- Email: [email protected]
− mice by improving endothelial dysfunction
Accepted Manuscript Longxuetongluo capsule inhibits atherosclerosis progression in high-fat diet-induced −/− ApoE mice by improving endothelial dysfunction Jiao Zheng, Binglin Liu, Qixing Lun, Xiaopan Gu, Bo Pan, Yunfang Zhao, Wei Xiao, Jun Li, Pengfei Tu PII:
S0021-9150(16)31271-0
DOI:
10.1016/j.atherosclerosis.2016.08.022
Reference:
ATH 14754
To appear in:
Atherosclerosis
Received Date: 5 April 2016 Revised Date:
5 July 2016
Accepted Date: 19 August 2016
Please cite this article as: Zheng J, Liu B, Lun Q, Gu X, Pan B, Zhao Y, Xiao W, Li J, Tu P, −/− Longxuetongluo capsule inhibits atherosclerosis progression in high-fat diet-induced ApoE mice by improving endothelial dysfunction, Atherosclerosis (2016), doi: 10.1016/j.atherosclerosis.2016.08.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Longxuetongluo capsule inhibits atherosclerosis
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progression in high-fat diet-induced ApoE−/− mice by
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improving endothelial dysfunction
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Jiao Zheng1, ¶, Binglin Liu 1, 2, ¶, Qixing Lun 1, 2, Xiaopan Gu 1, 2, Bo Pan 1, 2, Yunfang
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Zhao 1, Wei Xiao 3, Jun Li 1, *, Pengfei Tu 1, *
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Medicine, Beijing 100029, China
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Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese
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China
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Kanion Pharmaceutical Co. Ltd., Lianyungang 222001, China
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102,
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National Key Laboratory of Pharmaceutical New Technology for Chinese Medicine, Jiangsu
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*Corresponding authors: Modern Research Center for Traditional Chinese Medicine,
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Beijing University of Chinese Medicine, Beijing 100029, P. R. China. Tel/Fax: (86)-10-82802750
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(P. Tu); Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese
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Medicine, Beijing 100029, P. R. China. Tel/Fax: (86)-10-64286350 (J. Li).
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E-mail addresses: [email protected] (P. Tu) ;[email protected] (J. Li).
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These authors contributed equally to this work.
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ACCEPTED MANUSCRIPT Abbreviations
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CG: chow diet group; COX-2: cyclooxygenase-2; DMEM: Dulbecco’s modified
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Eagle’s medium; EG: ezetimibe-treated group; eNOS: endothelial nitric oxide
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synthase; FBS: fetal bovine serum; HDLc: high density lipoprotein cholesterol; HE:
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hematoxylin and eosin; HFD: high-fat diet; HG: HFD-induced group; HUVECs:
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human umbilical vein endothelial cells; IκB: inhibitor of kappa B; IKK: inhibitor of
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kappa B kinase; LTC: Longxuetongluo capsule; LTC100 (low), LTC200 (medium),
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and LTC300 (high): oral administration at doses of 100, 200, and 300 mg/kg/d,
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respectively; MAPK: mitogen-activated protein kinase; MCP-1: monocyte
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chemotactic protein-1; NF-κB: nuclear factor kappa B; NO: nitric oxide; OCT:
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optimum cutting temperature; Ox-LDL: oxidized low-density lipoprotein; ORO: oil
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Red O; PBS: phosphate buffered saline; SEM: standard error of the mean; TC: total
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cholesterol; TG: triglycerides; VCAM-1: vascular cell adhesion molecule-1.
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Keywords: Chinese dragon’s blood; Longxuetongluo capsule; anti-atherosclerosis;
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endothelial dysfunction.
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Abstract
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Background and aims: Chinese dragon’s blood has been used to treat blood stasis for
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thousands of years. Its total phenolic extract (Longxuetongluo capsule, LTC) is used
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for the treatment of ischemic stroke; however, its protective effect against
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atherosclerosis remains poorly understood. This paper aims to investigate the
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antiatherosclerotic effect of LTC and the underlying mechanisms in high-fat diet
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(HFD)-induced ApoE−/− mice.
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Methods: The levels of plasma lipid and areas of atherosclerotic lesions in the aortic
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sinus in ApoE−/− mice were evaluated. The effect of LTC on the nitric oxide (NO)
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production in oxidized low-density lipoprotein (ox-LDL)-stimulated human umbilical
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vein endothelial cells (HUVECs) was determined. The adhesion of monocytes to
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ox-LDL-stimulated HUVECs was further studied.
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Results: LTC at low, medium, and high doses markedly decreased the atherosclerotic
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lesion areas of the aortic sinus in HFD-induced ApoE−/− mice by 26.4% (p<0.05),
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30.1% (p<0.05), and 46.5% (p<0.01), respectively, although it did not improve the
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dyslipidemia. Furthermore, LTC restored the diminished NO production of
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ox-LDL-stimulated HUVECs (p<0.001) and inhibited the adhesion between
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monocytes and endothelial cells (p<0.01). LTC appeared to alleviate
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ox-LDL-stimulated dysfunction of HUVECs, and inhibit the adhesion of monocytes
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to HUVECs via the MAPK/IKK/IκB/NF-κB signaling pathway, thus decrease
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atherosclerotic lesions in the aortic sinus in HFD-induced ApoE−/− mice.
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Conclusions: These findings suggest the potential of LTC for use as an effective agent
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against atherosclerosis.
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1. Introduction Atherosclerosis is characterized by a disorder of lipid metabolism and deposition,
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the primary step of which is associated with inflammation [1]. Oxidized low-density
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lipoprotein (ox-LDL), which is believed to play a critical role in the process of
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atherogenesis [2], is known to enhance the expression of endothelial inflammatory
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factors [3], and inhibit the endothelial production of nitric oxide (NO) [4]. Decreased
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NO production is related to endothelial dysfunction [5]. The dysfunctional endothelial
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cells can increase the expression of adhesion molecules such as vascular cell adhesion
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molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1), which in turn
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mediate the recruitment of monocytes and their adhesion to the endothelium, thus
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promoting the development of atherosclerotic plaques [6].
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Chinese dragon’s blood, the red resin of Dracaena cochinchinensis (Lour.) S. C. Chen, has been used to improve perfusion, alleviate blood stasis, relieve pain, and
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stop bleeding in traditional Chinese medicine [7]. Its analgesic [8], wound-healing [9],
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antibacterial [10], and anti-inflammatory [8] properties are well documented. Phenolic
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compounds such as flavanones, flavans, dihydrochalcones, chalcones, stilbenes, and
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oligomeric flavonoids are considered to be its main active ingredients [11].
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Longxuetongluo capsule (LTC), which contains the total phenolic extract of Chinese
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dragon’s blood, was approved for the treatment of ischemic stroke by China Food and
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Drug Administration in June 2013 [19]. Previously, we found that LTC could
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improves erythrocyte function against lipid peroxidation and abnormal
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hemorheological parameters in high-fat diet (HFD)-induced ApoE−/− mice [12].
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Moreover, the anti-inflammatory activity of Chinese dragon’s blood has been
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demonstrated both in vivo [8] and in vitro [13]. However, the definitive evidence of
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the anti-atherosclerotic effect of LTC has not been proved in vivo, and its
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anti-inflammatory activity on human umbilical vein endothelial cells (HUVECs) has
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not been clearly investigated.
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The aim of this study was to investigate the anti-atherosclerotic effect of LTC in ApoE−/− mice with HFD-induced hypercholesterolemia, as well as the possible
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underlying mechanisms for its effect in improving effect on endothelial dysfunction
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via the regulation of nuclear factor kappa B (NF-κB) and mitogen-activated protein
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kinase (MAPK) signaling pathways.
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2. Materials and methods
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2.1. Reagents
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Ezetimibe was purchased from Schering-Plough Pte Ltd. (Lot: 2EZPA17005).
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Commercial total cholesterol (TC) and triglyceride (TG) kits for measurement of
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plasma lipids levels were purchased from Biosino Bio-technology and Science, Inc.
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(Beijing, China). Optimum cutting temperature (OCT) compound was purchased from
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Sakura Finetek USA, Inc. (Torrance, USA). The Oil Red O (ORO) was purchased
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from Sigma-Aldrich (St Louis, USA). Ox-LDL was purchased from Yiyuan
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Biotechnologies (Guangzhou, China). RPMI-1640, Dulbecco’s modified Eagle’s
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medium (DMEM), and fetal bovine serum (FBS) were purchased from Mediatech, Inc.
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(A Corning Subsidiary, Manassas, USA). Commercial kit for NO determination and
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the ECL chemiluminescence detection kit were purchased from Applygen
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Technologies, Inc. (Beijing, China). The fluorescent dye BCECF AM was purchased
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from Beyotime (Jiangsu, China). Primary antibodies against VCAM-1, MCP-1, and
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cyclooxygenase-2 (COX-2) were obtained from Abcam (Cambridge, UK). Primary
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antibody against endothelial nitric oxide synthase (eNOS) was obtained from BD
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Biosciences (San Jose, USA). Primary antibodies of NF-κB signaling pathway (p65,
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P-p65, IκBα, P-IκBα) and MAPK signaling pathway (ErK1/2, P-ErK1/2, p38, P-p38,
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JNK, P-JNK) were obtained from Cell Signaling Technology (Danvers, MA, USA).
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2.2. Medicinal materials
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LTC was provided by Jiangsu Kanion Pharmaceutical Co. Ltd. (Jiangsu, China).
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The quantitative and qualitative characterization of chemical components in LTC was
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performed as described previously [12].
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2.3. Animals
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Male ApoE−/− mice, weighing 19–23 g, were obtained from the Animal Center of Peking University Health Science Center. All experiments were approved by the
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Animal Research Committee of Peking University Health Science Center (Beijing,
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China). The Principles of Laboratory Animal Care (NIH Publication 85–23, revised
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1996) were followed. Mice were housed at 24 ± 1 °C and a relative humidity of 50 ±
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1% with a light/dark cycle of 12 h. Mice were randomly divided into six groups (n = 9
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for each group): the chow diet group (CG), the HFD-induced group (HG), the low,
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medium, high-doses LTC treatment groups (LTC100, LTC200, and LTC300,
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respectively), and the ezetimibe treatment group (EG). Ezetimibe, a potent cholesterol
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absorption inhibitor, was used as a positive control drug in the present study. Mice
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assigned to LTC100, LTC200, and LTC300 groups received 100, 200, and 300
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mg/kg/d LTC, orally, respectively; those in the EG group received 30 mg/kg/d
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ezetimibe, for 6 successive weeks. All mice were fed HFD (0.2% cholesterol and 15%
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fat added) for 1 week prior to the start of various treatments. The mice were fed with
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HFD or chow diet throughout this experiment. The food and water were available ad
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libitum in each group throughout this experiment.
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2.4. Food intake, body weight and composition measurements Food intake and body weight were measured per 2 weeks with a scale. For food consumption measurement, the amount of normal diet consumed was carefully
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monitored at 10:00 AM local time every morning. The mice were fasted for 6 h and
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the blood was collected and anticoagulated by heparin for the measurement of plasma
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lipids levels. Levels of TC and TG were assessed enzymatically using commercial kits,
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and high-density lipoprotein cholesterol (HDLc) was measured by the same TC kit
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after the precipitation of ApoB-lipoprotein with the 20% polyethylene glycol solution.
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2.5. Measurement of atherosclerotic lesions in the aortic sinus Lesions in the aortic sinus were analyzed according to the modified method [13, 14]. The mice were sacrificed by lethal intravenous injection of a sodium
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pentobarbital; the heart and adjoining aorta was perfused through the left ventricle
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with phosphate-buffered saline (PBS) for 10 min and subsequently fixed in
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phosphate-buffered formaldehyde (4%, pH 7.4). Hearts with ascending aortas were
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embedded in OCT compound and stored at −80 °C until further processing. Serial
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sections (7 µm thick) were prepared using a Leica cryostat starting from the
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appearance of the aortic valve to the ascending aorta until the point where valve cusps
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were no longer visible. The sections were stained with ORO and hematoxylin and
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eosin (HE) for the detection of atherosclerotic lesions and morphometric analysis,
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respectively. For the quantification of total atherosclerotic lesion area, digital images
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of the stained lesions were captured by a Leica icc50 HD microscope and analyzed by
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ImageJ software.
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2.6. Cell culture The SV40-transformed human endothelial cells (PUMC-HUVEC-T1) were
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purchased from China infrastructure of cell line resources and grown in DMEM
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containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Human
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monocytic cell line THP-1 cells were grown in RPMI-1640 containing 10% FBS, 100
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U/mL penicillin, and 100 µg/mL streptomycin. Cultures were maintained at 37 °C in a
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humidified atmosphere containing 5% CO2. HUVECs between passages 3 and 15
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were used in the current study.
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2.7. Measurement of NO production
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To assess the effect of LTC on ox-LDL-stimulated NO production in HUVECs, confluent HUVECs were detached by 0.125% trypsin and grown in 96-well plates. On
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attaining a confluence of 70–80%, cells were incubated with 50 µg/mL ox-LDL in 2%
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FBS DMEM in the presence or absence of various concentrations (1, 10, 20, and 40
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µg/mL) of LTC. After further incubation for 24 h, NO production was determined
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using a commercial kit. Cell culture supernatants (50 µL) were collected and mixed
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with 100 µL of Griess reagents in 96-well plates. The absorbance was measured at
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540 nm, and nitrite concentration was calculated by reference to a sodium nitrite
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standard curve.
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2.8. Cell adhesion assay
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HUVECs were cultured in 48-well plates for 18 h to achieve 90% confluence and
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subsequently stimulated with 20 µg/mL ox-LDL in the presence or absence of various
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concentrations (10, 20, and 40 µg/mL) of LTC for 24 h. THP-1 cells were labeled with 8
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37 °C for 30 min, and subsequently the cells (5 × 105 cells/mL) were seeded onto the
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confluent HUVECs and incubated for another 30 min at 37 °C [15]. Non-adherent
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THP-1 cells were removed by gently washing with PBS for three times; adherent
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THP-1 cells were examined under an inverted phase-contrast fluorescence
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microscope.
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2.9. Western blot analysis
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HUVECs were cultured in 6-well plates for 18 h to achieve 90% confluence, and then stimulated with 20 µg/mL ox-LDL in the presence or absence of various
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concentrations (10, 20, and 40 µg/mL) of LTC for 24 h. The cells were then washed
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with cold PBS and lysed using lysis buffer (0.05% bromophenolblue, 10% sodium
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dodecyl sulfate, 40% glycerol, 0.01 M dithiothreitol, and 0.2 M Tris-HCl, pH 6.8).
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Sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and Western blot analysis
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were performed as previously described [14]. After blocking in 4% (w/v) nonfat dry
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milk, membranes were incubated with primary antibodies in optimized dilutions at
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4 °C overnight. Membranes were then washed and incubated with a 1:7,000 dilution
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of second antibody at 4 °C for 3 h and examined with the ECL chemiluminescence
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detection kit.
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2.10. Statistical analysis Data were presented as means ± standard error of the mean (SEM). Between-group
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differences were evaluated by one-way analysis of variance (ANOVA), followed by
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Dunnett’s post hoc test or Student’s t-test for unpaired observations, if appropriate.
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Between-group differences with associated p values of <0.05 were considered 9
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statistically significant.
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3. Results
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3.1. LTC reduces atherosclerotic lesions in the aortic sinus, but had no effect on
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plasma lipid levels in the HFD-induced mice
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The TC, TG, and non-HDLc levels of the AopE−/− mice fed on HFD were
significantly increased as compared to those in the mice fed a chow diet (p<0.001,
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p<0.01, and p<0.001, respectively; Fig. 1A, a-d), while the HDLc level significantly
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decreased (p<0.01). Ezetimibe, a potent cholesterol absorption inhibitor, was used as a
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positive control drug in the present study. After six weeks treatment with ezetimibe
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(30 mg/kg/day), a significantly reduction in the plasma levels of TC, TG, and
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non-HDLc levels were observed as compared with those in untreated mice fed the
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same HFD (p<0.001, p<0.001, and p<0.001, respectively). However, the effect of
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LTC on lipid levels in the HFD-induced mice was not observed in all groups. As for
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the body weight, there was no difference among the groups. (Figure 1 A, e). Then the
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results of food intakes showed that the HFD treatment remarkably reduced the food
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intakes in of AopE−/− mice (p <0.001), and all doses of LTC had no influence on food
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intake (Figure 1A, f).
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The development of atherosclerotic lesions in the aortic sinus was quantified by
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ORO staining followed by histological analysis (Fig. 1B and C). The lesion areas of
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the AopE−/− mice fed with the HFD significantly increased compared with those in the
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chow diet-mice (p<0.001). Compared with the untreated mice fed with the same HFD,
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ezetimibe markedly reduced the size of the lesion areas by 62.4% (p<0.001),
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measured as 106815 ± 58753 µm2 vs. 283973 ± 121175 µm2. The lesion areas were
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significantly reduced in low (26.4%, p<0.05), medium (30.1%, p<0.05), and high 10
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(46.5%, p<0.01) doses of LTC-treated mice, measured as 209112 ± 38804 µm2,
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198634 ± 198634 µm2, and 151892 ± 44668 µm2, respectively.
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3.2. LTC increased NO production and the eNOS protein expression of HUVECs
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stimulated by ox-LDL
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Endothelial injury is the primary event in the pathogenesis of atherosclerosis [6]. Ox-LDL-induced endothelial injury plays a pivotal role in the initiation of
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atherogenesis [2]. NO production by vascular endothelial cells is known to induce
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vasodilation and has an antiatherogenic effect [16]. The effect of LTC on the
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production of NO in HUVECs was further studied. Stimulation with ox-LDL
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significantly decreased NO production by HUVECs, while LTC treatment was
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associated with increased production of NO in HUVECs that were stimulated by
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ox-LDL, in a dose-dependent manner (Fig. 2A). Since LTC significantly increased the
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NO production by ox-LDL-stimulated HUVECs, the effect of LTC on the expression
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of eNOS protein in stimulated HUVECs was investigated by Western blot analysis
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(Fig. 2B). The eNOS protein expression level was significantly decreased by ox-LDL
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treatment. LTC dose-dependently increased the same protein levels in
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ox-LDL-stimulated HUVECs. However, LTC didn’t affect the NO production and
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eNOS protein expression in normal HUVECs without ox-LDL treatment (Fig. 2C and
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D). These data suggested that LTC could up-regulate ox-LDL-stimulated eNOS
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expression in HUVECs.
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3.3. LTC inhibited adhesion of THP-1 cells to ox-LDL-stimulated HUVECs Ox-LDL is known to enhance the adhesion of human monocytes to endothelial cells [17], which is regarded as an initial step of atherogenesis [6]. In the present study, 11
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ox-LDL was investigated. Ox-LDL significantly increased the adhesion between
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THP-1 cells and HUVECs (p<0.01). This finding is consistent with the previous
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report [18]. LTC dose-dependently reduced the adhesion of THP-1 cells to the
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ox-LDL-stimulated HUVECs (Fig. 3).
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3.4. Effects of LTC on VCAM-1, MCP-1, and COX-2 protein expression in
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ox-LDL-stimulated HUVECs
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The VCAM-1 and MCP-1, which contribute to macrophage migration and adhesion to endothelial surface [6], are regarded to participate in the ox-LDL-simulated
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inflammatory signaling [19]. The induction of COX-2 expression is a hallmark
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finding of atherosclerosis that indicates its inflammatory origin [20]. VCAM-1,
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MCP-1, and COX-2 protein expression levels were markedly increased in
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ox-LDL-stimulated HUVECs as compared to that in untreated cells. However,
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LTC-treatment appeared to reduce the expression levels of VCAM-1 and COX-2
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protein in stimulated HUVECs in a dose-dependent manner. Furthermore, LTC
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treatment at high doses was associated with a significant decrease in MCP-1 protein
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expression (Fig. 4).
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3.4. LTC regulated NF-κB and MAPK signaling pathway in ox-LDL-stimulated
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HUVECs
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NF-κB is an essential factor in controlling inflammatory mediators [21]. It is
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known to be activated upon phosphorylation of inhibitor of kappa B (IκB), a reaction
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catalyzed by the cytoplasmic inhibitor of kappa B kinase (IKK) complex [21]. NO
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released from eNOS inhibits the cleavage of IκB and the consequent activation of 12
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of which is in turn regulated by MAPKs [23]. In the present study, LTC treatment
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appeared to inhibit the phosphorylation of NF-κB-p65 and IκBα in
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ox-LDL-stimulated HUVECs and suppresse the degradation of IκBα in the cytosolic
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extracts of stimulated HUVECs, which indicates a dose-dependent inhibitory effect of
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LTC treatment on the activation of NF-κB, possibly mediated by inducing the
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inhibition of phosphorylation and degradation of IκBα (Fig. 5A). Further investigation
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on the upstream of NF-κB showed that LTC dose-dependently decreased the
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phosphorylation of IKKα/β, and the expression of IKKα and IKKβ in
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ox-LDL-stimulated HUVECs (Fig. 5B). These findings suggest a potential inhibitory
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effect of LTC on the IKK/IκB/NF-κB inflammatory signaling pathway.
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Previous studies have shown that ErK1/2, p38, and JNK MAPKs partly regulated
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the activation of NF-κB [24]. Thus, the observation that LTC suppressed the NF-κB
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signaling pathway prompted us to explore whether MAPKs were involved in this
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process. Ox-LDL significantly induced the phosphorylation of ErK1/2 and p38
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MAPKs in HUVECs (Fig. 5C). The levels of phosphorylated ErK1/2 and p38 MAPKs
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in ox-LDL-stimulated HUVECs appeared to be suppressed by LTC in a
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dose-dependent manner. However, no significant change in phospho-JNK expression
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was observed in ox-LDL-stimulated HUVECs (data not shown). These results
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indicated that LTC-induced suppression of IKK/IκB/NF-κB inflammatory signaling
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pathway is mediated via the suppression of MAPKs activation (Fig. 5D).
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4. Discussion Increased plasma level of low-density lipoprotein is regarded as a major risk factor 13
ACCEPTED MANUSCRIPT of atherogenesis [25], and its oxidative modification plays as a critical role in the
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pathogenesis of atherosclerosis [2]. Lipid-lowering therapies reduce the occurrence of
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these clinical events [26]. Our results showed that ezetimibe treatment is associated
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with a significant decrease in plasma lipid levels in HFD-induced ApoE−/− mice,
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compared with HG mice, while LTC did not affect the plasma lipids levels. These data
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indicate that LTC had no lipid-lowering effect.
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Atherosclerosis is a risk factor for major coronary heart disease event. It is
generally accepted that the progression of atherosclerosis is associated with chronic
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inflammation in the vessel wall. Reports on the anti-inflammatory activity of Chinese
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dragon’s blood prompted us to investigate the antiatherosclerosis effect of LTC in vivo
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and in vitro. Our findings indicated that LTC reduced the lesion areas in the aortic
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sinus in vivo and appeared to reverse the endothelial dysfunction stimulated by
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ox-LDL in vitro.
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Ox-LDL is instrumental in inflammation and thus in the pathogenesis of
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atherosclerosis and endothelial dysfunction [2]. As an indicator of endothelial
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dysfunction, the reduction of the bioactivity (bioavailability) of NO could be
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attributed to the stimulation of ox-LDL [5]. In addition, dysfunctional endothelial
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cells release adhesion molecules, such as VCAM-1 and MCP-1, that promote the
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adhesion of monocyte to endothelial cells [15]. In the present study, LTC not only
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increased the production of NO in ox-LDL-stimulated HUVECs but also inhibited
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adhesions of monocyte to HUVECs. Further investigation indicated that LTC
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increased the expression of eNOS and decreased that of VCAM-1, MCP-1, and
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COX-2 in stimulated HUVECs. Our findings suggest that alleviation of the
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ox-LDL-stimulated dysfunction of HUVECs is attributable to its antiinflammatory
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effect that inhibits monocyte adhesion to HUVECs and exerts its antiatherosclerotic
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effect in ApoE−/− mice. The activated NF-κB is known to induce the expression of adhesion molecules [27], such as VCAM-1 and MCP-1, and inflammatory mediators, such as COX-2 [25].
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Furthermore, the activation of NF-κΒ is implicated in the ox-LDL-stimulated
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dysfunction in the endothelial progenitor cells [28] and bovine aortic endothelial cells
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[29]. Therefore, the decrease of the atherosclerotic lesion areas in the aortic sinus of
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LTC-treated ApoE−/− mice fed HFD prompted us to further investigate the potential
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role of the activation of NF-κB. We further investigated whether the effect of LTC in
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alleviating the dysfunction of HUVECs was mediated via the inhibition of the
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activation of NF-κB, which in turn suppresses the adhesion of monocyte to
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endothelial cells. In the present study, LTC could dose-dependently down-regulate the
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ox-LDL-stimulated degradation and phosphorylation of IκBα, thus inhibiting the
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activation of NF-κB-p65.
In addition, the decrease of eNOS is critically associated with endothelial
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dysfunction, the expression of which in endothelial cells may play a complementary
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role in the endothelial activation of NF-κB. The inhibition of endothelial NF-κB
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activation was shown to suppress the lipopolysaccharide-induced eNOS
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down-regulation in vascular tissues [30]. Furthermore, the production of NO by eNOS
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was shown to inhibit vascular inflammatory NF-κB activity via inducing IκB, and
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eNOS could regulate the expression of NF-κB [31]. The phosphorylation of IκB is
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regulated by the IKK complex, consisting of at least three subunits: IKKα, IKKβ, and
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IKKγ. IKKβ is considered to play a greater role than IKKα in NF-κB activation [32].
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Moreover, eNOS is associated with the IKK complex [32]. The IKK/IκB/NF-κB
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pathway was earlier reported to be involved in the palmitic acid-induced dysfunction
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of HUVECs [33]. In the present study, a significant increase in the expressions of
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ACCEPTED MANUSCRIPT IKKα, IKKβ, and the phospho-IKKα/β in stimulated HUVECs were observed, which
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is consistent with the results from a previous study on the ox-LDL-stimulated human
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primary coronary artery endothelial cells [34]. The inhibitory effect of LTC on the
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expressions of IKKα, IKKβ, and the phospho-IKKα/β further indicates that its effect
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on the alleviation of the ox-LDL-stimulated dysfunction of HUVECs may be
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mediated via the IKK/IκB/NF-κB signaling pathway.
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MAPKs, known as intracellular effectors of cytokine receptor stimulation, have been shown to regulate the IKK complex [35]. The exposure of human brain
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microvessel endothelial cells to ox-LDL is known to induce changes in MAPKs [36].
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Moreover, the activation of ErK1/2 MAPK in ox-LDL-stimulated human coronary
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artery endothelial cells has been reported to play a critical role in the expression of
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MCP-1 [37]. Therefore, we further investigated protein expressions of major MAPKs
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(ErK1/2, p38, and JNK) in ox-LDL-stimulated HUVECs. We observed an increase in
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the phosphorylation of two MAPKs (ErK1/2 and p38) increased in
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ox-LDL-stimulated HUVECs, which is consistent with a previous report [24]. LTC
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dose-dependently decreased the phosphorylation of both ErK1/2 and p38 MAPK in
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ox-LDL-stimulated HUVECs without having any effect on the ErK1/2 and p38
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MAPK. However, LTC did not significantly affect the JNK MAPK phosphorylation.
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Therefore, the significant decrease in phosphorylation of two MAPKs (ErK1/2 and
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p38) in ox-LDL-stimulated HUVECs suggests a wider pharmacological effect of LTC
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on the inflammatory signaling pathways. These findings suggest that the
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antiatherosclerotic effect of LTC is mediated via the MAPKs/IKK/IκB/NF-κB
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signaling pathway (Fig. 5).
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In the present study, LTC caused a significant decrease in the atherosclerotic lesion areas in the aortic sinus of the HFD-induced ApoE−/− mice. Further in vitro 16
ACCEPTED MANUSCRIPT investigation indicated that the effect of LTC in alleviating the ox-LDL-stimulated
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dysfunction of HUVECs was mediated via the MAPKs/IKK/IκB/NF-κB signaling
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pathway, thus inhibiting the adhesion of THP-1 cells to the HUVECs. These in vivo
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and in vitro finding indicate that the anti-atherosclerotic effect of LTC may be
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improve the inducible endothelial dysfunction via the MAPKs/IKK/IκB/NF-κB
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signaling pathway and the inhibition of monocyte adhesion to endothelial cells.
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Conflict of interest
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The authors declared they do not have anything to disclose regarding conflict of
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interest with respect to this manuscript.
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Financial support
This work was supported by grants from the National Natural Science Foundation
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of China (Nos. 81503286, 81573572, 81530097), and New Century Excellent Talents
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in University (No. NCET-13-0693).
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Figure legends
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Fig. 1. Effects of LTC on plasma lipids levels and atherosclerotic lesions in
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HFD-induced ApoE−/− mice. (A) Plasma levels of TC (a), TG (b), HDLc (c), and
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non-HDLc (d) in ApoE−/− mice. (n= 5–8). Food intake (e) and body weight per mouse
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(f) during the 6 week experiments. (B) LTC inhibits atherosclerotic lesions. (a) ORO
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and HE staining of atherosclerotic lesions at the same level of aortic sinus from
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HFD-induced ApoE−/− mice. (b) Quantitative analyses of the atherosclerotic lesion
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areas in the aortic sinus. The mean values from 5–8 mice are presented. # p<0.05 vs.
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CG; ## p<0.01 vs. CG; ### p<0.001 vs. CG; * p<0.05 vs. HG; ** p<0.01 vs. HG; ***
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p<0.001 vs. HG. The arrows indicate the sites of positive staining. TC, total
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cholesterol; TG, triglycerides; HDLc, high density lipoprotein cholesterol; HFD,
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high-fat diet; LTC, Longxuetongluo capsule; CG, chow diet group; HG, HFD-induced
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group; LTC100, low-dose LTC, LTC200, medium-dose LTC; LTC300, high-dose LTC;
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EG, ezetimibe treatment group.
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Fig. 2. Effects of LTC on NO production and eNOS expression in HUVECs. The
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NO production of HUVECs with ox-LDL-stimulated (A) or absence (C) after LTC
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treatment. Western blot analysis of eNOS in HUVECs with ox-LDL-stimulated (B) or
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absence (D) after LTC treatment. # p<0.05 vs. HUVECs in control; ** p<0.01 vs.
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HUVECs stimulated by ox-LDL; *** p<0.001 vs. HUVECs stimulated by ox-LDL.
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Fig. 3. LTC inhibited the adhesion of THP-1 cells to HUVECs stimulated by
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ox-LDL. (A) Adhesion of BCECF AM labeled THP-1 cells with untreated HUVECs.
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(B) Adhesion of BCECF AM labeled THP-1 cells to HUVECs stimulated with 20
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µg/mL ox-LDL. (C–E) Adhesion of BCECF AM labeled THP-1 cells to 24
ACCEPTED MANUSCRIPT ox-LDL-stimulated HUVECs treated with various concentrations (10, 20, and 40
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µg/mL, respectively) of LTC. (F) The quantitative analysis of relative fluorescence of
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BCECF AM labeled THP-1 cells adherent to HUVECs Each bar of the representative
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images represents 200 µm. Each bar represents means (± SEM) values from six
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experiments. # p<0.05 vs. HUVECs in control; ## p<0.01 vs. HUVECs in control; **
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p<0.01 vs. HUVECs stimulated by ox-LDL.
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Fig. 4. Effect of LTC on VCAM-1, MCP-1, and COX-2 expression levels in
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ox-LDL-stimulated HUVECs. Western blot analysis of VCAM-1 (A), MCP-1 (B),
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and COX-2 (C) in HUVECs stimulated with 20 µg/mL ox-LDL in the presence or
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absence of LTC at different concentrations for 24 h. # p<0.05 vs. HUVECs in control;
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* p<0.05 vs. HUVECs stimulated by ox-LDL; ** p<0.01 vs. HUVECs stimulated by
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ox-LDL; *** p<0.001 vs. HUVECs stimulated by ox-LDL.
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Fig. 5. LTC inhibited NF-κB signaling pathway through the regulation of the
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MAPK signaling pathway in ox-LDL-stimulated HUVECs. (A) LTC
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dose-dependently downregulated the phosphorylation of NF-κB-p65 and IκBα and the
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degradation of IκBα. (B) LTC decreased the protein expression of phospho-IKKα/β,
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IKKα, and IKKβ in a dose-dependent manner. (C) LTC downregulated the
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phosphorylation of ErK1/2 and p38, without having an effect on protein expression of
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ErK1/2, p38 and JNK and phospho-JNK. (D) The sketched signal cascade was
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involved in the relationship between the ox-LDL-stimulated endothelial dysfunction
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and the atherosclerotic lesions in the aortic sinus. # p<0.05 vs. HUVECs in control; ##
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p<0.01 vs. HUVECs in control; * p<0.05 vs. HUVECs stimulated by ox-LDL; **
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p<0.01 vs. HUVECs stimulated by ox-LDL; *** p<0.001 vs. HUVECs stimulated by
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ACCEPTED MANUSCRIPT ox-LDL. Red arrows and T-shape arrows indicate stimulatory and inhibitory effect of
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LTC, respectively.
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ACCEPTED MANUSCRIPT Highlights: Longxuetongluo capsule (LTC) delays atherosclerosis development. LTC increased NO production and the eNOS protein expression level of HUVECs stimulated by ox-LDL.
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LTC regulated NF-κB and MAPK signaling pathway in ox-LDL-stimulated HUVECs.
S0021-9150(16)31271-0
DOI:
10.1016/j.atherosclerosis.2016.08.022
Reference:
ATH 14754
To appear in:
Atherosclerosis
Received Date: 5 April 2016 Revised Date:
5 July 2016
Accepted Date: 19 August 2016
Please cite this article as: Zheng J, Liu B, Lun Q, Gu X, Pan B, Zhao Y, Xiao W, Li J, Tu P, −/− Longxuetongluo capsule inhibits atherosclerosis progression in high-fat diet-induced ApoE mice by improving endothelial dysfunction, Atherosclerosis (2016), doi: 10.1016/j.atherosclerosis.2016.08.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Longxuetongluo capsule inhibits atherosclerosis
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progression in high-fat diet-induced ApoE−/− mice by
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improving endothelial dysfunction
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Jiao Zheng1, ¶, Binglin Liu 1, 2, ¶, Qixing Lun 1, 2, Xiaopan Gu 1, 2, Bo Pan 1, 2, Yunfang
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Zhao 1, Wei Xiao 3, Jun Li 1, *, Pengfei Tu 1, *
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Medicine, Beijing 100029, China
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Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese
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China
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Kanion Pharmaceutical Co. Ltd., Lianyungang 222001, China
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102,
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National Key Laboratory of Pharmaceutical New Technology for Chinese Medicine, Jiangsu
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*Corresponding authors: Modern Research Center for Traditional Chinese Medicine,
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Beijing University of Chinese Medicine, Beijing 100029, P. R. China. Tel/Fax: (86)-10-82802750
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(P. Tu); Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese
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Medicine, Beijing 100029, P. R. China. Tel/Fax: (86)-10-64286350 (J. Li).
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E-mail addresses: [email protected] (P. Tu) ;[email protected] (J. Li).
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¶
These authors contributed equally to this work.
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ACCEPTED MANUSCRIPT Abbreviations
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CG: chow diet group; COX-2: cyclooxygenase-2; DMEM: Dulbecco’s modified
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Eagle’s medium; EG: ezetimibe-treated group; eNOS: endothelial nitric oxide
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synthase; FBS: fetal bovine serum; HDLc: high density lipoprotein cholesterol; HE:
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hematoxylin and eosin; HFD: high-fat diet; HG: HFD-induced group; HUVECs:
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human umbilical vein endothelial cells; IκB: inhibitor of kappa B; IKK: inhibitor of
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kappa B kinase; LTC: Longxuetongluo capsule; LTC100 (low), LTC200 (medium),
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and LTC300 (high): oral administration at doses of 100, 200, and 300 mg/kg/d,
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respectively; MAPK: mitogen-activated protein kinase; MCP-1: monocyte
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chemotactic protein-1; NF-κB: nuclear factor kappa B; NO: nitric oxide; OCT:
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optimum cutting temperature; Ox-LDL: oxidized low-density lipoprotein; ORO: oil
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Red O; PBS: phosphate buffered saline; SEM: standard error of the mean; TC: total
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cholesterol; TG: triglycerides; VCAM-1: vascular cell adhesion molecule-1.
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Keywords: Chinese dragon’s blood; Longxuetongluo capsule; anti-atherosclerosis;
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endothelial dysfunction.
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Abstract
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Background and aims: Chinese dragon’s blood has been used to treat blood stasis for
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thousands of years. Its total phenolic extract (Longxuetongluo capsule, LTC) is used
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for the treatment of ischemic stroke; however, its protective effect against
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atherosclerosis remains poorly understood. This paper aims to investigate the
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antiatherosclerotic effect of LTC and the underlying mechanisms in high-fat diet
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(HFD)-induced ApoE−/− mice.
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Methods: The levels of plasma lipid and areas of atherosclerotic lesions in the aortic
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sinus in ApoE−/− mice were evaluated. The effect of LTC on the nitric oxide (NO)
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production in oxidized low-density lipoprotein (ox-LDL)-stimulated human umbilical
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vein endothelial cells (HUVECs) was determined. The adhesion of monocytes to
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ox-LDL-stimulated HUVECs was further studied.
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Results: LTC at low, medium, and high doses markedly decreased the atherosclerotic
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lesion areas of the aortic sinus in HFD-induced ApoE−/− mice by 26.4% (p<0.05),
16
30.1% (p<0.05), and 46.5% (p<0.01), respectively, although it did not improve the
17
dyslipidemia. Furthermore, LTC restored the diminished NO production of
18
ox-LDL-stimulated HUVECs (p<0.001) and inhibited the adhesion between
19
monocytes and endothelial cells (p<0.01). LTC appeared to alleviate
20
ox-LDL-stimulated dysfunction of HUVECs, and inhibit the adhesion of monocytes
21
to HUVECs via the MAPK/IKK/IκB/NF-κB signaling pathway, thus decrease
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atherosclerotic lesions in the aortic sinus in HFD-induced ApoE−/− mice.
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Conclusions: These findings suggest the potential of LTC for use as an effective agent
24
against atherosclerosis.
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1. Introduction Atherosclerosis is characterized by a disorder of lipid metabolism and deposition,
4
the primary step of which is associated with inflammation [1]. Oxidized low-density
5
lipoprotein (ox-LDL), which is believed to play a critical role in the process of
6
atherogenesis [2], is known to enhance the expression of endothelial inflammatory
7
factors [3], and inhibit the endothelial production of nitric oxide (NO) [4]. Decreased
8
NO production is related to endothelial dysfunction [5]. The dysfunctional endothelial
9
cells can increase the expression of adhesion molecules such as vascular cell adhesion
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molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1), which in turn
11
mediate the recruitment of monocytes and their adhesion to the endothelium, thus
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promoting the development of atherosclerotic plaques [6].
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Chinese dragon’s blood, the red resin of Dracaena cochinchinensis (Lour.) S. C. Chen, has been used to improve perfusion, alleviate blood stasis, relieve pain, and
15
stop bleeding in traditional Chinese medicine [7]. Its analgesic [8], wound-healing [9],
16
antibacterial [10], and anti-inflammatory [8] properties are well documented. Phenolic
17
compounds such as flavanones, flavans, dihydrochalcones, chalcones, stilbenes, and
18
oligomeric flavonoids are considered to be its main active ingredients [11].
19
Longxuetongluo capsule (LTC), which contains the total phenolic extract of Chinese
20
dragon’s blood, was approved for the treatment of ischemic stroke by China Food and
21
Drug Administration in June 2013 [19]. Previously, we found that LTC could
22
improves erythrocyte function against lipid peroxidation and abnormal
23
hemorheological parameters in high-fat diet (HFD)-induced ApoE−/− mice [12].
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Moreover, the anti-inflammatory activity of Chinese dragon’s blood has been
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demonstrated both in vivo [8] and in vitro [13]. However, the definitive evidence of
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the anti-atherosclerotic effect of LTC has not been proved in vivo, and its
2
anti-inflammatory activity on human umbilical vein endothelial cells (HUVECs) has
3
not been clearly investigated.
4
The aim of this study was to investigate the anti-atherosclerotic effect of LTC in ApoE−/− mice with HFD-induced hypercholesterolemia, as well as the possible
6
underlying mechanisms for its effect in improving effect on endothelial dysfunction
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via the regulation of nuclear factor kappa B (NF-κB) and mitogen-activated protein
8
kinase (MAPK) signaling pathways.
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2.1. Reagents
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Ezetimibe was purchased from Schering-Plough Pte Ltd. (Lot: 2EZPA17005).
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Commercial total cholesterol (TC) and triglyceride (TG) kits for measurement of
14
plasma lipids levels were purchased from Biosino Bio-technology and Science, Inc.
15
(Beijing, China). Optimum cutting temperature (OCT) compound was purchased from
16
Sakura Finetek USA, Inc. (Torrance, USA). The Oil Red O (ORO) was purchased
17
from Sigma-Aldrich (St Louis, USA). Ox-LDL was purchased from Yiyuan
18
Biotechnologies (Guangzhou, China). RPMI-1640, Dulbecco’s modified Eagle’s
19
medium (DMEM), and fetal bovine serum (FBS) were purchased from Mediatech, Inc.
20
(A Corning Subsidiary, Manassas, USA). Commercial kit for NO determination and
21
the ECL chemiluminescence detection kit were purchased from Applygen
22
Technologies, Inc. (Beijing, China). The fluorescent dye BCECF AM was purchased
23
from Beyotime (Jiangsu, China). Primary antibodies against VCAM-1, MCP-1, and
24
cyclooxygenase-2 (COX-2) were obtained from Abcam (Cambridge, UK). Primary
25
antibody against endothelial nitric oxide synthase (eNOS) was obtained from BD
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Biosciences (San Jose, USA). Primary antibodies of NF-κB signaling pathway (p65,
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P-p65, IκBα, P-IκBα) and MAPK signaling pathway (ErK1/2, P-ErK1/2, p38, P-p38,
3
JNK, P-JNK) were obtained from Cell Signaling Technology (Danvers, MA, USA).
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2.2. Medicinal materials
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LTC was provided by Jiangsu Kanion Pharmaceutical Co. Ltd. (Jiangsu, China).
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The quantitative and qualitative characterization of chemical components in LTC was
8
performed as described previously [12].
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2.3. Animals
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Male ApoE−/− mice, weighing 19–23 g, were obtained from the Animal Center of Peking University Health Science Center. All experiments were approved by the
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Animal Research Committee of Peking University Health Science Center (Beijing,
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China). The Principles of Laboratory Animal Care (NIH Publication 85–23, revised
15
1996) were followed. Mice were housed at 24 ± 1 °C and a relative humidity of 50 ±
16
1% with a light/dark cycle of 12 h. Mice were randomly divided into six groups (n = 9
17
for each group): the chow diet group (CG), the HFD-induced group (HG), the low,
18
medium, high-doses LTC treatment groups (LTC100, LTC200, and LTC300,
19
respectively), and the ezetimibe treatment group (EG). Ezetimibe, a potent cholesterol
20
absorption inhibitor, was used as a positive control drug in the present study. Mice
21
assigned to LTC100, LTC200, and LTC300 groups received 100, 200, and 300
22
mg/kg/d LTC, orally, respectively; those in the EG group received 30 mg/kg/d
23
ezetimibe, for 6 successive weeks. All mice were fed HFD (0.2% cholesterol and 15%
24
fat added) for 1 week prior to the start of various treatments. The mice were fed with
25
HFD or chow diet throughout this experiment. The food and water were available ad
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libitum in each group throughout this experiment.
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2.4. Food intake, body weight and composition measurements Food intake and body weight were measured per 2 weeks with a scale. For food consumption measurement, the amount of normal diet consumed was carefully
6
monitored at 10:00 AM local time every morning. The mice were fasted for 6 h and
7
the blood was collected and anticoagulated by heparin for the measurement of plasma
8
lipids levels. Levels of TC and TG were assessed enzymatically using commercial kits,
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and high-density lipoprotein cholesterol (HDLc) was measured by the same TC kit
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after the precipitation of ApoB-lipoprotein with the 20% polyethylene glycol solution.
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2.5. Measurement of atherosclerotic lesions in the aortic sinus Lesions in the aortic sinus were analyzed according to the modified method [13, 14]. The mice were sacrificed by lethal intravenous injection of a sodium
15
pentobarbital; the heart and adjoining aorta was perfused through the left ventricle
16
with phosphate-buffered saline (PBS) for 10 min and subsequently fixed in
17
phosphate-buffered formaldehyde (4%, pH 7.4). Hearts with ascending aortas were
18
embedded in OCT compound and stored at −80 °C until further processing. Serial
19
sections (7 µm thick) were prepared using a Leica cryostat starting from the
20
appearance of the aortic valve to the ascending aorta until the point where valve cusps
21
were no longer visible. The sections were stained with ORO and hematoxylin and
22
eosin (HE) for the detection of atherosclerotic lesions and morphometric analysis,
23
respectively. For the quantification of total atherosclerotic lesion area, digital images
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of the stained lesions were captured by a Leica icc50 HD microscope and analyzed by
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ImageJ software.
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2.6. Cell culture The SV40-transformed human endothelial cells (PUMC-HUVEC-T1) were
4
purchased from China infrastructure of cell line resources and grown in DMEM
5
containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Human
6
monocytic cell line THP-1 cells were grown in RPMI-1640 containing 10% FBS, 100
7
U/mL penicillin, and 100 µg/mL streptomycin. Cultures were maintained at 37 °C in a
8
humidified atmosphere containing 5% CO2. HUVECs between passages 3 and 15
9
were used in the current study.
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2.7. Measurement of NO production
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To assess the effect of LTC on ox-LDL-stimulated NO production in HUVECs, confluent HUVECs were detached by 0.125% trypsin and grown in 96-well plates. On
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attaining a confluence of 70–80%, cells were incubated with 50 µg/mL ox-LDL in 2%
15
FBS DMEM in the presence or absence of various concentrations (1, 10, 20, and 40
16
µg/mL) of LTC. After further incubation for 24 h, NO production was determined
17
using a commercial kit. Cell culture supernatants (50 µL) were collected and mixed
18
with 100 µL of Griess reagents in 96-well plates. The absorbance was measured at
19
540 nm, and nitrite concentration was calculated by reference to a sodium nitrite
20
standard curve.
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2.8. Cell adhesion assay
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HUVECs were cultured in 48-well plates for 18 h to achieve 90% confluence and
24
subsequently stimulated with 20 µg/mL ox-LDL in the presence or absence of various
25
concentrations (10, 20, and 40 µg/mL) of LTC for 24 h. THP-1 cells were labeled with 8
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2
37 °C for 30 min, and subsequently the cells (5 × 105 cells/mL) were seeded onto the
3
confluent HUVECs and incubated for another 30 min at 37 °C [15]. Non-adherent
4
THP-1 cells were removed by gently washing with PBS for three times; adherent
5
THP-1 cells were examined under an inverted phase-contrast fluorescence
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microscope.
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2.9. Western blot analysis
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HUVECs were cultured in 6-well plates for 18 h to achieve 90% confluence, and then stimulated with 20 µg/mL ox-LDL in the presence or absence of various
11
concentrations (10, 20, and 40 µg/mL) of LTC for 24 h. The cells were then washed
12
with cold PBS and lysed using lysis buffer (0.05% bromophenolblue, 10% sodium
13
dodecyl sulfate, 40% glycerol, 0.01 M dithiothreitol, and 0.2 M Tris-HCl, pH 6.8).
14
Sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and Western blot analysis
15
were performed as previously described [14]. After blocking in 4% (w/v) nonfat dry
16
milk, membranes were incubated with primary antibodies in optimized dilutions at
17
4 °C overnight. Membranes were then washed and incubated with a 1:7,000 dilution
18
of second antibody at 4 °C for 3 h and examined with the ECL chemiluminescence
19
detection kit.
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2.10. Statistical analysis Data were presented as means ± standard error of the mean (SEM). Between-group
23
differences were evaluated by one-way analysis of variance (ANOVA), followed by
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Dunnett’s post hoc test or Student’s t-test for unpaired observations, if appropriate.
25
Between-group differences with associated p values of <0.05 were considered 9
ACCEPTED MANUSCRIPT 1
statistically significant.
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3. Results
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3.1. LTC reduces atherosclerotic lesions in the aortic sinus, but had no effect on
5
plasma lipid levels in the HFD-induced mice
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The TC, TG, and non-HDLc levels of the AopE−/− mice fed on HFD were
significantly increased as compared to those in the mice fed a chow diet (p<0.001,
8
p<0.01, and p<0.001, respectively; Fig. 1A, a-d), while the HDLc level significantly
9
decreased (p<0.01). Ezetimibe, a potent cholesterol absorption inhibitor, was used as a
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positive control drug in the present study. After six weeks treatment with ezetimibe
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(30 mg/kg/day), a significantly reduction in the plasma levels of TC, TG, and
12
non-HDLc levels were observed as compared with those in untreated mice fed the
13
same HFD (p<0.001, p<0.001, and p<0.001, respectively). However, the effect of
14
LTC on lipid levels in the HFD-induced mice was not observed in all groups. As for
15
the body weight, there was no difference among the groups. (Figure 1 A, e). Then the
16
results of food intakes showed that the HFD treatment remarkably reduced the food
17
intakes in of AopE−/− mice (p <0.001), and all doses of LTC had no influence on food
18
intake (Figure 1A, f).
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The development of atherosclerotic lesions in the aortic sinus was quantified by
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ORO staining followed by histological analysis (Fig. 1B and C). The lesion areas of
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the AopE−/− mice fed with the HFD significantly increased compared with those in the
22
chow diet-mice (p<0.001). Compared with the untreated mice fed with the same HFD,
23
ezetimibe markedly reduced the size of the lesion areas by 62.4% (p<0.001),
24
measured as 106815 ± 58753 µm2 vs. 283973 ± 121175 µm2. The lesion areas were
25
significantly reduced in low (26.4%, p<0.05), medium (30.1%, p<0.05), and high 10
ACCEPTED MANUSCRIPT 1
(46.5%, p<0.01) doses of LTC-treated mice, measured as 209112 ± 38804 µm2,
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198634 ± 198634 µm2, and 151892 ± 44668 µm2, respectively.
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3.2. LTC increased NO production and the eNOS protein expression of HUVECs
5
stimulated by ox-LDL
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Endothelial injury is the primary event in the pathogenesis of atherosclerosis [6]. Ox-LDL-induced endothelial injury plays a pivotal role in the initiation of
8
atherogenesis [2]. NO production by vascular endothelial cells is known to induce
9
vasodilation and has an antiatherogenic effect [16]. The effect of LTC on the
10
production of NO in HUVECs was further studied. Stimulation with ox-LDL
11
significantly decreased NO production by HUVECs, while LTC treatment was
12
associated with increased production of NO in HUVECs that were stimulated by
13
ox-LDL, in a dose-dependent manner (Fig. 2A). Since LTC significantly increased the
14
NO production by ox-LDL-stimulated HUVECs, the effect of LTC on the expression
15
of eNOS protein in stimulated HUVECs was investigated by Western blot analysis
16
(Fig. 2B). The eNOS protein expression level was significantly decreased by ox-LDL
17
treatment. LTC dose-dependently increased the same protein levels in
18
ox-LDL-stimulated HUVECs. However, LTC didn’t affect the NO production and
19
eNOS protein expression in normal HUVECs without ox-LDL treatment (Fig. 2C and
20
D). These data suggested that LTC could up-regulate ox-LDL-stimulated eNOS
21
expression in HUVECs.
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3.3. LTC inhibited adhesion of THP-1 cells to ox-LDL-stimulated HUVECs Ox-LDL is known to enhance the adhesion of human monocytes to endothelial cells [17], which is regarded as an initial step of atherogenesis [6]. In the present study, 11
ACCEPTED MANUSCRIPT the inhibitory effect of LTC on the adhesion of THP-1 cells to HUVECs stimulated by
2
ox-LDL was investigated. Ox-LDL significantly increased the adhesion between
3
THP-1 cells and HUVECs (p<0.01). This finding is consistent with the previous
4
report [18]. LTC dose-dependently reduced the adhesion of THP-1 cells to the
5
ox-LDL-stimulated HUVECs (Fig. 3).
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3.4. Effects of LTC on VCAM-1, MCP-1, and COX-2 protein expression in
8
ox-LDL-stimulated HUVECs
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The VCAM-1 and MCP-1, which contribute to macrophage migration and adhesion to endothelial surface [6], are regarded to participate in the ox-LDL-simulated
11
inflammatory signaling [19]. The induction of COX-2 expression is a hallmark
12
finding of atherosclerosis that indicates its inflammatory origin [20]. VCAM-1,
13
MCP-1, and COX-2 protein expression levels were markedly increased in
14
ox-LDL-stimulated HUVECs as compared to that in untreated cells. However,
15
LTC-treatment appeared to reduce the expression levels of VCAM-1 and COX-2
16
protein in stimulated HUVECs in a dose-dependent manner. Furthermore, LTC
17
treatment at high doses was associated with a significant decrease in MCP-1 protein
18
expression (Fig. 4).
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3.4. LTC regulated NF-κB and MAPK signaling pathway in ox-LDL-stimulated
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HUVECs
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NF-κB is an essential factor in controlling inflammatory mediators [21]. It is
23
known to be activated upon phosphorylation of inhibitor of kappa B (IκB), a reaction
24
catalyzed by the cytoplasmic inhibitor of kappa B kinase (IKK) complex [21]. NO
25
released from eNOS inhibits the cleavage of IκB and the consequent activation of 12
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2
of which is in turn regulated by MAPKs [23]. In the present study, LTC treatment
3
appeared to inhibit the phosphorylation of NF-κB-p65 and IκBα in
4
ox-LDL-stimulated HUVECs and suppresse the degradation of IκBα in the cytosolic
5
extracts of stimulated HUVECs, which indicates a dose-dependent inhibitory effect of
6
LTC treatment on the activation of NF-κB, possibly mediated by inducing the
7
inhibition of phosphorylation and degradation of IκBα (Fig. 5A). Further investigation
8
on the upstream of NF-κB showed that LTC dose-dependently decreased the
9
phosphorylation of IKKα/β, and the expression of IKKα and IKKβ in
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ox-LDL-stimulated HUVECs (Fig. 5B). These findings suggest a potential inhibitory
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effect of LTC on the IKK/IκB/NF-κB inflammatory signaling pathway.
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Previous studies have shown that ErK1/2, p38, and JNK MAPKs partly regulated
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the activation of NF-κB [24]. Thus, the observation that LTC suppressed the NF-κB
14
signaling pathway prompted us to explore whether MAPKs were involved in this
15
process. Ox-LDL significantly induced the phosphorylation of ErK1/2 and p38
16
MAPKs in HUVECs (Fig. 5C). The levels of phosphorylated ErK1/2 and p38 MAPKs
17
in ox-LDL-stimulated HUVECs appeared to be suppressed by LTC in a
18
dose-dependent manner. However, no significant change in phospho-JNK expression
19
was observed in ox-LDL-stimulated HUVECs (data not shown). These results
20
indicated that LTC-induced suppression of IKK/IκB/NF-κB inflammatory signaling
21
pathway is mediated via the suppression of MAPKs activation (Fig. 5D).
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4. Discussion Increased plasma level of low-density lipoprotein is regarded as a major risk factor 13
ACCEPTED MANUSCRIPT of atherogenesis [25], and its oxidative modification plays as a critical role in the
2
pathogenesis of atherosclerosis [2]. Lipid-lowering therapies reduce the occurrence of
3
these clinical events [26]. Our results showed that ezetimibe treatment is associated
4
with a significant decrease in plasma lipid levels in HFD-induced ApoE−/− mice,
5
compared with HG mice, while LTC did not affect the plasma lipids levels. These data
6
indicate that LTC had no lipid-lowering effect.
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Atherosclerosis is a risk factor for major coronary heart disease event. It is
generally accepted that the progression of atherosclerosis is associated with chronic
9
inflammation in the vessel wall. Reports on the anti-inflammatory activity of Chinese
10
dragon’s blood prompted us to investigate the antiatherosclerosis effect of LTC in vivo
11
and in vitro. Our findings indicated that LTC reduced the lesion areas in the aortic
12
sinus in vivo and appeared to reverse the endothelial dysfunction stimulated by
13
ox-LDL in vitro.
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Ox-LDL is instrumental in inflammation and thus in the pathogenesis of
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atherosclerosis and endothelial dysfunction [2]. As an indicator of endothelial
16
dysfunction, the reduction of the bioactivity (bioavailability) of NO could be
17
attributed to the stimulation of ox-LDL [5]. In addition, dysfunctional endothelial
18
cells release adhesion molecules, such as VCAM-1 and MCP-1, that promote the
19
adhesion of monocyte to endothelial cells [15]. In the present study, LTC not only
20
increased the production of NO in ox-LDL-stimulated HUVECs but also inhibited
21
adhesions of monocyte to HUVECs. Further investigation indicated that LTC
22
increased the expression of eNOS and decreased that of VCAM-1, MCP-1, and
23
COX-2 in stimulated HUVECs. Our findings suggest that alleviation of the
24
ox-LDL-stimulated dysfunction of HUVECs is attributable to its antiinflammatory
25
effect that inhibits monocyte adhesion to HUVECs and exerts its antiatherosclerotic
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effect in ApoE−/− mice. The activated NF-κB is known to induce the expression of adhesion molecules [27], such as VCAM-1 and MCP-1, and inflammatory mediators, such as COX-2 [25].
4
Furthermore, the activation of NF-κΒ is implicated in the ox-LDL-stimulated
5
dysfunction in the endothelial progenitor cells [28] and bovine aortic endothelial cells
6
[29]. Therefore, the decrease of the atherosclerotic lesion areas in the aortic sinus of
7
LTC-treated ApoE−/− mice fed HFD prompted us to further investigate the potential
8
role of the activation of NF-κB. We further investigated whether the effect of LTC in
9
alleviating the dysfunction of HUVECs was mediated via the inhibition of the
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activation of NF-κB, which in turn suppresses the adhesion of monocyte to
11
endothelial cells. In the present study, LTC could dose-dependently down-regulate the
12
ox-LDL-stimulated degradation and phosphorylation of IκBα, thus inhibiting the
13
activation of NF-κB-p65.
In addition, the decrease of eNOS is critically associated with endothelial
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dysfunction, the expression of which in endothelial cells may play a complementary
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role in the endothelial activation of NF-κB. The inhibition of endothelial NF-κB
17
activation was shown to suppress the lipopolysaccharide-induced eNOS
18
down-regulation in vascular tissues [30]. Furthermore, the production of NO by eNOS
19
was shown to inhibit vascular inflammatory NF-κB activity via inducing IκB, and
20
eNOS could regulate the expression of NF-κB [31]. The phosphorylation of IκB is
21
regulated by the IKK complex, consisting of at least three subunits: IKKα, IKKβ, and
22
IKKγ. IKKβ is considered to play a greater role than IKKα in NF-κB activation [32].
23
Moreover, eNOS is associated with the IKK complex [32]. The IKK/IκB/NF-κB
24
pathway was earlier reported to be involved in the palmitic acid-induced dysfunction
25
of HUVECs [33]. In the present study, a significant increase in the expressions of
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2
is consistent with the results from a previous study on the ox-LDL-stimulated human
3
primary coronary artery endothelial cells [34]. The inhibitory effect of LTC on the
4
expressions of IKKα, IKKβ, and the phospho-IKKα/β further indicates that its effect
5
on the alleviation of the ox-LDL-stimulated dysfunction of HUVECs may be
6
mediated via the IKK/IκB/NF-κB signaling pathway.
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MAPKs, known as intracellular effectors of cytokine receptor stimulation, have been shown to regulate the IKK complex [35]. The exposure of human brain
9
microvessel endothelial cells to ox-LDL is known to induce changes in MAPKs [36].
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Moreover, the activation of ErK1/2 MAPK in ox-LDL-stimulated human coronary
11
artery endothelial cells has been reported to play a critical role in the expression of
12
MCP-1 [37]. Therefore, we further investigated protein expressions of major MAPKs
13
(ErK1/2, p38, and JNK) in ox-LDL-stimulated HUVECs. We observed an increase in
14
the phosphorylation of two MAPKs (ErK1/2 and p38) increased in
15
ox-LDL-stimulated HUVECs, which is consistent with a previous report [24]. LTC
16
dose-dependently decreased the phosphorylation of both ErK1/2 and p38 MAPK in
17
ox-LDL-stimulated HUVECs without having any effect on the ErK1/2 and p38
18
MAPK. However, LTC did not significantly affect the JNK MAPK phosphorylation.
19
Therefore, the significant decrease in phosphorylation of two MAPKs (ErK1/2 and
20
p38) in ox-LDL-stimulated HUVECs suggests a wider pharmacological effect of LTC
21
on the inflammatory signaling pathways. These findings suggest that the
22
antiatherosclerotic effect of LTC is mediated via the MAPKs/IKK/IκB/NF-κB
23
signaling pathway (Fig. 5).
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In the present study, LTC caused a significant decrease in the atherosclerotic lesion areas in the aortic sinus of the HFD-induced ApoE−/− mice. Further in vitro 16
ACCEPTED MANUSCRIPT investigation indicated that the effect of LTC in alleviating the ox-LDL-stimulated
2
dysfunction of HUVECs was mediated via the MAPKs/IKK/IκB/NF-κB signaling
3
pathway, thus inhibiting the adhesion of THP-1 cells to the HUVECs. These in vivo
4
and in vitro finding indicate that the anti-atherosclerotic effect of LTC may be
5
improve the inducible endothelial dysfunction via the MAPKs/IKK/IκB/NF-κB
6
signaling pathway and the inhibition of monocyte adhesion to endothelial cells.
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Conflict of interest
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The authors declared they do not have anything to disclose regarding conflict of
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interest with respect to this manuscript.
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Financial support
This work was supported by grants from the National Natural Science Foundation
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of China (Nos. 81503286, 81573572, 81530097), and New Century Excellent Talents
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in University (No. NCET-13-0693).
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Figure legends
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Fig. 1. Effects of LTC on plasma lipids levels and atherosclerotic lesions in
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HFD-induced ApoE−/− mice. (A) Plasma levels of TC (a), TG (b), HDLc (c), and
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non-HDLc (d) in ApoE−/− mice. (n= 5–8). Food intake (e) and body weight per mouse
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(f) during the 6 week experiments. (B) LTC inhibits atherosclerotic lesions. (a) ORO
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and HE staining of atherosclerotic lesions at the same level of aortic sinus from
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HFD-induced ApoE−/− mice. (b) Quantitative analyses of the atherosclerotic lesion
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areas in the aortic sinus. The mean values from 5–8 mice are presented. # p<0.05 vs.
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CG; ## p<0.01 vs. CG; ### p<0.001 vs. CG; * p<0.05 vs. HG; ** p<0.01 vs. HG; ***
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p<0.001 vs. HG. The arrows indicate the sites of positive staining. TC, total
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cholesterol; TG, triglycerides; HDLc, high density lipoprotein cholesterol; HFD,
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high-fat diet; LTC, Longxuetongluo capsule; CG, chow diet group; HG, HFD-induced
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group; LTC100, low-dose LTC, LTC200, medium-dose LTC; LTC300, high-dose LTC;
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EG, ezetimibe treatment group.
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Fig. 2. Effects of LTC on NO production and eNOS expression in HUVECs. The
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NO production of HUVECs with ox-LDL-stimulated (A) or absence (C) after LTC
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treatment. Western blot analysis of eNOS in HUVECs with ox-LDL-stimulated (B) or
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absence (D) after LTC treatment. # p<0.05 vs. HUVECs in control; ** p<0.01 vs.
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HUVECs stimulated by ox-LDL; *** p<0.001 vs. HUVECs stimulated by ox-LDL.
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Fig. 3. LTC inhibited the adhesion of THP-1 cells to HUVECs stimulated by
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ox-LDL. (A) Adhesion of BCECF AM labeled THP-1 cells with untreated HUVECs.
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(B) Adhesion of BCECF AM labeled THP-1 cells to HUVECs stimulated with 20
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µg/mL ox-LDL. (C–E) Adhesion of BCECF AM labeled THP-1 cells to 24
ACCEPTED MANUSCRIPT ox-LDL-stimulated HUVECs treated with various concentrations (10, 20, and 40
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µg/mL, respectively) of LTC. (F) The quantitative analysis of relative fluorescence of
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BCECF AM labeled THP-1 cells adherent to HUVECs Each bar of the representative
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images represents 200 µm. Each bar represents means (± SEM) values from six
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experiments. # p<0.05 vs. HUVECs in control; ## p<0.01 vs. HUVECs in control; **
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p<0.01 vs. HUVECs stimulated by ox-LDL.
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Fig. 4. Effect of LTC on VCAM-1, MCP-1, and COX-2 expression levels in
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ox-LDL-stimulated HUVECs. Western blot analysis of VCAM-1 (A), MCP-1 (B),
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and COX-2 (C) in HUVECs stimulated with 20 µg/mL ox-LDL in the presence or
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absence of LTC at different concentrations for 24 h. # p<0.05 vs. HUVECs in control;
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* p<0.05 vs. HUVECs stimulated by ox-LDL; ** p<0.01 vs. HUVECs stimulated by
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ox-LDL; *** p<0.001 vs. HUVECs stimulated by ox-LDL.
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Fig. 5. LTC inhibited NF-κB signaling pathway through the regulation of the
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MAPK signaling pathway in ox-LDL-stimulated HUVECs. (A) LTC
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dose-dependently downregulated the phosphorylation of NF-κB-p65 and IκBα and the
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degradation of IκBα. (B) LTC decreased the protein expression of phospho-IKKα/β,
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IKKα, and IKKβ in a dose-dependent manner. (C) LTC downregulated the
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phosphorylation of ErK1/2 and p38, without having an effect on protein expression of
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ErK1/2, p38 and JNK and phospho-JNK. (D) The sketched signal cascade was
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involved in the relationship between the ox-LDL-stimulated endothelial dysfunction
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and the atherosclerotic lesions in the aortic sinus. # p<0.05 vs. HUVECs in control; ##
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p<0.01 vs. HUVECs in control; * p<0.05 vs. HUVECs stimulated by ox-LDL; **
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p<0.01 vs. HUVECs stimulated by ox-LDL; *** p<0.001 vs. HUVECs stimulated by
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ACCEPTED MANUSCRIPT Highlights: Longxuetongluo capsule (LTC) delays atherosclerosis development. LTC increased NO production and the eNOS protein expression level of HUVECs stimulated by ox-LDL.
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LTC regulated NF-κB and MAPK signaling pathway in ox-LDL-stimulated HUVECs.