Hydrogen-rich saline attenuates vascular smooth muscle cell proliferation and neointimal hyperplasia by inhibiting reactive oxygen species production and inactivating the Ras-ERK1/2-MEK1/2 and Akt pathways

  • Authors:
    • Yali Chen
    • Jinyao Jiang
    • Huibing Miao
    • Xingjuan Chen
    • Xuejun Sun
    • Yongjun Li
  • View Affiliations

  • Published online on: January 22, 2013     https://doi.org/10.3892/ijmm.2013.1256
  • Pages: 597-606
Metrics: HTML 0 views | PDF 0 views     Cited By (CrossRef): 0 citations


Hydrogen-rich saline has been reported to prevent neointimal hyperplasia induced by carotid balloon injury. The purpose of the present study was to further investigate the molecular mechanisms underlying this phenomenon. Daily injection of a hydrogen-rich saline solution (HRSS) in rats was employed to study the effect of hydrogen on balloon injury-induced neointimal hyperplasia and the neointima/media ratio was assessed. HRSS significantly decreased the neointima area and neointima/media ratio in a dose-dependent manner. In vitro effects of hydrogen on fetal bovine serum (FBS)-induced vascular smooth muscle cell (VSMC) proliferation were also investigated. Hydrogen-rich medium (HRM) inhibited rat VSMC proliferation and migration induced by 10% FBS. FBS-induced reactive oxygen species (ROS) production and activation of intracellular Ras, MEK1/2, ERK1/2, proliferative cell nuclear antigen (PCNA), Akt were significantly inhibited by HRM. In addition, HRM blocked FBS-induced progression from the G0/G1 to the S-phase and increased the apoptosis rate of VSMCs. These results showed that hydrogen-rich saline was able to attenuate FBS-induced VSMC proliferation and neointimal hyperplasia by inhibiting ROS production and inactivating the Ras-ERK1/2-MEK1/2 and Akt pathways. Thus, HRSS may have potential therapeutic relevance for the prevention of human restenosis.


Percutaneous transluminal coronary angioplasty (PTCA) is a procedure used to open stenotic coronary arteries due to cholesterol-laden atherosclerotic plaques (1). During PTCA, a cardiologist inserts a catheter carrying a deflated balloon in the femoral artery and up to the blocked site in the heart. The balloon is then inflated, allowing the blood to flow again (1). A long-term analysis showed that 10- and 20-year survival in PTCA patients was similar to survival in coronary artery bypass graft patients, but more interventions had to be repeated during the first year in PTCA patients (2). Indeed, restenosis, or blood vessel re-narrowing leading to restricted blood flow, still occurs in approximately 30% of patients within 6 months after undergoing PTCA. This complication represents a major disadvantage of PTCA therapies. Modern use of drug-eluting stents (DESs) has improved the outcome in these patients; however, 5–10% of patients are still affected by restenosis after DES placement (3).

Reactive oxygen species (ROS) and oxidative stress are involved in the pathogenesis of intimal thickening in atherosclerosis and restenosis (4,5). Ohsawa et al (6) demonstrated that hydrogen is a potent anti-oxidant, able to selectively scavenge and neutralize hydroxyl radicals (such as OH and ONOO) and able to protect the brain against ischemiareperfusion (I/R) injury and stroke. Hydrogen treatment is able to reduce a number of markers of oxidative stress (7) and a number of clinical and experimental studies demonstrated that hydrogen acts as a scavenging agent, selectively neutralizing ROS in vivo and exerting potent cellular protective effects (811). Hydrogen has been shown to protect against restenosis in models of myocardial I/R injury (12,13). Such results underline the use of hydrogen (H2) gas in therapeutic medical applications.

Although the use of H2 gas may pose certain safety issues, due to its inflammability, hydrogen-saturated saline (HRSS) has been shown to be safe, easy to administer and effective in carotid I/R injury (14), renal I/R injury (15,16), acute liver injury (9), carbon monoxide intoxication (17) and atherosclerotic lesions (18). The exact mechanisms by which hydrogen exerts its effects on the vascular wall remain unknown. Aside from its anti-oxidant effects, there are certain indications that hydrogen may directly interact with some specific pathways (14,19).

The present study examined the anti-proliferative effects of HRSS on abnormal vascular smooth muscle cells (VSMC) proliferation and investigated the mechanisms responsible for these effects.

Materials and methods

Preparation of hydrogen-rich saline and medium

HRSS and media (HRM) were produced as previously described (20). Briefly, hydrogen gas was dissolved in saline and in Dulbecco’s modified Eagle’s serum (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS). We obtained saturation using a pressure of 0.4 MPa for 6 h. HRSS and HRM were maintained at 4°C and fresh solutions were prepared each week. Gas chromatography was used to confirm the hydrogen levels by the method described by Ohsawa et al (6). Cells were cultured in closed culture flasks.

Rat balloon injury model

All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of the Hebei Medical University (Shijiazhuang, China). A total of 30 male Sprague-Dawley rats (weight, 280–320 g) were purchased from the animal center of the University and were divided into five groups: the sham group (no angioplasty) and the balloon-injured groups including the control group and 3 groups administered with HRSS at 2.5, 5, 10 ml/kg, intraperitoneally, respectively.

Rats were anaesthetized with 3.6% (w/v) chlorohydrate (1 ml/100 g, intraperitoneally). Left common carotid artery (CCA) was balloon-denuded as previously described (21,22). In brief, a median incision was performed on the anterior neck and left carotid arteries were isolated. A ligature in external carotid artery (ECA) was distally placed and the proximal ends of the CCA and internal carotid arteries (ICA) were clamped. An incision was performed in the ECA. Following blood removal, a 0.13 mm balloon catheter was delicately introduced into the CCA using the incision in the ECA. The balloon was inflated using saline to distend the CCA and pulled back to the ECA. The catheter was removed and the ECA’s proximal end was ligated. Clamps were removed to re-establish blood flow.

Rats were then housed (12 h light cycles) and fed for 2 weeks (free access to food and water), and were then sacrificed using a pentobarbital overdose. Sections from carotid arteries from both sides were excised and fixed using 4% paraformaldehyde. Histologically stained sections were used to assess the extent of neointimal formation using computed planimetry. The intima-to-media (I/M) area ratio was calculated as the mean of these determinations (23).

Cell culture

VSMCs were isolated from the thoracic aorta of 10–12-week-old male Sprague-Dawley rats (2325) and were cultured (37°C in a humidified 5% CO2 incubator) in DMEM with 10% FBS, penicillin 100 U/ml and streptomycin 100 μg/ml. Purity was confirmed by immunocytochemical localization of α-smooth muscle actin. Upon confluence, cells were sub-cultured using 0.5% (w/v) trypsin. Media were changed every 3 days and experiments were performed between 3 to 6 passages.

Cell proliferation assay
MTT assay

VSMCs (4×103 cells/well) were placed in a 96-well plate. Following incubation, sterile MTT (20 μl of 5 mg/ml) was added, and incubated for 4 h at 37°C; 150 μl of dimethyl sulfoxide (DMSO) was then added. Absorbance (490 nm) was measured using a microplate reader (26).

BrdU assay

VSMCs were synchronized by serum deprivation and incubated with 10% FBS for 24 h with or without HRM. BrdU labeling mixture was then added and cultures were incubated for 12 h at 37°C. Cell numbers were measured using a cell proliferation ELISA (Roche Molecular Biochemicals, Germany) (9,27).

Wound healing assay

The migration of VSMCs was evaluated by performing a cell-wounding assay according to a previously described method (28). Cells grown to 100% confluence on glass slides were scraped off the slides using a cell scraper. These cells were used to create a 3-mm-wide wound and were then incubated at 37°C for 24 h in DMEM containing 10% FBS. Cells were then fixed with methanol and stained with hexamethyl pararosaniline. Cell migration activity was expressed as the number of cells that migrated into the wound area in each field.

Flow cytometric analysis of the cell cycle

VSMCs were synchronized at the G0-phase by serum depletion for 48 h. After replenishment with fresh DMEM, the cells were preincubated with hydrogen-rich medium, and 10% FBS was added to allow progression of cell cycle. After 48 h, cells were trypsinized, centrifuged at 1,250 x g for 5 min. DNA was stained with propidium iodide (PI) (50 μg/ml) for 30 min at 37°C, and 5×103 cells were then analyzed by flow cytometry.

Flow cytometric analysis of cell apoptosis

To assess apoptosis, we suspended HRM-cultured cells in buffer (400 μl; 10 mM HEPES/NaOH; 140 mM NaCl; 2.5 mM CaCl2; pH 7.4) after a careful wash. FITC-conjugated Annexin V (5 μl; BD Biosciences, San Diego, CA, USA) was added and cells were incubated for 15 min at 4–8°C in the dark. PI (10 μl) was added. After 5 min, cells were washed again and 400 μl of buffer was added. Cells were counted using a FACSCalibur (BD Biosciences).

RT-PCR for Bax/Bcl-2

Total RNA was extracted using the TRIzol reagent method and cDNA was obtained using a cDNA synthesis kit (TransScript first-strand) according to the manufacturer’s instructions. Bcl-2 primers were: ATGGGGTGAAC TGGGGGAGGATTG (forward) and TTTCATATTTGTTT GGGGCAGGTC (reverse). Bax primers were: GAGAGGAT GGCTGGGGAGAC (forward) and GGTGAGCGAGGCGG TGAGGACT (reverse). DNA fragments were analyzed on a 1.5% agarose gel. Quantitative RNA data were expressed by normalizing band density to GAPDH (internal control).

Western blot analysis

The cells and arterial tissue were solubilized or homogenized in lysate buffer on ice. Homogenates were centrifuged at 12,000 x g for 30 min at 4°C, yielding a cell protein supernatant. Proteins were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA, USA). Samples were then probed with antibodies against: Ras, phosphorylated and nonphosphorylated ERK1/2, MEK1/2, proliferative cell nuclear antigen (PCNA), Akt and β-actin. Non-specific binding was blocked with 1.5% (w/v) evaporated skimmed milk (Difco, Franklin Lakes, NJ, USA) in TBS (154 mM NaCl, 10 mM Tris base). Anti-rabbit or anti-mouse secondary antibodies conjugated to IRDYe700DX and IRDYe800 (1:5,000; Rockland Immunochemicals, Gilbertsville, PA, USA) were used to probe primary antibodies. Protein bands were detected and quantified on an Odyssey 2-color infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). The integrated signal densities were normalized first to β-actin (the loading control) and subsequently expressed in terms of the fraction abundance relative to control cells or arterial tissues. These experiments were performed in triplicate.

Immunohistochemistry and image analysis

Immunohistochemistry was performed as previously described (29). The control and sham groups were treated with anti-PCNA (1:100) antibody on Day 14 following balloon injury. Sections were counterstained with hematoxylin. Staining intensities were determined by measurement of the integrated optical density (IOD) with light microscopy using a computer-based Image-Pro Morphometric system. Measurements were conducted by 2 independent observers in a double-blind manner.

Determination of reactive oxygen species
DCFH-DA assay for intracellular reactive oxygen species

VSMCs (1×105 cells) were incubated with 10% FBS in the presence or absence of HRM for 12 h. Cells were stained with 10 μM of DCFH-DA for 30 min at 37°C, detached with trypsin/EDTA, washed, re-suspended in PBS, and immediately analyzed by flow cytometry using a FACScan by fluorescence intensity (FL-1,525 nm) of 10,000 cells (23).

Determination of MDA concentration in VSMCs and hyperplasia neointima using TBARS production

Following incubation with HRM for 24 h, cells were collected and washed with cold PBS, and hyperplastic neointimal tissues were homogenized. Tissue samples (0.1 ml), 8.1% SDS (0.2 ml), 20% acetic acid buffer solution (1.5 ml, pH 3.5), 1% total bile acid (TBA) thiobarbituric solution (1.5 ml), and distilled water (1 ml) were added. The solution was heated in a 95°C water bath for 40 min and then allowed to cool; it was then centrifuged at 3,500 rpm/min for 15 min and measured at 532 nm using a spectrophotometer (14,30).

Determination of 8-OHdG in VSMCs and hyperplasia neointima

The 8-OHdG amounts were determined in VSMCs and hyperplastic neointimal tissues using a Bioxytech 8-OHdG-EIA kit. DNA was isolated using the DNAzol reagent and quantified. DNA (400 μg) was resuspended in 50 μl reaction mixture containing 100 mmol/l sodium acetate (pH 5.0) and 5 mmol/l MgCl2 and digested using DNaseI. Assay was performed according to the manufacturer’s instructions (Shanghai Lanji Biological Institute, China).

Statistical analysis

Data are provided as the means ± SEM. One-way analysis of variance (ANOVA) followed by the LSD-t-test were performed using SPSS 19.0 software. P<0.05 was considered to indicate a statistically significant difference.


ROS and Ras signaling pathways are involved in the effects of HRSS on neointimal hyperplasia

Balloon-induced intimal hyperplasia was evident in treated rats compared with rats of the control group (Fig. 1A). Results showed that 3 doses of HRSS were effective for prevention of neointimal formation in a dose-dependent manner (Fig. 1A). These findings showed neointimal area ratio reductions of 36.9, 52.2 and 71.8% (P<0.01) in HRSS-treated groups using 2.5, 5 ml and 10 ml/kg doses, respectively, compared with the balloon-injured control group (Fig. 1B).

Western blot analysis revealed that the activation of ERK1/2, MEK1/2 and Ras was strongly suppressed in a dose-dependent manner in 14-day HRSS-treated samples compared with control samples (P<0.01) (Fig. 1E).

PCNA-positive cells were abundant in the balloon injury group. However, PCNA immunostaining was much less apparent in cells treated with HRSS. Staining intensities were lower in HRSS-treated rats (P<0.01) (Fig. 1C and D).

Treatment with HRSS significantly inhibited the levels of carotid artery MDA and 8-OHdG in a dose-dependent manner (Fig. 2).

HRM inhibits FBS-induced VSMC proliferation and migration

MTT assay showed that VSMC viability in the HRSS group was significantly lower than in the control group (32.4±5.2 vs. 73.3±5.1%; P<0.01). Two VSMC cultures were treated with HRM for 48 h; hydrogen was then ceased for one culture, while it was continued in the other. Following the removal of hydrogen, cells resumed viability (Fig. 3A). Cells without hydrogen treatment exhibited a BrdU index of 64.1±11.1%, compared to 26.7±4.9% in the hydrogen group (Fig. 3B).

VSMC cell count was smaller in HRM-treated cells after 48 and 72 h (40 and 51% reductions, respectively; P<0.01) (Fig. 4A and B). VSMCs treated with FBS for 24 h showed greater mobility (FBS 58.3±13.9 vs. hydrogen 18.6±5.8 cells; P<0.01) (Fig. 4C and D).

HRM prevents FBS-induced S-phase entry in VSMCs

Treatment with HRM inhibited FBS-induced G1-S progression, as demonstrated by the increase in G0/G1 cells (68.8±2.2%) accompanied by concurrent decrease in S-phase cells (16.7±2.0%) (Fig. 5A). These results suggest that hydrogen may prevent FBS-induced S-phase entry in VSMCs via a G0-G1 blocking mechanism.

HRM increases the proportion of apoptotic cells in VSMCs

Proportion of early apoptotic cells (Fig. 6A and B; lower right) increased from 1.36% (FBS-treated) to 2.1% (FBS + hydrogen-treated) (P<0.05). These results also showed that the proportion of late apoptotic cells (Fig. 6A and B; upper right) increased from 6.96 to 13.47% (P<0.01).

Following HRM treatment, a significant increase in Bax/Bcl-2 ratio (P<0.01) indicated that VSMCs were, in fact, progressing towards apoptosis (Fig. 6C and D).

ROS is involved in the effect of HRM on FBS-stimulated VSMCs

FBS treatment significantly induced intracellular peroxide production (Fig. 7A and B). ROS generation in cells treated with HRM was 395.6±16.1, compared with 483.0±15.1 for controls (P<0.01).

The 8-OHdG levels in cells treated with HRM were 1.3±0.0 ng/ml, compared to 1.6±0.1 ng/ml in controls (P<0.01) (Fig. 7C). Fig. 7D shows that treatment with HRM significantly inhibited MDA generation (FBS 3.7±0.2 vs. hydrogen 2.9±0.1; P<0.01).

Ras-MEK1/2-ERK1/2 pathway and Akt phosphorylation are involved in the effect of HRM on FBS-stimulated VSMCs

Stimulation of cells with 10% FBS induced Ras activation. Cells treated with HRM exhibited a slight inhibition of FBS-induced Ras activation (Fig. 8A). Treatment of FBS-stimulated VSMCs with HRM markedly decreased phosphorylated MEK1/2 levels (Fig. 8C). Data showed that FBS also induced a profound increase in ERK1/2 activation. Treatment with HRM significantly inhibited FBS-stimulated phosphorylation of ERK1/2 (Fig. 8B). By contrast, total ERK1/2 protein levels were not altered by treatment with hydrogen. FBS induced a profound increase in Akt activation. The level of Akt phosphorylation following FBS stimulation was also significantly inhibited by treatment with HRM (Fig. 5E). As shown in Fig. 8D, FBS-induced PCNA expression was significantly inhibited by HRM by 50% (P<0.01).


In the process of opening the artery, PTCA may injure the vascular wall (31). VSMC proliferation in the arterial wall is crucial in the development of post-angioplasty restenosis and atherosclerosis (32,33). Currently, the most common approach to reduce neointimal formation and its consequences is the use of DES (34). However, DES presents a number of issues, such as incomplete stent coverage, inadequate stent placement or failure of the eluting drug to inhibit VSMC proliferation (3537). The current study demonstrates that hydrogen inhibits VSMC proliferation and migration and provides important mechanistic information; this inhibitory effect involves the blockage of the G1-S-phase cell cycle progression, thus increasing apoptosis and inhibition of Ras-MEK1/2-ERK1/2 and Akt signaling pathways. HRSS treatment also inhibits neointimal hyperplasia induced by balloon injury via suppression of the Ras-MEK1/2-ERK1/2 signaling pathway.

Oxidative stress is involved in vascular diseases (38,39). Initial observations focused on ROS derived from invading macrophages, indicating possible involvement in oxidative lipid modifications of vessel walls. ROS can attack circulating lipoproteins, contributing to atherosclerotic plaque development (40), and can attack vessel walls, maintaining a pro-inflammatory state (41). Ohsawa et al (18) used an apolipoprotein E knockout mouse model to demonstrate the role of hydrogen as an antioxidant. In their study, oral ingestion of HRSS for 6 months prevented atherosclerosis development by decreasing oxidative stress in the blood vessels. Moreover, controlled ROS production occurs in all vascular cells, where these compounds act as secondary messengers in the regulation of varied cellular functions. Notably, VSMC proliferation and stenosis following vascular injury was shown to coincide with elevated ROS levels in experimental animals (42,43).

The O2 and H2O2 ROS are primarily detoxified by antioxidant defense enzymes, unlike radical OH and ONOO species. Hydrogen gas has been demonstrated to selectively reduce these 2 ROS species (6). Thus, 8-OHdG formed by deoxyguanosine in DNA in the presence of hydroxyl free radicals may be a useful biomarker for intracellular oxidative stress (44). MDA is also a non-specific marker of lipid peroxidation. Assessment of 8-OHdG levels demonstrated that increasing ROS levels were inhibited by HRSS in vitro in FBS-treated VSMCs. Furthermore, increases in ROS levels in neointimal tissues in vivo were inhibited by HRSS. These findings are consistent with previous reports, including the report by Qin et al (14) that demonstrates hydrogen ability to reduce injury-induced excess superoxide anions and inflammation. Results from the present study also show that ROS reduction coincides with a decrease in VSMC proliferation and proliferative pathways. This moderate antioxidant ability indicates that hydrogen may cause only minimal disturbance to normal physiological functions involving ROS.

Upon exposure to growth factors or serum, protein-tyrosine kinase (PTK) receptors are activated, resulting in the activation of Ras/Raf/MAPK/MEK and downstream proteins. ERK also plays a central role in this pathway (45). It has also been suggested that ERK may play a pivotal role in VSMC proliferation control (46,47). Specifically, it may inhibit MAPK phosphorylation, thus markedly inhibiting VSMC growth in vitro (48). The activation of ERK1/2 can increase the expression of downstream transcription genes, such as cyclin D1, resulting in protein synthesis and cell proliferation (49). Therefore, confirmation that HRSS inhibits the ERK pathway partly explains the antiproliferative mechanisms of such treatments.

Furthermore, Ras, a major upstream signaling protein of Raf-MEK1/2-ERK1/2, is primarily stimulated by mitogenic factors (50). It plays a pivotal role in G1 progression and in the G1-S transition. Hydrogen treatment significantly and efficiently reduces phosphorylated ERK (active form) and phosphorylated MEK, resulting in a slight decrease in Ras expression, suggesting that HRSS is capable of effectively inhibiting the ERK pathway. This effect likely contributes to the observed anti-proliferative activity of hydrogen in VSMCs. HRSS effects on the ERK signaling cascade activated by balloon injury may also be associated with Ras-MEK1/2-ERK1/2 signaling pathway involvement in suppression of neointimal hyperplasia. Dose-dependent inhibition of ROS, VSMC proliferation and of ERK1/2, MEK1/2, and Ras activations were observed in cases of balloon injury treated with lercanidipine (51). Our results indicate that the same outcomes are achieved simply using hydrogen and that hydrogen acts through similar mechanisms. These results further support the ability of HRSS to inhibit the FBS- or injury-induced ERK1/2 signaling cascade, thus affecting VSMC proliferation both in vitro and in vivo, which has not previously been reported as a major hydrogen effect in a review on hydrogen’s medical properties (7). HRSS was also demonstrated to inhibit the PI3K pathway in VSMCs. PI3K activates Akt (serine/threonine kinase). In its active state, Akt promotes cell survival and growth (52); however, it remains unclear whether the PI3K pathway is affected by HRM treatment. If HRM can inhibit the PI3K pathway in VSMCs, this effect would also contribute to the anti-proliferative effect of HRM on VSMCs.

Suppressed PCNA levels in the G0/G1-phase were associated with hydrogen treatment, demonstrating the effect of hydrogen treatment on the regulation of VSMCs at the nuclear level. PCNA is synthesized in the early G1- and S-phase, making it a useful marker for proliferation (50,53). The anti-proliferative effect of HRM may result from its ability to block the entry of cells into the S-phase due to interference in the early G0/G1 transition phase. Preventing cell cycle transition between the G1- and S-phase in VSMCs may be beneficial in reducing cell proliferation and migration, as well as restenosis (54,55). The current evaluation of VSMC cell cycle alteration in response to treatment with HRM showed a considerable increase in cells in the G0/G1-phase and a concomitant decrease in S-phase cells, which may result from reduced proliferation, increased apoptosis, or a combination of the two. Additionally, HRM treatment revealed increased apoptosis rates. Cellular apoptotic events are known to be governed by levels of anti-apoptotic (Bcl-xL family) and pro-apoptotic proteins (Bax, Bak) (56), indicating that the significant reduction in Bcl-2/Bax ratio observed in VSMCs treated with HRM supports the involvement of hydrogen in the apoptotic pathway.

The use of FBS to induce VSMC growth in vitro could be questioned. However, a previous study also using rat VSMCs showed that 10% FBS was as effective as 20 ng/ml of plateled-derived growth factor (PDGF) to promote VSMC proliferation (51). Furthermore, lercanidipine had the same efficacy in FBS- and PDGF-induced VSMCs, and inhibited Ras-ERK1/2 signaling in the same way, suggesting that FBS and PDGF promote VSMC proliferation using the same pathways.

Significant evidence shows that enhanced VSMC proliferation is a fundamental feature of atherosclerosis pathogenesis. In conclusion, data presented in this report demonstrate that treatment with HRM can effectively inhibit VSMC proliferation and migration in vitro and protect neointimal formation in vivo following vascular injury by balloon angioplasty. These results demonstrate that HRM suppresses the proliferation of VSMCs by inhibiting the ROS, Ras-MEK1/2-ERK1/2 and P13K/Akt pathways. Thus, hydrogen treatment promotes cell cycle arrest in VSMCs at the G0/G1-phase and increases apoptosis rates. HRSS is therefore a potentially useful antioxidant that may prove clinically useful in patients undergoing coronary artery angioplasty, stenting or coronary artery bypass to prevent restenosis.



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March 2013
Volume 31 Issue 3

Print ISSN: 1107-3756
Online ISSN:1791-244X

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Chen, Y., Jiang, J., Miao, H., Chen, X., Sun, X., & Li, Y. (2013). Hydrogen-rich saline attenuates vascular smooth muscle cell proliferation and neointimal hyperplasia by inhibiting reactive oxygen species production and inactivating the Ras-ERK1/2-MEK1/2 and Akt pathways. International Journal of Molecular Medicine, 31, 597-606. https://doi.org/10.3892/ijmm.2013.1256
Chen, Y., Jiang, J., Miao, H., Chen, X., Sun, X., Li, Y."Hydrogen-rich saline attenuates vascular smooth muscle cell proliferation and neointimal hyperplasia by inhibiting reactive oxygen species production and inactivating the Ras-ERK1/2-MEK1/2 and Akt pathways". International Journal of Molecular Medicine 31.3 (2013): 597-606.
Chen, Y., Jiang, J., Miao, H., Chen, X., Sun, X., Li, Y."Hydrogen-rich saline attenuates vascular smooth muscle cell proliferation and neointimal hyperplasia by inhibiting reactive oxygen species production and inactivating the Ras-ERK1/2-MEK1/2 and Akt pathways". International Journal of Molecular Medicine 31, no. 3 (2013): 597-606. https://doi.org/10.3892/ijmm.2013.1256