Clinical and Experimental Pharmacology and Physiology (2015) 42, 502–509
doi: 10.1111/1440-1681.12375
ORIGINAL ARTICLE
Artemisinin inhibits tumour necrosis factor-a-induced vascular smooth muscle cell proliferation in vitro and attenuates balloon injury-induced neointima formation in rats Qian Cao,* Yan Jiang,† Jin Shi,* Xue Liu,* Jie Chen,* Tiesheng Niu* and Xiaodong Li* Departments of *Cardiology, and †Emergency Medicine, Shengjing Hospital of China Medical University, Shenyang, China
SUMMARY The aim of this study was to evaluate the effect of artemisinin (ART) on rat vascular smooth muscle cell (VSMC) proliferation induced by tumour necrosis factor (TNF)-a, cell cycle arrest, and apoptosis, and its effect on neointima formation after balloon injury of rat carotid artery. Primary rat VSMC were identified by immunofluorescence assay. The proliferation of VSMC induced by TNF-a was significantly inhibited by ART treatment in a dose-dependent manner. Treatment with 100-lM ART significantly reduced the expression of proliferating cell nuclear antigen. In contrast, the same treatment arrested the cell cycle in G0/G1 phase. Western blot analysis showed that the cell cycle-related proteins cyclin D1, cyclin E, cyclin-dependent kinase 2, and cyclin-dependent kinase 4 were downregulated by ART in TNF-a-stimulated VSMC. For apoptosis induced by ART, cleaved caspase-3/-9 was detected, and the pro-apoptotic protein Bcl-2-associated X protein was upregulated while the anti-apoptotic protein Bcl2 was downregulated. The results suggest that ART can effectively inhibit the proliferation of VSMC induced by TNF-a through the apoptotic induction pathway and cell cycle arrest. Also, balloon injury indicated that ART significantly inhibited neointima formation in the rat carotid arteries. Key words: apoptosis, artemisinin, cell cycle, neointima formation, proliferation, TNF-a, vascular smooth muscle cells.
INTRODUCTION Atherosclerosis, a chronic disease characterized by injury to vessel walls, is a major cause of human morbidity and mortality, and it is occurring at alarmingly increasing rates in both developed and developing countries.1 Current evidence suggests that the activation, proliferation, and anti-apoptotic effect of vascular
Correspondence: Dr Qian Cao, Department of Cardiology, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang 110004, China. Email: [email protected] Received 4 December 2014; revision 27 January 2015; accepted 15 February 2015. © 2015 Wiley Publishing Asia Pty Ltd
smooth muscle cells (VSMC) are absolutely essential in the early progress of atherogenesis.2 In the physiological state, VSMC are the major producers of extracellular matrix within the vessel wall, and they contract or relax to change the volume of blood vessels and local blood pressure.3 However, the transition into the earliest recognized form of an atherosclerotic lesion, termed a ‘pathological intimal thickening’, is associated with morphological and biochemical alterations to the VSMC and their extracellular matrix, leading to the release of growth factors such as tumour necrosis factor (TNF)-a, which is the major contributing factor in the pathogenesis of atherosclerosis.4 Thus, inhibition of VSMC proliferation induced by TNF-a, induction of apoptosis and cell cycle arrest, or reduction of neointima hyperplasia through drugs or other agents could prevent atherosclerosis and potentially delay or suppress its progress. Artemisinin (ART), a sesquiterpene lactone extracted from the Chinese plant Artemisia annua, is a potent anti-malarial drug that is effective in the treatment of severe and multidrug-resistant malaria.5 In addition, ART and its derivatives have been shown to possess pleiotropic characteristics such as anti-cancer,6 antiinflammatory,7 and anti-oxidant activities.8 Recently, several studies have indicated that ART has an effect on atherosclerosis. Wang et al.9 reported that ART significantly inhibited rats’ VSMC proliferation through induction of cell cycle arrest and apoptosis. Nonetheless, no study has investigated the effect of ART on neointima formation in the rat carotid artery. Moreover, whether ART can inhibit proliferation in VSMC induced by TNF-a through apoptosis or cell cycle arrest remains to be clarified, and the corresponding biological mechanism is still unclear. In this study, we investigated the inhibitory effects of ART in primary rat thoracic aortic VSMC proliferation induced by TNFa in vitro and the potential pathway of growth inhibition. At the same time, we evaluated the suppression effect of ART on neointima formation in rat carotid arteries in vivo, which may provide insights into the treatment of atherosclerosis using ART.
RESULTS Artemisinin inhibited proliferation induced by TNF-a and arrested cell cycle at G0/G1 phase in VSMC Artemisinin has been reported to induce growth arrest that involves selective changes in the expression and activity of cell
ART inhibits VSMC proliferation cycle components.10 To study the growth inhibition of ART in VSMC induced by TNF-a, we first identified the VSMC by immunofluorescence assay. It was found that 40 ,6-diamidino-2phenylindole and fluorescent a-actin were evenly distributed in the cells, indicating the VSMC (Fig. 1a). We tested the proliferation of VSMC after pretreatment with or without ART at different concentrations, and cells then underwent co-incubation with TNF-a by 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Data showed that after co-incubation with ART, the rate of VSMC proliferation induced by TNF-a decreased in a dose-dependent manner (Fig. 1b). The level of proliferationrelated protein proliferating cell nuclear antigen (PCNA) was also tested, and it was found that it was reduced to almost half after co-incubation with 100-lmol/L ART (Fig. 1c). Thus, ART suppresses the proliferation in TNF-a-induced VSMC. To investigate the pathway of growth inhibition induced by ART in TNF-a-stimulated VSMC, the cell cycle was tested by fluorescence-activated cell sorting analysis after staining with propidium iodide (PI). Data indicated that ART alone at 100 lmol/L did not affect the cell cycle of VSMC, while the G0/G1 phase was reduced from 65.32 2.55 to 40.00 2.55% after TNF-a stimulation in VSMC (Fig. 1d). However, the G0/G1 phase increased almost to the original level after co-incubation with 100-lmol/L ART (40.00 2.55 vs 59.97 2.64%) (Fig. 1d). These results suggested that ART may inhibit TNF-a-induced VSMC proliferation by interfering with cell mitosis through cell cycle arrest. Levels of the cell cycle-related proteins cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, and CDK4 were determined in VSMC induced by TNF-a with or without ART by western blot analysis. These proteins, especially cyclin E and CDK4, were significantly upregulated by TNF-a stimulation (Fig. 1e). However, when VSMC pretreated with TNF-a were co-incubated with ART, the high expression levels of these proteins remained almost at their original levels in a dose-dependent manner (Fig. 1e, lower panel). Thus, we concluded that ART inhibits cell proliferation through cell cycle arrest by downregulating cyclin D1, cyclin E, CDK2, and CDK4 in VSMC induced by TNF-a. Artemisinin induced apoptosis in TNF-a-stimulated VSMC Currently, an increasing number of investigations have reported that ART and its derivatives induce apoptosis in cancer cells.11,12 To evaluate the apoptosis induction of ART in TNF-a-stimulated VSMC, the levels of apoptotic cells were determined by fluorescence-activated cell sorting analysis (Fig. 2a). Data showed that ART and TNF-a alone did not induce apoptosis in VSMC. Interestingly, apoptotic cells were detected after pretreatment with ART at different concentrations followed by co-incubation with TNF-a in a dose-dependent manner (Fig. 2a, upper panel). The apoptotic index was 80.58 7.35% in the 100-lmol/L ART group treated with TNF-a, which was much higher than the ART group (11.73 2.47%) (Fig. 2a, lower panel). Data indicate that ART induces apoptosis in TNF-a-stimulated VSMC. To determine the mechanisms of apoptosis induction by ART in TNF-a-stimulated VSMC, we used western blot analysis to investigate levels of apoptosis-related proteins in VSMC induced by TNF-a with or without ART. As shown in Fig. 2b, the level of cleaved caspase-3 detected suggested that a caspase-mediated
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pathway led to apoptosis. Also, it was reported that ART and its derivative dihydroartemisinin induced apoptosis through an intrinsic pathway in Jurkat T-lymphoma cells.13 Therefore, we tested cleaved caspase-9 and found that it was upregulated after ART treatment. Activation of the apoptotic pathway is regulated by the balance between pro-apoptotic proteins, such as Bcl-2-associated X protein (Bax), and pro-survival Bcl-2 proteins, such as Bcl-2.14 Therefore, the levels of Bax and Bcl-2 were tested in VSMC induced by TNF-a with or without ART. It was found that the level of pro-apoptotic protein Bax was upregulated while the level of anti-apoptotic protein Bcl-2 was downregulated (Fig. 2b). Thus, upregulation of Bax and downregulation of Bcl-2 seem to account for the activation of the intrinsic apoptotic pathway induced by ART in TNF-a-stimulated VSMC. Artemisinin inhibits neointima formation after balloon injury of rat carotid artery To study the effect of ART on neointima formation after injury, balloon injury after treatment with ART was performed. As shown in Fig. 3a, at 35 days after balloon injury, model group showed abundant neointimal hyperplasia, whereas ART-treated animals showed significant suppression of neointimal hyperplasia, indicating the beneficial effect of ART on neointimal hyperplasia. Also, the increase in the lumen area of the 100-lmol/L ART group was larger than that of the model group (0.209 0.027 vs 0.176 0.021 mm2) (Fig. 3b). Moreover, the intimal area was reduced in half in the 100-lmol/L ART-treated group (from 0.047 0.015 to 0.019 0.007 mm2) (Fig. 3c). Similarly, the intimal-to-medial area ratio was significantly reduced in the ART-treated arteries compared to the model group (1.751 0.693 vs 0.784 0.306) (Fig. 3e). There were no significant differences in the medial area among the four groups (Fig. 3d). Western blot analysis was performed to test a possible contribution by cell cycle arrest to the decreased neointimal hyperplasia induced by ART. Data showed that balloon injury increased the expression levels of the cell cycle-related proteins cyclin D1, cyclin E, CDK2, and CDK4, but ART downregulated these protein levels (Fig. 3f). Thus, we conclude that ART suppresses neointima formation after balloon injury of rat carotid artery through the cell cycle arrest pathway, the specific mechanism of which needs to be further studied.
DISCUSSION It has been reported that the genesis of atherosclerotic lesions always involves a preponderance of VSMC.15,16 Atherosclerotic plaques develop at multiple sites within the arterial vasculature, locations termed ‘athero-susceptible’.17 Therefore, it is necessary to find an effective way to inhibit the abnormal proliferation of VSMC and neointima formation after injury to overcome atherosclerosis. In addition to its anti-malarial effect, ART has been shown, along with its derivatives, to affect other cellular biochemical processes,18,19 such as proliferation, angiogenesis, apoptosis, and cell cycle arrest. However, the exact anti-proliferation and apoptotic induction mechanisms of ART on VSMC are still unclear. In this study, we investigated the growth inhibition, apoptotic induction, and cell cycle arrest activities of ART in
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Fig. 1 Artemisinin (ART) inhibited proliferation and arrested cell cycle at G0/G1 phase in VSMC induced by TNF-a. (a) The identification of VSMC by immunofluorescence assay. (b) Growth inhibition of ART in VSMC induced by TNF-a was measured by 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Cells were first treated with 0, 25, 50, or 100-lmol/L ART for 2 h. Then VSMC were co-incubated with 10 ng/mL TNF-a for 24 h. (c) The expression level of cell proliferation-related protein PCNA by western blot analysis. (d) Cell cycle arrest induced by ART in TNF-a-stimulated VSMC by fluorescence-activated cell sorting analysis. (e) The expression level of cell cycle arrest-related protein. Levels of cyclin D1, cyclin E, CDK2 and CDK4 were tested and compared by western blot analysis. *P < 0.05 and †P < 0.01 versus TNF-a group. ‡P < 0.01 versus control group. CDK, cyclin-dependent kinase; con, control; DAPI, 40 ,6-diamidino-2-phenylindole; FL2-A, fluorescence channel 2-A; OD, optical density; PCNA, proliferating cell nuclear antigen; SMA, smooth muscle actin; TNF, tumour necrosis factor; VSMC, vascular smooth muscle cell.
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ART inhibits VSMC proliferation (a)
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Fig. 2 Artemisinin (ART) induced apoptosis in TNF-a-stimulated VSMC. (a) Apoptotic induction by ART in TNF-a-stimulated VSMC was tested by fluorescence-activated cell sorting analysis after staining with Annexin V-fluorescein-isothiocyanate. (a, upper panel) Apoptotic cells were more abundant in ART-treated cells than in TNF-a-stimulated VSMC. (a, lower panel) The index of apoptosis. (b) The expression levels of apoptosis-related protein. Levels of cleaved caspase-3/-9, Bcl-2, and Bax after TNF-a and/or ART treatment in VSMC were tested and compared by western blot analysis. *P < 0.01 versus TNF-a group. Bax, Bcl-2-associated X protein; con, control; FL1-A, fluorescence channel 1-A; TNF, tumour necrosis factor; VSMC, vascular smooth muscle cell.
TNF-a-stimulated VSMC of rats. We have found that ART inhibited the proliferation of VSMC induced by TNF-a by downregulating PCNA, arresting the cell cycle at G0/G1 phase, and inducing apoptosis in a dose-dependent manner in VSMC stimulated by TNF-a. Notably, western blot analysis indicated that cell cycle-related proteins were activated by ART, as was the caspase cascade during ART-induced apoptosis. Balloon injury indicated that ART inhibited neointima formation of the rat carotid arteries probably through the regulation of the cell cycle-related proteins cyclin D1, cyclin E, CKD2, and CDK4. Proliferation of VSMC is indeed important in the pathogenesis of atherosclerosis.20 We have tested the growth inhibition of ART in TNF-a-induced VSMC by 4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2-H-tetrazolium bromide assay. Western bolt analysis uncovered that ART inhibited the proliferation stimulated by TNF-a in VSMC by downregulating growth-related protein PCNA, which is known as a molecular marker for proliferation.21 Thus, ART inhibited the proliferation induced by TNF-a through downregulation of PCNA in VSMC.
For the cell cycle pathway, cyclins are positive regulators of cell cycle progression, in which cyclin E and cyclin D1 are primary regulators at the late G1 phase and can contribute to G1 phase progression and chromosomal instability.22,23 They are likely related to both tumour initiation and proliferation. Intriguingly, we found that the expression levels of cyclin D1 and cyclin E were downregulated by ART in a dose-dependent manner. Moreover, CDK are critical regulators of cell cycle progression.24 The assembly of protein complexes with different cyclin and/or CDK can regulate the cell cycle at different phases.25 The cyclin-CDK complexes phosphorylate specific protein substrates to move a cell through the distinct stages of the cell cycle.26 For instance, the G0/G1 transition is regulated by cyclin D-CDK4; G1/S transition is regulated by cyclin E-CDK2; and the G2/M transition is regulated by cyclin B-CDK2. In the current study, fluorescence-activated cell sorting with PI staining analysis revealed that there was an obvious percentage increase of G1-phase in VSMC after ART treatment. In addition, the expression levels of cyclin D1, cyclin E, CDK2, and CDK4
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Fig. 3 Artemisinin (ART) inhibits neointima formation after balloon injury of rat carotid artery. (a) Pictures of neointima formation after balloon injury in rat carotid artery with haematoxylin–eosin staining. (b) Lesion area of the carotid artery after balloon injury in the model, SO, and ART groups. (c) Intimal area of the carotid artery after balloon injury in the model and ART groups. (d) Medial area of the carotid artery after balloon injury in the model, SO, and ART groups. (e) The ratio between intimal and medial area after balloon injury in rat carotid artery. (f) The expression level of cell cycle arrestrelated protein. Levels of cyclin D1, cyclin E, CDK2, and CDK4 were tested and compared by western blot analysis. *P < 0.05 and †P < 0.01 versus model group. ‡P < 0.01 versus SO group. CDK, cyclin-dependent kinase; SO, sham-operated; TNF, tumour necrosis factor; VSMC, vascular smooth muscle cell.
were downregulated by ART in a dose-dependent manner, resulting in blocking the G0/G1 and G1/S checkpoint; this may eventually induce G1 phase cell cycle arrest and inhibit the proliferation of TNF-a-stimulated VSMC. We previously con-
firmed that ART inhibits the proliferation, migration, and inflammatory reaction induced by TNF-a in VSMC through the nuclear factor kappa B pathway.27 Moreover, it has also been reported that ART attenuates lipopolysaccharide-stimulated pro-inflamma-
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ART inhibits VSMC proliferation tory responses by inhibiting the nuclear factor kappa B pathway in microglia cells.28 Therefore, we suppose that ART suppresses TNF-a-induced cell proliferation through the cell cycle-dependent pathway probably mediated by the nuclear factor kappa B signal pathway in VSMC, the potential mechanism of which needs further investigation. In another anti-proliferation pathway, apoptosis is mediated by the caspase cascade.29 Handrick et al.13 reported that dihydroartemisinin, an ART derivative, induced apoptosis via a caspasedependent intrinsic pathway. In our study we found that both caspase-3 and caspase-9 were cleaved after ART treatment in TNF-a-stimulated VSMC. It is known that caspase-9 plays a central role in the intrinsic apoptotic pathway and caspase-3 activation.30 To the upstream signalling cascades, the cell’s decision to undergo apoptosis is determined by the interaction between prosurvival Bcl-2 proteins, such as Bcl-2, and pro-apoptotic proteins, such as Bax.31 Bcl-2 binds to Bax and inhibits Bax activation. In this study, we found that the level of Bax expression was upregulated after ART treatment. In contrast, the level of Bcl-2 expression was downregulated. Thus, we believe that ART targets Bcl-2 to suppress its inhibition of Bax by promoting the release of proapoptotic Bax from a Bax/Bcl-2 heterodimeric complex. The released Bax permits the formation of outer-mitochondrial membrane-spanning pores, which trigger activation of caspase-9; apoptosis was ultimately executed by caspase-3.32 The precise mechanisms by which ART causes cell cycle arrest and apoptosis need to be further elucidated. To examine the effect of ART on injured neointimal growth, we used a balloon injury model to evaluate the inhibition of ART on the injured carotid arteries. Neointima formation after balloon injury of the rat carotid artery is the best studied model of vascular remodelling after vascular injury. We found that rats with balloon injury showed marked neointima hyperplasia at day 35, and it was associated with enhanced expression levels of the cell cycle-related proteins cyclin D1, cyclin E, CDK2, and CDK4. It has been recognized that VSMC proliferation after arterial injury results in neointima formation, in which cyclin D1, cyclin E, CDK2, and CDK4 play important roles in cell cycle regulation.33 However, ART significantly suppresses neointima hyperplasia of rat carotid arteries after balloon injury. Also, western blot revealed that the expression levels of cell cycle-related proteins of injured carotid arteries were downregulated by ART in a dose-dependent manner. Taken together, it is conceivable that the inhibitory effects of ART on neointima formation are attributable to enhancement of cell cycle arrest in injured neointimal cells of rat carotid artery. Our study has some limitations. ART is considered to have pleiotropic characteristic with, among others, anti-malarial, anticancer, and anti-inflammatory biological and pharmacological activities. It has also been reported that the effectiveness of ART on malaria is due to its endoperoxide moiety, which reacts with haem and is abundant in malaria parasites, leading to the formation of carbon-based free radicals that cause the death of the parasite.6 However, the precise mechanism by which ART plays a vital role still needs to be clarified. In this study, we investigated the growth inhibition and apoptosis induction effect of ART mainly in VSMC. Then, we examined these effects on the apoptotic pathways in an in vivo model in combination with in vitro results.
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In summary, we demonstrated that ART inhibited TNF-a-stimulated VSMC proliferation through cell cycle arrest and the apoptotic pathway. Also, ART inhibited neointima formation after balloon injury of the rat carotid artery through cell cycle arrest. Our findings may open up a potential way to treat atherosclerosis with ART.
METHODS Animals We obtained 8-week-old adult male Sprague–Dawley rats (200– 250 g) from the Laboratory Animal Center of China Medical University. They were housed in a room at a constant temperature of 20 2°C with 12-h light/dark cycles and were fed a standard dry laboratory rat diet and water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee. Experimental groups Rats were randomly assigned into one of four experimental groups, each of which contained six animals; all animals, except those in the sham-operated group, underwent balloon injury to the left carotid arteries. The four groups were named per their daily treatment (administered orally) as follows: (i) sham-operated group, which received 100-mg/kg normal saline; (ii) model group, which received 100-mg/kg normal saline; (iii) 50-mg/kg ART group, which received 50-mg/kg ART; and (iv) 100 mg/kg ART group, which received 100-mg/kg ART. After 35 days of treatment, carotid arteries were harvested for further analysis. The treatment caused no differences in the bodyweights of the animals. Balloon injury of the carotid arteries Briefly, after the rats were anaesthetized with 10% chloral hydrate (3 mL/kg bodyweight, intraperitoneally), a midline incision was made in the neck to expose the left external carotid artery. A 2.0 Fogarty arterial embolectomy catheter (Medtronic, Troy, MN, USA) was introduced into the artery through an arteriotomy and passed into the common carotid artery to the aortic arch. The balloon was inflated and then slowly rotated while the catheter was pulled back towards the external carotid artery. This was repeated three times, and then the external carotid artery was ligated. The animals were allowed to recover and given 50, 100 mg/kg ART, or normal saline by oral administration. Excision of carotid arteries Rats were anaesthetized with 3% pentobarbital sodium (30 mg/ kg). The chest was opened and the left ventricle was pierced, while the inferior caval vein was opened for exsanguination. The animals were perfused with 300-mL cooled saline. From a median cervical section, both the right and left common carotid arteries were explored, prepared under operational microscope, and excised in full. The approximately 15 mm long pieces were cleared from the connective and fatty tissue. Two-thirds of the length of each vessel from every animal was locked in tissue-processing cassettes and fixed in 4% formalde-
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hyde solution for histology; the rest was quick-frozen in liquid nitrogen and stored in 70°C for further study.
sis for western blot was made by GEL-PRO-ANALYZER software (Media Cybernetics, Rockville, MD, USA).
Vascular smooth muscle cells isolation
Haematoxylin–eosin staining
Carotid aortas were removed and the adventitia was gently removed. After that, VSMC were obtained by sequential digestion of the aortas with collagenase type II (175 U/mL; Beyotime, Shanghai, China), then collagenase type II (175 U/mL; Beyotime), and elastase (0.5 mg/mL; Beyotime). Cells were cultured in Dulbecco’s minimum essential medium (Gibco, Grand Island, NY, USA) and identified by immunofluorescent assay.
Samples from the carotid arteries were isolated, fixed with 10% paraformaldehyde (Sinopharm Group, Shanghai, China), and embedded in paraffin wax. Sections were cut at 5 lm by a microtome, and deparaffinized tissue sections were subjected to staining with haematoxylin (Solarbio, Beijing, China) and eosin (Sinopharm Group) for histological examination (Semi-Automatic Cryostat Microtome #CM69001; Leica, Wetzlar, Germany). The slides were examined by light microscopy and photographed.
4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay Vascular smooth muscle cells were cultured with series of ART dilutions (25, 50, and 100 lmol/L) (Dalian Meilun Biotech, Dalian, China) or the same concentration of dimethylsulphoxide (control group) for 2 h. Then cells were co-incubated with 10-ng/ mL TNF-a (Pepro Tech, Rocky Hill, NJ, USA) for 24 h. After the addition of 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (Sigma-Aldrich, St Louis, MO, USA), the absorbance of the wells was measured with a microplate reader (BIOTEK, Winooski, VT, USA) at 490 nm, and the inhibition rate was calculated as (1Absorbanceexperiment/Absorbancevehicle control) 9 100.
Statistical analysis All data were subjected to ANOVA using GRAPHPAD Prism software (La Jolla, CA, USA). Data were presented as the mean SD from at least three experiments. Statistical comparisons were analysed by Bonferroni’s multiple comparison tests or ANOVA. P < 0.05 was considered as statistically significant.
ACKNOWLEDGEMENTS This study was supported by grants from the Natural Science Foundation of the Department of Science and Technology, Liaoning Province (No.: 2012010185-401) and the Foundation for Scientific Research and Public Welfare (No.: GY2014-A-020).
Fluorescence-activated cell sorting assay For apoptosis assay, cells were harvested, washed twice in cold phosphate-buffered saline, stained with PI, and then detected with an Annexin V-fluorescein-isothiocyanate apoptosis detection kit (KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s protocol. For cell cycle assay, cells were fixed with ice-cold 70% ethanol at a density of 1 9 105 cells/mL and treated with 200-lg/mL RNase (Beyotime, Haimen, China) for 30 min at 37°C. Then, PI (Beyotime) was added into the samples. Both the apoptotic cells and DNA content were quantitated by flow cytometry (Becton Dickinson, San Jose, CA, USA). Data from 10 000 events were analysed with CELLQUEST software (Becton Dickinson). Western blot analysis Vascular smooth muscle cells or carotid arteries excised were harvested and lysed in ice-cold radioimmuneprecipitation buffer (Beyotime) plus phenylmethanesulphonyl fluoride (Beyotime) for 30 min on ice. Protein content was determined using BCA Protein Assay Kit (Beyotime) per the manufacturer’s instructions. Next, 40-lg protein was separated with 8–12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The filters were hybridized with the appropriate primary antibodies to PCNA, cyclin D1, cyclin E, CDK2, CDK4, cleaved caspase-3/-9, Bax, Bcl-2, and b-actin (1 : 1000; WanLei Life Sciences, Shenyang, China) overnight at 4°C. Quantitative analy-
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doi: 10.1111/1440-1681.12375
ORIGINAL ARTICLE
Artemisinin inhibits tumour necrosis factor-a-induced vascular smooth muscle cell proliferation in vitro and attenuates balloon injury-induced neointima formation in rats Qian Cao,* Yan Jiang,† Jin Shi,* Xue Liu,* Jie Chen,* Tiesheng Niu* and Xiaodong Li* Departments of *Cardiology, and †Emergency Medicine, Shengjing Hospital of China Medical University, Shenyang, China
SUMMARY The aim of this study was to evaluate the effect of artemisinin (ART) on rat vascular smooth muscle cell (VSMC) proliferation induced by tumour necrosis factor (TNF)-a, cell cycle arrest, and apoptosis, and its effect on neointima formation after balloon injury of rat carotid artery. Primary rat VSMC were identified by immunofluorescence assay. The proliferation of VSMC induced by TNF-a was significantly inhibited by ART treatment in a dose-dependent manner. Treatment with 100-lM ART significantly reduced the expression of proliferating cell nuclear antigen. In contrast, the same treatment arrested the cell cycle in G0/G1 phase. Western blot analysis showed that the cell cycle-related proteins cyclin D1, cyclin E, cyclin-dependent kinase 2, and cyclin-dependent kinase 4 were downregulated by ART in TNF-a-stimulated VSMC. For apoptosis induced by ART, cleaved caspase-3/-9 was detected, and the pro-apoptotic protein Bcl-2-associated X protein was upregulated while the anti-apoptotic protein Bcl2 was downregulated. The results suggest that ART can effectively inhibit the proliferation of VSMC induced by TNF-a through the apoptotic induction pathway and cell cycle arrest. Also, balloon injury indicated that ART significantly inhibited neointima formation in the rat carotid arteries. Key words: apoptosis, artemisinin, cell cycle, neointima formation, proliferation, TNF-a, vascular smooth muscle cells.
INTRODUCTION Atherosclerosis, a chronic disease characterized by injury to vessel walls, is a major cause of human morbidity and mortality, and it is occurring at alarmingly increasing rates in both developed and developing countries.1 Current evidence suggests that the activation, proliferation, and anti-apoptotic effect of vascular
Correspondence: Dr Qian Cao, Department of Cardiology, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang 110004, China. Email: [email protected] Received 4 December 2014; revision 27 January 2015; accepted 15 February 2015. © 2015 Wiley Publishing Asia Pty Ltd
smooth muscle cells (VSMC) are absolutely essential in the early progress of atherogenesis.2 In the physiological state, VSMC are the major producers of extracellular matrix within the vessel wall, and they contract or relax to change the volume of blood vessels and local blood pressure.3 However, the transition into the earliest recognized form of an atherosclerotic lesion, termed a ‘pathological intimal thickening’, is associated with morphological and biochemical alterations to the VSMC and their extracellular matrix, leading to the release of growth factors such as tumour necrosis factor (TNF)-a, which is the major contributing factor in the pathogenesis of atherosclerosis.4 Thus, inhibition of VSMC proliferation induced by TNF-a, induction of apoptosis and cell cycle arrest, or reduction of neointima hyperplasia through drugs or other agents could prevent atherosclerosis and potentially delay or suppress its progress. Artemisinin (ART), a sesquiterpene lactone extracted from the Chinese plant Artemisia annua, is a potent anti-malarial drug that is effective in the treatment of severe and multidrug-resistant malaria.5 In addition, ART and its derivatives have been shown to possess pleiotropic characteristics such as anti-cancer,6 antiinflammatory,7 and anti-oxidant activities.8 Recently, several studies have indicated that ART has an effect on atherosclerosis. Wang et al.9 reported that ART significantly inhibited rats’ VSMC proliferation through induction of cell cycle arrest and apoptosis. Nonetheless, no study has investigated the effect of ART on neointima formation in the rat carotid artery. Moreover, whether ART can inhibit proliferation in VSMC induced by TNF-a through apoptosis or cell cycle arrest remains to be clarified, and the corresponding biological mechanism is still unclear. In this study, we investigated the inhibitory effects of ART in primary rat thoracic aortic VSMC proliferation induced by TNFa in vitro and the potential pathway of growth inhibition. At the same time, we evaluated the suppression effect of ART on neointima formation in rat carotid arteries in vivo, which may provide insights into the treatment of atherosclerosis using ART.
RESULTS Artemisinin inhibited proliferation induced by TNF-a and arrested cell cycle at G0/G1 phase in VSMC Artemisinin has been reported to induce growth arrest that involves selective changes in the expression and activity of cell
ART inhibits VSMC proliferation cycle components.10 To study the growth inhibition of ART in VSMC induced by TNF-a, we first identified the VSMC by immunofluorescence assay. It was found that 40 ,6-diamidino-2phenylindole and fluorescent a-actin were evenly distributed in the cells, indicating the VSMC (Fig. 1a). We tested the proliferation of VSMC after pretreatment with or without ART at different concentrations, and cells then underwent co-incubation with TNF-a by 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Data showed that after co-incubation with ART, the rate of VSMC proliferation induced by TNF-a decreased in a dose-dependent manner (Fig. 1b). The level of proliferationrelated protein proliferating cell nuclear antigen (PCNA) was also tested, and it was found that it was reduced to almost half after co-incubation with 100-lmol/L ART (Fig. 1c). Thus, ART suppresses the proliferation in TNF-a-induced VSMC. To investigate the pathway of growth inhibition induced by ART in TNF-a-stimulated VSMC, the cell cycle was tested by fluorescence-activated cell sorting analysis after staining with propidium iodide (PI). Data indicated that ART alone at 100 lmol/L did not affect the cell cycle of VSMC, while the G0/G1 phase was reduced from 65.32 2.55 to 40.00 2.55% after TNF-a stimulation in VSMC (Fig. 1d). However, the G0/G1 phase increased almost to the original level after co-incubation with 100-lmol/L ART (40.00 2.55 vs 59.97 2.64%) (Fig. 1d). These results suggested that ART may inhibit TNF-a-induced VSMC proliferation by interfering with cell mitosis through cell cycle arrest. Levels of the cell cycle-related proteins cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, and CDK4 were determined in VSMC induced by TNF-a with or without ART by western blot analysis. These proteins, especially cyclin E and CDK4, were significantly upregulated by TNF-a stimulation (Fig. 1e). However, when VSMC pretreated with TNF-a were co-incubated with ART, the high expression levels of these proteins remained almost at their original levels in a dose-dependent manner (Fig. 1e, lower panel). Thus, we concluded that ART inhibits cell proliferation through cell cycle arrest by downregulating cyclin D1, cyclin E, CDK2, and CDK4 in VSMC induced by TNF-a. Artemisinin induced apoptosis in TNF-a-stimulated VSMC Currently, an increasing number of investigations have reported that ART and its derivatives induce apoptosis in cancer cells.11,12 To evaluate the apoptosis induction of ART in TNF-a-stimulated VSMC, the levels of apoptotic cells were determined by fluorescence-activated cell sorting analysis (Fig. 2a). Data showed that ART and TNF-a alone did not induce apoptosis in VSMC. Interestingly, apoptotic cells were detected after pretreatment with ART at different concentrations followed by co-incubation with TNF-a in a dose-dependent manner (Fig. 2a, upper panel). The apoptotic index was 80.58 7.35% in the 100-lmol/L ART group treated with TNF-a, which was much higher than the ART group (11.73 2.47%) (Fig. 2a, lower panel). Data indicate that ART induces apoptosis in TNF-a-stimulated VSMC. To determine the mechanisms of apoptosis induction by ART in TNF-a-stimulated VSMC, we used western blot analysis to investigate levels of apoptosis-related proteins in VSMC induced by TNF-a with or without ART. As shown in Fig. 2b, the level of cleaved caspase-3 detected suggested that a caspase-mediated
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pathway led to apoptosis. Also, it was reported that ART and its derivative dihydroartemisinin induced apoptosis through an intrinsic pathway in Jurkat T-lymphoma cells.13 Therefore, we tested cleaved caspase-9 and found that it was upregulated after ART treatment. Activation of the apoptotic pathway is regulated by the balance between pro-apoptotic proteins, such as Bcl-2-associated X protein (Bax), and pro-survival Bcl-2 proteins, such as Bcl-2.14 Therefore, the levels of Bax and Bcl-2 were tested in VSMC induced by TNF-a with or without ART. It was found that the level of pro-apoptotic protein Bax was upregulated while the level of anti-apoptotic protein Bcl-2 was downregulated (Fig. 2b). Thus, upregulation of Bax and downregulation of Bcl-2 seem to account for the activation of the intrinsic apoptotic pathway induced by ART in TNF-a-stimulated VSMC. Artemisinin inhibits neointima formation after balloon injury of rat carotid artery To study the effect of ART on neointima formation after injury, balloon injury after treatment with ART was performed. As shown in Fig. 3a, at 35 days after balloon injury, model group showed abundant neointimal hyperplasia, whereas ART-treated animals showed significant suppression of neointimal hyperplasia, indicating the beneficial effect of ART on neointimal hyperplasia. Also, the increase in the lumen area of the 100-lmol/L ART group was larger than that of the model group (0.209 0.027 vs 0.176 0.021 mm2) (Fig. 3b). Moreover, the intimal area was reduced in half in the 100-lmol/L ART-treated group (from 0.047 0.015 to 0.019 0.007 mm2) (Fig. 3c). Similarly, the intimal-to-medial area ratio was significantly reduced in the ART-treated arteries compared to the model group (1.751 0.693 vs 0.784 0.306) (Fig. 3e). There were no significant differences in the medial area among the four groups (Fig. 3d). Western blot analysis was performed to test a possible contribution by cell cycle arrest to the decreased neointimal hyperplasia induced by ART. Data showed that balloon injury increased the expression levels of the cell cycle-related proteins cyclin D1, cyclin E, CDK2, and CDK4, but ART downregulated these protein levels (Fig. 3f). Thus, we conclude that ART suppresses neointima formation after balloon injury of rat carotid artery through the cell cycle arrest pathway, the specific mechanism of which needs to be further studied.
DISCUSSION It has been reported that the genesis of atherosclerotic lesions always involves a preponderance of VSMC.15,16 Atherosclerotic plaques develop at multiple sites within the arterial vasculature, locations termed ‘athero-susceptible’.17 Therefore, it is necessary to find an effective way to inhibit the abnormal proliferation of VSMC and neointima formation after injury to overcome atherosclerosis. In addition to its anti-malarial effect, ART has been shown, along with its derivatives, to affect other cellular biochemical processes,18,19 such as proliferation, angiogenesis, apoptosis, and cell cycle arrest. However, the exact anti-proliferation and apoptotic induction mechanisms of ART on VSMC are still unclear. In this study, we investigated the growth inhibition, apoptotic induction, and cell cycle arrest activities of ART in
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Fig. 1 Artemisinin (ART) inhibited proliferation and arrested cell cycle at G0/G1 phase in VSMC induced by TNF-a. (a) The identification of VSMC by immunofluorescence assay. (b) Growth inhibition of ART in VSMC induced by TNF-a was measured by 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Cells were first treated with 0, 25, 50, or 100-lmol/L ART for 2 h. Then VSMC were co-incubated with 10 ng/mL TNF-a for 24 h. (c) The expression level of cell proliferation-related protein PCNA by western blot analysis. (d) Cell cycle arrest induced by ART in TNF-a-stimulated VSMC by fluorescence-activated cell sorting analysis. (e) The expression level of cell cycle arrest-related protein. Levels of cyclin D1, cyclin E, CDK2 and CDK4 were tested and compared by western blot analysis. *P < 0.05 and †P < 0.01 versus TNF-a group. ‡P < 0.01 versus control group. CDK, cyclin-dependent kinase; con, control; DAPI, 40 ,6-diamidino-2-phenylindole; FL2-A, fluorescence channel 2-A; OD, optical density; PCNA, proliferating cell nuclear antigen; SMA, smooth muscle actin; TNF, tumour necrosis factor; VSMC, vascular smooth muscle cell.
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Fig. 2 Artemisinin (ART) induced apoptosis in TNF-a-stimulated VSMC. (a) Apoptotic induction by ART in TNF-a-stimulated VSMC was tested by fluorescence-activated cell sorting analysis after staining with Annexin V-fluorescein-isothiocyanate. (a, upper panel) Apoptotic cells were more abundant in ART-treated cells than in TNF-a-stimulated VSMC. (a, lower panel) The index of apoptosis. (b) The expression levels of apoptosis-related protein. Levels of cleaved caspase-3/-9, Bcl-2, and Bax after TNF-a and/or ART treatment in VSMC were tested and compared by western blot analysis. *P < 0.01 versus TNF-a group. Bax, Bcl-2-associated X protein; con, control; FL1-A, fluorescence channel 1-A; TNF, tumour necrosis factor; VSMC, vascular smooth muscle cell.
TNF-a-stimulated VSMC of rats. We have found that ART inhibited the proliferation of VSMC induced by TNF-a by downregulating PCNA, arresting the cell cycle at G0/G1 phase, and inducing apoptosis in a dose-dependent manner in VSMC stimulated by TNF-a. Notably, western blot analysis indicated that cell cycle-related proteins were activated by ART, as was the caspase cascade during ART-induced apoptosis. Balloon injury indicated that ART inhibited neointima formation of the rat carotid arteries probably through the regulation of the cell cycle-related proteins cyclin D1, cyclin E, CKD2, and CDK4. Proliferation of VSMC is indeed important in the pathogenesis of atherosclerosis.20 We have tested the growth inhibition of ART in TNF-a-induced VSMC by 4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2-H-tetrazolium bromide assay. Western bolt analysis uncovered that ART inhibited the proliferation stimulated by TNF-a in VSMC by downregulating growth-related protein PCNA, which is known as a molecular marker for proliferation.21 Thus, ART inhibited the proliferation induced by TNF-a through downregulation of PCNA in VSMC.
For the cell cycle pathway, cyclins are positive regulators of cell cycle progression, in which cyclin E and cyclin D1 are primary regulators at the late G1 phase and can contribute to G1 phase progression and chromosomal instability.22,23 They are likely related to both tumour initiation and proliferation. Intriguingly, we found that the expression levels of cyclin D1 and cyclin E were downregulated by ART in a dose-dependent manner. Moreover, CDK are critical regulators of cell cycle progression.24 The assembly of protein complexes with different cyclin and/or CDK can regulate the cell cycle at different phases.25 The cyclin-CDK complexes phosphorylate specific protein substrates to move a cell through the distinct stages of the cell cycle.26 For instance, the G0/G1 transition is regulated by cyclin D-CDK4; G1/S transition is regulated by cyclin E-CDK2; and the G2/M transition is regulated by cyclin B-CDK2. In the current study, fluorescence-activated cell sorting with PI staining analysis revealed that there was an obvious percentage increase of G1-phase in VSMC after ART treatment. In addition, the expression levels of cyclin D1, cyclin E, CDK2, and CDK4
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Fig. 3 Artemisinin (ART) inhibits neointima formation after balloon injury of rat carotid artery. (a) Pictures of neointima formation after balloon injury in rat carotid artery with haematoxylin–eosin staining. (b) Lesion area of the carotid artery after balloon injury in the model, SO, and ART groups. (c) Intimal area of the carotid artery after balloon injury in the model and ART groups. (d) Medial area of the carotid artery after balloon injury in the model, SO, and ART groups. (e) The ratio between intimal and medial area after balloon injury in rat carotid artery. (f) The expression level of cell cycle arrestrelated protein. Levels of cyclin D1, cyclin E, CDK2, and CDK4 were tested and compared by western blot analysis. *P < 0.05 and †P < 0.01 versus model group. ‡P < 0.01 versus SO group. CDK, cyclin-dependent kinase; SO, sham-operated; TNF, tumour necrosis factor; VSMC, vascular smooth muscle cell.
were downregulated by ART in a dose-dependent manner, resulting in blocking the G0/G1 and G1/S checkpoint; this may eventually induce G1 phase cell cycle arrest and inhibit the proliferation of TNF-a-stimulated VSMC. We previously con-
firmed that ART inhibits the proliferation, migration, and inflammatory reaction induced by TNF-a in VSMC through the nuclear factor kappa B pathway.27 Moreover, it has also been reported that ART attenuates lipopolysaccharide-stimulated pro-inflamma-
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ART inhibits VSMC proliferation tory responses by inhibiting the nuclear factor kappa B pathway in microglia cells.28 Therefore, we suppose that ART suppresses TNF-a-induced cell proliferation through the cell cycle-dependent pathway probably mediated by the nuclear factor kappa B signal pathway in VSMC, the potential mechanism of which needs further investigation. In another anti-proliferation pathway, apoptosis is mediated by the caspase cascade.29 Handrick et al.13 reported that dihydroartemisinin, an ART derivative, induced apoptosis via a caspasedependent intrinsic pathway. In our study we found that both caspase-3 and caspase-9 were cleaved after ART treatment in TNF-a-stimulated VSMC. It is known that caspase-9 plays a central role in the intrinsic apoptotic pathway and caspase-3 activation.30 To the upstream signalling cascades, the cell’s decision to undergo apoptosis is determined by the interaction between prosurvival Bcl-2 proteins, such as Bcl-2, and pro-apoptotic proteins, such as Bax.31 Bcl-2 binds to Bax and inhibits Bax activation. In this study, we found that the level of Bax expression was upregulated after ART treatment. In contrast, the level of Bcl-2 expression was downregulated. Thus, we believe that ART targets Bcl-2 to suppress its inhibition of Bax by promoting the release of proapoptotic Bax from a Bax/Bcl-2 heterodimeric complex. The released Bax permits the formation of outer-mitochondrial membrane-spanning pores, which trigger activation of caspase-9; apoptosis was ultimately executed by caspase-3.32 The precise mechanisms by which ART causes cell cycle arrest and apoptosis need to be further elucidated. To examine the effect of ART on injured neointimal growth, we used a balloon injury model to evaluate the inhibition of ART on the injured carotid arteries. Neointima formation after balloon injury of the rat carotid artery is the best studied model of vascular remodelling after vascular injury. We found that rats with balloon injury showed marked neointima hyperplasia at day 35, and it was associated with enhanced expression levels of the cell cycle-related proteins cyclin D1, cyclin E, CDK2, and CDK4. It has been recognized that VSMC proliferation after arterial injury results in neointima formation, in which cyclin D1, cyclin E, CDK2, and CDK4 play important roles in cell cycle regulation.33 However, ART significantly suppresses neointima hyperplasia of rat carotid arteries after balloon injury. Also, western blot revealed that the expression levels of cell cycle-related proteins of injured carotid arteries were downregulated by ART in a dose-dependent manner. Taken together, it is conceivable that the inhibitory effects of ART on neointima formation are attributable to enhancement of cell cycle arrest in injured neointimal cells of rat carotid artery. Our study has some limitations. ART is considered to have pleiotropic characteristic with, among others, anti-malarial, anticancer, and anti-inflammatory biological and pharmacological activities. It has also been reported that the effectiveness of ART on malaria is due to its endoperoxide moiety, which reacts with haem and is abundant in malaria parasites, leading to the formation of carbon-based free radicals that cause the death of the parasite.6 However, the precise mechanism by which ART plays a vital role still needs to be clarified. In this study, we investigated the growth inhibition and apoptosis induction effect of ART mainly in VSMC. Then, we examined these effects on the apoptotic pathways in an in vivo model in combination with in vitro results.
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In summary, we demonstrated that ART inhibited TNF-a-stimulated VSMC proliferation through cell cycle arrest and the apoptotic pathway. Also, ART inhibited neointima formation after balloon injury of the rat carotid artery through cell cycle arrest. Our findings may open up a potential way to treat atherosclerosis with ART.
METHODS Animals We obtained 8-week-old adult male Sprague–Dawley rats (200– 250 g) from the Laboratory Animal Center of China Medical University. They were housed in a room at a constant temperature of 20 2°C with 12-h light/dark cycles and were fed a standard dry laboratory rat diet and water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee. Experimental groups Rats were randomly assigned into one of four experimental groups, each of which contained six animals; all animals, except those in the sham-operated group, underwent balloon injury to the left carotid arteries. The four groups were named per their daily treatment (administered orally) as follows: (i) sham-operated group, which received 100-mg/kg normal saline; (ii) model group, which received 100-mg/kg normal saline; (iii) 50-mg/kg ART group, which received 50-mg/kg ART; and (iv) 100 mg/kg ART group, which received 100-mg/kg ART. After 35 days of treatment, carotid arteries were harvested for further analysis. The treatment caused no differences in the bodyweights of the animals. Balloon injury of the carotid arteries Briefly, after the rats were anaesthetized with 10% chloral hydrate (3 mL/kg bodyweight, intraperitoneally), a midline incision was made in the neck to expose the left external carotid artery. A 2.0 Fogarty arterial embolectomy catheter (Medtronic, Troy, MN, USA) was introduced into the artery through an arteriotomy and passed into the common carotid artery to the aortic arch. The balloon was inflated and then slowly rotated while the catheter was pulled back towards the external carotid artery. This was repeated three times, and then the external carotid artery was ligated. The animals were allowed to recover and given 50, 100 mg/kg ART, or normal saline by oral administration. Excision of carotid arteries Rats were anaesthetized with 3% pentobarbital sodium (30 mg/ kg). The chest was opened and the left ventricle was pierced, while the inferior caval vein was opened for exsanguination. The animals were perfused with 300-mL cooled saline. From a median cervical section, both the right and left common carotid arteries were explored, prepared under operational microscope, and excised in full. The approximately 15 mm long pieces were cleared from the connective and fatty tissue. Two-thirds of the length of each vessel from every animal was locked in tissue-processing cassettes and fixed in 4% formalde-
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hyde solution for histology; the rest was quick-frozen in liquid nitrogen and stored in 70°C for further study.
sis for western blot was made by GEL-PRO-ANALYZER software (Media Cybernetics, Rockville, MD, USA).
Vascular smooth muscle cells isolation
Haematoxylin–eosin staining
Carotid aortas were removed and the adventitia was gently removed. After that, VSMC were obtained by sequential digestion of the aortas with collagenase type II (175 U/mL; Beyotime, Shanghai, China), then collagenase type II (175 U/mL; Beyotime), and elastase (0.5 mg/mL; Beyotime). Cells were cultured in Dulbecco’s minimum essential medium (Gibco, Grand Island, NY, USA) and identified by immunofluorescent assay.
Samples from the carotid arteries were isolated, fixed with 10% paraformaldehyde (Sinopharm Group, Shanghai, China), and embedded in paraffin wax. Sections were cut at 5 lm by a microtome, and deparaffinized tissue sections were subjected to staining with haematoxylin (Solarbio, Beijing, China) and eosin (Sinopharm Group) for histological examination (Semi-Automatic Cryostat Microtome #CM69001; Leica, Wetzlar, Germany). The slides were examined by light microscopy and photographed.
4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay Vascular smooth muscle cells were cultured with series of ART dilutions (25, 50, and 100 lmol/L) (Dalian Meilun Biotech, Dalian, China) or the same concentration of dimethylsulphoxide (control group) for 2 h. Then cells were co-incubated with 10-ng/ mL TNF-a (Pepro Tech, Rocky Hill, NJ, USA) for 24 h. After the addition of 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (Sigma-Aldrich, St Louis, MO, USA), the absorbance of the wells was measured with a microplate reader (BIOTEK, Winooski, VT, USA) at 490 nm, and the inhibition rate was calculated as (1Absorbanceexperiment/Absorbancevehicle control) 9 100.
Statistical analysis All data were subjected to ANOVA using GRAPHPAD Prism software (La Jolla, CA, USA). Data were presented as the mean SD from at least three experiments. Statistical comparisons were analysed by Bonferroni’s multiple comparison tests or ANOVA. P < 0.05 was considered as statistically significant.
ACKNOWLEDGEMENTS This study was supported by grants from the Natural Science Foundation of the Department of Science and Technology, Liaoning Province (No.: 2012010185-401) and the Foundation for Scientific Research and Public Welfare (No.: GY2014-A-020).
Fluorescence-activated cell sorting assay For apoptosis assay, cells were harvested, washed twice in cold phosphate-buffered saline, stained with PI, and then detected with an Annexin V-fluorescein-isothiocyanate apoptosis detection kit (KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s protocol. For cell cycle assay, cells were fixed with ice-cold 70% ethanol at a density of 1 9 105 cells/mL and treated with 200-lg/mL RNase (Beyotime, Haimen, China) for 30 min at 37°C. Then, PI (Beyotime) was added into the samples. Both the apoptotic cells and DNA content were quantitated by flow cytometry (Becton Dickinson, San Jose, CA, USA). Data from 10 000 events were analysed with CELLQUEST software (Becton Dickinson). Western blot analysis Vascular smooth muscle cells or carotid arteries excised were harvested and lysed in ice-cold radioimmuneprecipitation buffer (Beyotime) plus phenylmethanesulphonyl fluoride (Beyotime) for 30 min on ice. Protein content was determined using BCA Protein Assay Kit (Beyotime) per the manufacturer’s instructions. Next, 40-lg protein was separated with 8–12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The filters were hybridized with the appropriate primary antibodies to PCNA, cyclin D1, cyclin E, CDK2, CDK4, cleaved caspase-3/-9, Bax, Bcl-2, and b-actin (1 : 1000; WanLei Life Sciences, Shenyang, China) overnight at 4°C. Quantitative analy-
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