP1-NC and VSMC+Ang-II+si MCPIP1 groups;6.EdU detects changes in cell proliferation ability,Hoechst33258 and flow cytometry were used to detect apoptosis;Flow cytometry to detect cell cycle changes;7.ELISA to detect the concentration changes of MCP-1,IL-1? and NF-?B in the cell?supernatant;8.qPCR,western blot and immunofluorescence were used to detect changes in MCPIP1 expression levels and localization in cells;Western blot was used to detect protein expression changes of MCPIP1,MCP-1,IL-1?,NF-?B,MMP2 and MMP9 in cells;9.All values are expressed as mean ± standard deviation(SD);statistical analysis was performed using SPSS 17.0(SPSS,Inc.,Chicago,IL,USA);T-test was used to assess differences between 2 groups groups and one-way ANOVA analysis of was used to evaluate the variance more than 2 groups;P<0.05 was considered statistically significant.Results:1.Ultrasound measurement of abdominal aorta diameter on AAA model mice on days 0,7,14,and 28 revealed that compared with the sham operation group,the abdominal aorta on days 7,14,and 28 in the AAA model group were significantly larger;HE staining of abdominal aortic smooth muscle actin and CD68 immunohistochemical analysis found that a large number of inflammatory cell infiltrations were observed in abdominal aortic smooth muscle actin of the AAA mouse model compared to the sham group.At the same time,immunohistochemical staining,qRT-PCR and western blot analysis revealed that compared with the sham operation group,the expression of MCPIP1 was significantly increased in the AAA model group.In addition,compared with the sham operation group,the expression levels of MCP1,IL-1? and NF-?B protein in the aneurysm tissue in the AAA model group were significantly increased;2.EVG staining analysis found that compared with the sham operation group,the middle layer of the aortic annulus of the aortic aneurysm in the AAA mouse model group was incomplete,the elastic annulus was partially ruptured,and the waveform was lost.Compared with the sham operation group,the red collagen fibers in the adventitial aneurysm were significantly reduced in the AAA mouse model group;CD31 immunohistochemistry and tunel staining analysis of vascular endothelial cell markers found that compared with sham operation,neovascularization was observed in the model group,but apoptosis of vascular smooth muscle cells increased significantly in AAA model mice.3.MCPIP1 immunofluorescence staining analysis found that compared with the control,the MCPIP1 fluorescence signal in VSMC cytoplasm induced by Ang-? was significantly enhanced;Further analysis found that compared with Ang-? model group and SiRNA-NC group,the fluorescence signal of MCPIP1 in the in SiMCPIPl group was significantly weakened.MCPIP1 expression levels in different groups detected by qRT-PCR and western blot revealed that compared with the Ang-? model group and the SiRNA-NC group,the expression of MCPIP1 was significantly reduced in the Si-MCIPIP1 group;eAt the same time,compared with the Ang-? model group and the SiRNA-NC group,the MCP-1 content in the cells of the Si-MCIPIP1 group was also significantly reduced;In addition,the study found that the expression levels of MMP-2 and MMP-9 were 16.4 times and 6.8 times higher than those of the control,respectively;Compared with the Ang-? and Si-NC groups,the expressions of MMP-2 and MMP-9 in the SiMCPIP1 group were reduced by 2.4 times and 6.8 times,but still 3.37 times for the NC group.4.Compared with the control group,the G0/G1 phase of VSMCs cells was significantly shortened and the proliferation was significantly increased after Ang-? treatment.Compared with the Ang-? group Si-NC group,the G0/G1 phase of VSMCs was significantly increased after MCPIP1 silencing and proliferative effect was significantly reduced;Apoptosis analysis of VSMCs showed that compared with controls,the apoptosis rate of VSMCs treated with Ang-? was significantly increased by about 4.44 times;compared with Si-NC and Ang-? groups,MCPIP1 was silent The apoptotic rate of VSMCs decreased by 1.48 times and 1.72 times,respectively.Conclusion:Increased inflammatory infiltration in vascular smooth muscle in AAA mouse models promotes MCPIP1 expression in smooth muscle cells in corresponding AAA models;The aortic wall elasticity of Ang-?-induced aneurysm mice was impaired;The up-regulation of MCPIP1 induced by Ang-? may be through increasing MMPs expression to degrade elasticity and collagen fibers,thereby destroying the elasticity of the aortic wall;Ang-?-induced upregulation of MCPIP1 breaks the imbalance between VSMC proliferation and apoptosis and thus destroys the elasticity of the aortic wall.Part ? Explores the role of MCPIP1 in the pathogenesis of abdominal aortic aneurysmsObjective:Through the study of autophagy activator RAPA-induced smooth muscle cell autophagy-related protein expression in different groups,changes in the proportion of autophagosomes and apoptosis to determine the role of MCPIP1 in the pathogenesis of abdominal aortic aneurysms.Methods:1.Culture and transfection of vascular smooth muscle cell in VSMCs control group,VSMCs+Ang-? group,VSMCs+Ang-II+siMCPIPl-NC group,VSMCs+Ang-?+siMCPIPl group,respectively;Transmission electron microscope detection of cells changes in the proportion of autophagosomes,immunofluorescence detection of autophagy LC3B expression differences,western Blot detection of autophagy-related proteins LC3B,p62,BNIP3 differential expression;2.Vascular smooth muscle cells were cultured and transfected.The experimental groups were_VSMCs+Ang-? group,VSMCs+Ang-II+siMCPIP1 group,VSMCs+Ang-?+si MCPIP1+1?M RAPA group;Transmission electron microscopy was used to detect autophagy,the expression of autophagy LC3B was detected by immunofluorescence,and the differential expression of autophagy-related proteins LC3B,p62,and BNIP3 was detected by western blot.Flow cytometry was used to detect apoptosis;3.All values are expressed as mean ± standard deviation(SD);statistical analysis was performed with SPSS 17.0(SPSS,Inc.Chicago,IL,USA);T-test was used to assess differences between 2 groups groups and one-way ANOVA analysis of was used to evaluate the variance more than 2 groups;P<0.05 was considered statistically significant.Result:1.Analysis of the expression of autophagy-related proteins found that compared with the control group,the expression levels of LC3BII and BNIP3 were significantly increased and the expression levels of LC3BI and P62 were significantly decreased in Ang-?-induced AAA cell models;Compared with the Ang-? and Si-NC control groups,the expression levels of LC3B? and BNIP3 in the SiMCPIP1 group were significantly reduced,while the expression levels of LC3BI and P62 were significantly increased.The analysis of the expression of autophagy LC3B using immunofluorescence found that:compared with the control group,the fluorescence signal of LC3B was significantly enhanced in the Ang-?-induced AAA cell model;Compared with the Ang-? and Si-NC control groups,the fluorescence signal of LC3B was significantly reduced in the SiMCPIP1 group.Transmission electron microscopy detected changes in the proportion of autophagosomes in Ang-?-induced cells:compared with the control group,autophagosomes in Ang-?-induced smooth muscle cells increased significantly,but compared with Ang-? and Si-NC controls Compared with the group,the number of autophagic bodies was significantly reduced in the SiMCPIP1 group.2.Analysis of the expression of autophagy-related proteins revealed that compared with the Ang-? group,the expression levels of LC3BII and BNIP3 in the SiMCPIP1 group were significantly reduced while the expression levels of LC3BI and P62 were significantly increased;The expression levels of LC3BII and BNIP3 were significantly increased while the expression levels of LC3BI and P62 were significantly decreased.The expression analysis of LC3B detected by immunofluorescence revealed that compared with the Ang-?group,the LC3B fluorescence signal in the SiMCPIP1 group was significantly reduced;The fluorescence signal of LC3B in RAPA group was significantly enhanced.The transmission electron microscope was used to detect the change in the proportion of autophagosomes in Ang-?-induced cells.Compared with the Ang-? group,the autophagosomes in smooth muscle cells induced by SiMCPIP1 were significantly weakened,but the autophagosomes were smaller after RAPA addition The number of body was significantly increased;the apoptosis analysis found that compared with the Ang-?group,the apoptosis of smooth muscle cells in the SiMCPIP1 group was significantly reduced,but the apoptosis was significantly increased after the addition of RAPA.Conclusion:The study of the expression of autophagy-related proteins,the proportion of autophagosomes,and apoptosis in smooth muscle cells in different groups induced by autophagy activator RAPA showed that SiMCPIPl can reduce the apoptosis of smooth muscle cells due to autophagy.Conclusion in whole study1.Analysis of the changes in the content of inflammatory factors IL-1?,NF-?B,MCP-1 and the expression of MCPIP1 in AAA showed that there was obvious inflammation infiltration in AAA,and the up-regulation of MCPIP1 expression caused by inflammatory response was significantly related to AAA.2.Through the functional analysis of MCPIP1 in VSMC,it was found that Ang-II-induced up-regulation of MCPIP1 broke the imbalance between VSMC proliferation and apoptosis and destroyed the elasticity of the aortic wall;3.The investigation of the expression of autophagy-related proteins,the proportion of autophagosomes and the apoptosis of smooth muscle cells in different groups induced by the autophagy activator RAPA showed that SiMCPIPl affects the occurrence and development of AAA by reducing the apoptosis of smooth muscle cells.