Abstract:

Objective To discuss the effect of forkhead box O1 （FOXO1） gene on the autophagy and apoptosis of the vascular smooth muscle cells in abdominal aortic aneurysm （AAA）， and to clarify its possible mechanism. Methods ^{The aneurysm tissue of nineteen AAA patients （AAA group） and adjacent normal aortic tissue of nineteen AAA patients （control group） were collected. Real-time fluorenscence quantitative PCR （RT-qPCR） method was used to detect the expression level of FOXO1 mRNA in aneurysm tissue of the subjects in two groups； transmission electron microscope was used to observe the autophagolysosome formation in aneurysm tissue of the subjects in two groups； Western blotting method was used to detect the expression levels of FOXO1 and autophagy-related proteins B cell lymphoma-2（Bcl-2）-binding protein（Beclin1）， microtubule-associated protein 1 light chain 3α （LC3）， and P62 proteins in aneurysm tissue of the subjects in two groups. The human aortic vascular smooth muscle cells （hVSMCs） were cultured in vitro and infected with FOXO1 siRNA （si-FOXO1） and its negative control （si-NC） lentivirus， then treated with 10 μmol·L－1 angiotensin Ⅱ （Ang Ⅱ） combined with autophagy activator rapamycin （Rap）. The cells were divided into blank control group， Ang Ⅱ group， Ang Ⅱ+ si-NC group， Ang Ⅱ + si-FOXO1 group， Ang Ⅱ+ si-NC + Rap group， and Ang Ⅱ+ si-FOXO1 + Rap group.CCK-8 assay was used to detect the proliferation activities of the cells in various groups； flow cytometry was used to detect the apoptotic rates of the cells in various groups； ELISA method was used to detect the levels of matrix metalloproteinase-2 （MMP-2） and matrix metalloproteinase-9 （MMP-9） in the cell supernatant in various groups； RT-qPCR method was used to detect the expression level of FOXO1 mRNA in the cells in various groups； Western blotting method was used to detect the expression levels of FOXO1， Bcl-2， Bcl-2 associated X prorein（Bax）， Cleaved-cysteinyl aspartate specific proteinase-3 （Cleaved caspase-3）， Beclin1， LC3， and P62 proteins in the cells in various groups. Results Compared with control group， the expression level of FOXO1 mRNA in aneurysm tissue of the subjects in AAA group was increased （P<0.05）， the number of autophagolysosomes was increased （P<0.05）， the levels of Beclin1 protein and the ratio of LC3 Ⅱ/LC3 Ⅰ were increased （P<0.05）， and the expression level of P62 protein was decreased （P<0.05）. Compared with blank control group， the proliferation activity of the hVSMCs in Ang Ⅱ group was decreased （P<0.05）， the apoptotic rate of the cells was increased （P<0.05）， the levels of MMP-2 and MMP-9 in the cell supernatant were increased （P<0.05）， the expression levels of Bax， Cleaved caspase-3， and Beclin1 proteins in the cells and the ratio of LC3 Ⅱ/LC3 Ⅰ were increased （P<0.05）， and the expression levels of Bcl-2 and P62 proteins were decreased （P<0.05）. Compared with Ang Ⅱ+ si-NC group， the proliferation activity of the hVSMCs in Ang Ⅱ+ si-FOXO1 group was increased （P<0.05）， the apoptotic rate of the cells was decreased （P<0.05）， the levels of MMP-2 and MMP-9 in the cell supernatant were decreased （P<0.05）， the expression levels of Bax， cleaved caspase-3， and Beclin1 proteins in the cells and the ratio of LC3 Ⅱ/LC3 Ⅰ were decreased （P<0.05）， and the expression levels of Bcl-2 and P62 proteins were increased （P<0.05）. Compared with Ang Ⅱ+ si-FOXO1 group， the apoptotic rate of the hVSMCs in Ang Ⅱ+ si-FOXO1 + Rap group was increased （P<0.05）， the levels of MMP-2 and MMP-9 in the cell supernatant were increased （P<0.05）， the expression level of Beclin1 protein in the cells and the ratio of LC3 Ⅱ/LC3 Ⅰ were decreased （P<0.05）， and the expression level of P62 protein was increased （P<0.05）. Conclusion Silencing the FOXO1 gene may increase the proliferation activity of the hVSMCs exposed to Ang Ⅱ by reducing the autophagy level and inhibiting the apoptosis， thus participating in the pathogenesis of AAA.}

Key words: Abdominal aortic aneurysm, Human vascular smooth muscle cell, Forkhead box transcription factor O1, Autophagy, Apoptosis