Thymoquinone suppresses platelet‐derived growth factor‐BB–induced vascular smooth muscle cell proliferation, migration and neointimal formation

Abstract The excessive proliferation and migration of vascular smooth muscle cells (VSMCs) are mainly responsible for vascular occlusion diseases, such as pulmonary arterial hypertension and restenosis. Our previous study demonstrated thymoquinone (TQ) attenuated monocrotaline‐induced pulmonary arterial hypertension. The aim of the present study is to systematically examine inhibitory effects of TQ on platelet‐derived growth factor‐BB (PDGF‐BB)–induced proliferation and migration of VSMCs in vitro and neointimal formation in vivo and elucidate the potential mechanisms. Vascular smooth muscle cells were isolated from the aorta in rats. Cell viability and proliferation were measured in VSMCs using the MTT assay. Cell migration was detected by wound healing assay and Transwell assay. Alpha‐smooth muscle actin (α‐SMA) and Ki‐67‐positive cells were examined by immunofluorescence staining. Reactive oxygen species (ROS) generation and apoptosis were measured by flow cytometry and terminal deoxyribonucleotide transferase–mediated dUTP nick end labelling (TUNEL) staining, respectively. Molecules including the mitochondria‐dependent apoptosis factors, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), PTEN/AKT and mitogen‐activated protein kinases (MAPKs) were determined by Western blot. Neointimal formation was induced by ligation in male Sprague Dawley rats and evaluated by HE staining. Thymoquinone inhibited PDGF‐BB–induced VSMC proliferation and the increase in α‐SMA and Ki‐67‐positive cells. Thymoquinone also induced apoptosis via mitochondria‐dependent apoptosis pathway and p38MAPK. Thymoquinone blocked VSMC migration by inhibiting MMP2. Finally, TQ reversed neointimal formation induced by ligation in rats. Thus, TQ is a potential candidate for the prevention and treatment of occlusive vascular diseases.


| INTRODUC TI ON
TQ has also been reported to attenuate chemotherapeutic agent-induced toxicity. 5 In addition, the inhibition of AKT phosphorylation was associated with PTEN upregulation and ROS generation. 6,7 The focus on TQ is increasing because of its efficacy and selectivity against cancer cells and lack of toxicity in normal tissues. 8,9 The effects of TQ in cardiovascular diseases have not been well identified. In our previous study, TQ was found to ameliorate monocrotaline-induced PAH in rats. 10 TQ inhibited small pulmonary arterial remodelling and pulmonary arterial VSMC proliferation in vivo. However, the effects of TQ on proliferation, migration and apoptosis of VSMCs in vitro and neointimal formation in vivo remain to be established.
Mechanistically, TQ has been shown to trigger apoptosis by increasing ratio of Bax and Bcl-2 followed by mitochondrial disruption and release of cytochrome C. 11 And consequently, TQ induces the activation of caspase 3 and the effector of apoptosis, poly(ADP-ribose) polymerase (PARP). 12 An increase in the Bax/ Bcl-2 ratio in response to TQ has been observed in MDA-MB231 human breast cancer. Thymoquinone was found to induce apoptosis by the deregulation of the mitogen-activated protein kinase (MAPK) pathways in multiple myeloma, 13 human prostate cancer cell lines 14 and squamous cell carcinoma. 15 In addition, AKT phosphorylation was blocked by TQ in breast tumours 6 and primary effusion lymphomas. 7 The inhibition of AKT activation was associated with PTEN up-regulation and ROS generation. In general, extensive evidence suggests the modulation of MAPK and AKT signalling pathways by TQ is strongly linked to its antiproliferative potential.
The aim of present study is to investigate inhibitory effects of TQ on platelet-derived growth factor (PDGF)-BB-induced proliferation, migration in rat aortic VSMCs and neointimal formation followed by ligation injury in rats, as well as the underlying mechanisms.

| Chemicals and reagents
Thymoquinone, bovine serum albumin (BSA) and the antibody against alpha-smooth muscle actin (α-SMA) were obtained from Sigma.
Thymoquinone was stored at 4°C and dissolved in olive oil. Foetal bovine serum (FBS) and DMEM were obtained from Life Technologies.

| Animals
Animal studies were carried out in accordance with the Guidelines

| Cell isolation and culture
The male Sprague Dawley rats (220-250 g) were maintained under pathogen-free conditions at the Wenzhou Medical University. These rats were purchased from Experimental Animal Center of Zhejiang Province (Hangzhou, Zhejiang, China). The rats were killed under euthanasia using overdose pentobarbital. Rat aortic VSMCs were isolated and cultured as described previously. 16 The cells were cultured in DMEM containing 20% FBS at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . The cells from passages 4 to 8 were used in all experiments.

| Cell viability and proliferation assay
Cell viability and proliferation were measured by the MTT assay.

| Scratch wound assay
Vascular smooth muscle cells were seeded in 6-well plates for 48 hours and reached 90%-100% confluence in culture plate wells.
Vascular smooth muscle cells were incubated with starvation medium (1% FBS) for 48 hours. After a linear wound was gently introduced in the centre of the cell monolayer using 200 µL tip, VSMCs were subjected to stimulation with or without 40 ng/mL PDGF-BB and TQ (5-15 μmol/L). Images were acquired using Leica Application Suite software, and cell migration was determined by the percentage of the wound closure area using the ImageJ software.

| Transwell migration assay
Cells were seeded into the upper chamber treated with TQ

| Detection of apoptosis
Vascular smooth muscle cells were seeded on coverslips and incubated with starvation in serum-free medium for 48 hours. Vascular smooth muscle cells were exposed to 40 ng/mL PDGF-BB and treated with TQ (5-15 μmol/L) or DMSO for 24 hours. After dewaxing and rehydrating with xylene and ethanol, VSMCs were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 1 hour at 25°C and blocked with 3% H 2 O 2 for 10 minutes and permeabilized with 0.1% Triton X-100 sodium citrate solution for 3 minutes. Apoptotic cells were labelled by terminal deoxyribonucleotide transferase-mediated dUTP nick end labelling (TUNEL) assay, and cell nuclei were labelled by DAPI. Images (magnification ×400) were obtained using fluorescence microscope (BX53, Olympus), and apoptotic cells was analysed with the ImageJ software.

| ROS analysis
Serum-starved VSMCs were stimulated with 40 ng/mL PDGF-BB in the presence or absence of TQ (5-15 μmol/L) for 24 hours. Cells were washed twice with 1 mL PBS. 500 μL of 0.25% trypsin was added to digest cells at 37°C with 5% CO 2 saturation humidity for 2-3 minutes. Then, the cells were collected and centrifuged (352g, 5 minutes). The cells were washed twice with 1 mL PBS and incubated with CM-H2DCFDA fluorescent probes at room temperature and dark for 30 minutes. The fluorescence intensity was measured using a flow cytometer (NovoCyte, ACEA).

| Immunofluorescence staining analysis
Vascular smooth muscle cells were seeded on coverslips and incubated with starvation in serum-free medium for 48 hours. The serum-starved cells were pre-treated with TQ (5-15 μmol/L) or DMSO, and 40 ng/mL PDGF-BB or none for 24 hours. The cells were fixed with cold 4% formaldehyde for 15 minutes and permeabilized with chilled 0.5% Triton X-100 for 10 minutes. Then, cells were blocked by 3% BSA in PBS for 1 hour at room temperature.
The cells were incubated with primary antibodies at 4°C overnight followed by incubation with secondary antibody for 1 hour at 37°C.
Cell nuclei were stained with DAPI, and immunofluorescence images (scale bar = 50 µm) were acquired using a confocal laser scanning microscope (Olympus).

| MMP gelatine zymography
Gelatine zymography was performed to assess MMP activity as previously described. 17 Briefly, serum-starved VSMCs were stimulated with 40 ng/mL PDGF-BB in the presence or absence of TQ (5-15 μmol/L) for 24 hours. The culture medium was treated with RIPA lysate buffer and collected. Then, proteins were separated with 12% SDS-PAGE gel. Separated gels were stained with 0.05% Coomassie brilliant blue for 1 hour. The densities of the clear bands were determined using Quantity One software (Bio-Rad).

| Western blot analysis
Cell lysates were prepared by using ice-cold RIPA lysis buffer containing PMSF and proteinase inhibitors for 30 minutes. Proteins were separated with 12% SDS-PAGE gel and transferred to PVDF membrane. After blocking with BSA, the membranes were incubated with specific primary antibodies. The membrane was incubated with horseradish peroxidase-conjugated secondary antibody. The activated proteins were normalized to β-actin or GAPDH. To determine the release of cytochrome C, mitochondrial and cytosol pellets were isolated and immunoblotted with primary antibody against cytochrome C. VDAC and GAPDH acted as mitochondrial and internal control, respectively. The optical density of bands was calculated with Quantity One software (Bio-Rad).

| Rat carotid ligation model
The rats were randomly assigned to the different treatment groups anaesthetized by intraperitoneal injection of pentobarbital (60 mg/ kg). The left common carotid artery was ligated with a 6-0 silk suture so that the common carotid artery blood flow was completely disrupted. The rats were treated with intraperitoneal administration of 8 or 16 mg/kg TQ (n = 6 in each group) daily for 2 weeks. Vehicletreated rats (3 mL of olive oil i.p., n = 6) were served as controls.
Carotid arteries were harvested 14 days after ligation, and subsequently fixed with 4% paraformaldehyde. Arteries were embedded in paraffinum for histological analysis.

| Histological analysis
Tissue was sectioned at 4 μm and stained with haematoxylin and eosin (HE) staining. The structure remodelling of the arteries was examined by light microscope (Nikon) at a magnification of 40× and 100× and measured by the ImageJ software.

| Statistical analysis
All data were expressed as mean ± standard deviation (SD).
Statistical analyses were performed with Student's t test or one-way Dunnett's analysis of variance. Statistical analyses were performed with GraphPad Prism software (version 5.0). All P < .05 was considered statistically significant.

| Effects of TQ on viability and proliferation of VSMCs
Firstly, we used the MTT method to detect effects of TQ on VSMC viability. As shown in Figure 1A We also evaluated the inhibitory effects of TQ proliferation on VSMCs by light microscope. In line with MTT assay, TQ significantly reduced VSMC proliferation ( Figure 1D). Together, these results indicated TQ (5-15 μmol/L) had no cytotoxicity and inhibited proliferation of VSMCs.

| Effects of TQ on migration of VSMCs, and the activity and expression of MMPs
To explore effects of TQ on VSMC migration, scratch wound assay and Transwell assay were conducted. Wound closure levels were increased after PDGF-BB stimulation for 24 hours (Figure 2A

| Effects of TQ on apoptosis of VSMCs
We investigated whether TQ exhibited a inhibition potential of proliferation of VSMCs, and we further investigated whether TQ

| Effects of TQ on ROS generation in VSMCs
ROS plays a important role in the inhibitory effects of TQ on tumour.
Thus, effects of TQ on ROS generation were detected by flow cytometry. PDGF-BB reduced ROS generation, which was abolished by TQ treatment in a dose-dependent manner ( Figure 6). The result suggested TQ induced apoptosis through ROS generation.

| Effects of TQ on apoptotic signals
We also determined whether TQ affected apoptotic signals in VSMCs.
PDGF-BB caused up-regulation of Bcl-2, cleaved caspase 3 and cleaved PARP and down-regulation of Bax, which were attenuated by TQ ( Figure 7A). Bax/Bcl-2 ratio was evaluated to reflect the apoptosis level, and the results indicated that cell apoptosis was inhibited by PDGF-BB but induced by TQ. Noticeably, PDGF-BB inhibited the release of cytochrome C from mitochondria to cytoplasm that was reversed by TQ treatment (Figure 7A,C,D). The results suggested TQ enhanced apoptosis in the mitochondria-dependent apoptosis pathway.

| Effects of TQ on PTEN/AKT and MAPK signals
Then, we investigated whether TQ regulated VSMC functions through PTEN/AKT or MAPK signalling pathways. As shown in Figure

| Effects of TQ on neointimal formation induced by ligation in rats
To demonstrate whether TQ ameliorated neointimal formation in vivo, rat carotid artery ligation model was employed and the neoin-  Thymoquinone has proved to trigger apoptosis in multiple cancer in vivo and in vitro. 12,24,25 The present study demonstrated that TQ attenuated PDGF-BB-induced apoptosis resistance in VSMCs. Then, we explored the apoptotic mechanism of TQ. The mechanism of potential of TQ involves differential triggering of ROS in cancer and normal cells. In line with anticancer effects, TQ reversed the decrease in ROS generation induced by PDGF-BB. In addition, the increase in cytochrome C in mitochondria and the reduction of cytochrome C in cytoplasm were reversed after TQ treatment. It has been well demonstrated that the pro-apoptotic factor, Bax, enhances cytochrome C release and activates caspase 3 to execute apoptotic programme, while Bcl-2, an anti-apoptosis molecule, inhibits Bax activation and cytochrome C release to suppress the apoptosis. 26 Our data showed MAPKs including p38MAPK, ERK1/2 and JNK are major pathways controlling cell differentiation, proliferation and death. 35 It has been established that p38MAPK phosphorylation was responsible for pathology of arterial remodelling and PAH models. 36 p38MAPK governed fibroblast proliferation and the hypoxic proliferative response, which also led to vascular remodelling. 37  to depend on the cell type. Our previous study showed TQ alleviated PAH via inhibition of p38MAPK activation. Consistent with the result, we found TQ blocked p38MAPK activation induced by the stimulation of PDGF-BB. Therefore, p38MAPK inhibition is the primary mechanism by which TQ inhibits VSMC proliferation and migration, and triggers apoptosis, leading to attenuate neointimal formation.

| D ISCUSS I ON
In summary, we firstly reported that TQ inhibited PDGF-BBmediated VSMC proliferation and migration, and induced apoptosis through mitochondria-dependent apoptosis pathway in vitro. In addition, TQ modulated ligation-induced neointimal formation in rats.
Mechanistically, inhibition of p38MAPK, but not PTNE/AKT, was involved in TQ's antiproliferative effects. Our investigation provides evidence that TQ has the potential to be a good candidate for the treatment of neointimal restenosis.

ACK N OWLED G EM ENTS
This work was supported by Science & Technology Bureau of Wenzhou (grant no. Y20170247).

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
NZ and CLZ designed the study, and wrote and revised the manuscript. NZ, YJX, XYZ, CHC, HC, WBJ and YW performed the experiments and analysed the data.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included in the article.