Alterations And Significance Of Th17/IL-17 In The Senescence Process Of Mouse Vascular Endothelial Cells

Background:Aging is a problem that people have been studying and trying to slow down since ancient times.In the medical field,the vascular system is one of the important components to maintain the life structure of the human body.It is of great significance to study the aging of the cardiovascular system.Vascular endothelial cells separate the blood components and play a protective role,while secreting angiotensin,and the surface platelet-dependent components play an important role in the body's vascular homeostasis and anticoagulation processes.After vascular endothelial aging,the morphology shows that the gap between cells becomes larger,the nucleus and volume increase,and the cell morphology is irregular.The vascular structure of aging is no longer complete,and it is easy to cause vasodilation and thrombosis,resulting in the accumulation of inflammatory substances,so that feedback regulation increases aging.The aging of the vascular endothelium has an important relationship with cardiovascular disease.Helper T cells 17,?Th17?is a newly discovered CD4⁺helper T cell different from Th1,Th2 in 2007,which can secrete a variety of cytokines,interleukin-17?IL-17?is a major effector of Th17 cells,and Th17 plays an important role in innate immunity and acquiredness.And participate in many important physiological or pathological processes,such as defense against extracellular bacterial infections,mediating chronic inflammation,immune rejection of organ transplants,autoimmune diseases and onset of tumors.Current research indicates that it plays an important role in diseases such as inflammatory reactions and autoimmune diseases.At the same time,IL-17/Th17 can also affect the secretion and expression of many inflammatory factors.Depending on the expression of CD44 and CD62L,they can be divided into memory T cells(CD4⁺CD44^highCD62L^low)and primary T cells(CD4⁺CD44^lowCD62L^high).Aging involves some immune cells including CD4⁺T lymphocytes,while Th17 cells secrete interleukin-17,which induces the inflammatory factor IL-6 to participate in the inflammatory response,thus involving the hypothesis of inflammatory aging,linking Th17 to aging.Aging is often accompanied by an increase in the incidence and severity of many diseases,including infections,cancer and some autoimmune diseases,leading to a decline in quality of life.These diseases are all involved in the immune component,and aging has a certain correlation with immunity.Vascular endothelial cell dysfunction and apoptosis are involved in the pathogenesis of atherosclerosis,and endothelial damage is considered to be the first trigger for atherosclerosis.In addition,endothelial cell dysfunction and apoptosis are major determinants of plaque erosion and thrombosis,leading to ACS triggering.Thus,vascular endothelial aging is linked in series with cardiovascular disease.The role and mechanism of IL-17/Th17 in vascular endothelial aging has not been reported in detail.In this study,the expression of Th17/IL-17 in serum and spleen of of different ages mice was examined,and the primary endothelial cells of mouse aorta were cultured to analyze the pathway changes involved in the aging process of primary endothelial cells and other indicators change.At the same time,the expression of IL-17/IL-17RA in vascular arteries of was detected.The effect of Th17/IL-17 on senescence of vascular endothelial cells was analyzed,and its role and significance in aging process were explored,which provided a new direction for cardiovascular aging.Objective:To study the pathway changes involved in the aging process of mouse primary vascular endothelial cells,analyze the changes of vascular tissue.The changes of vascular tissue and the expression of Th17/IL-17 in spleen and serum were analyzed in mice at different ages of mice,and analyze its influence and significance in vascular endothelial cell senescence.Methods:?1?In this study,healthy young mice,middle-aged mice and aged mice were firstly studied.Each group of 20 patients was tested for serum IL-17 concentration in all age groups,as well as related cytokines IL-6.The sensitized-associated secretory phenotype?SASP?factor IL-1?and the atherosclerosis biochemical marker Ox-LDL were detected by enzyme-linked immuno-sorbent assay?ELISA?.The correlation between Th17/IL-17 and the expression levels of these SASP-related factors and inflammatory markers was analyzed.The multi-parameter flow cytometry technique was used to label cells with different fluorochrome antibodies,and the ratio of Th17cells was obtained by stimulating and inducing lymphocytes of spleen cell suspension obtained from mouse spleen;detection of CD44 and CD62L in mice Expression in Th17 cells;?2?HE staining and immunohistochemistry were performed in mice of different ages to observe the changes of arterial blood vessels in aging and the expression of IL17/17RA and endothelial aging index VCAM-1 on blood vessels.Happening.?3?The primary aorta cells of mouse aorta were isolated and cultured,and the purity was identified by flow cytometry and immunohistochemistry.The senescence of primary cells was detected by?-galactosidase staining,and the Senescence-associated heterochromatic foci?SAHF?level of senile endothelial cells was detected..The primary endothelial cells were collected and protein extracted,and the expression of senescence protein in endothelial cells was detected by western-blot method,and the main signal pathways involved in aging were analyzed.Results:?1?The expression of serum IL-17?210.82±27.68 pg/ml?and th17 of spleen stimulation in aged mice?39.49±3.81%?was significantly higher than that in healthy young mice?88.78±14.35 pg/ml,23.53±4.74%?and healthy middle-aged mice?128.47±16.63 pg/ml,27.28±3.86%??both P<0.05?.?2?The expression of Th17cells,Memory T cells(CD4⁺CD44^highCD62L+^low)in young mice,middle-aged mice and aged mice were?82.57±6.578%?,?76.38±6.45%?,and?78.54±6.87%?,respectively?P>0.05?.The initial T cell expression rate of Th17 cells(CD4⁺CD44^highCD62L^lowIL-17⁺/CD4⁺IL17⁺T cells)was observed in young mice?5.82±2.61%?,middle-aged mice?7.73±3.26%?,and aged mice?6.52±2.96%?.)there was no statistically significant difference in expression between the groups?all P>0.05?.Th17 cells in each group were mainly memory T cells.?3?Serum IL-6,IL-1?and atherosclerosis-related ox-LDL concentrations were significantly higher in aged mice than in young and middle-aged mice,and correlation analysis showed that IL-17/Th17 There is a positive correlation with these indicator changes.?4?Mouse aortic HE staining and immunohistochemistry results:Aortic HE staining results showed that the endothelial cells of young and middle-aged mice were arranged neatly,the cells were closely connected,and the subendothelial space was small.The old mice artery is thicker than the young and middle-aged mice,the intima is rough,the endothelium is widened,and the arrangement is loose.The results of aortic immunohistochemistry showed that the expression levels of IL-17/IL-17RA and endothelial aging index VCAM-1 in older mice were significantly higher than those in young and middle-aged mice.?5?The primary cultured endothelial cells were isolated and cultured.The results of senescence staining showed that the expression of positive cells and the expression of senescence-associated chromosomal?SAHF?were significantly increased in the primary vascular endothelial cells of aged mice.Western-Blot results showed that the expression of aging protein such as P16,P19,P21,P53 and Rb in primary endothelial cells was significantly enhanced.Conclusion:?1?Th17 expression rate in old mice,Serum IL-17,IL-6 concentration and SASP factors IL-1?concentrations were significantly higher than young and middle-aged mice;There was a significant positive correlation between th17 and other factors concentration levels.?2?The positive rate of primary vascular endothelial senescence and the expression rate of SAHF in aged mice were significantly higher than those in young and middle-aged mice.?3?The expression of VCAM-1 in the aorta IL-17 and receptor IL-17RA and vascular endothelial aging index increased significantly in aging.Thus,the expression of Th17/IL-17 is significantly enhanced in the aging process,which is closely related to the senescence of mouse vascular endothelial cells,providing a new research point for aging and cardiovascular and cerebrovascular diseases.