Nicotine Affects The Functions Of Vascular Smooth Muscle Cells And Atherosclerosis By Autophagy

Background/Aims:Currently,cardiovascular diseases have been become the important diseases to threaten human health,in which atherosclerosis（AS）is one of the most common diseases.AS causes several diseases,including atherosclerotic heart diseases,stroke and perivascular diseases.Cigarette smoking is the risk factor of AS.Nicotine,the main toxic substance in cigarette smoking,is associated with the development of AS.Recent studies showed that nicotine could induce the phenotype switching of vascular smooth muscles（VSMCs）.Autophagy has been reported to regulate the formation of atherosclerotic plaque,but the connection between nicotine and autophagy remains elusive.Our study aimed to elucidate the role of autophagy on nicotine-induced effects on vascular smooth muscle cells and AS,as well as the underlying mechanisms.Methods:Mouse model of AS was established by the Apoe^-/-mice fed with the high fat diet for 12 weeks,subcutaneously injected with nicotine every day.The degree of atherosclerotic plaque was detected by HE of the aortic root and oil red O staining of the whole aorta.The amount of vascular smooth muscle cells was shown by the immunohistochemical staining ofα-SMA.Cultured VSMCs treated with nicotine,and western blotting were used to evaluate the protein markers LC3 II/I and p62,as well GFP-LC3 assays were used to assess the formation of autophagosome respectively.Using of the autophagy inducer/inhibitors to find out the effects of autophagy on nicotine-induced VSMCs alterations.Transwell migration and scratch assays were performed to assess VSMCs migration under different conditions.Western blotting was used to detect the protein expression of LC3II/I,P62,collagen I,α-SMA,SM22α,osteopontin,Beclin-1,p-IKBαand p-p65.The immunofluorescence assays were used to detect the expression ofα-SMA.DCFH-DA probe was used to test the oxidative stress in cells by the flow cytometry and fluorescence microscopes.Results:Nicotine aggravated AS,enhanced the levels of autophagy in the whole aorta and increased the amount of VSMCs in atherosclerotic plaque.In vitro study showed that nicotine increased the autophagy in VSMCs and induced the phenotype switching,migration and expression of collagen I.Autophagy inhibitors,at some extent,reversed the nicotine-induced effects in VSMCs.Applicated with HEX,non-selective nicotinic acetylcholine receptor antagonists,the nicotine-induced autophagy and phenotypic switching of VSMCs could be reversed.Flow cytometry and fluorescence microscopy of DCFH-DA probe showed that nicotine induced oxidative stress in VSMCs and using NAC,the oxidative oxygen species scavenger,reversed the nicotine-induced autophagy of VSMCs,as well as the related migration ability.These results showed the oxidative stress was the intermediate part of nicotine-induced autophagy in VSMCs.In addition,NAC,in some extent,inhibited the activation of NF-κB by nicotine.Using the inhibitor of NF-κB signaling pathway BAY 11-7082 and si-RNA of p65 reversed the nicotine-induced autophagy in VSMCs,showed that NF-κB signaling pathway involved in above process.Conclusions:Our study elucidated that nicotine exposure induced autophagy in VSMCs and AS,which promoted its phenotypic transition.The above process was mainly mediated by promoting oxidative stress and then activating NF-κB signaling pathway through nicotinic acetylcholine receptors in VSMCs.