Arsenic trioxide ameliorates atherosclerosis by inhibiting CD36-induced endocytosis and TLR4/NF-κB-induced inflammation in macrophage and ApoE-/- mice

Background and Purpose Inflammation and lipid accumulation are key events in atherosclerosis progression. Arsenic trioxide (ATO) has been reported to prevent vascular restenosis by promoting smooth muscle apoptosis and rapid initialization. However, its specific role and mechanism underlying its role in atherosclerosis remain unknown. Herein, we evaluated whether ATO suppresses atherosclerotic plaque development and instability. Experimental Approach ApoE-/- mice were fed a high-fat diet for 3 months and treated with ATO every alternate day for 30 days. The carotid artery and serum samples were collected to determine atherosclerotic lesion size, histological features, and related protein and lipid profiles. In vitro, RAW264.7 or THP-1 cells were stimulated using oxidized low-density lipoprotein (ox-LDL) or LPS to explore the anti-inflammatory and anti-pyroptosis effects of ATO. Key Results ATO reduced atherosclerotic lesion formation and plasma lipid levels in ApoE-/- mice. Additionally, it reduced the levels of various pro-inflammatory factors, including IL-6 and TNFα, in the serum and aortic plaques, but increased the IL-10 level. Mechanistically, ATO promotes the CD36-mediated internalization of ox-LDL, which may explain the reduction in blood lipid levels. Further, ATO reduced TLR4 expression in plaques and macrophages and inhibited LPS-induced p65 nuclear translocation and IκB-α degradation. Conclusion and Implications ATO has the potential atheroprotective effects, especially in macrophages. The mechanisms include inhibition of CD36-mediated foam cell formation, inflammatory responses, and pyroptosis via the suppression of TLR4/NF-κB and NLRP3 activation. Our findings provide evidence for the potential atheroprotective value of ATO. Arsenic trioxide ameliorates atherosclerosis by inhibiting CD36-induced endocytosis and TLR4/NF-κB-induced inflammation in macrophage and ApoE -/- mice Running title: ATO suppresses ox-LDL uptake, inflammation, and atherosclerosis Authors: Xiaoyi Zou 2 *, Zhaoying Li 1,2 *, Xin Wan 2,3, Song Sun 2,3, Shanjie Wang 1,2, Yinan Qu 5, Yun Zhang 6, Liming Yang 4 †, Shaohong Fang 1,2 † 1 Department of Cardiology, The 2nd Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China. 2 The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin, Heilongjiang Province, China. 3 Department of Neurobiology, School of Basic Medical Sciences, Harbin Medical University, Harbin, Heilongjiang Province, China. 4 Department of Pathophysiology, Harbin Medical University-Daqing, Daqing 163319, China. 5 The Second XiangYa Hospital of Central South University, Changsha, Hunan Province, China. 6 Univ Texas MD Anderson Canc Ctr, Dept Clin Canc Prevent, Houston, TX 77030 USA *These authors contributed equally to this work: Xiaoyi Zou, Zhaoying Li †Correspondence: Liming Yang: Department of Pathophysiology, Harbin Medical University-Daqing, Daqing 163319, China Tel: E-mail: [email protected] Shaohong Fang: 246 Xuefu Road, Nangang District, 150086 Harbin, China, Tel: +86 451 8660 7221, E-mail: [email protected] Word coun t: 3214 Acknowledgments: These studies were funded by National Natural Science Foundation Projects to Dr. Shaohong Fang (No.81870353,No.82170262); and Supported by The 2nd Affiliated Hospital of Harbin Medical University,Harbin (No. CX2016-21). Dr.Zou was granted by Key Laboratory of Myocardial Ischemia,Ministry of Education (No.KF201809). Author contributions XYZ, ZYL, LMY, and SHF designed experiments. XYZ drafted the manuscript. ZYL, LMY and SHF edited the manuscript. XW, SS, YNQ and XYZ performed the animal experiments; ZYL, SJW and YZ performed experiments in vitro. LMY provided crucial reagents. SHF and LMY take responsibility for accuracy of the analysis of the whole experiment. Conflicts of interest: The authors have declared that no competing interest exists. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions. Ethics Approval Statement Animals were euthanised by cervical dislocation. Animal experiments were performed according to guidelines approved by the Institutional Animal Care Committee and in compliance with the guidelines from Directive 2010/63/EU of the European. Parliament with approval from the Harbin Medical University Ethics Review Board.

Background and Purpose Inflammation and lipid accumulation are key events in atherosclerosis progression. Arsenic trioxide (ATO) has been reported to prevent vascular restenosis by promoting smooth muscle apoptosis and rapid intimalisation. However, its specific role and mechanism underlying its role in atherosclerosis remain unknown. Herein, we evaluated whether ATO suppresses atherosclerotic plaque development and instability. Experimental Approach ApoE -/- mice were fed a high-fat diet for 3 months and treated with ATO every alternate day for 30 days. Carotid artery and serum samples were collected to determine atherosclerotic lesion size, histological features, and related protein and lipid profiles. In vitro, RAW264.7 or THP-1 cells were stimulated using oxidised low-density lipoprotein (ox-LDL) or LPS to explore the anti-inflammatory and anti-pyroptosis effects of ATO. Key Results ATO reduced atherosclerotic lesion formation and plasma lipid levels in ApoE -/- mice. Additionally, it reduced the levels of various pro-inflammatory factors, including IL-6 and TNFα, in the serum and aortic plaques, but increased the IL-10 level. Mechanistically, ATO promotes the CD36-mediated internalization of ox-LDL, which may explain the reduction in blood lipid levels. Further, ATO reduced TLR4 expression in plaques and macrophages and inhibited LPS-induced p65 nuclear translocation and IκB-α degradation.

and Implications ATO has the potential atheroprotective effects, especially in macrophages. The mechanisms include inhibition of CD36-mediated foam cell formation, inflammatory responses, and pyroptosis via the suppression of TLR4/NF-κB and NLRP3 activation. Our findings provide evidence for the potential atheroprotective value of ATO.

Arsenic trioxide, lipid metabolism, macrophage, pyroptosis, NLRP3 inflammasome Abbreviations As 2 O 3, arsenic trioxide; ApoE, apolipoprotein E; IL-6, interleukin 6; IL-10, interleukin 10; TNF-α, tumor necrosis factor α; ALT/GPT, glutamic-pyruvic transaminase; AST/GOT, glutamic oxalacetic transaminase; H&E, hematoxylin and eosin, OX-LDL, oxidized low density lipoprotein, LPS, Lipopolysaccharides,

Atherosclerosis and associated acute coronary syndrome are leading causes of morbidity and mortality worldwide (Herrington et al., 2016). Despite its complex aetiology, atherosclerosis is considered a progressive inflammatory disease caused by abnormal lipid metabolism in which macrophages play a central role by inducing inflammatory responses and foam cell formation (Seneviratne et al., 2017). Oxidised low-density lipoprotein (ox-LDL) plays an important role in the formation of atherosclerotic plaques and is internalised by surface scavenger receptors, mainly, CD36 (Nicholson et al., 2001). Notably, Toll-like receptor 4 (TLR4) and CD36 can respond to ox-LDL, thereby triggering an intracellular signalling cascade that leads to the activation of the downstream factors MyD88 and NF-κB (Chen et al., 2017). NF-κB activation is responsible for the pro-inflammatory effects of angiotensin II (Ang II) in the blood vessel wall; it activates the production of the pro-inflammatory cytokine IL-6. IL-6 is an important pathogenic factor of atherosclerosis; it interacts with the renin-angiotensin system (Wassmann et al., 2004) (5). The atherogenic effect of macrophage inflammasomes, mainly, NLRP3 (NLR Family Pyrin Domain Containing 3), is also being extensively investigated (Hoseini et al., 2018; Tang et al., 2018). Activated NLRP3 recruits caspase-1, thereby converting the inflammatory factor IL-1β into its mature form, ultimately leading to pyroptosis. IL-1β is a potent atherogenic cytokine that promotes the secretion of many other cytokines and chemokines (Seneviratne et al., 2017). Further, IL-1β induces the expression of adhesion molecules, endothelin-1, and inducible nitric oxide synthase in endothelial cells. The nuclear translocation of p65 promotes pro-IL1β transcription (Kauser et al., 1998). In addition, Western diet-fed Ldlr -/- mice transplanted with bone marrow cells lacking NLRP3 showed reduced lesion size, along with reduced IL-1 and IL-18 levels (Duewell et al., 2010). Currently, statins are the most effective treatment for lowering lipoprotein levels and preventing major cardiovascular events. However, statins have shown severe withdrawal responses in clinical trials. For example, in patients with hypercholesterolemia, atorvastatin withdrawal rapidly increased the activation of pro-inflammatory and thrombogenic pathways (Güleç Başer et al., 2018), while acute simvastatin withdrawal after myocardial infarction was associated with an increased rebound in the CRP (C-reactive protein) levels (Babu et al., 2011). Therefore, it is necessary to develop a safer and more effective method for protection against vascular inflammation. Arsenic-based compounds have been used to treat diseases for more than 2,000 years. Among such compounds, arsenic trioxide (As 2 O 3, ATO) has garnered attention due to its therapeutic activity. In the past two decades, the therapeutic efficacy of ATO against acute promyelocytic leukaemia (APL) has been approved by US Food and Drug Administration (FDA) (Au et al., 2003; Zhao et al., 2021). Moreover, ATO has also shown efficacy against other diseases, such as multiple myeloma (Munshi, 2001), and various solid tumours (Subbarayan et al., 2014) due to its anti-proliferative properties. Although ATO use is limited by its toxicity, its combination with nanotechnological strategies could increase its bioavailability while reducing systemic toxicity, as has been observed with various conventional chemotherapy drugs (Akhtar et al., 2017). Many researches showed ATO-eluting stents inhibited in-stent restenosis at the responding concentration of ATO (Zhang et al., 2017). Mechanistically, ATO exerts anti-tumour effects by inhibiting the secretion of various pro-inflammatory cytokines and NF-κB transcription (Zhang et al., 2016). Nevertheless, the atheroprotective role of ATO in atherosclerosis, especially, in macrophages, is largely unknown. In this study, we investigated the potential atheroprotective nature of ATO in atherosclerosis and the mechanism whereby it disturbs macrophage lipid metabolism and inflammatory responses. Animals Male ApoE -/- C57BL/6 mice (8-weeks-old) were purchased from Vital River Inc. (Beijing, China). All interventions and animal care methods were conducted according to the Guidelines and Policies for Animal Surgery offered by the Animal Ethics Committee of the Second Affiliated Hospital of Harbin Medical University (Harbin, China). The mice were kept in a temperature-controlled facility (temperature: 24 degrees Celsius to 25 degrees Celsius, humidity: 55%) with a 12 h light/12 h dark photoperiod, and food and water were freely available. ApoE -/- mice were randomized into two groups and treated as follows: control (saline, ip.) and ATO (2.5 mg kg -1 d -1, ip.). Mice were maintained on a Western diet for four months, consisting of 78.85% of basic mice maintain feed, 21% of fat and 0.15% of cholesterol. At the last month, mice were fed with intraperitoneal administration of the drugs once every two days for one month, a total of 15 times. All of the animals were killed at the end of the testing period. Blood was collected from the retro-orbital vein and serum was separated. Blood urea acid (UA) and creatine Kinase(CK) levels as well as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined on a Hitachi 917 Roche Analyzer (Meylan, France) using dedicated reagents. The investigator was blinded to the group allocation and assessing the outcome during the experiments. Cell culture Raw264.7 (RRID:CVCL_0493) and THP-1 (RRID:CVCL_0006) were obtained from China Center for Type Culture Collection. The cell lines used in this study were authenticated using short tandem repeat (STR) analysis and regularly tested for mycoplasma. Raw264.7 were seeded in 6-well plates (1×10 6 per well) and cultured with Dulbecco’s Modified Eagle’s medium (HYCLONE, Cat#SH30022.01B), 10% fetal bovine serum (ScienCell, Cat#0500, USA). After 24h, Culture media was then renewed and cells were treated by ox-LDL (Yiyuan Biotechnologies, Cat#2018-05-15,) at a concentration of 50 μg ml -1 with or without As 2 O 3 (2.5 μM or 5.0 μM) for 24 h. THP-1 were seeded in 6-well plates (1×10 6 per well) and cultured with 1640 medium (Gibco, Cat#88365), 10% fetal bovine serum and PMA(100nM). After 12 h, culture media was renewed and cells were treated by ox-LDL at a concentration of 50 μg ml -1 with or without As 2 O 3 (2.5 μM or 5.0 μM)for 24 h. Cells were then collected for detection. RNA extraction and real-time PCR Total RNA was prepared using Trizol reagent (Thermo Fisher, USA) according to the manufacturer’s instructions. RNA purity and concentration were measured using a spectrophotometer. One microgram of total RNA was reverse transcribed into cDNA using an RT Easy II First Strand cDNA Synthesis Kit (Roche, Cat#04379012001). Then, cDNA(18 ng) was amplified in a Real-Time PCR Easy (SYBR Green I) (MCE, Cat#HY-K0501) on Bio-Rad Sequence Detection system (Bio-Rad, USA). The following primers were listed in the supplementary table S1. Measurements of cytokines Serum were collected and determined via ELISA according to the manufacturer’s instructions. Serum levels of IL-6, IL-10, and TNF-α were measured by IL-6 (R&D systems, Cat#DY406-05), IL-10 (R&D systems, Cat#DY417-05) and TNF-α (R&D systems, Cat#DY410-05) detection kits. The levels of IL-1β in the THP-1 cell supernate were measured by IL-1β ELISA kit (BOSTOR, Cat#EK0392). NF-κB p65 detection in subcellular compartments RAW264.7 cell was seeded in 6-well plates (1×10 6 per well) and cultured with DMEM, with 10% fetal bovine serum. After 24 h, Culture media was then renewed and cells were treated with or without As 2 O 3 (2.5 μM or 5.0 μM ) for 2-6 h or BAY11-7082(5mM, MCE, Cat#HY-13453) for 40 min, followed by LPS (200 ng mL -1, Sigma, Cat#L2880) stimulation for another 30 min. The treated cells were washed with PBS and suspended in lysis buffer A (10 mmol L -1 HEPES, pH 7.6; 10 mmol L -1 KCl, 1 mmol L -1 dithiothreitol, 0.1 mmol L -1 EDTA and 0.5 mmol L -1 PMSF) for 10 min on ice. Cytosolic fractions were separated using pipettes after centrifugation at 12 000× g for 10 min at 4 °C. The remaining unclear fractions were lysed again with lysis buffer B (20 mmol L -1 HEPES, pH 7.6; 1 mmol L -1 EDTA, 1 mmol L -1, DTT, 0.5 mmol L -1 PMSF, 25% glycerol and 0.4 mol L -1 NaCl) and centrifuged at 12 000× g for 20 min. As mentioned above, anti-NF-κB p65 (1:1000 dilution, Biolegend, Cat#622602，RRID:AB_315956) primary antibody was used to detect the cytosolic and nuclear proteins by western blotting. GAPDH and PCNA were used as internal controls for the cytoplasmic and nuclear fractions. Western blot analysis After treatments, the cells were lysed in RIPA buffer containing protease and phosphatase inhibitors on ice. The concentration of protein was tested using the bicinchoninic acid (BCA) protein assay. Protein samples (30 μg) were separated by 6%, 10% or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to 0.22-μm PVDF membranes, followed by blocking for 2 h at room temperature with 5% dried skimmed milk in Tris-buffered saline with 0.05% Tween 20. The membranes were incubated with primary antibodies at 4 ℃ overnight, including CD36 (1:1000, proteintech, Cat#18836-1-AP, RRID:AB_10597244), TLR4 (1:1000, santa, Cat#sc-10741, RRID:AB_2240715), IκBα (1:1000, CST, Cat#4812), PCNA (1:10000, proteintech, Cat#60097-1-lg, RRID:AB_2236728), NLRP3 (1:3000, CST, Cat#15101S, RRID:AB_2722591), cleaved-IL1β (1:1000, CST, Cat#83186S, RRID:AB_2800010), cleaved-caspase-1 (1:1000,CST, Cat#4199T RRID:AB_1903916), GAPDH (1:1000, ZSGB-BIO, Cat#ta-08, RRID:AB_2747414). Subsequently, the membrane was incubated with Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:8000) for 2 h at room temperature. Immunoreactivity were visualized by chemiluminescence method using ChemiDocTM MP Imaging System (Tanon, China). The protein bands were quantified using a Bio-Rad Chemi EQ densitometer and Bio-Rad Quantity One software(RRID:SCR_014280) and normalized to GAPDH or PCNA. Oil Red O staining of whole aortas for en face analysis For atherosclerotic lesion analysis, nine mice per group were evaluated. The aortas from the base of the ascending aorta to the iliac bifurcation were separated, and the aortic roots with the heart were harvested. For en face aorta analyses, the aortic tree was perfused with PBS, and the aorta was then dissected en bloc from the root to the iliac bifurcation by removing minor branching arteries and fat tissue. The aortic lumen was opened with a longitudinal incision and then immediately fixed in 4% paraformaldehyde. After 24 h of fixation, aortic lipids were stained with Oil Red O (Solarbio, Cat#G1261), and the stained aortas were photographed. The percentage of aortic area stained with Oil Red O was determined and quantified with Image‐Pro Plus 6.0 (Image Metrology, Copenhagen, Denmark, RRID: SCR_007369). Atherosclerotic lesion analysis and immunofluorescence staining of aortic sinus The heart and proximal aorta were collected and embedded in optimum cutting temperature compound. Sections were stained with Oil Red O and hematoxylin for analysis of plaque sizes. Masson Trichrome staining was used to analysis collagen content. For immunofluorescence staining, slides were fixed in cold methanol and permeabilized using 0.3% Triton-X. Then, slides were blocked using 5% bovine serum albumin for 30 min and incubated overnight with primary antibodies. Alexa-488/647 Alexa647(Abcam, Cat#ab150075, RRID:AB_2752244), Alexa488(Abcam, Cat#ab150077, RRID:AB_2630356) conjugated secondary antibodies were used for detection. The isotype antibodies were used as controls. Slides were counterstained with DAPI(Beyotime, Cat#P0012, China). Foam Cell Formation Resident mouse peritoneal macrophages were obtained from male ApoE -/- mice fed a WD for 16 weeks. Sterile ice-cold phosphate-buffered saline (PBS) was injected into the cavity of each mouse by peritoneal lavage. This fluid was carefully collected and centrifuged at 1,000 rpm for 6 min. After centrifugation, the supernatant was then withdrawn, and the cell pellet was resuspended in RPMI 1640 medium (containing 100 IU ml -1 of penicillin, 100 μg ml -1 of streptomycin and 100 μg ml -1 of l-glutamine) and plated in 6-well tissue culture plates (Costar) at 1 × 10 6 cells per well. Cells were incubated in a humidified CO2 (5%) incubator at 37 °C for 2–3 h to allow adherence, and non-adherent cells were rinsed away with pre-warmed RPMI 1640 and 2 ml of complete RPMI 1640 medium (supplemented with 10% fetal bovine serum) was added. Medium with all additions were replaced daily and macrophages were used within 5 days from harvesting. Then, peritoneal macrophages were stimulated with 50 μg mL -1 of oxidized LDL (ox‐LDL) in the presence or absence of As 2 O 3 (2.5 μM or 5.0 μM ) for 24 hours. Cells were then fixed in 4% paraformaldehyde for 15 min, washed with PBS, and incubated with a 0.5% working solution of Oil Red O for 15 min. Images were captured using Nikon microscope equipped with a digital camera (Tokyo, Japan). Statistical analysis GraphPad Prism 6.0 version software(RRID:SCR_002798) was used for statistical analysis. All experiments were repeated at least five times. Comparisons between two groups were performed using Student’s t-test. The variance is similar between the groups that are being statistically compared，data were considered statistically significant if p value was less than 0.05.

As 2 O 3 (ATO, Sigma, Cat#311383-125G), Lipopolysaccharides(LPS, Sigma, Cat#L2880, Sigma), oxidized low-density lipoprotein (ox-LDL, yiyuanBiotech, Cat#YB-002, China), PMA (Sigma, Cat#P1585, USA) BAY11-7082(MCE, Cat#HY-13453, USA). CD36 (proteintech, Cat#18836-1-AP, RRID:AB_10597244), TLR4 (santa, Cat#sc-10741, RRID:AB_2240715), IκBα (CST, Cat#4812), PCNA (proteintech, Cat#60097-1-lg, RRID:AB_2236728), NLRP3 (CST, Cat#15101S, RRID:AB_2722591), cleaved-IL1β (CST, Cat#83186S, RRID:AB_2800010), cleaved-caspase-1 (CST, Cat#4199T RRID:AB_1903916), GAPDH (ZSGB-BIO, Cat#ta-08, RRID:AB_2747414). CD68(Abcam, Cat#ab53444, RRID:AB_869007), CD36(Invitrogen, Cat#MA514112, RRID:AB_11007635), Alexa647(Abcam, Cat#ab150075, RRID:AB_2752244), Alexa488(Abcam, Cat#ab150077, RRID:AB_2630356), DAPI (Beyotime, Cat#P0012, China). FITC-Annexin V/PI (BD,Cat#556547, RRID:AB_2869082), APC-CD3 (Biolegend, Cat#100235, RRID:AB_2561455),Percp-cy5.5-B220 (Biolegend, Cat#103235, RRID:AB_893356), PE-Ly6G (Biolegend, Cat#127607, RRID:AB_1186104). RT Easy II First Strand cDNA Synthesis Kit (Roche, Cat#04379012001, Roche), Real-Time PCR Easy (SYBR Green I) (MCE, Cat#HY-K0501), IL-6 (R&D systems, Cat#DY406-05), IL-10 (R&D systems, Cat#DY417-05) and TNF-α (R&D systems, Cat# DY410-05), IL-1β ELISA kit (BOSTOR, Cat#EK0392).

ATO attenuates atherosclerotic plaque progression and destabilisation in ApoE -/- mice To investigate the role of ATO in protecting against atherosclerosis, ApoE -/- mice were divided into two groups and fed a high-fat diet for 3 months. One group was treated with ATO (2.5 mg kg -1 ), and the other group was treated with vehicle (ATO and control groups, respectively; Figure 1A). ATO significantly reduced atherosclerosis development, as demonstrated by Oil Red O staining of both the aortic sinus and en face preparations of the entire aorta (Figure 1B, D, G, J). In addition, the lesion area and the proportion of necrotic cores in plaques significantly reduced after ATO treatment (Figure 1C, E, F), but the necrotic core area decreased slightly (Figure 1I). In addition, ATO increased the collagen content in plaques (Figure 1H, K). We further determined the effects of ATO on plaque composition. ATO reduced macrophage accumulation, as indicated by CD68 immunostaining (Figure 1L, M). To determine whether the dose we used in this study was safe, we examined the histological features of various tissues and organs (Figure S1A, B). We also examined the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea acid (UA), and creatine Kinase (CK) in ApoE -/- mouse plasma, apoptosis in peripheral lymphoid tissues, and the proportion of peripheral white blood cells (Figure S1D-G, S2). The results showed that the appropriate dose we used (2.5 mg kg -1 ) did not cause significant serological changes, histopathological changes, or extensive white blood cell apoptosis. Further, there were no significant changes in the body weight of ApoE -/- mice before and after ATO treatment (Figure S1C). These results indicate that the dose of ATO used in vivo (2.5 mg kg -1 ) attenuated atherosclerotic plaque progression but did not cause significant toxicity in ApoE -/- mice. Furthermore, our previous study has shown how the ATO dose affects the viability of macrophages (Fang et al., 2021). Based on these results, we chose the dose of ATO as 2.5 mg kg -1 in vivo and the doses of 2.5 and 5.0 μM in vitro in this experiment. ATO decreases serum lipid levels in ApoE -/- mice and CD36-mediated ox-LDL uptake in macrophages Excessive cholesterol accumulation in macrophages results in the transformation of macrophages into foam cells, which eventually causes atherosclerosis (Akıl et al., 2014; Kavurma et al., 2017). Preventing excessive cholesterol accumulation in macrophages is one atheroprotective strategy. Indeed, high CD36 expression can facilitate foam cell formation (Zhao et al., 2016). To investigate whether ATO regulates lipid metabolism by regulating CD36 expression in macrophages, we first determined the serum lipid levels of ApoE -/- mice treated with ATO and those left untreated (Figure 2A-E). After 4 weeks of ATO therapy, serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and ox-LDL levels decreased significantly compared with those in the controls. However, high-density lipoprotein (HDL) levels were slightly, but not significantly, lower in the ATO group (Figure 2A-E). These data indicate that the protective effect of ATO against atherosclerosis in ApoE -/- mice may be attributed to altered serum lipid metabolism. Compared with those in the untreated model group, CD36 mRNA and protein levels in the aortic tissue were significantly decreased by ATO treatment (Figure 2F, I, L). When we determined the CD36 expression in lesion areas by immunofluorescence staining, we observed that CD36 expression in macrophages (CD68 + ) was substantially reduced by ATO treatment (Figure 2G, H). We also evaluated the ATO-mediated promotion of CD36-mediated ox-LDL internalization in vitro . Peritoneal macrophages of ApoE -/- mice were treated with ox-LDL (50 μg mL -1 ), and either 2.5 or 5.0 μΜ ATO for 24 h. Oil red O staining showed that both ATO doses significantly decreased ox-LDL uptake by peritoneal macrophages (Figure 2M, N). Moreover, both ATO doses significantly reduced CD36 protein and mRNA levels in RAW264.7 cells (Figure 2J, K, O). Taken together, these data suggest that ATO protects against foam cell formation by decreasing CD36-mediated cholesterol uptake in vivo and in vitro, which may be the mechanism by which ATO regulates blood lipid metabolism. ATO inhibits inflammatory factor expression in ApoE -/- mice and macrophages Mounting evidence points towards a role of inflammation in plaque progression and vulnerability (Kataoka et al., 2015). We analysed inflammatory cytokine expression in carotid plaques in control and ATO-treated mice. Our results showed that IL-6 and TNFα mRNA levels were significantly decreased, whereas IL-10 mRNA expression was increased in the ATO group (Figure 3D-F). A similar tendency was observed for inflammatory cytokines in the serum (Figure 3A-C). These results suggest that ATO attenuates atherosclerosis by inhibiting the expression and secretion of inflammatory factors in the entire body and lesions of ApoE -/- mice. Consistently, ATO significantly reduced the IL-6 and TNFα mRNA levels in ox-LDL treated RAW264.7 cells (Figure 3G, H), while increasing the IL-10 mRNA levels (Figure 3I). These data suggest that ATO reduces the expression and secretion of inflammatory factors. ATO inhibits the activation of TLR4/NF-κB signalling in macrophages Previous studies showed that TLR4 promotes the transcription of inflammatory cytokines and causes plaque instability by activating downstream signalling pathways (such as the NF-κB pathway) in the inflammatory site of atherosclerosis (Tak et al., 2001; Hansson et al., 2015). Before activation, p65 binds to its inhibitor, IκB-α, in the cytoplasm. After activation, IκB-α is phosphorylated and the released p65 is transferred to the nucleus, where it binds to target DNA and regulates the expression of inflammation-related genes. To determine whether ATO inhibits TLR4/NF-κB signalling pathway activation, we extracted the aortic tissue protein from ApoE -/- model mice after ATO treatment. We observed that ATO significantly reduced the TLR4 protein levels in the aorta (Figure 4A,D). Consistently, ATO (2.5 and 5.0 μM) significantly reduced the TLR4 protein levels in ox-LDL-treated RAW264.7 cells (Figure 4B, E). Next, we examined the intracellular localisation of p65 following treatment with or without BAY-11, an IκB-α degradation inhibitor, and ATO. The results showed that in RAW264.7 cells, ATO treatment (2.5 μM) for 4–6 hours significantly inhibited LPS-induced p65 translocation into the nucleus, which increased its accumulation in the cytosol (Figure 4C, F, G, I). Interestingly, BAY-11 (5 μM) treatment for 40 min showed a similar trend (Figure 5C, F, G,I), indicating that ATO may inhibit IκB-α degradation. To further evaluate this observation, we examined IκB-α protein expression in LPS-treated RAW264.7 cells in the presence or absence of ATO. The results showed that compared with the control group, the LPS-stimulated groups showed significantly reduced IκB-α levels. However, treatment with ATO (2.5 μM) for 4–6 h significantly increased the IκB-α levels in RAW264.7 cells (Figure 5H, J). Taken together, these data suggest that ATO inhibits TLR4 activation and inhibits nuclear p65 translocation, which may occur through the inhibition of IκB-α degradation(Figure 5K). ATO reduces pyroptosis in the aorta of ApoE -/- mice and macrophages Previous studies showed that apoptosis caused by NLRP3 inflammasomes promotes the development of atherosclerosis. Caspase-1 activation, which converts IL-1β into its mature form, is a critical step (Zhang et al., 2018). First, we investigated whether the expression of pyroptosis-related proteins in the aorta was altered by ATO. Notably, the NLRP3 levels in the aorta were significantly reduced, caspase-1 activation was inhibited, and the abundance of the mature form of IL1β (c-IL-1β) was reduced (Figure 5A, D, B, E). Consistently, the NLRP3 and IL-1β mRNA expression levels in the aorta were significantly reduced in ATO-treated mice fed a high-fat diet (Figure 5F). Furthermore, we performed CD68/IL-1β and CD68/TUNEL double staining of aortic sinuses isolated from ApoE -/- mice. The IL-1β levels in, and proportion of TUNEL-positive cells among, macrophages (CD68 + ) were significantly reduced after ATO treatment in mice fed a high-fat diet (Figure 5K, L, Figure S1H, I).To elucidate the relationship between ATO and pyroptosis, we used THP-1 cells for the in vitro experiments. We treated these cells with ox-LDL (50 μg mL -1 ) to mimic the atherosclerotic environment in vitro . The results showed that ox-LDL significantly increased the expression levels of NLRP3, C-cas1, and C-IL-1β (Figure 5C, G, H,I,J,M), while ATO treatment (2.5 or 5.0 μM) reversed this increase. Consistently, THP-1 cells treated with ATO (2.5 or 5.0 μM) showed similar changes in IL-1β levels in the cell supernatant (Figure 5N). Taken together, these data indicate that ATO inhibits caspase-1 activation, thereby reducing the secretion of mature IL-1β and inhibiting the pyrolysis of macrophages in aortic lesions of ApoE -/- mice(Figure 5O).

and Conclusions At present, atherosclerosis and subsequent acute myocardial infarction are the most frequent causes of death worldwide (Hansson et al., 2011; Grines, 2015). Therefore, the development of drugs that target lipid accumulation and the subsequent inflammatory cascade observed during the progression of this disease is urgently required. Over the last two decades, statins have emerged as effective drugs for reducing atherosclerotic lipoprotein levels and preventing major cardiovascular events. However, various adverse reactions, including rhabdomyolysis, hepatotoxicity, peripheral neuropathy, impaired myocardial contractility, and autoimmune diseases, have been reported to occur after the use of statins. Therefore, it is necessary to re-evaluate the existing statin treatment guidelines (Bellosta et al., 2018). In recent years, clinical and laboratory studies have focused on the use of ATO as a heart stent coating. These studies found that ATO-eluting stents can significantly reduce the area and thickness of the neointima (Shen et al., 2013). Another study found that placing ATO-eluting stents in the coronary arteries of pigs effectively inhibited local inflammation (Zhao et al., 2018).These findings suggest that the anti-inflammatory effects of ATO may represent an effective protective strategy for atherosclerosis. In this study, for the first time, we indicated that ATO could suppress the progression and instability of atherosclerotic plaques by reducing CD36-mediated ox-LDL internalisation, inhibiting inflammatory responses and pyroptosis in macrophages and plaques. Accordingly, our results have revealed a pivotal role of ATO in suppressing the development of atherosclerosis, suggesting the use of ATO as a potential pharmacological atheroprotective strategy. In our previous studies, we confirmed that ATO promoted autophagy in macrophages without affecting their viability in vitro (Fang et al., 2021) . In addition, we confirmed that an appropriate ATO dose would not cause liver and kidney damage or toxic changes in various organs; therefore, our chosen dosage was not significantly toxic and might be clinically useful. In the present study, we first subjected ApoE -/- model mice with atherosclerosis to treatment with ATO. The results showed that ATO significantly reduced atherosclerosis development and altered the plaque composition by reducing macrophage accumulation and increasing collagen content. This evidence highlights the potent effects of ATO against atherosclerosis. The TC, TG, and LDL levels significantly affect the incidence of major vascular events and the development of atherosclerosis in humans (Landray et al., 2014). Lowering TC and LDL levels can prevent atherosclerosis progression (Sarwar et al., 2007). Additionally, many studies demonstrated that elevated TG levels represent an independent risk factor for coronary heart disease. Recently, CD36 has been reported to serve as an effective pharmacological target as it promotes lipid accumulation in the vascular wall and the internalisation of ox-LDL in macrophages (Febbraio et al., 2000; Koelwyn et al., 2018). Moreover, a study of medullary chimeras showed that atherosclerotic lesion formation was significantly reduced in mice receiving CD36-null macrophages, while the reintroduction of CD36 in macrophages doubled the area of atherosclerotic lesions (Febbraio et al., 2004). These studies suggest that lipid metabolism and CD36 expression are important therapeutic targets for atherosclerosis. Our results indicated that ATO markedly reduced the TC, TG, and LDL levels in ApoE -/- mice. In addition, ATO inhibited CD36 expression at the transcriptional and translational levels in vivo and in vitro ; thus, ATO reduced foam cell formation. These results suggest ATO inhibits atherosclerotic plaque progression by regulating blood lipid homeostasis and CD36 expression. However, in addition to CD36, previous studies showed that other members of the scavenger receptor family, including ABCA1, ABCG1, and SR-B1, can prevent foam cell formation and the development of atherosclerosis by promoting cholesterol efflux from macrophages (Navab et al., 2011; Hazen et al., 2012; Huang et al., 2019; Ouimet et al., 2019). Whether ATO can act on other scavenger receptor family members remains to be explored. Our study demonstrates that ATO treatment suppresses inflammatory factor expression and reduces inflammatory responses. Previous studies have indicated that pro-inflammatory cytokines are involved in all stages of atherosclerosis, and vulnerable plaques are rich in inflammatory cells. Moreover, IL-6 signal transduction occurs through multiple mechanisms, including the release of other pro-inflammatory cytokines, which stimulates acute phase protein secretion and prethrombotic mediator release. Another study demonstrates that TNFα blockers have a beneficial effect on preventing the progression of subclinical atherosclerosis and arterial stiffness (Tam et al., 2014; Tie et al., 2015). Here, we show that ATO reduces IL-6 and TNF-α expression and increases IL-10 expression significantly in vivo and in vitro, which further emphasizes the anti-inflammatory and atheroprotective nature of ATO. TLR4 is a classic pattern recognition receptor for activating macrophages, and its role in the occurrence and development of atherosclerotic lesions has been extensively studied (Li et al., 2017). Previous studies showed that TLR4 and CD36 have a synergistic effect in mediating ox-LDL-induced inflammation. Activated TLR4 triggers the activation of downstream signalling molecules, leading to the nuclear translocation of p65 and the transcription of genes encoding various pro-inflammatory cytokines, including TNF, IL-6, and IL-12 p40 (Monaco et al., 2004). Here, we provided the first evidence that ATO inhibited inflammatory responses in macrophages by modulating the TLR4/NF-κB signalling pathway. In this study, we demonstrated that ATO significantly reduced TLR4 expression in vivo and in vitro . In vitro, ATO inhibited the nuclear translocation of p65 by inhibiting IκB-α degradation, similar to the action of BAY-11, an IκB-α degradation inhibitor. Our results suggest that the suppression of TLR4/NF-κB signalling pathway activation may be the specific mechanism by which ATO inhibits atherosclerotic inflammation in macrophages. Duewell et al. transplanted NLRP3 -/- and IL-1α/β -/- bone marrow cells into LDL-deficient mice and found that the knockdown of these inflammatory components reduced the formation of atherosclerotic lesions (Christ et al., 2018). Similarly, caspase-1 deficiency significantly reduces macrophage infiltration and the formation of atherosclerotic lesions in ApoE -/- mice (Usui et al., 2012). Other studies showed that NF-κB activation triggered IL-1β and NLRP3 transcription and ox-LDL could induce IL-1β release (Latz et al., 2018; Zhang et al., 2018). Based on these premises, we proposed whether ATO played an atheroprotective role by inhibiting the pyroptosis caused by NLRP3 inflammasome activation. Firstly, we observed that macrophages in ApoE -/- model mice aortic tissue showed characteristics of pyroprosis. In addition, we demonstrated that ATO inhibited NLRP3 expression at the transcriptional and translational levels in vivo and in vitro . Expression of mature caspase-1 and IL-1β, which were downstream effector molecules of NLRP3, were also reduced by ATO treatment in vivo and in vitro . Taken together, these results indicate that ATO inhibits IL-1β secretion and caspase-1 activation by NLRP3 inflammasomes. Hence, we demonstrate the underlying protective effects of ATO against atherosclerosis and provide mechanistic insights into its atheroprotective effects. However, this study had a few limitations. Firstly, whether ATO inhibits NLRP3 transcription by inhibiting the TLR4/NF-κB signalling pathway lacks direct evidence and is worthy of further investigation. Secondly, in this study, we focused on the role of ATO in regulating lipid metabolism and inflammatory responses in atherosclerosis. However, other mechanisms, such as those underlying macrophage activation or other processes related to the atherosclerosis progression, need further investigation. Thirdly, although our chosen dosage is not significantly toxic and may be clinically useful in this experimental period, it remains unknown whether the side effects of ATO are cumulative over time. Herein, whether long-term treatment of ATO has side effects needs further observation. Lastly, there are important differences between animals and humans, and thus, additional work is needed to fully explore whether the results obtained herein are also applicable in humans. In summary, we confirmed that ATO had protective effects against the progression of atherosclerosis and instability of atherosclerotic plaques in ApoE -/- model mice. Thus, the use of ATO to inhibit macrophage lipid endocytosis and inflammatory responses may represent a potential atheroprotective strategy.

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(C) Representative images of hematoxylin and eosin staining of lesion areas of aortic sinus for ApoE -/- mice treated with vehicle or ATO(As 2 O 3 ). Bar=100 µm. (D) Quantitation of Oil Red O + areas of the entire aorta. Values are mean ± SEM from 9 mice. (E) Quantitation of lesion areas of the two groups. Values are mean ± SEM from 9 mice. (F) Quantitation of necrotic core areas relative to total plague area. Values are mean ± SEM from 9 mice. (G) Cryosections of the aortic sinus stained with Oil Red O; hematoxylin was used as a counterstain. Bar=100 µm. (H) Representative histological findings in masson’s trichrome stained of the aortic root in treated with vehicle or ATO(As 2 O 3 ). Collagen fibers are indicated in blue. Bar=100 µm. (I) Quantitation of necrotic core areas. Values are mean ± SEM from 9 mice. (J) Quantitation of Oil Red O + areas relative to total plague area. Values are mean ± SEM from 9 mice in each group. (K) Quantification of collagen content using a masson’s trichrome staining. (L) Representative photomicrographs of immunofluorescent staining for macrophage(CD68 + ) in aortic sinus sections. Bar = 100 μm. (M) Percentage of CD68 + -stained area relative to total lesion area, quantified with ImageJ software. Values are mean ± SEM from 5 mice in each group. The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs control group. Figure 2. Arsenic trioxide (As 2 O 3, ATO) regulate serum lipid metabolism in ApoE -/- mice and decrease CD36-mediated ox-LDL up-take in macrophages. (A-E) The effects of As 2 O 3 treatment on plasma cholesterol levels. The concentration of total cholesterol(A), triglycerides(B), HDL cholesterol(C), LDL cholesterol(D), and ox-LDL(E), values are mean ± SEM from 6 mice in each group. (F) The mRNA levels of CD36 in the aortic root of ApoE -/- mice treated with vehicle or As 2 O 3 were determined via RT‐PCR analysis. (G) Representative images of CD68 macrophage (green), CD36 staining (red) and CD36 on macrophages (CD68/CD36 merge) in the aortic root. Bar=200 µm. (H) Quantification of CD36 on CD68 positive macrophages in the aortic root. n = 5 mice in each group. (I, L) The protein levels of CD36 in the aortic root of ApoE -/- mice treated with vehicle or As 2 O 3 were revealed by western blot analysis. GAPDH was used as an internal control. (J, K) The protein levels of CD36 in the RAW264.7 cell were revealed by western blot analysis after being treated with ox-LDL (50 μg ml -1 ) in the presence or absence of As 2 O 3 (2.5μM, 5.0 μM) for 24 h. GAPDH was used as an internal control. (M-N) RAW264.7 cell were treated with ox-LDL (50 μg ml -1 ) in the presence or absence of As 2 O 3 (2.5μM,5.0 μM) for 24h. Area of the average size of lipid deposits in RAW264.7 cell. (O) CD36 mRNA expression in RAW264.7 cells was analyzed by RT-qPCR after being treated with ox-LDL (50 μg ml -1 ) in the presence or absence of As 2 O 3 (2.5μM,5.0 μM) for 24 h. The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs control group. # p <0.05, ## p <0.01 vs oxLDL-stimulated group. Figure 3. Arsenic trioxide (As 2 O 3, ATO) inhibits inflammatory factor expression and macrophage inflammatory polarization in ApoE -/- mice. (A) IL-6, (B)TNFα, and (C) IL-10 mRNA levels in serum of ApoE -/- mice treated with vehicle or As 2 O 3 were measured by ELISA assays. (D) IL-6, (E)TNFα, and (F) IL-10 levels in the aortic root of ApoE -/- mice treated with vehicle or As 2 O 3 were determined via RT‐PCR analysis. (G-I) RAW264.7 macrophages were induced by addition of ox-LDL (50 μg ml -1 ) without or with As 2 O 3 (2.5μM,5.0 μM) for 24h. (G) IL-6, (H)TNFα, and (I) IL-10 mRNA levels in cell lysate were determined via RT‐PCR analysis. n = 5 mice in each group. The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs control group. # p <0.05, ## p <0.01 vs ox-LDL-stimulated group. Figure 4. Arsenic trioxide (As 2 O 3, ATO) inhibits activation of TLR4/NF-κB inflammatory pathway in macrophages. (A) The protein level of TLR4 in the aortic root of ApoE -/- mice treated with vehicle or As 2 O 3 were revealed by western blot analysis. GAPDH was used as an internal control. (B) RAW264.7 cell was induced by addition of ox-LDL (50 μg ml -1 ) without or with As 2 O 3 (2.5μM,5.0 μM) for 24h. The protein level of TLR4 in cell lysate was revealed by western blot analysis. GAPDH was used as an internal control. (C) RAW264.7 cell was pretreated with or without BAY-11(5mM, 30min) or As 2 O 3 (2.5μM,5.0 μM,2-6 h) and then the cells were incubated with LPS(200 ng ml -1 ) for 30 min. Nuclear extracts were used to analyze p65 translocation by Western blot. PCNA was used as an internal control to nuclear fractions. (D) Quantitative analysis of TLR4 protein levels in the aortic root. GAPDH was used as an internal control. (E) Quantitative analysis of TLR4 protein levels in RAW264.7 cell lysate. GAPDH was used as an internal control. (F) Quantitative analysis of p65 protein levels in nuclear fractions of RAW264.7 cell.PCNA was used as an internal control. (G) RAW264.7 cell was pretreated with or without BAY-11(5mM, 30min) or As 2 O 3 (2.5,5.0 μM, 2-6 h) and then the cells were incubated with LPS (200 ng ml -1 ) for 30 min. Cytoplasmic extracts were used to analyze p65 translocation by Western blot. GAPDH and was used as an internal control to cytoplasmic fractions. (I) Quantitative analysis of p65 protein levels in cytoplasmic fractions of RAW264.7 cell. GAPDH was used as an internal control. (H-J) RAW264.7 cell was pretreated with or without As2O3 (2.5,5.0 μM, 2-6 h) and then the cells were incubated with LPS (200 ng ml -1 ) for 30 min. Expression of IκBα in RAW264.7 cells was determined by western blot (H) followed by quantitative analysis (J). (K) Schematic diagram of ATO acting on TLR4/ NF-κB signaling pathway. n = 5 mice in each group. The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs control group. # p <0.05, ## p <0.01 vs LPS or ox-LDL-stimulated group. Figure 5. Arsenic trioxide (As 2 O 3, ATO) reduces the characteristics of pyroptosis in the aorta of ApoE -/- mice and inhibits macrophage pyroptosis. (A-B) The protein levels of NLRP3, C-IL-1β and C-caspase-1 in the aortic root of ApoE -/- mice treated with vehicle or As 2 O 3 were revealed by western blot analysis. GAPDH was used as an internal control. (C)THP-1 cell was pretreated with PMA(100μM，12 h) and then the cells were incubated by addition of ox-LDL (50 μg ml -1 ) without or with As 2 O 3 (2.5,5.0 μM) for 24h. The protein levels of NLRP3, C-IL-1β and C-caspase-1 in the THP-1 cells were revealed by western blot analysis.(D-E) Quantitative analysis of NLRP3(D), C-IL-1β and C-caspase-1(E) protein levels in the aortic root. GAPDH was used as an internal control. (F) The mRNA levels of NLRP3 and IL-1β in the aortic root of ApoE -/- mice treated with vehicle or As 2 O 3 were determined via RT‐PCR analysis. (G-I) Quantitative analysis of NLRP3(G), C-IL-1β(H)and C-caspase-1(I) protein levels in THP-1 cell lysate. GAPDH was used as an internal control. (J)The mRNA levels of NLRP3 in Cell lysate were determined via RT‐PCR analysis. (K-L) Representative images of CD68 macrophage (green),IL-1β staining (red) and IL-1β on macrophages (IL-1β/CD36 merge) in the aortic root(K). Bar=200 µm. Quantification of IL-1β on CD68 positive macrophages in the aortic root(L). (M) The mRNA levels of IL-1β in Cell lysate were determined via RT‐PCR analysis. (N) THP-1 cell was pretreated with PMA(100μM，12 h) and then the cells were incubated by addition of ox-LDL (50 μg ml -1 ) without or with As 2 O 3 (2.5,5.0 μM) for 24h. IL-1β levels in culture supernatants were quantified by ELISA. (O) Schematic diagram of ATO acting on NLRP3 signaling pathway. n = 5 mice in each group. The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs control group. # p <0.05, ## p <0.01 vs ox-LDL-stimulated group. Figure 6. Proposed model describing the mechanism of atheroprotection by ATO. The black arrows represent promotion; The red arrows with flat end represent inhibition. Figure Supplement. TableS1: Primers used in this study. Figure S1. (A-B) Haematoxylin staining of ApoE -/- mice heart, liver, spleen, lung and kidney tissues. (C) Body weight was measured every day from day 1. (D-G) After the last treatment, serum levels of ALT(D), AST(E), UA(F) and CK(G) were determined. (H) Quantification of CD68 on IL-1β positive macrophages in the aortic root.(I) Quantification of CD68 on Tunel positive macrophages in the aortic root.n = 10 mice in each group. The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs control group. Figure S2. (A)Flow cytometry was used to detect the apoptosis of spleen and lymph node cells in ApoE -/- mice, where annexin-V + PI - represents early apoptosis. (B)Flow cytometry was used to detect the percentages of T, B, and monocytes in peripheral blood of ApoE -/- mice, and were characterized by CD3, Ly6G, and B220, respectively. (C) Quantification of lymphocytes in PBMC of control and ATO groups. (D) Quantification of apoptosis cells in lymph node and spleen between the control and ATO groups. n = 5 mice in each group. Bullet point summary: What is already known? 1. ATO-eluting stents can significantly reduce the area and thickness of the neointima. 2. Dysfunction of lipid metabolism and pro-inflammatory responses are involved in all stages of atherosclerosis. What this study adds? 1. A potential atheroprotective agent, ATO, which ameliorates atherosclerosis by suppressing lipid endocytosis and macrophage dysfunction. 2. ATO suppresses CD36 expression, and TLR4/NF-κB and NLRP3 inflammasome activation in macrophages, reducing inflammatory responses. What is the clinical significance? 1. ATO presents a potential promising therapeutic agent for atherosclerosis treatment. Supplementary Material File (primer table.docx) - Download - 13.31 KB Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 238views 161downloads Citations Download citation Xiaoyi Zou, Zhaoying Li, Xin Wan, et al. Arsenic trioxide ameliorates atherosclerosis by inhibiting CD36-induced endocytosis and TLR4/NF-κB-induced inflammation in macrophage and ApoE-/- mice. Authorea. 31 January 2024. DOI: https://doi.org/10.22541/au.170669100.03220590/v1 DOI: https://doi.org/10.22541/au.170669100.03220590/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.