Acrolein-induced apoptosis of smooth muscle cells through NEAT1- Bmal1/Clock pathway and a protection from asparagus extract * Lijun Chen a, 1 , Xiaoyue Wu a, 1 , Falak Zeb a , Yunxiang Huang b , Jing An b , Pan Jiang a , Aochang Chen a , Chuyue Xu a , Qing Feng a, * a Department of Nutrition and Food Hygiene, School of Public Health, Nanjing Medical University, Nanjing, 211166, China b Hebei Province Asparagus Industry Technology Research Institute, Qinhuangdao, 066004, China article info Article history: Received 1 July 2019 Received in revised form 8 September 2019 Accepted 4 December 2019 Available online xxx Keywords: Acrolein Asparagus extract NEAT1 Circadian clock Atherosclerosis abstract Apoptosis of vascular smooth muscle cells (VSMCs) accelerates manifestation of plaque vulnerability in atherosclerosis. Long noncoding RNA NEAT1 participates in the proliferation and apoptosis of cells. In addition, circadian clock genes play a significant role in cell apoptosis. However, whether acrolein, an environmental pollutant, affects the apoptosis of VSMCs by regulating NEAT1 and clock genes is still elusive. We established VSMCs as an atherosclerotic cell model in vitro. Acrolein exposure reduced survival rate of VSMCs, and raised apoptosis percentage through upregulating the expression of Bax, Cytochrome c and Cleaved caspase-3 and downregulating Bcl-2. Asparagus extract (AE), as a dietary supplementation, was able to protect VSMCs against acrolein-induced apoptosis. Expression of NEAT1, Bmal1 and Clock was decreased by acrolein, while was ameliorated by AE. Knockdown of NEAT1, Bmal1 or Clock promoted VSMCs apoptosis by regulating Bax, Bcl-2, Cytochrome c and Caspase-3 levels. Correspondingly, overexpression of NEAT1 inhibited the apoptosis. We also observed that silence of NEAT1 repressed the expression of Bmal1/Clock and vice versa. In this study, we demonstrated that VSMCs apoptosis induced by acrolein was associated with downregulation of NEAT1 and Bmal1/Clock. AE alleviated the effects of proapoptotic response and circadian disorders caused by acrolein, which shed a new light on cardiovascular protection. © 2019 Elsevier Ltd. All rights reserved. 1. Introduction Atherosclerosis is considered as a chronic inflammatory disease, during which macrophages, smooth muscle cells and endothelial cells in the vessel wall play vital roles (Gimbrone and Garcia- Cardena, 2016; Johnson, 2014; Tabas and Bornfeldt, 2016). Vascular smooth muscle cells (VSMCs) proliferation may be bene- ficial throughout atherogenesis, and contributes to the formation of a protective fibrous cap in advanced lesions (Bennett et al., 2016). The apoptosis of VSMCs has been implicated in a thinner fibrous cap and atherosclerotic plaque rupture (Clarke et al., 2006). VSMC- specific apoptosis induced by human diphtheria toxin exerts a thin fibrous cap and aggravating plaque in a mouse model (Clarke et al., 2008). Similarly, the rates of VSMC apoptosis are increased in p53 /  /ApoE /- mice with increased aortic plaque formation (Mercer et al., 2005). A previous study also reveals that VSMC apoptosis promotes hypothyroidism-associated atherosclerosis in ApoE /- mice (Wang et al., 2014). Acrolein, highly electrophilic alpha, beta-unsaturated aldehyde, is a ubiquitous environmental pollutant. It is one of the most harmful components in cigarettes, along with nicotine and carbon monoxide (Csordas and Bernhard, 2013). Combustion smoke of coal, gasoline, diesel, wood and plastic contains large quantities of acrolein (Takahashi, 2015). In addition, acrolein has also been found in foods and beverages, such as cheese, donuts, potatoes, fish, beer and wine (Henning et al., 2017). Epidemiological and animal studies have verified that acrolein exposure increases cardiovascular dis- ease risk, especially in the pathogenesis of atherosclerosis (DeJarnett et al., 2014). Acrolein has been reported to activate the death receptor and mitochondrial pathway of apoptosis in CHO cells (Tanel and Averill-Bates, 2005, 2007). Acrolein can also induce apoptosis of human endothelial-like EAhy926 cell and car- diomyocyte H9c2 cell (Luo et al., 2018; Xu et al., 2018). However, * This paper has been recommended for acceptance by Wen Chen. * Corresponding author. E-mail address: qingfeng@njmu.edu.cn (Q. Feng). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/envpol https://doi.org/10.1016/j.envpol.2019.113735 0269-7491/© 2019 Elsevier Ltd. All rights reserved. Environmental Pollution 258 (2020) 113735