| BackgroundWith the continuous improvement of people’s living standards,atherosclerotic diseases have become one of the major diseases threatening human health.Population-based studies have shown that regular physical exercise can prevent and treat atherosclerotic disease.However,the molecular mechanisms underlying the anti-atherosclerotic effect of physical exercise are not fully understood.Neuropeptide Y（NPY）,a 36-amino-acid residue peptide,is the most abundant neuropeptide in the heart and is widely found in all sympathetic nerves that innervate the cardiovascular system.In addition to its neuronal origin,NPY can be synthesized and released in a variety of vascular cells,including endothelial cells（ECs）,immune cells,and megakaryocytes/platelets.Previous studies identified that NPY and its receptors are involved in the progress of atherosclerosis.NPY can induce long-lasting vasoconstriction,promote vascular smooth muscle cells（VSMCs）proliferation and hypertrophy,stimulate endothelial cells proliferation and differentiation into capillary-like tubes,and aggravate inflammatory response by activated macrophages and promoted macrophages migration and adhesion,all of which can aggravate the progress of atherosclerosis.In addition,many studies have shown that NPY and its receptors were highly up-regulated in human and animal plaques compared to normal vascular tissue.Compelling evidence has identified that physical exercise can alter sympathetic activation of the cardiovascular system,which is the main source of peripheral NPY.Additionally,it has been demonstrated that physical exercise can alter the expression of NPY in the brain and peripheral areas.Therefore,we speculate that the anti-atherosclerotic effect of physical exercise is associated with the change of NPY expression.To confirm our hypothesis,ApoE-/-mice were fed a high fat chow diet to establish the mouse model of atherosclerosis.Meanwhile,ApoE-/-mice were randomly divided into different exercise model group for eight weeks to observe the development of atherosclerotic plaque and the expression of NPY.In addition,we further explore the underlying mechanisms of NPY on macrophages migration in vitro to confirm the results of animal experience.Objective1.To observe if the anti-atherosclerotic effects of physical activity are mediated by alterations the NPY-related pathway in ApoE-/-mice.2.To investigate the potential mechanism of NPY effect on macrophages migration.Methods1.The effect and mechanism of physical exercise effect on atherosclerosis in ApoE-/-mice.ApoE-/-mice were fed a high fat diet and randomly divided into high frequency exercise,low frequency exercise,and sedentary groups.The mice in the high frequency exercise group ran 5 days a week,while those in the low frequency exercise group ran only one or two days a week.The sedentary mice were kept in cages throughout the study.After 8 weeks,plasma was collected to measure lipid concentrations and NPY.One portion of whole aorta were stained with Oil Red O to measure the percentage lesion area.The other whole aorta were used to measure the mRNA expression of NPY,its receptors,and inflammatory cytokines by qRT-PCR.10μm-thick frozen sections of the aortic root were used to measure atherosclerotic burden and plaque stability by histological and morphological analysis.Hematoxylin eosin（HE）staining,Oil Red O staining and Masson staining were used to detect plaque burden,lipid deposition and collagen content in the aortic root respectively.In addition,the content of macrophage,SMC,NPY,and NPY Y1 receptor were detected by immunohistochemistry.2.The mechanism of NPY effect on macrophages migration.Raw264.7 macrophage cells were cultured and treated with different concentrations of NPY to observe the expression of matrix metalloproteinase-8（MMP-8）by qRT-PCR and Western blot.To clarify its signal transduction pathway,macrophages were first treated with NPY receptor antagonist and ERK1/2 inhibitor for 60 min,and then further treated with NPY.Using qRT-PCR to measure MMP-8 mRNA levels after incubated with NPY and different antagonist in macrophages,and Western blot to detect the protein expression of MMP-8,T-ERK1/2,and P-ERK1/2.In addition,migration assay was used to determine the role of NPY on macrophages migration.Results1.Physical exercise attenuated atherosclerotic plaque growth and enhanced atherosclerotic plaque stability in ApoE-/-mice.HE and Oil Red O staining results showed a significant decrease in plaque burden in the two exercise groups compared to the sedentary group.In addition,physical exercise significantly reduced plaque lipid and macrophage content and increased plaque collagen and SMCs content,which are associated with enhancing the stability of atherosclerotic plaques.However,high frequency exercise and low frequency exercise showed no difference in preventing atherosclerotic plaque formation and enhancing atherosclerotic plaque stability in our study.Moreover,physical exercise had no effect on the body weight,serum glucose concentration,or serum lipid concentration in ApoE-/-mice.2.The anti-atherosclerotic effects of physical exercise may be mediated alterations in the NPY-related pathway in ApoE-/-mice.The serum ELISA results showed that high frequency exercise and low frequency exercise groups significantly decreased plasma NPY level compare to sedentary group.Interestingly,the level of plasma NPY was significantly correlated with plaque burden.qRT-PCR results also showed that physical exercise significantly down-regulated aortic NPY,NPY1R,NPY2R,NPY5R,and DPPIV mRNA expression.Immunohistochemistry confirmed that the expression of NPY and Y1 receptor in the two exercise groups showed a significantly decreased in plaque compare to the sedentary group,especially in plaque areas with high macrophage expression.In addition,the expression of inflammatory cytokines in the aorta was significantly lower in the two exercise groups than in the sedentary group.As the Y1receptor has been shown to be involved in the release of inflammatory cytokines in macrophages,we may speculate that exercise reduces the expression of Y1Rs in macrophages,thus inhibiting the expression of inflammatory cytokines in atherosclerotic plaques.3.NPY promotes macrophage migration by up-regulating MMP-8 expression.NPY induced MMP-8 mRNA and protein expression in a dose-dependent manner in Raw264.7 cell,and this effect was abolished by Y1 receptor antagonist and ERK1/2 inhibitor.In addition,NPY increased ERK1/2 activation,which was also greatly inhibited by Y1receptor antagonist.Furthermore,NPY promoted macrophages migration across type I collagen,and this effect was attenuated by the co-treatment with TIMP-1,Y1 receptor antagonist,and ERK1/2 inhibitor.Conclusions1.High frequency exercise and low frequenc exercise were equally effective at preventing atherosclerotic plaque growth and enhancing plaque stability under high fat diet conditions.2.The anti-atherosclerotic effect of physical exercise may be mediated by inhibited macrophage migration and the activity of macrophages through NPY and NPY1R,thereby inhibiting inflammatory response.3.NPY increased MMP-8 expression in macrophages and promoted microphages migration by activating ERK 1/2 pathway via Y1 receptor. |
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