Inhibitory Effect And Related Mechanism Of Tangeretin On Abdominal Aortic Aneurysm Of Mice

Objectiv Abdominal aortic aneurysm（AAA）is a chronic inflammatory vascular disease,which was mediated by inflammatory cells.The rupture of an advanced aneurysm is the main cause of the high mortality rate of AAA patients.At present,the main treatment of AAA was surgical.But at the same time,the high risk of surgery was inevitable.To find safer and more effective therapeutic drugs had been the centre of attention.Tangeretin interfered with the expression of Notch1 protein by participating in the Notch signaling pathway.Tangeretin played an important role in inhibiting inflammatory response,blocking oxidative stress and anti-tumor.According to reports,despite Notch1signaling involves in the pathological process of AAA,but it’s still not reported what impact done tangeretin could had on the progression of AAA.And on this basis we carried out the experimental study and explored the influence of tangeretin on the progression of mouse abdominal aortic aneurysm（AAA）model.Methods 1 Mouse AAA model and its intervention experiment:7-8 week male Apo E^-/-mice were perfused with angiotensin II（angiotensin,Ang II）and fed with high fat to construct an AAA model.The AAA mice proved by ultrasound were randomly divided into control group（normal saline）and treatment group（Tangeretin）,each group has 10mice.Mice in treatment group were gavaged Tangeretin（100mg/kg）once a day,while control group with the same amount of saline.After 4 weeks,aneurysm tissues were harvested and embedded in paraffin for continuous paraffin section.Masson and elastic fiber staining（EVG）methods were performed to assess the changes in collagen deposition and elastic fiber rupture of aorta wall and collagen deposition respectively in each group.Immunofluorescence staining（IF）methods were used to detect the expression levels of macrophages and vascular smooth muscle cells as well as the expression of MMP-2 and MMP-9 in aortic wall,and the effect of Tangeretin on ERK1/2 signaling pathway in mouse aortic tissue were detected by Western blot（WB）.2 In vitro cell experiments:Gained primary vascular smooth muscle cells of mice by trypsin digestion and tissue block crawling after the operation of thoracic aorta dissection.Then each group given the corresponding stimulator after the homogenous starvation treatment.Q-PCR methods were used to detect the expression levels of MMP-9 in cell proteins.3Collection of serum from AAA patients and healthy persons:We taked blood samples from 10 AAA patients who were admitted to hospital within 24 hours and 10 healthy persons were selected as the normal control group.Patients with tumors,renal failure,fibrosis,and inflammatory diseases were excluded.We then isolated white blood cells and extracted proteins.Western blot was used to detect the expression of Notch1and p-ERK.Results 1 The animals experiments show that the abdominal aortic dilatation was significantly reduced after the intervention of hesperidin（1.812±0.1436mm vs.2.549±0.2650mm,n=10）,there was statistically significant.Compared with control group,Tangeretin alleviates the destruction of elastic fibers in the arterial wall.The elastic fibers showed normal wavy curvature and collagen was more evenly arranged,besides,we could saw the relatively complete arterial wall structure which had fewer macrophages and more vascular smooth muscle cells.The fluorescence intensity of MMP-2/9 was decreased（P<0.05）.The decreased phosphorylation levels of ERK1/2 protein as well as the down-regulation of MMP-2 and MMP-9 expression in the abdominal aortic wall tissue were also found by western blot and immunofluorescence respectively（P<0.05）.2The cell experiments show that compared with control group,the increased expression levels of P-ERK1/2 and MMP-9 m RNA.The test results of the Tangeretin and AngⅡmixed liquors group was slightly higher than the Tangeretin group.The expression levels of P-ERK1/2 and MMP-9 m RNA of the Jagged1 and AngⅡmixed liquors group was greater than AngⅡgroup.The activity of genes was inhibited of the Tangeretin and AngⅡmixed liquors group so that reduced the expression level P-ERK1/2 and MMP-9 m RNA（P<0.05）.There was no significant difference in gene expression between the Jagged1 and AngⅡmixed liquors group and the Tangeretin and the Jagged1 and AngⅡmixed liquors group（P>0.05）.3 Western blot methods were performed to test the expression of Notch-1 and p-ERK protein in the serum of AAA patients.Compared with healthy control group,the above test items were increased significantly in AAA patients（P<0.05）.Conclusion Notch-1 signaling inhibitor Tangeretin can attenuate the progression of mouse AAA by inhibiting the ERK 1/2 signaling pathway,decreasing the expression of MMP-2/9 and inhibiting the aggregation of inflammatory factors,which could maintain the normal shape and function of elastic fibers and enhanced the content of collagen in extracellular matrix.Figure[8];Table[1];Reference[73]...