The Studies On Effects Of Interleukin-16 On Extracellular Matrix Remodeling And Atherosclerotic Plaque Stability

III Objective:Rupture of atherosclerosis(AS)plaques increases the risk of ischemic heart disease and stroke and is a common cause of sudden death.Clinically high levels of circulating or intraplaque interleukin-16(Interleukin-16,IL-16)are associated with a reduced incidence of cardiovascular and cerebrovascular events.The specific mechanism based on the effect of IL-16 on the stability of atherosclerotic plaques is not clear.The objective of this study was to use ApoE-/-mice as atherosclerosis animal models and knock out IL-16 gene on the basis of ApoE-/-,aiming to study the effect of IL-16 deletion on the stability of atherosclerotic plaques.Methods:IL-16 was knocked out in ApoE-/-mice to generate IL-16-/-ApoE-/-mice,and ApoE-/--ApoE-/-mice were subjected to high-fat treatment for 24 weeks to detect their body weight and blood lipid changes.Carotid artery frozen section oil red O staining,Masson staining to detect ApoE-/-,IL-16-/-ApoE-/-mouse lipid deposition and collagen content,immunohistochemical staining to detect CD68 and α-SMA content in plaque,Sirius red staining to detect fibrous cap thickness in plaque,HE staining to detect necrotic nucleus size in plaque.Western blot detected the expression of TIMP3,collagen I,p JAK2,and p STAT6 proteins in the aortic root of mice.q RT-PCR detected the expression of m RNA of TIMP3 and collagen I at the root of the aorta.Gelatin enzyme spectroscopy detected the expression of MMP2/9 enzyme activity in the aortic root,in situ fluorescent enzyme spectroscopy staining detected the activity of MMPs enzyme in the plaque of the aortic root,and double fluorescence staining detected the expression of TIMP3 and α-SMA in the plaque of the aortic root.Primary mouse vascular smooth muscle cells were extracted in vitro,and Western blot detected the expression of JAK1/2/3,STAT1/3/5/6 phosphorylation and TIMP3 after IL-16 recombinant protein treatment,and added JAK and STAT related inhibitors to detect the expression of downstream proteins and the expression of TIMP3 protein after CD4 treatment.In vitro tail vein injection of lentivirus-mediated TIMP3 overexpression vector,plaque lesions were detected after 24 weeks of high-lipid treatment.Results:ApoE-/-,IL-16-/-ApoE-/-There was no significant difference in body weight and blood lipids,and pathological section staining analysis showed that compared with ApoE-/-mice,IL-16-/-ApoE-/-mouse carotid plaque had large necrotic nuclei,thinner fibrous cap,more lipid content,reduced collagen content,increased CD68 content,and reduced α-SMA content,which improved the fragility index of plaque.At the same time,the necrotic nucleus/ratio of plaque in the aortic root increased,indicating that the loss of IL-16 reduced the stability of the plaque.It was further found that IL-16 deletion reduced the expression of TIMP3 and collagen I in mice aortic root plaques,promoted the increase of MMP2/9 enzyme activity in plaques and the degradation of matrix proteins such as collagen and elastic fibers.In vitro experiments showed that IL-16 promoted the expression of p JAK2,p STAT6 and TIMP3 proteins in primary mouse vascular smooth muscle cells,while inhibiting JAK2/STAT6 signaling activity decreased the expression of TIMP3.In addition,inhibition or different concentrations of CD4 treatment can reduce or promote intracellular TIMP3 expression,respectively.This indicates that IL-16 regulates the expression of TIMP3 through the CD4/JAK2/STAT6 signaling pathway.Finally,TIMP3 was overexpressed in IL-16-/-ApoE-/-mice,and after 24 weeks of high-fat induction,the stability of carotid artery and aortic root plaque was significantly improved.Conclusions:IL-16 regulates the expression of TIMP3 in smooth muscle cells and promotes the stability of atherosclerotic plaques through the CD4/JAK2/STAT6 pathway,indicating that IL-16 is a regulator of new extracellular matrix(ECM)remodeling and plaque phenotype.