The Role Of Iars Gene In The Remodeling Of Large Vessel Wall And Its Related Mechanisms

Part One Expression of Tars and PI3k/Akt signaling pathway in human acute aortic dissection specimensObject:To investigate the expression of Iars,PI3K/Akt signaling pathway,MMP2,MMP9,SM22a and OPN in the middle of the aorta of patients with AD.Methods:15 cases of acute Stanford type A aortic dissection were collected as the experimental group,and 15 cases of DCD donor ascending aortic wall were taken as the control group.The expression of Iars,PI3K,p-PI3K,Akt,p-Akt,MMP2,MMP9,SM22a and OPN protein in the aorta samples were detected by immunohistochemical staining and Western-bolt method.RT-PCR was used to detect the expression of the above mRNA.HE staining and masson staining were used to observe the morphological changes of aortic wall.Results:Western blot and immunohistochemical staining showed that the expression of Iars,MMP2,MMP9 and OPN in the AD group was significantly higher than that of DCD group,while PI3K,p-PI3K,Akt,p-Akt and SM22a Expression was significantly reduced.RT-PCR results showed the same trend.HE staining and masson staining showed that the dissolution and collagen fibers deposition in the aortic wall of the AD group were significantly higher than those in the DCD group and the number of VSMCs was significantly decreased.Conclusion:The VSMCs in the middle of the Aortic wall from differentiation to dedifferentiation,accompanied by increased expression of matrix metalloproteinases,and Iars gene expression increased,while PI3K and Akt expression and phosphorylation were weakened,suggesting that Iars and PI3K/Akt signaling pathways may play important roles in the process of arterial wall medial lesions.Part Two Effects of Tars Gene Expression on VSMC Function and PI3K/Akt Signaling PathwayObject:To investigate the effect of Iars gene expression on PI3K/Akt signaling pathway and MMPs and the function of VSMCs.Methods:The Iars gene overexpression plasmid was constructed and transfected into HA-SMC cells.Cells were divided into blank control group(group A),GFP plasmid transfection group(group B)and Iars gene overexpression plasmid transfection group(group C).The levels of SM22a,OPN,Iars,PI3K,p-PI3K,Akt,p-Akt,MMP2 and MMP9 in HA-SMC were detected by Western-blot.mRNA expression of Iars,PI3K,Akt,MMP2 and MMP9 were detected by RT-PCR.The proliferation of HA-SMC was detected by MTT assay.The migration of HA-SMC was detected by Transwell method.Apoptosis was detected by flow cytometry.Cellular immunofluorescence technique was used to compare the expression of smooth muscle phenotype markers SM22a and OPN.Results:The expression of Iars gene,the expression of PI3K,p-PI3K,Akt,p-Akt and SM22a in HA-SMC cells in Iars overexpression group was significantly lower than that in normal control group and GFP plasmid transfection group,while the expressions of OPN and Iars,MMP2 and MMP9 were significantly increased,and the expression of Iars,MMP2 and MMP9 mRNA was up-regulated and the expression of PI3K and Akt mRNA was down-regulated by RT-PCR.MTT and Transwell results suggested that the proliferation and migration ability of HA-SMC were decreased,and the results of flow cytometry showed that the apoptosis was increased.Cell immunofluorescence also confirmed that the fluorescence intensity of OPN was enhanced and the fluorescence intensity of SM22a was decreased.Conclusion:Overexpression of Iars gene result to differentiated transformation of HA-SMC,increased MMPs,while proliferation and migration capacity decreased,and increased levels of apoptosis,which may involve the mechanism of Iars by regulating the PI3K/Akt signaling pathway and the impact of activity HA-SMC cells.Part Three The PI3K/Akt signaling pathway mediates the regulation of VSMC function by IarsObject:Blocking the PI3K/Akt signaling pathway to investigate the effect of Iars gene expression on VSMC function.Methods:Subcultured HA-SMC cells were treated with PI3K/Akt signaling pathway antagonist Dactolisib.The cells were divided into four groups:Blank control group(group A),Iars gene overexpression plasmid transfection group(group B),Dactolisib treatment group(group C)and Iars gene overexpression plasmid transfection+Dactolisib treatment group(group D).The levels of SM22a,OPN,Iars,PI3K,p-PI3K,Akt,p-Akt,MMP2 and MMP9 in HA-SMC were detected by Western-blot.The mRNA expression of Iars,PI3K,Akt,MMP2 and MMP9 were detected by RT-PCR..The proliferation of HA-SMC was detected by MTT assay.The migration of HA-SMC was detected by Transwell method.Apoptosis was detected by flow cytometry.Results:The expression of SM22a,PI3K,p-PI3K,Akt,p-Akt expression was significantly decreased in Iars gene overexpression plasmid transfection group,Iars gene overexpression plasmid transfection + Dactolisib treatment group and Dactolisib treatment group,while the expression of OPN,MMP2 and MMP9 was significantly increased.The expression of SM22a,OPN,MMP2 and MMP9 expression was no significant differences between Iars gene overexpression plasmid transfection +Dactolisib treatment group and Dactolisib treatment group.The expression of Iars had no difference in normal group and Dactolisib treatment group.After blocking the pI3k/Akt signaling pathway,VSMC proliferation and migration decreased,while apoptotic cells increased.Conclusion:After blocked PI3K/Akt signaling pathway,VSMC phenotype transformed to dedifferentiation type,expression of extracellular matrix metalloproteinase increased,cell proliferation and migration capacity decreased and apoptosis increased,while Iars gene expression is not affected,suggesting that PI3K/Akt signaling pathway mediates the process of Iars gene regulation of VSMC function.