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NF-κB signaling pathway in vitro
Author’s Accepted Manuscript β-elemene inhibits Monocyte-endothelial cells interactions via Reactive oxygen species/MAPK/NF-κB signaling pathway in vitro Meng Liu, Lifei Mao, Abdelkader Daoud, Waseem Hassan, Liangliang Zhou, Jiawei Lin, Jun Liu, Jing Shang www.elsevier.com
PII: DOI: Reference:
S0014-2999(15)30263-6 http://dx.doi.org/10.1016/j.ejphar.2015.09.032 EJP70239
To appear in: European Journal of Pharmacology Received date: 21 May 2015 Revised date: 15 September 2015 Accepted date: 21 September 2015 Cite this article as: Meng Liu, Lifei Mao, Abdelkader Daoud, Waseem Hassan, Liangliang Zhou, Jiawei Lin, Jun Liu and Jing Shang, β-elemene inhibits Monocyte-endothelial cells interactions via Reactive oxygen species/MAPK/NFκB signaling pathway in vitro, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.09.032 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 galley proof before it is published in its final citable 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.
β-elemene inhibits Monocyte-endothelial cells interactions via Reactive oxygen species/MAPK/NF-κB signaling pathway in vitro
Meng Liu a,c, Lifei Mao a, Abdelkader Daoud a,d, Waseem Hassan a, Liangliang Zhou a, Jiawei ＊
Lin a, Jun Liu a,b ,Jing Shang a,b a
National Center for Drug Screening & State Key Laboratory of Natural Medicines, China Pharmaceutical
University, Jiangsu Province, 210009, P. R. China. b
Jiangsu Key Laboratory of TCM Evaluation and Translational Research, China Pharmaceutical University, 24
Tong jia xiang, Nanjing 210009, Jiang Su Province, P.R. China. c
Cancer Hospital Affiliated to Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China
d
Département de Pharmacie, Faculté de Médecine, Université Abou Bekr Belkaid, Tlemcen, Algérie
＊
Corresponding author: Jing Shang
National Center for Drug Screening & State Key Laboratory of Natural Medicines, China Pharmaceutical
University, Nanjing, Jiang Su Province, P.R. China. Telephone number: +86-25-83271043; Fax number:
+86-25-83271142; E-mail address: [email protected]
Abstract The recruitment of monocytes to the active endothelial cells is an early step in the formation of
atherosclerotic lesions; therefore, the inhibition of monocyte-endothelial cells interactions may serve as a potential therapeutic strategy for atherosclerosis. Recent studies suggest that β-elemene can protect against
atherosclerosis in vivo and vitro; however, the mechanism underlying the anti-atherosclerotic effect by
1 / 29
β-elemene is not clear yet. In this study, we aimed to investigate the effects of β-elemene on the
monocyte-endothelial cells interactions in the initiation of atherosclerosis in vitro. Our results showed that β-elemene protects human umbilical vein endothelial cells (HUVECs) from hydrogen peroxide-induced
endothelial cells injury in vitro. Besides, this molecule inhibits monocyte adhesion and transendothelial migration
across inflamed endothelium through the suppression of the nuclear factor-kappa B-dependent expression of cell adhesion molecules. Further, β-elemene decreases generation of reactive oxygen species (ROS) and prevents the
activation of mitogen-activated protein kinase (MAPK) signaling pathway in HUVECs. In conclusion, this study would provide a new pharmacological evidence of the significance of β-elemene as a future drug for prevention
and treatment of atherosclerosis.
Keywords β-elemene; Atherosclerosis; Human umbilical vein endothelial cells; Cell adhesion; NF-κB
1. Introduction The recruitment of circulating monocytes to the sites of arterial injuries and adhere to endothelium and transmigrate into the arterial wall are an initial step of the progression of atherosclerosis (Clapp et al., 2004; Erdogan et al., 2007; Lee et al., 2013; Mestas and Ley, 2008). Accumulated evidence demonstrates that monocyte infiltration plays a crucial role in the pathophysiology of coronary artery diseases including atherosclerosis, which can not only initiate and propagate the accumulation of monocyte-derived macrophages but also produce inflammatory mediators that destabilize atherosclerotic plaques (Kartikasari et al., 2009; Zhu et al., 2013). The monocyte-endothelium cells interactions consist of consecutive processes of monocytes adhesion 2 / 29
and infiltration through the endothelium. During early stages of atherosclerosis, endothelial cells elicit the up-regulation of vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 levels, which mediate the monocyte adhesion to activated endothelial cells and finally induce the progression of atherosclerosis (Kim et al., 2008). Many of stimulators including H2O2, oxidized low-density lipoprotein (ox-LDL) and homocysteine have been indicated to promote the expression levels of CAMs in endothelial cells resulting in endothelial cells activation, dysfunction and injury (Boulden et al., 2006; Cernuda-Morollon and Ridley, 2006; Coyle et al., 2006; Wilson, 2003). Hence, the inhibition of monocyte-endothelial cells interactions may represent a therapeutic approach against atherosclerosis and other related disorders (Fuentes and Palomo, 2014). Oxidative stress-induced disruption of redox states could lead to the activation of several redox-sensitive pathways including MAPK and NF-κB. These can result in the elevation of adhesion molecules, chemokines and cytokines expression and ultimately contribute to endothelial cells activation and chronic inflammation that induces leukocytes recruitment and infiltration (Kyaw et al., 2004). In vitro experiments have sufficiently proved that oxidative stressors (e.g. H2O2 and ox-LDL) could mimic the oxidative environment and lead to endothelial cells activation and elevation of adhesion molecule and chemokine expression (Song et al., 2014). H2O2 can up-regulate CAMs’ expression leading to monocytes adhesion to endothelial cells through the activation of NF-κB and MAPK signaling pathways and ultimately induce endothelial dysfunction (Jin et al., 2014). Accordingly, studying the molecular recognition and signaling pathway might help us understand the mechanisms underlying pharmacological inhibition of monocyte- endothelial cells interactions implicated 3 / 29
in atherosclerosis. In conclusion, the suppression of reactive oxygen species (ROS) generation, NF-κB translocation and MAPK signal pathways can be a very useful strategy to inhibit CAMs’ expression and monocyte-endothelial cells interactions. β-elemene (Fig. 1A), a sesquiterpenes compound extracted from the Curcuma Wenyujin (Guo, 1983), is a traditional Chinese herbal medicine that has been shown to inhibit tumor cell growth in vivo and in vitro. Because of its proven safety, this compound has been applied in clinics for the treatment of malignant effusions and some solid tumors (Li et al., 2005; Wang et al., 2005a; Wang et al., 2005b). Previous studies have shown that β-elemene derivatives have a significant antioxidant activity and cytoprotective effects against oxidative damage in HUVECs (Chen et al., 2014). In addition, another study indicated that β-elemene can inhibit smooth muscle proliferation/migration and inhibit neointima formation in vivo (Wu et al., 2011). However the effect of β-elemene on H2O2-induced monocyte-endothelial cells interactions and the underlying molecular mechanisms remain unknown. Therefore, the aim of this study is to investigate the potential inhibition effect of β-elemene on monocyte-endothelial cells interactions and unveil the possible mechanism for preventing atherosclerosis.
2. Materials and methods 2.1. Materials β-elemene [99.3% purity, CSPC Yuanda (Dalian) Pharmaceutical Co., Ltd. Dalian Liaoning China,1 mg/l]. DAPI were from Life Technologies Corporation (USA), Hydrogen peroxide (H2O2), Pyrrolidinedithiocarbamate (PDTC), Dimethylsulfoxide (DMSO), 3-(4,5-dimethyl– thiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT), β-actin antibody, and horseradish peroxidase4 / 29
conjugated secondary antibodies were purchased from Sigma-Aldrich (MO, USA). The Takara quantitative RT-PCR kit and SYBR Green Premix Ex Taq were products of Takara Biomedical Inc. (Shiga, Japan). Antibodies against p-p38, p38, p-ERK1/2, ERK1/2, p-JNK (Thr183/Tyr185), JNK, p-Iκβα (Ser32), Iκβα, NF-κB p65, Histone H3 were from Cell Signaling Technology (MA, USA), Anti-Cytochrome b245 Light Chain antibody was from Abcam (Cambridge, UK). ICAM-1(G-5): sc-8439, VCAM-1(E-10): sc-13160 was from Santa Cruz Biotechnology (Santa Cruz, CA). BCA protein assay kit, phenylmethylsulfonyl fluoride (PMSF) and cell lysis buffer for Western and IP, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF/AM), Reactive oxygen species and Superoxide dismutase (SOD) Assay Kit were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Other reagents were of the highest obtainable quality.
2.2. Cell culture Human umbilical vein endothelial cells (HUVECs) were from Nanjing Key Gen Biotech Co. Ltd (Nanjing, China). HUVECs were cultured in DMEM (Invitrogen) containing 10% heat-inactivated fetal bovine serum (FBS) at 37°C in 5% CO2 humid incubator. Human peripheral blood monocyte (THP-1) cells were grown in RPMI-1640 medium with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin sulfate.
2.3. Cell viability assay HUVECs were seeded in 96-well plates at a density of 5×103 cells/well. After 24 h 5 / 29
growth, HUVECs were pre-treated with β-elemene (0.5, 5, 50 μM) for 24 h at 37°C in 5% CO2 incubator. Then, HUVECs were treated with 0.5 mM H2O2 for 2 h. After that MTT (0.5%, 20 μl) was added to the medium and cells were further incubated for 4 h. The supernatant was removed and 100μl DMSO was added to dissolve the precipitate. Finally, the absorbance was measured spectrophotometrically at 570 nm.
2.4. Detection of OH· and H2O2 in HUVECs The effects of β-elemene on H2O2-induced hydroxyl radical (OH·) and hydrogen peroxide (H2O2) production were measured with commercially available kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. Briefly, OH· was generated by the Fenton reaction and treated with a chromogenic substrate nitrotetrazolium blue chloride (NBT) to yield a stable colored substance, which was measured at 450 nm. The reaction product of H2O2 and molybdic acid was determined at 405 nm using the fluorescence microplate reader (Xing et al., 2014) (TECAN Safire2TM). The scavenging rate (%) = [(O.D control – O.D sample) / O.D control] ×100.
2.5. Reactive oxygen species contents analysis The intracellular generation of reactive oxygen species was measured by the fluorescent probe, 2’, 7’-dichlorodihydrofluorescein diacetate (DCFH-DA). Briefly, HUVECs were incubated with β-elemene (0.5, 5, 50 μM) and PDTC (20μM) for 24 h and then stimulated with 0.5 mM H2O2 for 2h. After that, HUVECs were labeled with 10μM DCFH-DA and incubated for 20 min, followed by washed three times with medium. The fluorescence microplate reader (TECAN 6 / 29
Safire2TM) was used to determine the fluorescence. The results of reactive oxygen species were expressed as the percentage of the control group fluorescence intensity.
2.6. Measurement of intracellular Superoxide Dismutase The superoxide dismutase (SOD) activity was detected using a commercial kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. Briefly, HUVECs were seeded and then stimulated with β-elemene and H2O2. After that, cells were washed with Phosphate Buffered Saline (PBS) and then lysed. The lysate was centrifuged at 12,000 g for 15 min at 4°C. The supernatants were collected to measure the SOD activity.
2.7. Quantitative real-time PCR HUVECs were incubated with β-elemene (0.5, 5, 50 μM) for 24 h and then stimulated with 0.5mM H2O2 for 2h. Total RNA was extracted using TRIZOL reagent (Invitrogen, U.S.A). First strand cDNA was synthesized with PrimeScript RT Master Mix kit (Takara, Japan) according to the manufacturer’s instructions. The quantitative real-time PCR was performed on an iQ5 multicolor real-time PCR detection system (Bio-Rad, Hercules, Calif., U.S.A) by using SYBR Premix Ex TaqTM (Takara, Japan) according to the manufacturer’s instructions. To investigate the effects of β-elemene on atherosclerosis, the mRNA expression of p22 phox, VCAM-1 and ICAM-1 was examined. Primer sequences were as follows: p22 phox , 5’- AGA AGT ACA TGA CCG CCG TGG -3’ (forward) and 5’- AGT AGG TAG ATG CCG CTC GCA AT -3’ (reverse); VCAM-1 , 5’- GAT ACA ACC GTC TTG GTC AGC CC 7 / 29
-3’ (forward) and 5’- CGC ATC CTT CAA CTG GCC TT -3’ (reverse); ICAM-1 , 5’- TAT GGC AAC GAC TCC TTC T -3’ (forward) and 5’- CAT TCA GCG TCA CCT TGG -3’ (reverse) and β-actin, 5’-CGC AAA GAC CTG TAC GCC AAC-3’ (forward) and 5’-CAC GGA GTA CTT GCG CTC AGG-3’ (reverse). p22phox, VCAM-1 and ICAM-1 transcript levels were calculated by the 2-ΔΔCT method.
2.8. Western blotting analysis After treatment, HUVECs were lysed by RIPA lysis buffer. The lysates were subjected to centrifugation at 14,000 g for 10 min at 4°C and the supernatants were used for quantification of total protein concentration by using the BCA Protein Assay Kit (Beyotime Biotechnology, Nanjing, China). Protein samples were separated using electrophoresis on SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, U.S.A). Monoclonal antibodies against p22phox, p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, p38, p-Iκβα, Iκβα, p65 NF-κB, ICAM-1, VCAM-1 and β-actin were incubated at 4°C overnight. After that the membranes were incubated with secondary antibodies. Protein bands were analyzed using a chemiDoc XRS imaging system (Bio-Rad) and Quantity One (Bio-Rad) software program.
2.9. Immunofluorescence confocal microscopy HUVECs were seeded onto Class Bottom Cell Culture Dish (NEST) the day before treatment and processed for immunofluorescence. In brief, cells were pretreated with 50 μM β-elemene for 1h, and then exposure to 0.5 mM H2O2 for 2h. After those HUVECs were washed thrice with PBS for 5 min, cells were incubated with 4% paraformaldehyde on ice for 20 min to fix them and again 8 / 29
washed three times. After that, cells were incubated with Triton X-100 for 20 min. Then, cells were blocked with PBS containing 5% bovine serum albumin (BSA) for 1h, and incubated with p65 NF-κB antibody (1:400) at 37°C for 1h. After washing with PBS, HUVECs were stained with FITC-conjugated anti-rabbit IgG antibody (1:100) for 1h. Then the cells were stained with diamidino-phenyl-indole (DAPI) for 20 min. Pictures were captured with Olympus IX81 confocal microscope.
2.10. Monocyte adhesion assay HUVECs were pretreated with 50 μM β-elemene and 20 μM PDTC in 24-well plates for 24h and subsequently stimulated with 0.5 mM H2O2 for 2 h. THP-1 cells were labeled with 10 μg/ml BCECF-AM for 30min at 37°C. Labeled THP-1 cells were washed with PBS and re-suspended in RPMI 1640 medium containing 2% FBS. Then, HUVECs were co-cultured with the BCECF-AM -labeled THP-1 cells (2×106/well) for 30 min at 37°C. HUVECs were washed twice with PBS to remove non-adhering THP-1 cells and fluorescence levels were analyzed using a fluorescence microplate reader (TECAN Safire2TM) at 485 nm/520 nm. The percentage of adherent monocytes was calculated by the formula: % adherence = (adherent signal/total signal) × 100.
2.11. Transendothelial migration assay The transendothelial migration assay was measured by a fluorescence-based assay. In brief, HUVECs were placed on 24-Well Millcell (with 8.0 μm PET; MILLIPORE) at a density of 2.5 × 104 cells/insert. Because monolayers were not visible on these inserts, 9 / 29
manual readings of transendothelial electrical resistance (TEER) were taken with a voltmeter (MILLIPORE-ERS USA) to confirm monolayer formation. Thereafter, HUVECs were pretreated with 50μM β-elemene and 20μM PDTC for 24h and then co-incubated with 0.5 mM H2O2 for 2 h. The inserts were washed thoroughly and properly in every treatment, and labeled THP-1 monocytes (2.5× 105 cells/insert loaded with 10 μg/ml of the fluorescent dye BCECF/AM for 45 min) were placed on the upper chamber, and migration was continued to 6 h at 37°C. Migration was monitored by taking the fluorescence using a fluorescence plate reader (TECAN Safire2TM) when the labeled monocytes found in the lower chamber. Additionally, the data were shown as the percentage of input (right y - axis), which indicates the percentage of cells that migrated from the number of cellular input.
2.12. Statistical analysis Statistical analysis was achieved by using graph pad prism Version 5.0c (Graph Pad Software). Quantitative data are expressed as mean ± S.D. Data were analyzed, as relevant, by unpaired two-tailed Student’s t-test or by one-way ANOVA with Turey’s post hoc test. P value of 0.01 was accepted as statistically significant.
3. Results 3.1. Effect of β-elemene on H2O2-induced cytotoxicity Cell viability assay was used to assess the protective effect of β-elemene against H2O2-induced cellular toxicity. The results showed that the inhibitory rates of β-elemene (0.5, 5, 50 μM) on the H2O2-induced cytotoxicity were 59.7%, 75.1% and 86.2%, respectively (Fig. 1B). 10 / 29
The β-elemene at 0.5, 5, 50 μM was then used in all subsequent experiments. These results also suggested that β-elemene can protect endothelial cells from H2O2-induced cytotoxicity.
3.2. Inhibition of monocyte adhesion and transendothelial migration by β-elemene Monocytes adhesion toward vascular endothelial cells is the initial step in migration across the inflammation endothelial cells (Muller, 2009). Therefore monocytes adhesion, transendothelial migration and the role of NF-κB signaling pathway in these processes were investigated. As shown in Fig. 2A and 2B, monocytes adhesion increased to 40.0% when endothelial cells were stimulated with 0.5 mM H2O2. Pre-treatment of HUVECs with 50 μM β-elemene and 20 μM PDTC resulted in 24.0 % and 38.7 % decrease in cellular adhesion compared with H2O2 treated group. Afterwards we studied monocyte migration to determine whether β-elemene can also affect monocyte infiltration into the endothelium. The findings showed that the stimulation of endothelial cells with H2O2 caused an 81.7% increase in cell migration. Pretreatment with 50 μΜ β-elemene and 20 μM PDTC in the above treatment conditions produced 38.3% and 53.5% decrease in transendothelial migration compared with H2O2 treated group (Fig. 2 C). Taken together, these results suggest that β-elemene inhibits H2O2-induced monocyte adhesion and transendothelial migration and this effect may be mediated via suppression of NF-κB signaling pathway in HUVECs.
3.3. Inhibition of cell adhesion molecule expression by β-elemene We also investigated the effect of β-elemene on ICAM-1 and VCAM-1expression 11 / 29
induced by H2O2 in vitro. The mRNA expression and protein levels of ICAM-1 and VCAM-1 in HUVECs were increased after stimulation with H2O2 as shown in Fig 3A and 3B. After treatment with β-elemene, ICAM-1 and VCAM-1 mRNA expression were reduced by 88% and 84% respectively (Fig. 3A). Furthermore, the inhibition rates of VCAM-1 and ICAM-1 protein levels by β-elemene treatment were 74% and 79% (Fig. 3B). As shown in Fig. 3C, the results demonstrated that the protein levels of ICAM-1 and VCAM-1 were significantly reduced by PDTC. This finding was similar to that obtained from the treatment of HUVECs with 50 μM β-elemene. Hence, these results suggest that the inhibition effects of β-elemene on the protein levels of ICAM-1 and VCAM-1 in HUVECs may be mediated through suppression of NF-κB signaling pathway.
·
3.4. Inhibition of OH and H2O2 production by β-elemene ·
As shown in Fig. 4A and 4B, OH and H2O2 production was increased after H2O2 induction, however pretreatment with β-elemene inhibited these production in a dose-dependent manner. The ·
results pointed that β-elemene inhibits OH and H2O2 production and showed the scavenging ·
activates of OH and H2O2.
3.5. β-elemene attenuated reactive oxygen species generation and inhibited the over-expression of p22phox in HUVECs The results showed that the level of reactive oxygen species was markedly increased in HUVECs induced by H2O2 (Fig. 5A). After pre-treatment with β-elemene, the relative fluorescence intensity was decreased to 20.0%, 39.7% and 51.1% in a dose-dependent manner 12 / 29
compared with the H2O2 treated group. Meanwhile, the reactive oxygen species level was inhibited to 64.6% when cells were treated with 20 μM PDTC compared with the H2O2 treated group. This result revealed that PDTC inhibits the NF-κB activation by its anti-oxidation property (Yokoo and Kitamura, 1996). According to these findings, β-elemene also increased dose-dependently SOD activity (17.37±1.85, 18.41±2.71 and 20.43 ± 1.51 U /mgprot for 0.5, 5, 50 μM β-elemene, respectively in Fig. 5B). NADPH oxidase, which has a p22phox modulatory subunit, has been widely regarded as a main source of reactive oxygen species generation in endothelial cells (Jin et al., 2014). Our results showed that pretreatment with β-elemene (0.5, 5, 50 μM) could down-regulate the mRNA expression of p22phox in a dose-dependent manner as indicated in Fig. 5C (13.1%, 44.0%, 79.5%, compared with the H2O2 treated group). Moreover, the inhibitory effect of β-elemene on the protein level of p22phox was also confirmed by western blotting as Fig. 5D shows (14.2%, 23.8%, 29.0%, compared with the H2O2 treated group).
3.6. The modulation of NF-κB signaling pathway by β-elemene and its anti- inflammation effect Oxidative stress and persistent inflammation are critical pathologic events in atherosclerosis. Reactive oxygen species serves as the common intracellular messengers to activate multiple redox-sensitive signaling pathways including MAPK and NF-κB and plays a key role in the development of many vascular diseases including atherosclerosis (Griendling et al., 2000a). H2O2, which belongs to reactive oxygen species family, is an activator of NF-κB in endothelial cells. NF-κB plays a critical role in the inflammatory response progress by up-regulating the expression levels of VCAM-1, ICAM-1 and 13 / 29
E-selectin. Furthermore, the up-regulation of CAMs’ expression provides a mechanism for the monocyte adhesion and transendothelial migration. Our data revealed that the phosphorylation levels of Iκβ and the nuclear location of NF-κB were increased in H2O2-induced HUVECs, but this effect was decreased when HUVECs were pre-treated with β-elemene (0.5, 5, 50 μM) as indicated in Fig. 6A and 6B. Using immunofluorescence microscopy analysis, we demonstrated NF-κB translocation from the cytoplasm to the nucleus after H2O2 stimulation (Fig. 6C); this translocation was efficiently inhibited by pretreatment with β-elemene in HUVECs. In addition, these results were consistent with western blotting analysis and showed that β-elemene inhibits H2O2-induced NF-κB translocation. The effects of β-elemene on MAPK signaling pathway were analyzed and the ERK1/2, JNK, and p38 activation was measured. Our data showed that ERK 1/2, JNK and p38 were sufficiently activated after 2 h incubation with 0.5 mM H2O2. Meanwhile, the activation of ERK 1/2, JNK and p38 was markedly decreased by β-elemene in a dose-dependent manner (Fig. 7A and 7B). Total protein levels of ERK 1/2, JNK, and p38 were not altered upon H2O2 and/or β-elemene treatment. These results indicated that β-elemene specifically plays its role via modulation of NF-κB and ERK1/2 /JNK/p38 MAPK signaling pathways.
4. Discussion Monocytes recruitment and infiltration is a central mechanism in the early steps of atherosclerosis, which can lead to chronic inflammation and the development and progression of atherosclerosis. This process is initiated by the activation of endothelial cells and the up-regulation 14 / 29
of cell adhesion molecules and chemokines. β-elemene, which is derived from a traditional Chinese herbal, has been shown to have an anti-atherosclerotic effect; however, the molecular mechanisms by which β-elemene inhibits the monocyte-endothelial cells interactions associated with atherosclerosis remain unknown. The aims of the present study were to investigate the the effect of β-elemene on monocytes adhesion and migration during atherosclerosis and elucidate the probable mechanisms. Additionally the responses of the vascular endothelium to oxidative stress conditions play a significant role in the regulation of monocytes adhesion and infiltration into the endothelium. Previously, we reported that β-elemene derivatives have significant cytoprotective and anti-oxidative effects against oxidative damage induced by H2O2 in HUVECs (Chen et al., 2014). Consistent with previous reports, our results indicate firstly that β-elemene can inhibit H2O2-induced monocytes adhesion and transendothelial migration. Furthermore β-elemene inhibits the endothelial activation (attenuates oxidative stress, improved nitric oxide bioavailability and decreased ICAM-1 and VCAM-1 expression). These data suggest that its inhibition effects on monocyte-endothelial cells interactions might be due to the suppression of NF-κB-dependent ICAM-1 and VCAM-1 expression via modulation of reactive oxygen species generation and MAPK signaling pathway activation. Atherosclerosis is generally regarded as a chronic inflammatory process of the arterial wall. The endothelial cells activation state is regulated directly or indirectly by the expression of pro-inflammatory genes including ICAM-1, VCAM-1 and MCP-1(Kunsch and Medford, 1999). These pro-inflammatory genes facilitate monocyte-endothelial cells interactions and initiate early stages of atherosclerosis. Many evidences have revealed that H2O2 can cause 15 / 29
endothelial cells activation and induce adhesion molecules and chemokines expression in vitro (Xing et al., 2014). Here, we demonstrate for the first time that β-elemene can directly regulate the expression of adhesion molecules (ICAM-1 and VCAM-1). Meanwhile it inhibits THP-1 monocyte adhesion and transendothelial migration across endothelial cells and prevents a critical early step of atherosclerosis. Furthermore, these results are similar to those observed in response to NF-κB inhibitor (PDTC) treatment which suggests that the inhibition of H2O2-induced monocyte- endothelial cells interactions can be mediated by suppression of NF-κB signaling pathway. Reactive oxygen species play an important role in the endothelial cells activation and pathogenesis of cardiovascular disease including atherosclerosis (Wilson, 2003). Therefore, preventing reactive oxygen species formation, by targeting specific sources of superoxide anion and NADPH oxidases, might be beneficial against atherosclerosis (Guzik and Harrison, 2006). ·
Reactive oxygen species such as OH , O2- and H2O2 have been demonstrated to be implicated in the pathphysiology of atherosclerosis (Bedard and Krause, 2007). Our current results show that ·
β-elemene inhibits OH and H2O2 production and further confirm the direct reactive oxygen species scavenging capacity of β-elemene. Besides, the anti-oxidative mechanism of β-elemene is probably not due to the direct reaction between β-elemene and H2O2. Further, the up-regulation of antioxidant enzymes could also be another reason for the inhibitory effect of β-elemene on intracellular reactive oxygen species. Therefore we studied the effect of β-elemene on the expression of heme oxygenase (HO)-1 in HUVECs after treatment with H2O2. β-elemene showed a slight effect on the expression and activity of HO-1. NADPH oxidase is a major source of reactive oxygen species generation in vascular 16 / 29
endothelial cells. P22phox subunit is an essential component of all NOX family members of NADPH oxidases (Griendling et al., 2000b). In our previous study, we reported that β-elemene derivatives can prevent endothelial cells from H2O2-induced cytotoxicity by effectively suppressing reactive oxygen species generation (Chen et al., 2014). Additionally, the study showed that SOD enzymes could remove reactive oxygen species through metabolic conversion and exert anti-atherosclerotic effects by the inhibition of oxidative alterations (Han et al., 2008). Our current study indicates that the β-elemene treatment can dose-dependently inhibit the cytotoxicity induced by H2O2 and increase the SOD activation along with a decrease of reactive oxygen species generation. Other studies have shown that global knockout of Nox2 results in a significant decrease in aortic atherosclerotic burden (Judkins et al., 2010). Our further study confirmed that the NADPH oxidase subunit p22phox is down-regulated at both mRNA and protein levels by pre-treatment with β-elemene, which may explain the continuously decreased reactive oxygen species levels. Collectively, our present study demonstrates that β-elemene exerts its anti-atherosclerotic effects through the down-regulation the expression of NADPH oxidase subunit p22phox and the increase of SOD activation, which suppress the overproduction of reactive oxygen species. Moreover, recent studies suggest that the source of reactive oxygen species and the signaling pathways implicated may represent important therapeutic targets (Schramm et al., 2012). Recently, NF-κB has been identified as a redox-sensitive transcription factor, which can translocate from the cytoplasm to the nucleus. It plays key roles in up-regulating the expression of adhesion molecules including ICAM-1 and VCAM-1 in endothelial cells. Data showed that IκB protein is oxidation-sensitive: antioxidants can prevent agonist-stimulated 17 / 29
IκB phosphorylation and degradation (Barchowsky et al., 1995). Conversely, H2O2 increases nuclear translocation of NF-κB, contributing to the induction of genes responsive to this transcription factor (Spiecker et al., 1998). Consistent with these findings, our results show that β-elemene not only effectively suppresses H2O2-stimulated phosphorylation of IκBα in a dose-dependent manner but also blocks nuclear translocation of p65-NF-κB in HUVECs. It has also been shown that β-elemene exerts protective effect on endothelial cells by regulating redox-sensitive signaling pathway. Here, the elevation of intracellular reactive oxygen species levels and ICAM-1 and VCAM-1 expression induced by H2O2 could be attenuated by pretreatment of β-elemene and PDTC in a similar pattern. These results indicate that the inhibition effect of β-elemene on CAMs’ expression is largely dependent on the NF-κB signaling pathway. Therefore, the effects of β-elemene on CAMs’ expression and atherogenic processes may be through interfering with reactive oxygen species /NF-κB signaling pathways. To sum up, our data indicate that the inhibitory effects of β-elemene on monocyte-endothelial cells interactions might be due to the down-regulation of CMAs’ expression via inhibition of reactive oxygen species/NF-κB signaling pathways. Additionally, several reports have shown that MAPKs such as BMK1, JNK, and p38 may be regulated by oxidative stress (Breton-Romero and Lamas, 2014). Moreover, the activation of MAPK signaling pathway has been reported to promote CAMs’ expression in vitro (Surapisitchat et al., 2001). In the present study, we investigated the probable implication of MAPK signaling pathway in the effects of β-elemene on H2O2-mediated CAMs’ expression. We found that β-elemene inhibits the H2O2-induced activation of ERK1/2, JNK and p38 MAPK signaling pathways in HUVECs which demonstrates the involvement of these signaling pathways in 18 / 29
β-elemene effects on CAMs’ expression. In neonatal rat ventricular myocytes, ERK1/2, p38 MAPK and JNK have been demonstrated to be activated by H2O2 (Clerk et al., 1998). Therefore, we speculate that the effect of β-elemene on CAM’s expression and monocyte-endothelial cells interactions might be related to an interference with the reactive oxygen species/MAPK signaling pathways. In conclusion, our present study demonstrates that β-elemene blocks H2O2-induced monocyte-endothelial cells interactions through the inhibition of NF-κB-dependent CAMs’ expression, reactive oxygen species generation and MAPK activation which may explain its anti-atherogentic effects. Our findings confirmed the cytoprotective action of β-elemene and its suppressive effect on monocytes adhesion and infiltration to endothelial cells in atherosclerosis. Hence, β-elemene may be viewed as a promising candidate to become a novel therapeutic agent for the treatment of atherosclerosis. However, further in vivo studies are required in order to confirm our present findings.
Acknowledgments This study was supported by “National Science and Technology Infrastructure Program of China” (2012BAI30B001), “Mega-Projects of Science Research for the 12th Five-Year Plan of China” (2011ZX09401007), “Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University” (JKGZ201108), and “Jiangsu Yanjiusheng Chuangxin Plan” (CXLX11-0782).
References 19 / 29
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Figures Legends Fig. 1. Effect of β-elemene on H2O2-induced cytotoxicity in HUVECs. (A) Chemical structure of β-elemene. (B) After treatment with different concentrations of β-elemene for 24 h, HUVECs were incubated with 0.5 mM H2O2 for 2 h. MTT assay was used to analyze cells viability (n=3). ＊
P＜0.01 versus controls,
＃
P＜0.01 versus H2O2 groups.
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Fig. 2. Effect of β-elemene on H2O2-induced THP-1 monocyte adhesion and migration across HUVECs. HUVECs were incubated with 50 μM β-elemene and 20 μM PDTC for 24h prior to treatment with or without 0.5mM H2O2 for 2 h. Fluorescence-labeled THP-1 monocytes were added to the HUVECs and incubated for 6 h. (A) The adhered monocytes were photographed under a fluorescence microscope. (B) and (C) The numbers of monocytes adhered and migrated across endothelial cells were measured by a fluorescence plate reader at 488 nm/535 nm (n=3). ＊
P＜0.01 versus controls,
＃
P＜0.01 versus H2O2 groups.
Fig. 3. Effect of β-elemene on mRNA and protein levels of ICAM-1 and VCAM-1 in HUVECs. HUVECs were pre-treated with different concentration of β-elemene for 24 h and then incubated with 0.5 mM H2O2 for 2 h. (A) The mRNA expression levels of ICAM-1 and VCAM-1 in HUVECs were detected by Real-time PCR (n=3). (B) The protein levels of ICAM-1 and VCAM-1 in HUVECs were examined by western blotting (n=3). (C) HUVECs were pre-incubated with 50μM β-elemene and 20mM PDTC for 24 h prior to treatment with 0.5mM H2O2 for 2h; the protein levels of ICAM-1 and VCAM-1 were measured by western blotting (n=3). versus controls,
＊
P＜0.01
＃
P＜0.01 versus H2O2 groups.
Fig. 4. Inhibitory effect of β-elemene on OH· and H2O2 levels. HUVECs were pre-treated with or without different concentrations of β-elemene for 24 h and then stimulated with 0.5mM H2O2 for 2h. (A) The scavenging OH· activity of β-elemene (n=3). (B) The scavenging H2O2 activity of β-elemene (n=3).
＊
P＜0.01 versus controls,
＃
P＜0.01 versus H2O2 groups.
25 / 29
Fig. 5. Effect of β-elemene on H2O2-stimulated reactive oxygen species generation, SOD activity and p22phox expression in HUVECs. HUVECs were treated with 0.5mM H2O2 for 2 h after in the presence of β-elemene at different concentrations or 20μM PDTC for 24h and then incubation with DCFH-DA for 20min. (A) SOD activity in HUVECs were detected according to the manufacturer’s instructions (n=3). (B) The fluorescence intensity of DCFH-DA oxidation was examined with a fluorescence microplate reader. Data were expressed as % of increase intensity (n=3). (C) The mRNA expressions of p22phox was determined by Real-time PCR (n=3). (D) The protein levels of p22phox was detected by western blotting analysis (n=3). β-actin was used as a control protein.
＊
P＜0.01 versus controls,
＃
P＜0.01 versus H2O2 groups.
Fig. 6. Effect of β-elemene on H2O2-induced NF-κB translocation in HUVECs. Cells were pretreated with or without different concentrations of β-elemene for 1 h and then stimulated with 0.5mM H2O2 for 2 h. (A) Effect of β-elemene on IκB phosphorylation; the expression in HUVECs was analyzed by western blotting (n=3). (B) The separated nuclear and cytoplasm fractions of p65-NF-κB protein levels were analyzed by western blotting analysis (n=3). β-actin was used as a control protein. (C) The localization of p65-NF-κB was detected by immunocytochemical analysis. Green indicates p65-NF-κB protein and blue fluorescence indicates the nuclei. Scar bars = 20 μm. ＊
P＜0.01 versus controls,
＃
P＜0.01 versus H2O2 groups.
Fig. 7. Effect of β-elemene on H2O2-stimulated MAPK phosphorylation in HUVECs. (A) Western blotting analyses of ERK1/2, JNK, p38, p-ERK1/2, p-JNK and p-p38 expressions were performed after HUVECs were treated with or without different concentrations of β-elemene for 1 h before 26 / 29
incubation with 0.5mM H2O2 for 2 h. β-actin was utilized as a control protein (n=3). (B) Data from (A) and other similar experiments were quantified by densitometry (n=3). controls,
＃
P＜0.01 versus H2O2 groups.
Figure 1
Figure 2
Figure 3 27 / 29
＊
P＜0.01 versus
Figure 4
Figure 5
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Figure 6
Figure 7
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PII: DOI: Reference:
S0014-2999(15)30263-6 http://dx.doi.org/10.1016/j.ejphar.2015.09.032 EJP70239
To appear in: European Journal of Pharmacology Received date: 21 May 2015 Revised date: 15 September 2015 Accepted date: 21 September 2015 Cite this article as: Meng Liu, Lifei Mao, Abdelkader Daoud, Waseem Hassan, Liangliang Zhou, Jiawei Lin, Jun Liu and Jing Shang, β-elemene inhibits Monocyte-endothelial cells interactions via Reactive oxygen species/MAPK/NFκB signaling pathway in vitro, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.09.032 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 galley proof before it is published in its final citable 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.
β-elemene inhibits Monocyte-endothelial cells interactions via Reactive oxygen species/MAPK/NF-κB signaling pathway in vitro
Meng Liu a,c, Lifei Mao a, Abdelkader Daoud a,d, Waseem Hassan a, Liangliang Zhou a, Jiawei ＊
Lin a, Jun Liu a,b ,Jing Shang a,b a
National Center for Drug Screening & State Key Laboratory of Natural Medicines, China Pharmaceutical
University, Jiangsu Province, 210009, P. R. China. b
Jiangsu Key Laboratory of TCM Evaluation and Translational Research, China Pharmaceutical University, 24
Tong jia xiang, Nanjing 210009, Jiang Su Province, P.R. China. c
Cancer Hospital Affiliated to Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China
d
Département de Pharmacie, Faculté de Médecine, Université Abou Bekr Belkaid, Tlemcen, Algérie
＊
Corresponding author: Jing Shang
National Center for Drug Screening & State Key Laboratory of Natural Medicines, China Pharmaceutical
University, Nanjing, Jiang Su Province, P.R. China. Telephone number: +86-25-83271043; Fax number:
+86-25-83271142; E-mail address: [email protected]
Abstract The recruitment of monocytes to the active endothelial cells is an early step in the formation of
atherosclerotic lesions; therefore, the inhibition of monocyte-endothelial cells interactions may serve as a potential therapeutic strategy for atherosclerosis. Recent studies suggest that β-elemene can protect against
atherosclerosis in vivo and vitro; however, the mechanism underlying the anti-atherosclerotic effect by
1 / 29
β-elemene is not clear yet. In this study, we aimed to investigate the effects of β-elemene on the
monocyte-endothelial cells interactions in the initiation of atherosclerosis in vitro. Our results showed that β-elemene protects human umbilical vein endothelial cells (HUVECs) from hydrogen peroxide-induced
endothelial cells injury in vitro. Besides, this molecule inhibits monocyte adhesion and transendothelial migration
across inflamed endothelium through the suppression of the nuclear factor-kappa B-dependent expression of cell adhesion molecules. Further, β-elemene decreases generation of reactive oxygen species (ROS) and prevents the
activation of mitogen-activated protein kinase (MAPK) signaling pathway in HUVECs. In conclusion, this study would provide a new pharmacological evidence of the significance of β-elemene as a future drug for prevention
and treatment of atherosclerosis.
Keywords β-elemene; Atherosclerosis; Human umbilical vein endothelial cells; Cell adhesion; NF-κB
1. Introduction The recruitment of circulating monocytes to the sites of arterial injuries and adhere to endothelium and transmigrate into the arterial wall are an initial step of the progression of atherosclerosis (Clapp et al., 2004; Erdogan et al., 2007; Lee et al., 2013; Mestas and Ley, 2008). Accumulated evidence demonstrates that monocyte infiltration plays a crucial role in the pathophysiology of coronary artery diseases including atherosclerosis, which can not only initiate and propagate the accumulation of monocyte-derived macrophages but also produce inflammatory mediators that destabilize atherosclerotic plaques (Kartikasari et al., 2009; Zhu et al., 2013). The monocyte-endothelium cells interactions consist of consecutive processes of monocytes adhesion 2 / 29
and infiltration through the endothelium. During early stages of atherosclerosis, endothelial cells elicit the up-regulation of vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 levels, which mediate the monocyte adhesion to activated endothelial cells and finally induce the progression of atherosclerosis (Kim et al., 2008). Many of stimulators including H2O2, oxidized low-density lipoprotein (ox-LDL) and homocysteine have been indicated to promote the expression levels of CAMs in endothelial cells resulting in endothelial cells activation, dysfunction and injury (Boulden et al., 2006; Cernuda-Morollon and Ridley, 2006; Coyle et al., 2006; Wilson, 2003). Hence, the inhibition of monocyte-endothelial cells interactions may represent a therapeutic approach against atherosclerosis and other related disorders (Fuentes and Palomo, 2014). Oxidative stress-induced disruption of redox states could lead to the activation of several redox-sensitive pathways including MAPK and NF-κB. These can result in the elevation of adhesion molecules, chemokines and cytokines expression and ultimately contribute to endothelial cells activation and chronic inflammation that induces leukocytes recruitment and infiltration (Kyaw et al., 2004). In vitro experiments have sufficiently proved that oxidative stressors (e.g. H2O2 and ox-LDL) could mimic the oxidative environment and lead to endothelial cells activation and elevation of adhesion molecule and chemokine expression (Song et al., 2014). H2O2 can up-regulate CAMs’ expression leading to monocytes adhesion to endothelial cells through the activation of NF-κB and MAPK signaling pathways and ultimately induce endothelial dysfunction (Jin et al., 2014). Accordingly, studying the molecular recognition and signaling pathway might help us understand the mechanisms underlying pharmacological inhibition of monocyte- endothelial cells interactions implicated 3 / 29
in atherosclerosis. In conclusion, the suppression of reactive oxygen species (ROS) generation, NF-κB translocation and MAPK signal pathways can be a very useful strategy to inhibit CAMs’ expression and monocyte-endothelial cells interactions. β-elemene (Fig. 1A), a sesquiterpenes compound extracted from the Curcuma Wenyujin (Guo, 1983), is a traditional Chinese herbal medicine that has been shown to inhibit tumor cell growth in vivo and in vitro. Because of its proven safety, this compound has been applied in clinics for the treatment of malignant effusions and some solid tumors (Li et al., 2005; Wang et al., 2005a; Wang et al., 2005b). Previous studies have shown that β-elemene derivatives have a significant antioxidant activity and cytoprotective effects against oxidative damage in HUVECs (Chen et al., 2014). In addition, another study indicated that β-elemene can inhibit smooth muscle proliferation/migration and inhibit neointima formation in vivo (Wu et al., 2011). However the effect of β-elemene on H2O2-induced monocyte-endothelial cells interactions and the underlying molecular mechanisms remain unknown. Therefore, the aim of this study is to investigate the potential inhibition effect of β-elemene on monocyte-endothelial cells interactions and unveil the possible mechanism for preventing atherosclerosis.
2. Materials and methods 2.1. Materials β-elemene [99.3% purity, CSPC Yuanda (Dalian) Pharmaceutical Co., Ltd. Dalian Liaoning China,1 mg/l]. DAPI were from Life Technologies Corporation (USA), Hydrogen peroxide (H2O2), Pyrrolidinedithiocarbamate (PDTC), Dimethylsulfoxide (DMSO), 3-(4,5-dimethyl– thiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT), β-actin antibody, and horseradish peroxidase4 / 29
conjugated secondary antibodies were purchased from Sigma-Aldrich (MO, USA). The Takara quantitative RT-PCR kit and SYBR Green Premix Ex Taq were products of Takara Biomedical Inc. (Shiga, Japan). Antibodies against p-p38, p38, p-ERK1/2, ERK1/2, p-JNK (Thr183/Tyr185), JNK, p-Iκβα (Ser32), Iκβα, NF-κB p65, Histone H3 were from Cell Signaling Technology (MA, USA), Anti-Cytochrome b245 Light Chain antibody was from Abcam (Cambridge, UK). ICAM-1(G-5): sc-8439, VCAM-1(E-10): sc-13160 was from Santa Cruz Biotechnology (Santa Cruz, CA). BCA protein assay kit, phenylmethylsulfonyl fluoride (PMSF) and cell lysis buffer for Western and IP, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF/AM), Reactive oxygen species and Superoxide dismutase (SOD) Assay Kit were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Other reagents were of the highest obtainable quality.
2.2. Cell culture Human umbilical vein endothelial cells (HUVECs) were from Nanjing Key Gen Biotech Co. Ltd (Nanjing, China). HUVECs were cultured in DMEM (Invitrogen) containing 10% heat-inactivated fetal bovine serum (FBS) at 37°C in 5% CO2 humid incubator. Human peripheral blood monocyte (THP-1) cells were grown in RPMI-1640 medium with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin sulfate.
2.3. Cell viability assay HUVECs were seeded in 96-well plates at a density of 5×103 cells/well. After 24 h 5 / 29
growth, HUVECs were pre-treated with β-elemene (0.5, 5, 50 μM) for 24 h at 37°C in 5% CO2 incubator. Then, HUVECs were treated with 0.5 mM H2O2 for 2 h. After that MTT (0.5%, 20 μl) was added to the medium and cells were further incubated for 4 h. The supernatant was removed and 100μl DMSO was added to dissolve the precipitate. Finally, the absorbance was measured spectrophotometrically at 570 nm.
2.4. Detection of OH· and H2O2 in HUVECs The effects of β-elemene on H2O2-induced hydroxyl radical (OH·) and hydrogen peroxide (H2O2) production were measured with commercially available kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. Briefly, OH· was generated by the Fenton reaction and treated with a chromogenic substrate nitrotetrazolium blue chloride (NBT) to yield a stable colored substance, which was measured at 450 nm. The reaction product of H2O2 and molybdic acid was determined at 405 nm using the fluorescence microplate reader (Xing et al., 2014) (TECAN Safire2TM). The scavenging rate (%) = [(O.D control – O.D sample) / O.D control] ×100.
2.5. Reactive oxygen species contents analysis The intracellular generation of reactive oxygen species was measured by the fluorescent probe, 2’, 7’-dichlorodihydrofluorescein diacetate (DCFH-DA). Briefly, HUVECs were incubated with β-elemene (0.5, 5, 50 μM) and PDTC (20μM) for 24 h and then stimulated with 0.5 mM H2O2 for 2h. After that, HUVECs were labeled with 10μM DCFH-DA and incubated for 20 min, followed by washed three times with medium. The fluorescence microplate reader (TECAN 6 / 29
Safire2TM) was used to determine the fluorescence. The results of reactive oxygen species were expressed as the percentage of the control group fluorescence intensity.
2.6. Measurement of intracellular Superoxide Dismutase The superoxide dismutase (SOD) activity was detected using a commercial kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. Briefly, HUVECs were seeded and then stimulated with β-elemene and H2O2. After that, cells were washed with Phosphate Buffered Saline (PBS) and then lysed. The lysate was centrifuged at 12,000 g for 15 min at 4°C. The supernatants were collected to measure the SOD activity.
2.7. Quantitative real-time PCR HUVECs were incubated with β-elemene (0.5, 5, 50 μM) for 24 h and then stimulated with 0.5mM H2O2 for 2h. Total RNA was extracted using TRIZOL reagent (Invitrogen, U.S.A). First strand cDNA was synthesized with PrimeScript RT Master Mix kit (Takara, Japan) according to the manufacturer’s instructions. The quantitative real-time PCR was performed on an iQ5 multicolor real-time PCR detection system (Bio-Rad, Hercules, Calif., U.S.A) by using SYBR Premix Ex TaqTM (Takara, Japan) according to the manufacturer’s instructions. To investigate the effects of β-elemene on atherosclerosis, the mRNA expression of p22 phox, VCAM-1 and ICAM-1 was examined. Primer sequences were as follows: p22 phox , 5’- AGA AGT ACA TGA CCG CCG TGG -3’ (forward) and 5’- AGT AGG TAG ATG CCG CTC GCA AT -3’ (reverse); VCAM-1 , 5’- GAT ACA ACC GTC TTG GTC AGC CC 7 / 29
-3’ (forward) and 5’- CGC ATC CTT CAA CTG GCC TT -3’ (reverse); ICAM-1 , 5’- TAT GGC AAC GAC TCC TTC T -3’ (forward) and 5’- CAT TCA GCG TCA CCT TGG -3’ (reverse) and β-actin, 5’-CGC AAA GAC CTG TAC GCC AAC-3’ (forward) and 5’-CAC GGA GTA CTT GCG CTC AGG-3’ (reverse). p22phox, VCAM-1 and ICAM-1 transcript levels were calculated by the 2-ΔΔCT method.
2.8. Western blotting analysis After treatment, HUVECs were lysed by RIPA lysis buffer. The lysates were subjected to centrifugation at 14,000 g for 10 min at 4°C and the supernatants were used for quantification of total protein concentration by using the BCA Protein Assay Kit (Beyotime Biotechnology, Nanjing, China). Protein samples were separated using electrophoresis on SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, U.S.A). Monoclonal antibodies against p22phox, p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, p38, p-Iκβα, Iκβα, p65 NF-κB, ICAM-1, VCAM-1 and β-actin were incubated at 4°C overnight. After that the membranes were incubated with secondary antibodies. Protein bands were analyzed using a chemiDoc XRS imaging system (Bio-Rad) and Quantity One (Bio-Rad) software program.
2.9. Immunofluorescence confocal microscopy HUVECs were seeded onto Class Bottom Cell Culture Dish (NEST) the day before treatment and processed for immunofluorescence. In brief, cells were pretreated with 50 μM β-elemene for 1h, and then exposure to 0.5 mM H2O2 for 2h. After those HUVECs were washed thrice with PBS for 5 min, cells were incubated with 4% paraformaldehyde on ice for 20 min to fix them and again 8 / 29
washed three times. After that, cells were incubated with Triton X-100 for 20 min. Then, cells were blocked with PBS containing 5% bovine serum albumin (BSA) for 1h, and incubated with p65 NF-κB antibody (1:400) at 37°C for 1h. After washing with PBS, HUVECs were stained with FITC-conjugated anti-rabbit IgG antibody (1:100) for 1h. Then the cells were stained with diamidino-phenyl-indole (DAPI) for 20 min. Pictures were captured with Olympus IX81 confocal microscope.
2.10. Monocyte adhesion assay HUVECs were pretreated with 50 μM β-elemene and 20 μM PDTC in 24-well plates for 24h and subsequently stimulated with 0.5 mM H2O2 for 2 h. THP-1 cells were labeled with 10 μg/ml BCECF-AM for 30min at 37°C. Labeled THP-1 cells were washed with PBS and re-suspended in RPMI 1640 medium containing 2% FBS. Then, HUVECs were co-cultured with the BCECF-AM -labeled THP-1 cells (2×106/well) for 30 min at 37°C. HUVECs were washed twice with PBS to remove non-adhering THP-1 cells and fluorescence levels were analyzed using a fluorescence microplate reader (TECAN Safire2TM) at 485 nm/520 nm. The percentage of adherent monocytes was calculated by the formula: % adherence = (adherent signal/total signal) × 100.
2.11. Transendothelial migration assay The transendothelial migration assay was measured by a fluorescence-based assay. In brief, HUVECs were placed on 24-Well Millcell (with 8.0 μm PET; MILLIPORE) at a density of 2.5 × 104 cells/insert. Because monolayers were not visible on these inserts, 9 / 29
manual readings of transendothelial electrical resistance (TEER) were taken with a voltmeter (MILLIPORE-ERS USA) to confirm monolayer formation. Thereafter, HUVECs were pretreated with 50μM β-elemene and 20μM PDTC for 24h and then co-incubated with 0.5 mM H2O2 for 2 h. The inserts were washed thoroughly and properly in every treatment, and labeled THP-1 monocytes (2.5× 105 cells/insert loaded with 10 μg/ml of the fluorescent dye BCECF/AM for 45 min) were placed on the upper chamber, and migration was continued to 6 h at 37°C. Migration was monitored by taking the fluorescence using a fluorescence plate reader (TECAN Safire2TM) when the labeled monocytes found in the lower chamber. Additionally, the data were shown as the percentage of input (right y - axis), which indicates the percentage of cells that migrated from the number of cellular input.
2.12. Statistical analysis Statistical analysis was achieved by using graph pad prism Version 5.0c (Graph Pad Software). Quantitative data are expressed as mean ± S.D. Data were analyzed, as relevant, by unpaired two-tailed Student’s t-test or by one-way ANOVA with Turey’s post hoc test. P value of 0.01 was accepted as statistically significant.
3. Results 3.1. Effect of β-elemene on H2O2-induced cytotoxicity Cell viability assay was used to assess the protective effect of β-elemene against H2O2-induced cellular toxicity. The results showed that the inhibitory rates of β-elemene (0.5, 5, 50 μM) on the H2O2-induced cytotoxicity were 59.7%, 75.1% and 86.2%, respectively (Fig. 1B). 10 / 29
The β-elemene at 0.5, 5, 50 μM was then used in all subsequent experiments. These results also suggested that β-elemene can protect endothelial cells from H2O2-induced cytotoxicity.
3.2. Inhibition of monocyte adhesion and transendothelial migration by β-elemene Monocytes adhesion toward vascular endothelial cells is the initial step in migration across the inflammation endothelial cells (Muller, 2009). Therefore monocytes adhesion, transendothelial migration and the role of NF-κB signaling pathway in these processes were investigated. As shown in Fig. 2A and 2B, monocytes adhesion increased to 40.0% when endothelial cells were stimulated with 0.5 mM H2O2. Pre-treatment of HUVECs with 50 μM β-elemene and 20 μM PDTC resulted in 24.0 % and 38.7 % decrease in cellular adhesion compared with H2O2 treated group. Afterwards we studied monocyte migration to determine whether β-elemene can also affect monocyte infiltration into the endothelium. The findings showed that the stimulation of endothelial cells with H2O2 caused an 81.7% increase in cell migration. Pretreatment with 50 μΜ β-elemene and 20 μM PDTC in the above treatment conditions produced 38.3% and 53.5% decrease in transendothelial migration compared with H2O2 treated group (Fig. 2 C). Taken together, these results suggest that β-elemene inhibits H2O2-induced monocyte adhesion and transendothelial migration and this effect may be mediated via suppression of NF-κB signaling pathway in HUVECs.
3.3. Inhibition of cell adhesion molecule expression by β-elemene We also investigated the effect of β-elemene on ICAM-1 and VCAM-1expression 11 / 29
induced by H2O2 in vitro. The mRNA expression and protein levels of ICAM-1 and VCAM-1 in HUVECs were increased after stimulation with H2O2 as shown in Fig 3A and 3B. After treatment with β-elemene, ICAM-1 and VCAM-1 mRNA expression were reduced by 88% and 84% respectively (Fig. 3A). Furthermore, the inhibition rates of VCAM-1 and ICAM-1 protein levels by β-elemene treatment were 74% and 79% (Fig. 3B). As shown in Fig. 3C, the results demonstrated that the protein levels of ICAM-1 and VCAM-1 were significantly reduced by PDTC. This finding was similar to that obtained from the treatment of HUVECs with 50 μM β-elemene. Hence, these results suggest that the inhibition effects of β-elemene on the protein levels of ICAM-1 and VCAM-1 in HUVECs may be mediated through suppression of NF-κB signaling pathway.
·
3.4. Inhibition of OH and H2O2 production by β-elemene ·
As shown in Fig. 4A and 4B, OH and H2O2 production was increased after H2O2 induction, however pretreatment with β-elemene inhibited these production in a dose-dependent manner. The ·
results pointed that β-elemene inhibits OH and H2O2 production and showed the scavenging ·
activates of OH and H2O2.
3.5. β-elemene attenuated reactive oxygen species generation and inhibited the over-expression of p22phox in HUVECs The results showed that the level of reactive oxygen species was markedly increased in HUVECs induced by H2O2 (Fig. 5A). After pre-treatment with β-elemene, the relative fluorescence intensity was decreased to 20.0%, 39.7% and 51.1% in a dose-dependent manner 12 / 29
compared with the H2O2 treated group. Meanwhile, the reactive oxygen species level was inhibited to 64.6% when cells were treated with 20 μM PDTC compared with the H2O2 treated group. This result revealed that PDTC inhibits the NF-κB activation by its anti-oxidation property (Yokoo and Kitamura, 1996). According to these findings, β-elemene also increased dose-dependently SOD activity (17.37±1.85, 18.41±2.71 and 20.43 ± 1.51 U /mgprot for 0.5, 5, 50 μM β-elemene, respectively in Fig. 5B). NADPH oxidase, which has a p22phox modulatory subunit, has been widely regarded as a main source of reactive oxygen species generation in endothelial cells (Jin et al., 2014). Our results showed that pretreatment with β-elemene (0.5, 5, 50 μM) could down-regulate the mRNA expression of p22phox in a dose-dependent manner as indicated in Fig. 5C (13.1%, 44.0%, 79.5%, compared with the H2O2 treated group). Moreover, the inhibitory effect of β-elemene on the protein level of p22phox was also confirmed by western blotting as Fig. 5D shows (14.2%, 23.8%, 29.0%, compared with the H2O2 treated group).
3.6. The modulation of NF-κB signaling pathway by β-elemene and its anti- inflammation effect Oxidative stress and persistent inflammation are critical pathologic events in atherosclerosis. Reactive oxygen species serves as the common intracellular messengers to activate multiple redox-sensitive signaling pathways including MAPK and NF-κB and plays a key role in the development of many vascular diseases including atherosclerosis (Griendling et al., 2000a). H2O2, which belongs to reactive oxygen species family, is an activator of NF-κB in endothelial cells. NF-κB plays a critical role in the inflammatory response progress by up-regulating the expression levels of VCAM-1, ICAM-1 and 13 / 29
E-selectin. Furthermore, the up-regulation of CAMs’ expression provides a mechanism for the monocyte adhesion and transendothelial migration. Our data revealed that the phosphorylation levels of Iκβ and the nuclear location of NF-κB were increased in H2O2-induced HUVECs, but this effect was decreased when HUVECs were pre-treated with β-elemene (0.5, 5, 50 μM) as indicated in Fig. 6A and 6B. Using immunofluorescence microscopy analysis, we demonstrated NF-κB translocation from the cytoplasm to the nucleus after H2O2 stimulation (Fig. 6C); this translocation was efficiently inhibited by pretreatment with β-elemene in HUVECs. In addition, these results were consistent with western blotting analysis and showed that β-elemene inhibits H2O2-induced NF-κB translocation. The effects of β-elemene on MAPK signaling pathway were analyzed and the ERK1/2, JNK, and p38 activation was measured. Our data showed that ERK 1/2, JNK and p38 were sufficiently activated after 2 h incubation with 0.5 mM H2O2. Meanwhile, the activation of ERK 1/2, JNK and p38 was markedly decreased by β-elemene in a dose-dependent manner (Fig. 7A and 7B). Total protein levels of ERK 1/2, JNK, and p38 were not altered upon H2O2 and/or β-elemene treatment. These results indicated that β-elemene specifically plays its role via modulation of NF-κB and ERK1/2 /JNK/p38 MAPK signaling pathways.
4. Discussion Monocytes recruitment and infiltration is a central mechanism in the early steps of atherosclerosis, which can lead to chronic inflammation and the development and progression of atherosclerosis. This process is initiated by the activation of endothelial cells and the up-regulation 14 / 29
of cell adhesion molecules and chemokines. β-elemene, which is derived from a traditional Chinese herbal, has been shown to have an anti-atherosclerotic effect; however, the molecular mechanisms by which β-elemene inhibits the monocyte-endothelial cells interactions associated with atherosclerosis remain unknown. The aims of the present study were to investigate the the effect of β-elemene on monocytes adhesion and migration during atherosclerosis and elucidate the probable mechanisms. Additionally the responses of the vascular endothelium to oxidative stress conditions play a significant role in the regulation of monocytes adhesion and infiltration into the endothelium. Previously, we reported that β-elemene derivatives have significant cytoprotective and anti-oxidative effects against oxidative damage induced by H2O2 in HUVECs (Chen et al., 2014). Consistent with previous reports, our results indicate firstly that β-elemene can inhibit H2O2-induced monocytes adhesion and transendothelial migration. Furthermore β-elemene inhibits the endothelial activation (attenuates oxidative stress, improved nitric oxide bioavailability and decreased ICAM-1 and VCAM-1 expression). These data suggest that its inhibition effects on monocyte-endothelial cells interactions might be due to the suppression of NF-κB-dependent ICAM-1 and VCAM-1 expression via modulation of reactive oxygen species generation and MAPK signaling pathway activation. Atherosclerosis is generally regarded as a chronic inflammatory process of the arterial wall. The endothelial cells activation state is regulated directly or indirectly by the expression of pro-inflammatory genes including ICAM-1, VCAM-1 and MCP-1(Kunsch and Medford, 1999). These pro-inflammatory genes facilitate monocyte-endothelial cells interactions and initiate early stages of atherosclerosis. Many evidences have revealed that H2O2 can cause 15 / 29
endothelial cells activation and induce adhesion molecules and chemokines expression in vitro (Xing et al., 2014). Here, we demonstrate for the first time that β-elemene can directly regulate the expression of adhesion molecules (ICAM-1 and VCAM-1). Meanwhile it inhibits THP-1 monocyte adhesion and transendothelial migration across endothelial cells and prevents a critical early step of atherosclerosis. Furthermore, these results are similar to those observed in response to NF-κB inhibitor (PDTC) treatment which suggests that the inhibition of H2O2-induced monocyte- endothelial cells interactions can be mediated by suppression of NF-κB signaling pathway. Reactive oxygen species play an important role in the endothelial cells activation and pathogenesis of cardiovascular disease including atherosclerosis (Wilson, 2003). Therefore, preventing reactive oxygen species formation, by targeting specific sources of superoxide anion and NADPH oxidases, might be beneficial against atherosclerosis (Guzik and Harrison, 2006). ·
Reactive oxygen species such as OH , O2- and H2O2 have been demonstrated to be implicated in the pathphysiology of atherosclerosis (Bedard and Krause, 2007). Our current results show that ·
β-elemene inhibits OH and H2O2 production and further confirm the direct reactive oxygen species scavenging capacity of β-elemene. Besides, the anti-oxidative mechanism of β-elemene is probably not due to the direct reaction between β-elemene and H2O2. Further, the up-regulation of antioxidant enzymes could also be another reason for the inhibitory effect of β-elemene on intracellular reactive oxygen species. Therefore we studied the effect of β-elemene on the expression of heme oxygenase (HO)-1 in HUVECs after treatment with H2O2. β-elemene showed a slight effect on the expression and activity of HO-1. NADPH oxidase is a major source of reactive oxygen species generation in vascular 16 / 29
endothelial cells. P22phox subunit is an essential component of all NOX family members of NADPH oxidases (Griendling et al., 2000b). In our previous study, we reported that β-elemene derivatives can prevent endothelial cells from H2O2-induced cytotoxicity by effectively suppressing reactive oxygen species generation (Chen et al., 2014). Additionally, the study showed that SOD enzymes could remove reactive oxygen species through metabolic conversion and exert anti-atherosclerotic effects by the inhibition of oxidative alterations (Han et al., 2008). Our current study indicates that the β-elemene treatment can dose-dependently inhibit the cytotoxicity induced by H2O2 and increase the SOD activation along with a decrease of reactive oxygen species generation. Other studies have shown that global knockout of Nox2 results in a significant decrease in aortic atherosclerotic burden (Judkins et al., 2010). Our further study confirmed that the NADPH oxidase subunit p22phox is down-regulated at both mRNA and protein levels by pre-treatment with β-elemene, which may explain the continuously decreased reactive oxygen species levels. Collectively, our present study demonstrates that β-elemene exerts its anti-atherosclerotic effects through the down-regulation the expression of NADPH oxidase subunit p22phox and the increase of SOD activation, which suppress the overproduction of reactive oxygen species. Moreover, recent studies suggest that the source of reactive oxygen species and the signaling pathways implicated may represent important therapeutic targets (Schramm et al., 2012). Recently, NF-κB has been identified as a redox-sensitive transcription factor, which can translocate from the cytoplasm to the nucleus. It plays key roles in up-regulating the expression of adhesion molecules including ICAM-1 and VCAM-1 in endothelial cells. Data showed that IκB protein is oxidation-sensitive: antioxidants can prevent agonist-stimulated 17 / 29
IκB phosphorylation and degradation (Barchowsky et al., 1995). Conversely, H2O2 increases nuclear translocation of NF-κB, contributing to the induction of genes responsive to this transcription factor (Spiecker et al., 1998). Consistent with these findings, our results show that β-elemene not only effectively suppresses H2O2-stimulated phosphorylation of IκBα in a dose-dependent manner but also blocks nuclear translocation of p65-NF-κB in HUVECs. It has also been shown that β-elemene exerts protective effect on endothelial cells by regulating redox-sensitive signaling pathway. Here, the elevation of intracellular reactive oxygen species levels and ICAM-1 and VCAM-1 expression induced by H2O2 could be attenuated by pretreatment of β-elemene and PDTC in a similar pattern. These results indicate that the inhibition effect of β-elemene on CAMs’ expression is largely dependent on the NF-κB signaling pathway. Therefore, the effects of β-elemene on CAMs’ expression and atherogenic processes may be through interfering with reactive oxygen species /NF-κB signaling pathways. To sum up, our data indicate that the inhibitory effects of β-elemene on monocyte-endothelial cells interactions might be due to the down-regulation of CMAs’ expression via inhibition of reactive oxygen species/NF-κB signaling pathways. Additionally, several reports have shown that MAPKs such as BMK1, JNK, and p38 may be regulated by oxidative stress (Breton-Romero and Lamas, 2014). Moreover, the activation of MAPK signaling pathway has been reported to promote CAMs’ expression in vitro (Surapisitchat et al., 2001). In the present study, we investigated the probable implication of MAPK signaling pathway in the effects of β-elemene on H2O2-mediated CAMs’ expression. We found that β-elemene inhibits the H2O2-induced activation of ERK1/2, JNK and p38 MAPK signaling pathways in HUVECs which demonstrates the involvement of these signaling pathways in 18 / 29
β-elemene effects on CAMs’ expression. In neonatal rat ventricular myocytes, ERK1/2, p38 MAPK and JNK have been demonstrated to be activated by H2O2 (Clerk et al., 1998). Therefore, we speculate that the effect of β-elemene on CAM’s expression and monocyte-endothelial cells interactions might be related to an interference with the reactive oxygen species/MAPK signaling pathways. In conclusion, our present study demonstrates that β-elemene blocks H2O2-induced monocyte-endothelial cells interactions through the inhibition of NF-κB-dependent CAMs’ expression, reactive oxygen species generation and MAPK activation which may explain its anti-atherogentic effects. Our findings confirmed the cytoprotective action of β-elemene and its suppressive effect on monocytes adhesion and infiltration to endothelial cells in atherosclerosis. Hence, β-elemene may be viewed as a promising candidate to become a novel therapeutic agent for the treatment of atherosclerosis. However, further in vivo studies are required in order to confirm our present findings.
Acknowledgments This study was supported by “National Science and Technology Infrastructure Program of China” (2012BAI30B001), “Mega-Projects of Science Research for the 12th Five-Year Plan of China” (2011ZX09401007), “Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University” (JKGZ201108), and “Jiangsu Yanjiusheng Chuangxin Plan” (CXLX11-0782).
References 19 / 29
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Figures Legends Fig. 1. Effect of β-elemene on H2O2-induced cytotoxicity in HUVECs. (A) Chemical structure of β-elemene. (B) After treatment with different concentrations of β-elemene for 24 h, HUVECs were incubated with 0.5 mM H2O2 for 2 h. MTT assay was used to analyze cells viability (n=3). ＊
P＜0.01 versus controls,
＃
P＜0.01 versus H2O2 groups.
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Fig. 2. Effect of β-elemene on H2O2-induced THP-1 monocyte adhesion and migration across HUVECs. HUVECs were incubated with 50 μM β-elemene and 20 μM PDTC for 24h prior to treatment with or without 0.5mM H2O2 for 2 h. Fluorescence-labeled THP-1 monocytes were added to the HUVECs and incubated for 6 h. (A) The adhered monocytes were photographed under a fluorescence microscope. (B) and (C) The numbers of monocytes adhered and migrated across endothelial cells were measured by a fluorescence plate reader at 488 nm/535 nm (n=3). ＊
P＜0.01 versus controls,
＃
P＜0.01 versus H2O2 groups.
Fig. 3. Effect of β-elemene on mRNA and protein levels of ICAM-1 and VCAM-1 in HUVECs. HUVECs were pre-treated with different concentration of β-elemene for 24 h and then incubated with 0.5 mM H2O2 for 2 h. (A) The mRNA expression levels of ICAM-1 and VCAM-1 in HUVECs were detected by Real-time PCR (n=3). (B) The protein levels of ICAM-1 and VCAM-1 in HUVECs were examined by western blotting (n=3). (C) HUVECs were pre-incubated with 50μM β-elemene and 20mM PDTC for 24 h prior to treatment with 0.5mM H2O2 for 2h; the protein levels of ICAM-1 and VCAM-1 were measured by western blotting (n=3). versus controls,
＊
P＜0.01
＃
P＜0.01 versus H2O2 groups.
Fig. 4. Inhibitory effect of β-elemene on OH· and H2O2 levels. HUVECs were pre-treated with or without different concentrations of β-elemene for 24 h and then stimulated with 0.5mM H2O2 for 2h. (A) The scavenging OH· activity of β-elemene (n=3). (B) The scavenging H2O2 activity of β-elemene (n=3).
＊
P＜0.01 versus controls,
＃
P＜0.01 versus H2O2 groups.
25 / 29
Fig. 5. Effect of β-elemene on H2O2-stimulated reactive oxygen species generation, SOD activity and p22phox expression in HUVECs. HUVECs were treated with 0.5mM H2O2 for 2 h after in the presence of β-elemene at different concentrations or 20μM PDTC for 24h and then incubation with DCFH-DA for 20min. (A) SOD activity in HUVECs were detected according to the manufacturer’s instructions (n=3). (B) The fluorescence intensity of DCFH-DA oxidation was examined with a fluorescence microplate reader. Data were expressed as % of increase intensity (n=3). (C) The mRNA expressions of p22phox was determined by Real-time PCR (n=3). (D) The protein levels of p22phox was detected by western blotting analysis (n=3). β-actin was used as a control protein.
＊
P＜0.01 versus controls,
＃
P＜0.01 versus H2O2 groups.
Fig. 6. Effect of β-elemene on H2O2-induced NF-κB translocation in HUVECs. Cells were pretreated with or without different concentrations of β-elemene for 1 h and then stimulated with 0.5mM H2O2 for 2 h. (A) Effect of β-elemene on IκB phosphorylation; the expression in HUVECs was analyzed by western blotting (n=3). (B) The separated nuclear and cytoplasm fractions of p65-NF-κB protein levels were analyzed by western blotting analysis (n=3). β-actin was used as a control protein. (C) The localization of p65-NF-κB was detected by immunocytochemical analysis. Green indicates p65-NF-κB protein and blue fluorescence indicates the nuclei. Scar bars = 20 μm. ＊
P＜0.01 versus controls,
＃
P＜0.01 versus H2O2 groups.
Fig. 7. Effect of β-elemene on H2O2-stimulated MAPK phosphorylation in HUVECs. (A) Western blotting analyses of ERK1/2, JNK, p38, p-ERK1/2, p-JNK and p-p38 expressions were performed after HUVECs were treated with or without different concentrations of β-elemene for 1 h before 26 / 29
incubation with 0.5mM H2O2 for 2 h. β-actin was utilized as a control protein (n=3). (B) Data from (A) and other similar experiments were quantified by densitometry (n=3). controls,
＃
P＜0.01 versus H2O2 groups.
Figure 1
Figure 2
Figure 3 27 / 29
＊
P＜0.01 versus
Figure 4
Figure 5
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Figure 6
Figure 7
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