AST Ⅳ inhibits H2O2-induced human umbilical vein endothelial cell apoptosis by suppressing Nox4 expression through the TGF-β1/Smad2 pathway

  • Authors:
    • Yuhong Ma
    • Weizu Li
    • Yanyan Yin
    • Weiping Li
  • View Affiliations

  • Published online on: April 17, 2015     https://doi.org/10.3892/ijmm.2015.2188
  • Pages: 1667-1674
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Abstract

Endothelial cell apoptosis plays an important role in the pathophysiological mechanisms of vascular complications in diabetes mellitus (DM). NADPH oxidase 4 (Nox4)-dependent reactive oxygen species (ROS) aggregation is the main cause of vascular endothelial cell apoptosis. The transforming growth factor-β1 (TGF-β1)/Smad2 signaling pathway is involved in the apoptosis of several types of cells. However, the association between vascular endothelial cell apoptosis and Nox4, and the involvement of the TGF-β1/Smad2 signaling pathway in vascular endothelial cell apoptosis remain unclear. In the present study, we aimed to investigate the role of Nox4-dependent ROS production and to determine the involvement of the TGF-β1/Smad2 signaling pathway in endothelial cell apoptosis induced by oxidative stress which causes vascular injury in DM. We demonstrated that hydrogen peroxide (H2O2) increased Nox4-dependent-ROS aggregation, as well as the expression of TGF-β1, Smad2, Bax and caspase-3, decreased Bcl-2 expression and increased the apoptosis of human umbilical vein endothelial cells (HUVECs). Treatment with diphenyliodonium (DPI), a specific inhibitor of Nox4 or astragaloside Ⅳ (AST Ⅳ), a monomer located in an extract of astragaloside, decreased Nox4 expression and the levels of ROS, decreased TGF-β1 and Smad2 expression, altered the expression of apoptosis-related genes and decreased the apoptosis of HUVECs. Treatment with LY2109761, a selective inhibitor of the TGF-β1/Smad2 pathway, produced results similar to those of DPI; however, LY2109761 had no effect on Nox4 expression and ROS levels. Taken together, the findings of the present study suggest that H2O2 contributes to HUVEC apoptosis by inducing Nox4-dependent ROS aggregation and activating the TGF-β1/Smad2 signaling pathway. Our data indicate that the protective effects of AST Ⅳ against vascular endothelial cell apoptosis in DM are mainly associated with the decrease in Nox4 expression through the TGF-β1/Smad2 signaling pathway. Furthermore, the inhibition of the activation of the TGF-β1/Smad2 signaling pathway may be another potential therapeutic strategy in the treatment of DM.

Introduction

Diabetes mellitus (DM) is a complex metabolic syndrome, the prevalence of which is rapidly increasing worldwide. Among the pathophysiological mechanisms of diabetes, vascular complications are a main cause of morbidity and mortality in diabetic patients (1). Oxidative stress is the main pathophysiological mechanism of macrovascular injury and contributes to endothelial cell injury. The aggregation of reactive oxygen species (ROS) frequently damages the cytoplasm, lipids and proteins, thus resulting in vascular endothelial cell apoptosis (25). Cell apoptosis is the initial step in macrovascular injury and is critical to the development and progression of cardiovascular diseases (6). NADPH oxidase 4 (Nox4), a subunit of NADPH oxidase, is abundantly expressed in several types of tissue and generates free radicals in vascular endothelial cells (7,8). The inhibition of Nox4 activity has been shown to prevent adipose-derived stem cell apoptosis (9). Nevertheless, the molecular mechanisms responsible for Nox4-induced endothelial cell apoptosis remain unclear.

Transforming growth factor-β1 (TGF-β1) plays a role in the apoptosis and proliferation of a variety of cells (1012). Smad2, a downstream cytokine of TGF-β1, is activated following the activation of TGF-β1. Activated Smad2 then translocates to the nucleus and modulates the transcription of TGF-β1 target genes (13,14). Previous studies have demonstrated that TGF-β1/Smad2 possesses potent proliferative activity in various cell types (1517), whereas others have demonstrated that it induces apoptosis in a number of cells (1820). Nevertheless, to the best of our knowledge, few studies have investigated whether the TGF-β1/Smad2 pathway is involved in vascular endothelial cell apoptosis in DM.

Astragaloside IV (AST IV), which is used in traditional Chinese medicine, is a monomer located in an extract of astragaloside (Fig. 1). Our previous studies confirmed that AST IV has pharmacological effects, including anti-inflammatory and antioxidant effects in some diseases (2123). However, to the best of our knowledge, the protective effects of AST IV against vascular injury in DM in vitro have not been investigated to date.

In the present study, we aimed to investigate the role of Nox4-dependent ROS production and whether the TGF-β1/Smad2 signaling pathway plays a critical role in endothelial cell apoptosis in vitro induced by oxidative stress, which causes vascular injury in DM, and whether AST IV inhibits hydrogen peroxide (H2O2)-induced HUVEC apoptosis by suppressing Nox4 expression through the TGF-β1/Smad2 pathway.

Materials and methods

Cell culture and administration

Human umbilical vein endothelial cells (HUVECs; China Center for Type Culture Collection, Wuhan, China) were incubated in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/l D-glucose supplemented with 10% fetal bovine serum (FBS) (both from HyClone, Logan, UT, USA), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C. The HUVECs were subcultured at a 1:2 ratio interval of 2 days. Vascular injury associated with DM in vitro was mimicked by incubation with H2O2 100 μmol/l for 18 h to induce damage to the HUVECs, as previously described (24). The cells were treated with each agent in the medium for 1 h prior to exposure to H2O2.

The cells were divided into 3 groups as follows: i) the control group: cells were left untreated; ii) the model group: cells were treated with H2O2 100 μmol/l for 18 h; and iii) the AST IV group: cells were treated with AST IV for 1 h and then treated with H2O2. In addition, some cells were treated with diphenyliodonium (DPI, a specific inhibitor of Nox4; from Sigma, St. Louis, MO, USA) or LY2109761 (a selective inhibitor of TGF-β1/Smad2; from MedChem Express, LLC, Princeton, NJ, USA).

MTT assay

The half maximal effective concentration (EC50) of AST IV in preventing the H2O2-induced damage to HUVECs was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma). In brief, the cells were seeded at a density of 1×104 cells/well in 96-well plates. They were then exposed to serial dilutions of AST IV in DMEM-high glucose medium and allowed to grow for the indicated periods of time. Following treatment, the cells were incubated with 100 μl DMEM-high glucose medium containing 5 mg/ml MTT. Following incubation for 4 h at 37°C, the supernatants were discarded, MTT crystals were dissolved in 100 μl dimethyl sulfoxide (DMSO) and the optical density (OD) was measured at 570 nm using a Bio-Rad microplate reader (Bio-Rad, Hercules, CA, USA).

ROS assay

Intracellular ROS production was detected using a probe, the redox-sensitive fluorophore carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Sigma) as the following steps. After delivery, the cells were washed with phosphate-buffered saline (PBS) and incubated with 20 μmol/l H2DCFDA in the dark for 30 min. The cells were then briefly exposed to 0.5 g/l trypsin. The deactivation of trypsin was accomplished by the addition of PBS supplemented with 3% FBS. All cells were examined using a FACScan flow cytometer (Beckman Coulter, Miami, FL, USA) and the data were processed using FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, USA) (25).

Annexin-V and propidium iodide (PI) staining to detect apoptosis

Following treatment, apoptosis was determined according to the following steps: the cells were collected by 0.25% ethylenediaminetetraacetic acid (EDTA)-free trypsin to digest the cells followed by centrifugation (at 1,500 rpm) to collect the cells; the cells then were suspended with 500 μl binding buffer, and the concentration was then adjusted to 1×106 followed by the addition of 5 μl Annexin V-FITC staining fluid, gentle blending and incubation at 4°C in the dark for 15 min; the cells were then treated with PI dyeing liquid at 4°C in the dark for 5 min. Finally the cells were immediately examined using a FACScan flow cytometer (Beckman Coulter). Annexin V-FITC detection was carried out using the FL-1 and PI detection using the FL-2 channel.

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed using the First-Strand cDNA Synthesis kit (Life Technology, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cDNA were used as the template for the qPCR amplification using oligo primers, and the internal control for the qPCR reaction was glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer sequences of the Nox4, TGF-β1, Smad2, Bax, Bcl-2 and caspase-3 genes (Shanghai Sangon Biotech, Shanghai, China) are presented in Table I. qPCR was performed using an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). qPCR was performed with a total volume of 12 μl in each well, containing 5 μl of SYBR-Green® PCR Master Mix (Applied Biosystems), 5 μl of cDNA, and 1 μmol forward primers and 1 μmol reverse primers. Each sample was run in triplicate in a separate tube. The qPCR conditions were conducted as follows: 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 1 min, and extension at 72°C for 1 min. Initial heating at 95°C for 10 min and a final extension at 72°C for 7 min was performed for all qPCR reactions. The cycle threshold (CT) values from all the qPCR experiments were calculated using the 2−ΔΔCT method.

Table I

Sequence of primers used for RT-qPCR.

Table I

Sequence of primers used for RT-qPCR.

GeneSequence forward primer 5′→3′Sequence reverse primer 5′→3′Length (bp)
Nox4 TGGACCTTTGTGCCTGTACTGT TGAGGATGACTTATGACCGAAA89
Smad2 CTTTTGTTGTGTAAGCTCTCACTG GACCTTCTACCACTTTCAGAGTTG243
TGF-β1 TGGACACGCAGTACAGCAAG GCCCACGTAGTACACGATGG119
Bax TGGCAGCTGACATGTTTTCTGAC TCACCCAACCACCCTGGTCTT195
Bcl-2 TTTGAGTTCGGTGGGGTCATG TCACTTGTGGCTCAGATAGGC269
Caspase-3 AGAGGGGATCGTTGTAGAAGTC ACAGTCCAGTTCTGTACCACG81
GAPDH TCCCTGAGCTGAACGGGAAG GGAGGAGTGGGTGTCGCTGT217

[i] qPCR, quantitative polymerase chain reaction; Nox4, NADPH oxidase 4; TGF-β1, transforming growth factor-β1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Western blot analysis

Following treatment, the cells were lysed using RIPA buffer (Beyotime, Shanghai, China) containing 1% phenylmethanesulfonyl fluoride (PMSF; Sigma) on ice. The total protein concentrations were measured using the BCA Protein Assay kit (Beyotime). Proteins were separated by 8 or 10% SDS-PAGE and then transferred onto PVDF membranes (Millipore Corp., Billerica, MA, USA). Subsequently, the membranes were blocked with 5% non-fat milk at room temperature (RT) for 1 h, and incubated with primary antibodies as follows: anti-Nox4 (ab109225; Abcam, Cambridge, UK), anti-TGF-β1 (3712S; Cell Signaling Technology Inc., Beverly, MA, USA), anti-Smad2 (ab33875; Abcam), anti-Bax (sc-6236), anti-Bcl-2 (sc-783) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-caspase 3 (9665S; Cell Signaling Technology Inc.) and anti-β-actin (A1978; Sigma) antibodies overnight at 4°C. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary anti-mouse, anti-rabbit antibodies (1:2,000 dilution) for 1 h at RT. Moreover, the proteins were visualized using an ECL advanced western blot detection kit (Pierce, Thermo, Rockford, IL, USA). Densitometric measurements of band intensity in the western blot analysis were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

All experiments were carried out 3 times. All quantified results are expressed as the means ± SEM in graphical representation. Data analysis of all results was carried out using one-way analysis of variance (ANOVA) followed by Fisher’s LSD-based post-hoc analysis. All P-values were two sided and considered significant when P<0.05. Statistical analyses were performed with SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).

Results

EC50 of AST IV in HUVECs treated with H2O2

MTT (Sigma) assay was performed to determine the EC50 of AST IV in inhibiting H2O2-induced damage to HUVECs. The cells were treated with AST IV at 5, 10, 25, 50, 100, 200, 400 and 800 μmol/l for 1 h prior to treatment with H2O2 and cell viability was then examined by MTT assay. The results revealed that the EC50 of AST IV in inhibiting H2O2-induced injury to HUVECs was 100 μmol/l [calculated by regression equation (x, drug concentration; y, cell survival rate)].

Protective effect of AST IV against H2O2-induced HUVEC apoptosis

To assess the protective effects of AST IV against H2O2-induced HUVEC apoptosis, the mRNA and protein expression levels of Nox4, TGF-β1, Smad2, Bcl-2, Bax and caspase-3 were determined; the intercellular ROS level and the apoptotic rate were also determined. Nox4 expression in the model group was significantly increased in comparison to the control group (P<0.01; Fig. 2B and C). The geometric mean fluorescence intensity indicating ROS production in the control group was lower, whereas that in the model group was markedly increased (P<0.01; Fig. 2A). In comparison with the control group, the expression of TGF-β1/Smad2 was markedly elevated in the model group (P<0.01). In addition, the expression of Bax (pro-apoptotic gene) was lower and that of Bcl-2 (anti-apoptotic gene) was higher in the control group compared to the model group (P<0.01; Fig. 2B and C). The expression of caspase-3, a main terminal shear enzyme involved in apoptosis, was barely detectable in the control group; by contrast, its expression was upregulated in the model group (P<0.01; Fig. 2B and C). In comparison with the control group, the apoptotic rate was significantly increased in the model group (P<0.01; Fig. 2D). However, treatment with AST IV100 μmol/l reversed these effects (P<0.01).

Expression of Nox4 is an important promoting event in the onset of H2O2-induced HUVEC apoptosis

To explore the molecular mechanisms responsible for H2O2-induced HUVEC apoptosis, the HUVECs were treated with diphenyliodonium (DPI; 10 μmol, a Nox4 inhibitor) prior to treatment with H2O2. We found that treatment with DPI or AST IV decreased the expression of Nox4 (Fig. 3B and C), as well as the intercellular ROS levels in the HUVECs treated with H2O2 (Fig. 3A). Our results revealed that DPI or AST decreased TGF-β1/Smad2 expression in the HUVECs damaged by H2O2 (Fig. 3B and C). Our results also revealed that treatment with DPI or AST IV decreased Bax and caspase-3 expression, and increased Bcl-2 expression (Fig. 3B and C). In addition, treatment with DPI or AST IV decreased the HUVEC apoptotic rate (Fig. 3D). The exposure of the cells to AST IV at 100 μmol/l had a similar effect to that of treament with DPI (Fig. 3).

Inhibition of the TGF-β1/Smad2 signaling pathway decreases H2O2-induced HUVEC apoptosis, but not oxidative stress

To further investigate the role of the TGF-β1/Smad2 signaling pathway in H2O2-induced HUVEC apoptosis, LY2109761 (0.1 μmol/l), a selective inhibitor of TGF-β1/Smad2, was used to suppress the activation of the TGF-β1/Smad2 pathway. The results revealed that TGF-β1 and Smad2 mRNA and protein expression was detected at extremely low levels in the control group, whereas the overexpression of TGF-β1 and Smad2 was observed in the model group; there was a statistically significant difference between the control group and the model group (P<0.01; Fig. 4B and C). However, treatment with LY2109761 decreased TGF-β1 and Smad2 expression, as well as Bax and caspase-3 expression, and increased Bcl-2 expression (P<0.01; Fig. 4B and C). In addition, treatment with LY2109761 decreased HUVEC apoptosis (P<0.01; Fig. 4D), but had no effect on Nox4 expression and the ROS levels (P>0.05; Fig. 4A–C). Treatment with AST IV at 100 μmol/l significantly ameliorated these risk factors; it downregulated Nox4 expression (Fig. 4B and C), decreased ROS levels (Fig. 4A), decreased TGF-β1, Smad2, Bax and caspase-3 expression (Fig. 4B and C) and upregulated Bcl-2 expression (Fig. 4B and C) (P<0.05 and P<0.01 compared to model group), further decreasing HUVEC apoptosis (Fig. 4D).

Discussion

As is known, Nox4, a subunit of NADPH oxidase, mainly catalyzes and generates intracellular ROS in vascular endothelial cells (26). The elevation of intracellular ROS production results in pathophysiological changes, including vascular inflammation in DM (27,28). This study confirmed that incubation with H2O2 at 100 μmol/l for 18 h induced Nox4 expression and ROS generation, and that the aggregation of ROS in HUVECs led to the development of endothelial cell disorders, finally resulting in vascular complications in vitro, as those observed in DM (28,29). On the one hand, increased ROS freely transmits the cell membrane and induces membrane lipid peroxidation and DNA damage. On the other hand, ROS augments the cell oxidative reaction system and induces cell apoptosis (3033). In this study, following treatment with DPI, a compound which inhibits Nox4 generation, or AST IV markedly suppressed Nox4 expression, significantly decreased the generation of intracellular ROS, markedly decreased the expression of apoptosis-related genes and the apoptotic rate of the HUVECs, as demonstrated in previous studies using other agents (3442). Therefore, the expression of Nox4 is an important promoting event in the onset of H2O2-induced HUVEC apoptosis. AST IV may thus inhibit H2O2-induced HUVEC apoptosis by suppressing Nox4 expression.

Moreover, apoptosis, or programmed cell death, is associated with the activation of multiple genes and multiple signaling pathways. The TGF-β signaling pathway is associated with oxidative stress and the apoptotic process (4345). In this study, our results also revealed that the TGF-β1/Smad2 pathway was activated in the H2O2-treated HUVECs. TGF-β1 stimulates cell responses by signaling through the canonical Smad protein pathway, as well as using alternative pathways involving Smads, mitogen-activated protein kinases (MAPKs), protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K). Activated Smad2, a downstream effector of TGF-β1 signaling, then promotes cell apoptosis (4648). In this study, treatment with LY2109761, a selective TGF-β1/Smad2 pathway inhibitor, produced results similar to those obtained with DPI; however, LY2109761 had no effect on Nox4 expression and ROS levels. Nevertheless, AST IV decreased Nox4 expression and ROS levels, decreased TGF-β1 and Smad2 expression, decreased Bax and caspase-3 expression, and increased Bcl-2 expression and decreased HUVEC apoptosis.

Taken together, these results suggest that AST IV exerts an anti-inflammatory effect by decreasing the apoptosis of HUVECs induced by H2O2 through the inhibition of the activation of the TGF-β1/Smad2 signaling pathway. A previous study also demonstrated that AST IV possessed strong antioxidant capabilities by scavenging and neutralizing free radicals, as well as anti-inflammatory properties by inhibiting ROS formation and accumulation (23). Nevertheless, the findings of this study indicate that AST IV exerts effects similar to those of DPI, but not LY2109761. Thus, the protective effects of AST IV against vascular injury in DM in vitro, are mainly related to the decrease in Nox4 expression. Moreover, our results also suggest that potential therapeutic strategies to combat anti-vascular complications in DM may be developed through the manipulation of the redox status in DM. Furthermore, the inhibition of the activation of the TGF-β1/Smad2 signaling pathway may be another potential therapeutic strategy in the treatment of DM. However, the signaling pathways related to apoptosis are complex, multiple, and a number of pathways interact with each other. In this study, we only investigated the TGF-β/Smad signaling pathway; further investigations are warranted to investigate the other pathways involved. At the present time, the pharmacological effects of AST IV on vascular injury in DM need to be explored and further studies are required to determine the role of other signaling pathways.

Acknowledgments

This study was supported by a grant form the National Natural Science Foundation of China (no. 81173624).

Abbreviations:

DM

diabetes mellitus

ROS

reactive oxygen species

TGF-β1

transforming growth factor-β1

AST IV

astragaloside IV

HUVECs

human umbilical vein endothelial cells

H2O2

hydrogen peroxide

EC50

half maximal effective concentration

MTT

3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

OD

optical density

H2DCFDA

2′,7′-dichloro dihydrofluorescein diacetate

PBS

phosphate-buffered saline

FBS

fetal bovine serum

EDTA

ethylenediaminetetraacetic acid

qPCR

quantitative polymerase chain reaction

CT

cycle threshold

PMSF

phenylmethanesulfonyl fluoride

RT

room temperature

ANOVA

one-way analysis of variance

DPI

diphenyliodonium

References

1 

Haring R, Wallaschofski H, Nauck M, Felix SB, Schmidt CO, Dörr M, Sauer S, Wilmking G and Völzke H: Total and cardiovascular disease mortality predicted by metabolic syndrome is inferior relative to its components. Exp Clin Endocrinol Diabetes. 118:685–691. 2010. View Article : Google Scholar : PubMed/NCBI

2 

Erdei N, Bagi Z, Edes I, Kaley G and Koller A: H2O2 increases production of constrictor prostaglandins in smooth muscle leading to enhanced arteriolar tone in type 2 diabetic mice. Am J Physiol Heart Circ Physiol. 292:H649–H656. 2007. View Article : Google Scholar

3 

Farah R, Shurtz-Swirski R and Lapin O: Intensification of oxidative stress and inflammation in type 2 diabetes despite antihyperglycemic treatment. Cardiovasc Diabetol. 7:202008. View Article : Google Scholar : PubMed/NCBI

4 

Hamed S, Brenner B and Roguin A: Nitric oxide: A key factor behind the dysfunctionality of endothelial progenitor cells in diabetes mellitus type-2. Cardiovasc Res. 91:9–15. 2011. View Article : Google Scholar

5 

Yiu KH and Tse HF: Specific role of impaired glucose metabolism and diabetes mellitus in endothelial progenitor cell characteristics and function. Arterioscler Thromb Vasc Biol. 34:1136–1143. 2014. View Article : Google Scholar : PubMed/NCBI

6 

Zernecke A, Bidzhekov K, Noels H, et al: Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection. Sci Signal. 2:ra812009. View Article : Google Scholar : PubMed/NCBI

7 

Lambeth JD: NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol. 4:181–189. 2004. View Article : Google Scholar : PubMed/NCBI

8 

Spillmann F, Van Linthout S, Miteva K, Lorenz M, Stangl V, Schultheiss HP and Tschöpe C: LXR agonism improves TNF-α-induced endothelial dysfunction in the absence of its cholesterol-modulating effects. Atherosclerosis. 232:1–9. 2014. View Article : Google Scholar : PubMed/NCBI

9 

Scioli MG, Cervelli V, Arcuri G, Gentile P, Doldo E, Bielli A, Bonanno E and Orlandi A: High insulin-induced down-regulation of Erk-1/IGF-1R/FGFR-1 signaling is required for oxidative stress-mediated apoptosis of adipose-derived stem cells. J Cell Physiol. 229:2077–2087. 2014. View Article : Google Scholar : PubMed/NCBI

10 

Camelo A, Dunmore R, Sleeman MA and Clarke DL: The epithelium in idiopathic pulmonary fibrosis: Breaking the barrier. Front Pharmacol. 4:1732014. View Article : Google Scholar : PubMed/NCBI

11 

Herman-Edelstein M, Weinstein T and Gafter U: TGFβ1-dependent podocyte dysfunction. Curr Opin Nephrol Hypertens. 22:93–99. 2013. View Article : Google Scholar

12 

Kamato D, Burch ML, Piva TJ, Rezaei HB, Rostam MA, Xu S, Zheng W, Little PJ and Osman N: Transforming growth factor-β signalling: Role and consequences of Smad linker region phosphorylation. Cell Signal. 25:2017–2024. 2013. View Article : Google Scholar : PubMed/NCBI

13 

Blanco FF, Sanduja S, Deane NG, Blackshear PJ and Dixon DA: Transforming growth factor β regulates P-body formation through induction of the mRNA decay factor tristetraprolin. Mol Cell Biol. 34:180–195. 2014. View Article : Google Scholar :

14 

Bohanon FJ, Wang X, Ding C, Ding Y, Radhakrishnan GL, Rastellini C, Zhou J and Radhakrishnan RS: Oridonin inhibits hepatic stellate cell proliferation and fibrogenesis. J Surg Res. 190:55–63. 2014. View Article : Google Scholar : PubMed/NCBI

15 

Li YC, An YS, Wang T and Zang HR: Analysis of transforming growth factor β signaling in chronic rhinosinusitis. Chin Med J (Engl). 126:3340–3343. 2013.

16 

Wang X, Chu J, Wen CJ, Fu SB, Qian YL, Wo Y, Wang C and Wang DR: Functional characterization of TRAP1-like protein involved in modulating fibrotic processes mediated by TGF-β/Smad signaling in hypertrophic scar fibroblasts. Exp Cell Res. 332:202–211. 2015. View Article : Google Scholar : PubMed/NCBI

17 

Wang Z, Song Y, Tu W, He X, Lin J and Liu F: β-2 spectrin is involved in hepatocyte proliferation through the interaction of TGFβ/Smad and PI3K/AKT signalling. Liver Int. 32:1103–1111. 2012. View Article : Google Scholar : PubMed/NCBI

18 

Janeesh PA and Abraham A: Robinin modulates doxorubicin-induced cardiac apoptosis by TGF-β1 signaling pathway in Sprague Dawley rats. Biomed Pharmacother. 68:989–998. 2014. View Article : Google Scholar : PubMed/NCBI

19 

Liu T, Peng YF, Jia C, Yang BH, Tao X, Fang X and Zhong W: Effect of HGF on the apoptosis of rat corpus cavernosum smooth muscle cells induced by TGFβ1. Andrologia. Nov 11–2014.Epub ahead of print. View Article : Google Scholar

20 

Merle P and Trepo C: Molecular mechanisms underlying hepatocellular carcinoma. Viruses. 1:852–872. 2009. View Article : Google Scholar : PubMed/NCBI

21 

Li WZ, Li WP, Zhang W, Yin YY, Sun XX, Zhou SS, et al: Protective effect of extract of Astragalus on learning and memory impairments and neurons’ apoptosis induced by glucocorticoids in 12-month-old male mice. Anat Rec (Hoboken). 294:1003–1014. 2011. View Article : Google Scholar

22 

Yin YY, Li WP, Gong HL, Zhu FF, Li WZ and Wu GC: Protective effect of astragaloside on focal cerebral ischemia/reperfusion injury in rats. Am J Chin Med. 38:517–527. 2010. View Article : Google Scholar : PubMed/NCBI

23 

Sun L, Li W, Li W, Xiong L, Li G and Ma R: Astragaloside IV prevents damage to human mesangial cells through the inhibition of the NADPH oxidase/ROS/Akt/NF-κB pathway under high glucose conditions. Int J Mol Med. 34:167–176. 2014.PubMed/NCBI

24 

Ruan Y, Wu S, Zhang L, Chen G and Lai W: Retarding the senescence of human vascular endothelial cells induced by hydrogen peroxide: Effects of 17beta-estradiol (E2) mediated mitochondria protection. Biogerontology. 15:367–375. 2014. View Article : Google Scholar : PubMed/NCBI

25 

Wardman P: Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: Progress, pitfalls, and prospects. Free Radic Biol Med. 43:995–1022. 2007. View Article : Google Scholar : PubMed/NCBI

26 

Yan F, Wang Y, Wu X, Peshavariya HM, Dusting GJ, Zhang M and Jiang F: Nox4 and redox signaling mediate TGF-β-induced endothelial cell apoptosis and phenotypic switch. Cell Death Dis. 5:e10102014. View Article : Google Scholar

27 

Anderson TJ: Assessment and treatment of endothelial dysfunction in humans. J Am Coll Cardiol. 34:631–638. 1999. View Article : Google Scholar : PubMed/NCBI

28 

Hadi HA, Carr CS and Al Suwaidi J: Endothelial dysfunction: Cardiovascular risk factors, therapy, and outcome. Vasc Health Risk Manag. 1:183–198. 2005.

29 

Tousoulis D, Briasoulis A, Papageorgiou N, Tsioufis C, Tsiamis E, Toutouzas K and Stefanadis C: Oxidative stress and endothelial function: Therapeutic interventions. Recent Patents Cardiovasc Drug Discov. 6:103–114. 2011. View Article : Google Scholar

30 

Bramlage CP, Müller GA, Tampe B, et al: The role of bone morphogenetic protein-5 (BMP-5) in human nephrosclerosis. J Nephrol. 24:647–655. 2011. View Article : Google Scholar : PubMed/NCBI

31 

Gray SP, Di Marco E, Okabe J, et al: NADPH oxidase 1 plays a key role in diabetes mellitus-accelerated atherosclerosis. Circulation. 127:1888–1902. 2013. View Article : Google Scholar : PubMed/NCBI

32 

Liu XQ, Sheng R and Qin ZH: The neuroprotective mechanism of brain ischemic preconditioning. Acta Pharmacol Sin. 30:1071–1080. 2009. View Article : Google Scholar : PubMed/NCBI

33 

Urbański K, Nowak M and Guzik TJ: Oxidative stress and vascular function. Postepy Biochem. 59:424–431. 2013.In Polish.

34 

Dao J, Zhu L, Luo R, Hu C, Wang Y, Li H, Lu K, Liu J, Lin J and Cheng G: Molecular characterization of SjBIRP, another apoptosis inhibitor, from Schistosoma japonicum. Parasitol Res. 113:4065–4071. 2014. View Article : Google Scholar : PubMed/NCBI

35 

Han HJ, Kwon HY, Sohn EJ, Ko H, Kim B, Jung K, Lew JH and Kim SH: Suppression of E-cadherin mediates gallotannin induced apoptosis in Hep G2 hepatocelluar carcinoma cells. Int J Biol Sci. 10:490–499. 2014. View Article : Google Scholar : PubMed/NCBI

36 

Kvansakul M and Hinds MG: Structural biology of the Bcl-2 family and its mimicry by viral proteins. Cell Death Dis. 4:e9092013. View Article : Google Scholar : PubMed/NCBI

37 

Martel C, Wang Z and Brenner C: VDAC phosphorylation, a lipid sensor influencing the cell fate. Mitochondrion. 19:69–77. 2014. View Article : Google Scholar : PubMed/NCBI

38 

Qiao D, Xu J, Le C, Huang E, Liu C, Qiu P, Lin Z, Xie WB and Wang H: Insulin-like growth factor binding protein 5 (IGFBP5) mediates methamphetamine-induced dopaminergic neuron apoptosis. Toxicol Lett. 230:444–453. 2014. View Article : Google Scholar : PubMed/NCBI

39 

Wang G, Jiang MY, Meng Y, Song HR and Shi W: Cellular mechanisms of a new pyrazinone compound that induces apoptosis in SKOV-3 cells. Asian Pac J Cancer Prev. 15:797–802. 2014. View Article : Google Scholar : PubMed/NCBI

40 

Eachkoti R, Reddy MV, Lieu YK, Cosenza SC and Reddy EP: Identification and characterisation of a novel heat shock protein 90 inhibitor ONO4140. Eur J Cancer. 50:1982–92. 2014. View Article : Google Scholar : PubMed/NCBI

41 

Hou XQ, Wu DW, Zhang CX, et al: Bushen Yizhi formula ameliorates cognition deficits and attenuates oxidative stress related neuronal apoptosis in scopolamine induced senescence in mice. Int J Mol Med. 34:429–439. 2014.PubMed/NCBI

42 

Tharaheswari M, Jayachandra Reddy N, Kumar R, Varshney KC, Kannan M and Sudha Rani S: Trigonelline and diosgenin attenuate ER stress, oxidative stress-mediated damage in pancreas and enhance adipose tissue PPARγ activity in type 2 diabetic rats. Mol Cell Biochem. 396:161–174. 2014. View Article : Google Scholar : PubMed/NCBI

43 

Isfort RJ, Cody DB, Stuard SB, et al: The combination of epidermal growth factor and transforming growth factor-beta induces novel phenotypic changes in mouse liver stem cell lines. J Cell Sci. 110:3117–3129. 1997.

44 

Lee HS: Mechanisms and consequences of TGF-β overexpression by podocytes in progressive podocyte disease. Cell Tissue Res. 347:129–140. 2012. View Article : Google Scholar :

45 

Wakui H, Dejima T, Tamura K, et al: Activation of angiotensin II type 1 receptor-associated protein exerts an inhibitory effect on vascular hypertrophy and oxidative stress in angiotensin II-mediated hypertension. Cardiovasc Res. 100:511–519. 2013. View Article : Google Scholar : PubMed/NCBI

46 

He C, Zhu H, Zhang W, Okon I, Wang Q, Li H, Le YZ and Xie Z: 7-Ketocholesterol induces autophagy in vascular smooth muscle cells through Nox4 and Atg4B. Am J Pathol. 183:626–637. 2013. View Article : Google Scholar : PubMed/NCBI

47 

Li J, Li X, Xu W, Wang S, Hu Z, Zhang Q, et al: Antifibrotic effects of luteolin on hepatic stellate cells and liver fibrosis by targeting AKT/mTOR/p70S6K and TGFbeta/Smad signalling pathways. Liver Inter. 5–Aug;2014.Epub ahead of print.

48 

Martínez-Palacián A, del Castillo G, Suárez-Causado A, García-Álvaro M, de Morena-Frutos D, Fernández M, Roncero C, Fabregat I, Herrera B and Sánchez A: Mouse hepatic oval cells require Met-dependent PI3K to impair TGF-β-induced oxidative stress and apoptosis. PLoS One. 8:e531082013. View Article : Google Scholar

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June-2015
Volume 35 Issue 6

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

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Copy and paste a formatted citation
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Spandidos Publications style
Ma Y, Li W, Yin Y and Li W: AST Ⅳ inhibits H2O2-induced human umbilical vein endothelial cell apoptosis by suppressing Nox4 expression through the TGF-β1/Smad2 pathway. Int J Mol Med 35: 1667-1674, 2015.
APA
Ma, Y., Li, W., Yin, Y., & Li, W. (2015). AST Ⅳ inhibits H2O2-induced human umbilical vein endothelial cell apoptosis by suppressing Nox4 expression through the TGF-β1/Smad2 pathway. International Journal of Molecular Medicine, 35, 1667-1674. https://doi.org/10.3892/ijmm.2015.2188
MLA
Ma, Y., Li, W., Yin, Y., Li, W."AST Ⅳ inhibits H2O2-induced human umbilical vein endothelial cell apoptosis by suppressing Nox4 expression through the TGF-β1/Smad2 pathway". International Journal of Molecular Medicine 35.6 (2015): 1667-1674.
Chicago
Ma, Y., Li, W., Yin, Y., Li, W."AST Ⅳ inhibits H2O2-induced human umbilical vein endothelial cell apoptosis by suppressing Nox4 expression through the TGF-β1/Smad2 pathway". International Journal of Molecular Medicine 35, no. 6 (2015): 1667-1674. https://doi.org/10.3892/ijmm.2015.2188