The Role And Significance Of Th17/IL-17 In Vascular Endothelial Cell Senescence And Acute Cerebral Infarction

Background:Vascular endothelial cells(VECs)are the barrier between the vascular system and the blood,which play a key role in maintaining the integrity of blood vessels and regulating the homeostasis of vascular balance.In the aging state of VECs,the expression changes of inflammatory factors,coagulation factors,anti-oxidation factors,etc which modulates endothelium-dependent vasoconstriction and diastolic function affect blood vessel function and promoting thrombosis.Acute Cerebral Infarction(ACI)is a neurological disease caused by blood emboli.It has a high occurrence rate in the elderly,which severely affects the health of the elderly.Atherosclerotic plaque rupture is the main pathological basis of ACI.Vascular endothelial cell senescence is an important initial event in vascular dysfunction and related diseases such as AS and ACI.Aging vascular endothelial cells have the same characteristics as other senescent cells,but also have their own characteristics,such as increasedexpressionofvascularcelladhesionmolecule-1(VCAM-1),endothelin-1(ET-1)and decreased expression of endothelial nitric oxide synthase(eNOS).A large number of clinical and basic studies have shown that there are multiple immune cell-mediated chronic inflammatory reactions in the process of AS,in which CD4~+lymphocyte subsets,such as Th1 cells,Th2 cells,and many cytokines are involved.Th17 cell is a newly discovered subtype of CD4~+cell which has high specificity to secrete pro-inflammatory cytokine IL-17.So it is named Th17 cell.Th17 cells have a strong pro-inflammatory effect and are closely related to the development of inflammation and autoimmune diseases.According to whether CD45 subtype are expressed or not,T cells are also divided into primary T cells and memory T cells.Initial T cells express CD45RA as unsensitized T cells,and memory T cells express CD45RO,which highly expresses adhesion molecules when the antigen is again When stimulated,a strong immune response occurs.VECs are an important part of the inner wall of blood vessels,and VEC senescence is an important factor in the development of AS.Th17 cell play an important role in the occurrence of AS and ACI,however,its role in VEC senescence has not been reported.Therefore,in our study,we first detected the expression changes of Th17/IL-17 in healthy at different ages and elderly ACI patients,and analyzed the effects of Th17/IL-17 on VEC senescence using cellular experiment in vitro.We hope to explore the new mechanism of ACI occurrence and to provide a new target for the prevention and treatment of ACI.Objective:To investigate the expression of Th17 cells in healthy people of different ages and elderly patients with ACI,to clarify the role and significance of Th17/IL-17 in VEC senescence and ACI,and to explore its mechanism preliminarily.Methods:(1)We first chose the subjects including 30 healthy young people,33 healthy middle-aged people,34 healthy elderly people,and 36 elderly ACI patients.Surface and cytoplasma antigens were labelled by multicolor immunofluorescence labeling method,and the frequency of peripheral Th17 cells in different groups was detected by multiparameter flow cytometry.In addition,the expression of CD45RO and CD45RA in Th17 cells at different populations were analyzed.RORγt,Th17 cell transcription factor,expression level in peripheral blood mononuclear cells of different groups was detected by RT-PCR.The levels of serum Th17-relatedcytokines(IL-17,IL-6),vascular endothelial cell senescence markers(ET-1,VCAM-1)and inflammatory markers(ox-LDL,HCY)in different groups were measured by Enzyme-linked immune-sorbent assay(ELISA).Correlations of Th17/IL-17 levels to vascular endothelial cell senescence markers(ET-1,VCAM-1)and inflammatory markers(ox-LDL,HCY)expression levels were analyzed.(2)The effect of recombinant human IL-17(rIL-17)on VEC senescence process was analyzed by rIL-17 induction assay of human umbilical vein endothelial cells(HUVEC)in vitro.Firstly,HUVECs were divided into control group and rIL-17 intervention group.HUVECs were cultured on cell slides for 12 h.rIL-17 was added to the rIL-17-induced group,while the control group was treated with the same amount of PBS and continued to culture for 72 h.Then,the senescence of HUVECs was detected by galactosidase staining.Cells of control and intervention groups were collected and RNA was extracted.The expression of VCAM-1,eNOS and VCAM-1 in HUVECs was detected by RT-PCR.Results:(1)The expression rate of Th17 cells,the concentration of related cytokines(IL-17)and the expression of Th17 cell transcription factor RORγt were significantly higher in the healthy elderly group than in the healthy youth group and healthy middle-aged group(all P<0.05)In addition,the expression of Th17-related marker were also significantly higher in elderly ACI group than in the healthy elderly group(all P<0.01)(2)There were no significant differences in all the 3 groups for the expression rate of CD45RO in Th17 cells(81.94±6.21%in healthy young group;78.42±5.18%in healthy middle-aged group;83.21±4.72%in healthy elderly group;85.42±7.56%in elderly ACI group)and the expression rate of CD45RA(5.94±1.21%in healthy young group;7.42±2.38%in healthy middle-aged group;6.71±2.03%in healthy elderly group,5.12±1.65%in elderly ACI group,all P>0.05),which indicated that Th17 cells in all the 3 groups were expressed highly the memory markers CD45RO.(3)Serum concentrations of VEC senescence markers(ET-1 and VCAM-1)and inflammatory markers(OX-LDL,HCY)in healthy elderly patients were significantly higher than those in healthy youth and healthy middle-aged patients(all P<0.05),while the levels of above indicators in the elderly ACI group were significantly higher than those in the healthy elderly group(all P<0.05).(4)The expression of Th17 cells was positively correlated with the serum levels of endothelial cell senescence markers(ET-1,VCAM-1)and inflammatory markers(OX-LDL,HCY).IL-17 concentration in serum was also a significant positive correlated with the concentration of ET-1,VCAM-1,ox-LDL and HCY(all P<0.01).(5)Cellular experiment results showed that the positive rate of galactosidase staining in rIL-17-induced group was significantly higher than that in control group(P<0.01).(6)The expression levels of ET-1 and VCAM-1 in HUVECs were significantly higher in the rIL-17-induced group than in the control group(all P<0.01),while eNOS levels were significantly lower in the rIL-17-induced group than in the control group(all P<0.05).Conclusions:(1)With the increase of age,Th17/IL-17 expression levels,serum concentrations of endothelial cell senescence markers(ET-1 and VCAM-1)in healthy people were also increased obviously.The above indicators in elderly patients with ACI were increased significantly than those in the healthy elderly.The expression level of Th17/IL-17 was positively correlated with serum concentrations of ET-1 and VCAM-1.(2)IL-17 can induce VEC senescence.(3)With the increase of age,the serum levels of inflammatory markers(ox-LDL,HCY)in healthy people were also increased.The levels of ox-LDL and HCY in elderly patients with ACI were increased significantly than those in the healthy elderly.The expression level of Th17/IL-17 was positively correlated with serum concentrations of ox-LDL and HCY.Thus,Th17/IL-17 can induce VEC senescence andplay an important role in the occurrence of ACI.