Skip to main content Accessibility help
×
Home
Hostname: page-component-768dbb666b-l8xdn Total loading time: 1.297 Render date: 2023-02-05T05:57:10.041Z Has data issue: true Feature Flags: { "useRatesEcommerce": false } hasContentIssue true

Effects of sodium selenite on c-Jun N-terminal kinase signalling pathway induced by oxidative stress in human chondrocytes and c-Jun N-terminal kinase expression in patients with Kashin–Beck disease, an endemic osteoarthritis

Published online by Cambridge University Press:  07 March 2016

XiaoXia Dai
Affiliation:
Key Laboratory of Environment and Genes Related to Diseases of Education Ministry, Health Science Center, Institute of Endemic Diseases, Xi’an Jiaotong University, No.76 Yanta West Road, Xi’an, Shaanxi 710061, People’s Republic of China
YuanYuan Li
Affiliation:
School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, Shaanxi 710049, People’s Republic of China
RongQiang Zhang
Affiliation:
Key Laboratory of Environment and Genes Related to Diseases of Education Ministry, Health Science Center, Institute of Endemic Diseases, Xi’an Jiaotong University, No.76 Yanta West Road, Xi’an, Shaanxi 710061, People’s Republic of China
Yan Kou
Affiliation:
School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, Shaanxi 710049, People’s Republic of China
XiaoYan Mo
Affiliation:
School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, Shaanxi 710049, People’s Republic of China
JunLing Cao
Affiliation:
Key Laboratory of Environment and Genes Related to Diseases of Education Ministry, Health Science Center, Institute of Endemic Diseases, Xi’an Jiaotong University, No.76 Yanta West Road, Xi’an, Shaanxi 710061, People’s Republic of China
YongMin Xiong*
Affiliation:
Key Laboratory of Environment and Genes Related to Diseases of Education Ministry, Health Science Center, Institute of Endemic Diseases, Xi’an Jiaotong University, No.76 Yanta West Road, Xi’an, Shaanxi 710061, People’s Republic of China
*
*Corresponding author: Y. M. Xiong, fax +86 29 82655032, email xiongym@xjtu.edu.cn
Rights & Permissions[Opens in a new window]

Abstract

The c-Jun N-terminal kinases (JNK) are members of the mitogen-activated protein kinase family and are activated by environmental stress. Se plays an important role in the biological pathways by forming selenoprotein. Selenoproteins have been shown to exhibit a variety of biological functions including antioxidant functions and maintaining cellular redox balance, and compromise of such important proteins would lead to oxidative stress and apoptosis. We examined the expression levels of JNK in Kashin–Beck disease (KBD) patients, tested the potential protective effects of sodium selenite on tert-butyl hydroperoxide (tBHP)-induced oxidative injury and apoptosis in human chondrocytes as well as its underlying mechanism in this study. We produced an oxidative damage model induced by tBHP in C28/I2 human chondrocytes to test the essential anti-apoptosis effects of Se in vitro. The results indicated that the expression level of phosphorylated JNK was significantly increased in KBD patients. Cell apoptosis was increased and molecule expressions of the JNK signalling pathway were activated in the tBHP-injured chondrocytes. Na2SeO3 protected against tBHP-induced oxidative stress and apoptosis in cells by increasing cell viability, reducing reactive oxygen species generation, increasing Glutathione peroxidase (GPx) activity and down-regulating the JNK pathway. These results demonstrate that apoptosis induced by tBHP in chondrocytes might be mediated via up-regulation of the JNK pathway; Na2SeO3 has an effect of anti-apoptosis by down-regulating the JNK signalling pathway.

Type
Full Papers
Copyright
Copyright © The Authors 2016 

Kashin–Beck disease (KBD) is a chronic, endemic osteoarthritis (OA) that occurs in a limited endemic area in China, Central China, from Southeastern Siberia to North China and North Korea( Reference Sokoloff 1 ). Degradation of the matrix and cell necrosis in the growth plate and articular surface are the basic pathological features, and this can result in growth retardation, secondary osteoarthrosis and disability in the advanced stages of the disease( Reference Yao, Pei and Kang 2 , Reference Pasteels, Liu and Hinsenkamp 3 ). The cause of KBD remains unknown( Reference Schepman, Engelbert and Visser 4 ), but endemic deficiency of Se( Reference Tan, Zhu and Wang 5 ), serious cereal contamination by mycotoxin-producing fungi( Reference Chasseur, Suetens and Nolard 6 ) and high humic acid levels in drinking water( Reference Peng, Wang and Wang 7 ) are considered to contribute to KBD. Low dietary levels of Se are thought to be the most important environmental factor causing the disease( Reference Jirong, Huiyun and Zhongzhe 8 ), and populations from KBD-affected areas often show a deficiency of Se( Reference Xiong 9 , Reference Yamamuro 10 ). Researchers believe that multiple heterogeneous factors are involved in the aetiology of KBD( Reference Suetens, Moreno-Reyes and Chasseur 11 ). Recent findings of differential expressions of genes between KBD and normal controls mainly in chondrocyte metabolism and apoptosis, signal transduction, oxidative stress and cytokines have been reported( Reference Wang, Guo and Duan 12 Reference Han, Guo and Tan 14 ). Furthermore, the chondrocyte necrosis mediated by oxidative stress in KBD cartilage damage has also been reported( Reference Wang, Wei and Luo 15 ).

The c-Jun N-terminal kinases (JNK) are members of a larger group of serine/threonine protein kinases known as the mitogen-activated protein kinase (MAPK) family( Reference Davis 16 ). JNK are involved in cell proliferation, differentiation and apoptosis( Reference Dhanasekaran and Reddy 17 , Reference Liu and Lin 18 ). Activated JNK phosphorylates nuclear substrates such as c-Jun, a component of the activator protein 1 (AP-1) transcription factor family, which mediate nuclear events that lead to cell death. Thus, a blockade of JNK activation prevents cell death( Reference Pulverer, Kyriakis and Avruch 19 ). Similar to other degenerative articular cartilage diseases such as OA and rheumatoid arthritis (RA), apoptosis is also a possible pathogenic mechanism in KBD. Chondrocytes play a central role in maintaining cartilage homoeostasis. The vitality of articular cartilage is also critical and it can be judged on the basis of the capacity of chondrocytes to resist apoptosis( Reference Aigner, Kim and Roach 20 ). In addition, investigations have revealed that the JNK pathway is involved in the pathogenesis of articular cartilage degradation in OA and RA. JNK and the key upstream activators of JNK – mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7) – are expressed and activated in OA synovial tissues( Reference Han, Boyle and Chang 21 ). JNK are also expressed and activated in OA chondrocytes( Reference Fan, Soder and Oehler 22 Reference Mengshol, Vincenti and Coon 24 ). JNK and the upstream kinases (MKK4 and MKK7) are highly activated in isolated RA fibroblast-like synoviocytes and in the rheumatoid synovial lining layer and synovial mononuclear cell infiltrates( Reference Han, Boyle and Aupperle 25 Reference Schett, Tohidast-Akrad and Smolen 27 ).

In the present study, we examined the expressions of the JNK signalling molecules in KBD patients, evaluated the protective effect of Na2SeO3 in tert-butyl hydroperoxide (tBHP)-induced chondrocyte apoptosis and investigated the potential mechanisms underlying these effects.

Methods

Patients and blood sample collection

This study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Ethics Committee of the Xi’an Jiaotong University College of Medicine. Written informed consent was obtained from all the subjects. Patients with KBD were selected on the basis of the clinical criteria for the diagnosis of KBD in China (diagnostic code GB16395-1996)( Reference Yang, Wang and Liu 28 ). In all, 110 KBD patients aged between 40 and 70 years were from the KBD-endemic areas of Linyou, Changwu and Yongshou counties, Shaanxi province, China. In all, 160 healthy control subjects aged between 35 and 70 years were from non-KBD areas and had no history of joint diseases such as genetic bone and cartilage diseases, OA and RA. In all, twenty people from each group were selected by simple random sampling method as experimental subjects. In all the comparisons mentioned, the groups were age and sex matched. Blood samples were drawn from the antecubital vein of all subjects following an overnight fast into tubes containing EDTA for immediate protein extraction or storage at −20°C. Protein extracts were prepared, and protein expression levels of JNK and phosphorylated JNK (p-JNK) in whole blood from KBD patients and healthy controls were detected by Western blot.

Cell culture

C28/I2 chondrocytes, a human cell line (kindly provided by Dr Mary B. Goldring from the Harvard Institutes of Medicine, Boston, MA, USA), were cultured in Dulbecco’s modified Eagle’s medium/F12 with a ratio of 1:1 (v/v) (Hyclone) containing 10 % fetal calf serum (Hyclone) in 5 % CO2 and 95 % humidified air atmosphere at 37°C.

Cell viability and proliferation assay

The methylthiazolyl tetrazolium (MTT) assay was used to assess cell viability by the mitochondrial-dependent reduction to formazan. Absorbance at 490 nm was used to quantify the amount of MTT, which was assumed to correlate to the number of viable cells. C28/I2 chondrocytes seeded on ninety-six-well culture plates at a density of approximately 5×104 cells/well were pre-protected with various concentrations of Na2SeO3 (0·05, 0·1 and 0·15 μg/ml) for 24 h. To evaluate the protective effect of Na2SeO3, cells were subsequently treated with 300 μmol/l tBHP for 24 h. The protective effect against oxidative stress was measured using the MTT assay. MTT reagent (0·5 mg/ml MTT in PBS) was diluted into complete medium (dilution ratio of 1:10) and added to each well. After incubating in a CO2 incubator for 4 h, the medium was aspirated from each well and 200 μl dimethyl sulphoxide (DMSO) (Sigma) was added. The absorbance was measured in a microplate reader (Thermo Electron Corporation) at 490 nm. Cell viability was expressed as a percentage of the control (0·1 % DMSO). Assays were performed in triplicate in three independent experiments and the results are presented as mean values and standard deviations. A preliminary study with increasing concentrations of tBHP (100–500 μmol/l) for different time points (6–24 h) was performed, and the results showed that the appropriate concentration and tBHP treatment time for further studies were 300 μmol/l and 24 h, respectively.

Hoechst staining

C28/I2 chondrocytes were plated onto 60-mm-diameter dishes (1·0×106 cells/dish) and allowed to grow for 24 h. After incubation with various concentrations of Na2SeO3 (0·00, 0·05, 0·1 and 0·15 μg/ml) for 24 h and then tBHP for 24 h, chondrocytes were fixed in 2 % glutaraldehyde for 4 h. Cells were washed twice with PBS and stained with 1 mg/ml Hoechst 33342 (Sigma-Aldrich) diluted in PBS (137 mm-NaCl, 12 mm-phosphate, 2·7 mm-KCl, pH 7·4) for 30 min under ice-cold conditions in the dark. Cells were then washed twice with PBS. Apoptosis, with condensed and fragmented nuclei, was observed with a fluorescence microscope using appropriate filters for blue fluorescence at 200× magnification (BX51T-32H01; Olympus).

Determination of reactive oxygen species

Cellular reactive oxygen species (ROS) levels were measured using a 5-(and-6)-chloromethyl-2-,7-dichlorofluorescin diacetate (DCHF-DA) fluorescent probe (Sigma)( Reference Wang and Joseph 29 ). C28/I2 chondrocytes were pre-treated or untreated with various concentrations of Na2SeO3 (0·05, 0·1 and 0·15 μg/ml) for 24 h. After 24 h, cells were treated with the 300 μmol/l tBHP for 24 h. At the end of the treatment, cells were incubated in the presence of 25 m-DCFH for 50 min. After incubation, dichlorofluorescein was measured using a fluorescence spectrophotometer (PerkinElmer) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Glutathione peroxidase activity

Glutathione peroxidase (GPX) activity in cell lysate was measured using a kit (Nanjing Jiancheng Bioengineering Institute) based on the method of Hafeman et al. ( Reference Hafeman, Sunde and Hoekstra 30 ) – a coupled assay using H2O2 and dithio-bis-nitrobenzoic acid. One unit of GPX is defined as the amount of enzyme that catalyses the oxidation of 1 nmol NADPH/min at 37°C, whereas heated samples with inactivated enzymes were used as non-enzymatic controls to eliminate interference from endogenous reduced GPX. GPX activity was assessed by dinitrobenzoic acid colourimetry and read at a wavelength of 412 nm.

Western blotting

Total protein from blood samples in KBD patients and healthy controls was extracted using a Western and IP Cell Lysis Kit (Beyotime). C28/I2 chondrocytes were scraped in RIPA lysis buffer plus a protease inhibitor cocktail (Sigma) (50 mm-TRIS, 150 mm-NaCl, 1 mm-EDTA, 1 % Triton X-100, 0·1 % SDS, 1 % sodium deoxycholate, 1 mm-PMSF, 1 mm-Na3VO4, 1 mm-NaF, 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 μg/ml pepstatin). Protein concentration of samples was determined with the bicinchoninic acid assay method. Equivalent amounts of sample protein were separated in 10 % SDS-PAGE gel and transferred to nitrocellulose membrane (Millipore) using a semi-dry transfer method. The membrane was then blocked with 5 % milk in TRIS-buffered saline solution (TBS) with 0·1 % Tween-20 and incubated with primary antibodies at 4°C overnight (JNK 1:1000; p-JNK 1:1000; α-tubulin 1:5000; c-jun 1:1000; activating transcription factor 2 (ATF2) 1:1000; p-c-jun 1:1000; mitogen-activated protein kinase kinase kinase 1 (MEKK1) 1:500; B-cell lymphoma 2 (Bcl-2) 1:1000; and β-actin 1:1000, all from Santa Cruz), followed by incubation with the matching horseradish peroxidase-conjugated IgG antibody (1:8000; Pierce Company) at room temperature for 1 h. Bands were developed with enhanced chemiluminescence (ECL) (Amersham ECL Prime Western Blotting Detection Reagent, RPN2232; GE HealthCare) and exposure to X-ray film. The immunoblots were visualised and quantified by gel imaging system (GDS-8000 type; UVP).

Statistical analysis

Data are presented as mean values and standard deviations unless otherwise indicated. Student’s t test was used to compare two groups. P≤0·05 was considered to be statistically significant.

Results

Phosphorylated c-Jun N-terminal kinase protein expression was greater in Kashin–Beck disease patients compared with healthy controls

The protein expression levels of JNK and p-JNK in whole blood from KBD patients and healthy controls were detected by Western blot. Western blotting demonstrated that JNK phosphorylation was significantly increased (normalised to α-tubulin) in KBD patients compared with that in the healthy controls (P<0·05) (Fig. 1, right panel), but the levels of total JNK protein in KBD patients remained unaltered (Fig. 1, left panel).

Fig. 1 Increased protein expression of phosphorylated c-Jun N-terminal kinase (p-JNK) in whole blood of Kashin–Beck disease (KBD) patients compared with healthy control subjects. (a) Protein extracts from patient and healthy control whole blood samples were prepared and analysed by immunoblotting with JNK, p-JNK and α-tubulin antibodies. (b) Signal intensity was then quantified, and the results of the densitometric analysis are shown as mean values and standard deviations represented by vertical bars for p-JNK expression relative to α-tubulin (right panel) and mean values and standard deviations represented by vertical bars for JNK expression relative to α-tubulin (left panel). One experiment is representative of three independent experiments. p-JNK protein expression levels in the KBD group were significantly higher than that in the control group (P<0·05). K, samples from KBD patients; C, samples from control subjects. * P<0·05.

Effect of tert-butyl hydroperoxide on chondrocyte survival

Chondrocytes were exposed to increasing concentrations of tBHP (100–500 μmol/l) at different time points (6–24 h), and cell viability was determined by the MTT assay (Fig. 2(a) and (b)). tBHP induced a time- and dose-dependent inhibition of cellular proliferation in chondrocytes. We chose the exposure time of 24 h and the concentration of 300 μmol/l for the subsequent experiment because the cell survival rate reached 40–60 % of the normal control group at this point.

Fig. 2 Effects of different concentrations of tert-butyl hydroperoxide (tBHP) on the cellular viability of chondrocytes were estimated by methylthiazolyl tetrazolium (MTT) reduction. (a) Cells were incubated in absence or presence of several tBHP concentrations for different time periods (6–24 h: , 6 h; , 12 h; , 18 h; , 24 h). (b) Chondrocytes were treated with various doses of tBHF for 24 h and viability was determined by MTT. Values are means and standard deviations represented by vertical bars. One experiment is representative of four independent experiments. Mean value was significantly different from that of the control group: * P<0·05, ** P<0·01 (chondrocytes untreated with tBHP).

Protective effect of Na2SeO3 on tert-butyl hydroperoxide-induced cytotoxicity

Exposure to tBHP induced about 50 % decrease in cell viability (Fig. 3). However, pre-treatment with 0·05 and 0·1 µg/ml Na2SeO3 for 24 h significantly increased cell viability, indicating that pre-treated cells were protected against oxidative injury.

Fig. 3 Protection against tert-butyl hydroperoxide (tBHP)-induced oxidative stress by Na2SeO3. Cell viability was determined by the methylthiazolyl tetrazolium (MTT) assay 24 h after exposure to tBHP, following a 24 h pre-treatment with Na2SeO3. Treatment with tBHP alone was seen to significantly decrease cell viability. Na2SeO3 showed protection against tBHP-induced cell toxicity. Values are means and standard deviations represented by vertical bars. One experiment is representative of four independent experiments. C, control group; O, tBHP injury group (tBHP300 mmol/l); OS1, low Se pre-protection group (0·05 mg/ml Na2SeO3+300 mmol/l tBHP); OS2, middle Se pre-protection group (0·1 mg/ml Na2SeO3+300 mmol/l tBHP); OS3, high Se pre-protection group (0·15 mg/ml Na2SeO3+300 mmol/l tBHP). ** P<0·01.

We next addressed whether tBHP reduces cell viability of chondrocytes by enhancing apoptosis. We first measured apoptosis induced by tBHP by Hoechst 33342 staining. Fig. 4 shows the chondrocyte apoptosis detected by Hoechst 33342 staining and the protective effects of different Na2SeO3 concentrations in C28/I2 cells. Compared with the control group, apoptotic nuclei were obviously increased in the tBHP injury group. Pre-treatment with different concentrations of Na2SeO3 for 24 h decreased apoptotic nuclei in the three Se treatment groups. The middle Se pre-protection group (0·1 mg/ml Na2SeO3) exhibited the lowest apoptotic nuclei numbers among the three groups. Our results indicate that tBHP injury could induce apoptosis, whereas Na2SeO3 could reduce apoptosis in chondrocytes.

Fig. 4 The apoptosis of C28/I2 human chondrocytes detected by Hoechst 33342 staining and the protective effects of different Na2SeO3 concentrations on C28/I2 cells. Tert-butyl hydroperoxide (tBHP) injury obviously increased apoptosis of C28/I2 chondrocytes, whereas pre-treatment with Na2SeO3 reduced apoptosis. One experiment is representative of three independent experiments. C, control group; O, tBHP injury group (tBHP 300 mmol/l); OS1, low Se pre-protection group (0·05 mg/ml Na2SeO3+300 mmol/l tBHP); OS2, middle Se pre-protection group (0·1 mg/ml Na2SeO3+300 mmol/l tBHP); OS3, high Se pre-protection group (0·15 mg/ml Na2SeO3+300 mmol/l tBHP).

Western blot analysis of the apoptosis-related proteins further confirmed the occurrence of apoptosis. As shown in Fig. 5, quantification of band intensity showed that the chondrocytes treated with tBHP (300 μmol/l) for 24 h enhanced the expression levels of pro-apoptotic proteins p-c-jun, c-jun and ATF2 compared with the control group (P<0·01 or 0·05) and produced a decrease in the anti-apoptotic protein Bcl-2 (P<0·01). Compared with the tBHP injury group, the expression levels of p-c-jun, c-jun and ATF2 protein in the three Na2SeO3 pre-protection groups showed a down-regulated trend. The expression levels of p-c-jun and ATF2 in the high Se pre-protection group significantly decreased (P<0·01). The expression levels of p-c-jun, c-jun and ATF2 in the middle Se pre-protection group significantly decreased (P<0·05). The expression levels of p-c-jun and ATF2 in the low Se pre-protection group significantly decreased (P<0·05). The expression level of Bcl-2 in the low Se pre-protection significantly increased (P<0·05).

Fig. 5 The expressions of apoptosis-related proteins in C28/I2 cells using Western blots. (a) Protein extracts were prepared and analysed by immunoblotting with antibodies recognising phosphorylated (p)-c-Jun and β-actin. Signal intensity was then quantified and the results of the densitometric analysis are shown as mean values and standard deviations represented by vertical bars for p-c-Jun expression relative to β-actin (right panel). (b) Protein extracts were prepared and analysed by immunoblotting with c-Jun and actin antibodies. Signal intensity was then quantified and the results of the densitometric analysis are shown as mean values and standard deviations represented by vertical bars for c-Jun expression relative to β-actin (right panel). (c) Protein extracts were analysed by immunoblotting with activating transcription factor 2 (ATF2) and β-actin antibodies. The results of the densitometric analysis are shown as mean values and standard deviations represented by vertical bars for ATF2 expression relative to β-actin (right panel). (d) The results of immunoblotting with antibodies recognising B-cell lymphoma 2 (Bcl-2) and β-actin and densitometric analysis are shown as mean values and standard deviations represented by vertical bars for Bcl-2 expression relative to β-actin (right panel). For all, one experiment is representative of four independent experiments. C, control group; O, tBHP injury group; OS1, low Se pre-protection group; OS2, middle Se pre-protection group; OS3, high Se pre-protection group. * P<0·05, ** P<0·01.

Effect of Na2SeO3 on reactive oxygen species generation

To evaluate the cellular oxidative stress generated from tBHP, the intracellular ROS production was measured. Cells treated with tBHP showed a significant increase in ROS generation compared with untreated controls (Fig. 6(a), P<0·05). However, pre-treatment of chondrocytes with Na2SeO3 significantly reduced ROS generation in the presence of tBHP compared with the tBHP injury group (P<0·05 or 0·01).

Fig. 6 Intracellular reactive oxygen species (ROS) levels and activity of glutathione peroxidase (GPX). (a) ROS levels after pre-treatment with various concentrations of Na2SeO3 for 24 h followed by treatment with tert-butyl hydroperoxide (tBHP) (300 mmol/l) for 24 h. Treatment with tBHP significantly increased the ROS levels. Pre-treatment with Na2SeO3 significantly decreased the ROS levels. Values are means and standard deviations represented by vertical bars. One experiment is representative of four independent experiments. * P<0·05, ** P<0·01. (b) Activity of GPX. Values are means and standard deviations represented by vertical bars. *P<0·05. DCF, 5-(and-6)-chloromethyl-2-,7-dichlorofluorescin.

Effect of Na2SeO3 on glutathione peroxidase activity

The cellular antioxidant enzyme system plays an important role in the defence against oxidative stress, and changes in the activity of antioxidant enzymes can be used as biomarkers of antioxidant response. Therefore, the effect of Na2SeO3 on GPX activity in C28/I2 chondrocytes was evaluated (Fig. 6(b)). Treatment with tBHP induced a remarkable decrease in GPX activity compared with the control (P<0·05). However, pre-treatment with Na2SeO3 increased GPX activity. The low Se pre-protection group prevented GPX activity depletion to near control levels. The middle Se pre-protection group and the high Se pre-protection groups showed significantly higher GPX activity.

Effect of Na2SeO3 on c-Jun N-terminal kinase signalling pathway

The JNK pathway plays important roles in the stimulation of apoptotic signalling as well as inflammatory diseases. As shown in Fig. 7(a) and (c), both p-JNK and MEKK1 protein levels were significantly increased in tBHP-treated cells. Densitometry analysis showed that p-JNK and MEKK1 levels, when normalised to β-actin levels, were significantly increased in the tBHP injury group (P<0·01) compared with the control group. tBHP treatment for 24 h did not significantly affect JNK protein expression in chondrocytes (Fig. 7(b), P>0·05). Compared with the tBHP injury group, the expression levels of p-JNK and MEKK1 in the Se pre-protection group significantly decreased (P<0·01 or 0·05).

Fig. 7 Effect of Na2SeO3 on the c-Jun N-terminal kinase (JNK) signalling pathway. (a–c) Western blotting and densitometric analysis results of mitogen-activated protein kinase kinase kinase 1 (MEKK1), JNK and phosphorylated JNK (p-JNK). It showed that tert-butyl hydroperoxide (tBPH) injury for 24 h increased MEKK1 and p-JNK levels but not JNK expression in chondrocytes. Pre-treatment with various concentrations of Na2SeO3 for 24 h significantly decreased the expression levels of MEKK1 and p-JNK. One experiment is representative of four independent experiments. Values are means and standard deviations represented by vertical bars. C, control group; O, tBHP injury group; OS1, low Se pre-protection group; OS2, middle Se pre-protection group; OS3, high Se pre-protection group. Values are statistically significant at * P<0·05 and ** P<0·01.

Discussion

In this study, we demonstrated that JNK phosphorylation was up-regulated in KBD patients and tBHP-treated chondrocytes; tBHP treatment reduced cell viability, proliferation of chondrocytes, induced apoptosis and produced oxidative stress. Moreover, we showed that tBHP decreased anti-apoptotic protein Bcl-2 and increased levels of phosphorylated p-c-jun, c-jun and ATF2. tBHP treatment also up-regulated JNK signalling. In the parallel experiments, Se supplementation elicited protective effects on chondrocytes, including the inhibition of cell viability, apoptosis and oxidative injury, the attenuation of JNK signalling pathways, the reduction of phosphorylated c-jun, c-jun and ATF2 protein expressions and an increase in Bcl-2 protein expression. These findings are in accordance with a previous report( Reference Han, Guo and Tan 14 ) indicating the involvement of JNK pathway in KBD via ATF2 on cartilage and chondrocytes.

The mammalian JNK are encoded by three distinct genes (Jnk1, Jnk2 and Jnk3)( Reference Davis 16 ). The classical JNK pathway is activated following the exposure of cells to extracellular stresses such as UV irradiation, hyperosmolarity and heat shock( Reference Karin and Gallagher 31 ). The range of initiating signals has been expanded to include a diversity of stimuli. After JNK are activated, they subsequently phosphorylate a variety of substrates that regulate a wide range of cellular functions( Reference Bogoyevitch and Kobe 32 ). The JNK were originally identified by their ability to phosphorylate both Ser63 and Ser73 within the transactivation domain of the transcription factor c-Jun, which potentiates its transcriptional activity( Reference Guma, Ronacher and Firestein 33 ). JNK phosphorylates and regulates the activity of transcription factors other than c-Jun, including ATF2, Elk-1, p53 and c-Myc as well as non-transcription factors such as members of the Bcl-2 family( Reference Bogoyevitch and Kobe 32 ). Several reports suggest a critical role of JNK in arthritis( Reference Han, Boyle and Aupperle 25 , Reference Guma, Kashiwakura and Crain 34 , Reference Kallunki, Deng and Hibi 35 ). The inflammatory process in patients with OA and RA involves activation of the JNK signalling pathway. In thermal stress-induced activation of chondrocytes, the suppression of JNK activity helps maintain the capacity of chondrocytes for proteoglycan synthesis( Reference Chu, Kaplan and Fu 36 ). Notably, JNK signalling in RA synovial tissue is activated early in the course of disease( Reference de Launay, van de Sande and de Hair 37 ). Furthermore, studies have shown that JNK inhibitors are potential therapeutic agents for the management of a variety of inflammatory disorders( Reference Manning and Davis 38 , Reference Arkin and Whitty 39 ). In this study, we found that the expression level of p-JNK was significant higher in KBD patients compared with controls. Accordingly, the major pathological changes in KBD patients are chondrocyte apoptosis and death in the deep layers of the affected cartilage and the apoptosis and necrosis mediated by oxidative stress, which could be ameliorated by Se supplementation( Reference Tan, Zhu and Wang 5 , Reference Wang, Wei and Luo 15 , Reference Wang, Guo and Zuo 40 ). In order to further explore the mechanism of JNK pathway up-regulation in chondrocyte apoptosis and the protective effects of Se, we conducted a C28/I2 chondrocytes experiment. Our results demonstrated that chondrocyte apoptosis increased and the JNK pathway was up-regulated in the tBHP treatment group, which is similar to observations in KBD patients. Chondrocyte apoptosis decreased and the JNK pathway was down-regulated in the Se treatment group. These findings suggest that up-regulation of the JNK pathway may play an important role in chondrocyte apoptosis and necrosis. Furthermore, Se supplementation can ameliorate cell apoptosis by regulating the JNK pathway.

In addition, MEKK1 protein levels were significantly increased in the tBHP injury group of chondrocytes. Of the three MAPK families, JNK is particularly interesting because it efficiently phosphorylates c-Jun. Among the components of the MAPK family, MEKK1 is a 196-kDa serine–threonine kinase that is activated in response to cytokines and various stresses. MEKK1 preferentially activates the JNK pathway and also influences the activity of the extracellular-signal-regulated kinases (ERK) pathway( Reference Lange-Carter, Pleiman and Gardner 41 Reference Yujiri, Ware and Widmann 43 ). The roles of MEKK1 and downstream JNK have been extensively studied in many research areas. For instance, MEKK1 is required for JNK and c-Jun activation in the corneal epithelia of MEKK1−/− mice( Reference Zhang, Deng and Kao 44 ). MEKK1 is also critical for JNK activation in response to pro-inflammatory stimuli and cell migration in MEKK1−/− mouse embryo fibroblasts (MEF)( Reference Xia, Makris and Su 45 ). On the basis of our studies using tBHP-injured chondrocytes, MEKK1 protein level was significantly increased during phosphorylated JNK activation in cultured chondrocytes. This study suggests that MEKK1 is a crucial activator of the JNK pathway in chondrocytes.

Se has been supplied for the past few decades to the residents of affected areas in China for the prevention of KBD. Se is an essential element for humans, animals and other species. Se is incorporated into proteins not simply though ionic association, as most metals are, but is covalently bonded within the amino acid selenocysteine, the twenty-first amino acid( Reference Berry, Tujebajeva and Copeland 46 ). Se deficiency induces dysfunction of selenoproteins. Selenoproteins have been shown to exhibit a variety of biological functions including antioxidant functions, maintaining cellular redox balance and heavy metal detoxification, and compromise of such important proteins would lead to oxidative stress and apoptosis( Reference Steinbrenner and Sies 47 , Reference Bellinger, Raman and Reeves 48 ). Moreover, there is a significantly lower Se level in the grains in KBD areas when compared with non-KBD areas, suggesting a close relationship between Se and KBD occurrence( Reference Ge and Yang 49 ). In this study, we found that tBHP induced the inhibition of cellular proliferation, excessive oxidative stress in chondrocytes, leading to chondrocyte apoptosis. The protein expression levels of p-JNK, MEKK1, p-c-jun, c-jun and ATF2 in the tBHP treatment group were higher than that in the control group and Bcl-2 was lower than that in the control group. Supplementation with Se caused a large increase in GPX activity and level of Bcl-2 in the low Se protection treatment and an obvious decrease in p-JNK, MEKK1, p-c-jun, c-jun and ATF2 in the low and middle Se protection groups. It is likely that tBHP-induced oxidative stress, which in turn led to tissue degeneration similar to that observed in KBD patients. On the contrary, Se can partly block tBHP-induced oxidative and chondrocyte apoptosis because of its antioxidant functions. These findings suggest that oxidative damage could lead to activation of the JNK signalling pathway, whereas Na2SeO3 supplementation could inhibit the activation and minimise oxidative damage. Therefore, Na2SeO3 exerts its anti-apoptosis effect to protect cartilage cells from oxidative stress injury by the inhibition of JNK activation.

In conclusion, elevated expression levels of p-JNK were observed in KBD patients. Chondrocyte apoptosis induced by oxidative stress might be mediated via activation of the JNK signalling pathway. Thus, the activation of the JNK pathway may play an important role in chondrocyte apoptosis. Se exhibits anti-apoptotic effects by down-regulating the JNK signalling pathway. These findings provide the experimental evidence to elucidate the role of JNK pathway in the pathogenesis of KBD.

Acknowledgements

The authors thank the volunteers who took part in the study, as well as Jianhong Zhu (The Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of China) and Yuexiang Yu (Shaanxi Provincial Institute for Endemic Disease Control, People’s Republic of China) for their assistance with sample collection.

The present study was financially supported by the National Science Foundation of China (no. 81172610, 81573104).

The authors’ responsibilities were as follows: Y. M. Xiong takes responsibility for the integrity of the study as a whole, from inception to finished article. X. X. Dai, Y. Y. Li, R. Q. Zhang, Y. Kou, X. Y. Mo and J. L. Cao were responsible for acquisition of the data, data analysis and interpretation of the data. All the authors read and approved the final version of the manuscript.

The authors declare that they have no conflicts of interest.

References

1. Sokoloff, L (1989) The history of Kashin-Beck disease. N Y State J Med 89, 343351.Google ScholarPubMed
2. Yao, Y, Pei, F & Kang, P (2011) Selenium, iodine, and the relation with Kashin-Beck disease. Nutrition 27, 10951100.CrossRefGoogle ScholarPubMed
3. Pasteels, JL, Liu, FD, Hinsenkamp, M, et al. (2001) Histology of Kashin-Beck lesions. Int Orthop. 25, 151153.CrossRefGoogle ScholarPubMed
4. Schepman, K, Engelbert, RHH, Visser, MM, et al. (2011) Kashin-Beck disease: more than just osteoarthrosis – a cross-sectional study regarding the influence of body function-structures and activities on level of participation. Int Orthop 35, 767776.CrossRefGoogle ScholarPubMed
5. Tan, J, Zhu, W, Wang, W, et al. (2002) Selenium in soil and endemic diseases in China. Sci Total Environ 284, 227235.CrossRefGoogle Scholar
6. Chasseur, C, Suetens, C, Nolard, N, et al. (1997) Fungal contamination in barley and Kashin-Beck disease in Tibet. Lancet 350, 1074.CrossRefGoogle ScholarPubMed
7. Peng, A, Wang, WH, Wang, CX, et al. (1999) The role of humic substances in drinking water in Kashin-Beck disease in China. Environ Health Perspect 107, 293296.CrossRefGoogle Scholar
8. Jirong, Y, Huiyun, P, Zhongzhe, Y, et al. (2012) Sodium selenite for treatment of Kashin-Beck disease in children: a systematic review of randomized controlled trials. Osteoarthritis Cartilage 20, 605613.CrossRefGoogle Scholar
9. Xiong, G (2001) Diagnostic, clinical and radiological characteristics of Kashin-Beck disease in Shaanxi Province, PR China. Int Orthop 25, 147150.CrossRefGoogle ScholarPubMed
10. Yamamuro, T (2001) Kashin-Beck disease: a historical overview. Int Orthop 25, 134137.CrossRefGoogle ScholarPubMed
11. Suetens, C, Moreno-Reyes, R, Chasseur, C, et al. (2001) Epidemiological support for a multifactorial aetiology of Kashin-Beck disease in Tibet. Int Orthop 25, 180187.CrossRefGoogle ScholarPubMed
12. Wang, WZ, Guo, X, Duan, C, et al. (2009) Comparative analysis of gene expression profiles between the normal human cartilage and the one with endemic osteoarthritis. Osteoarthritis Cartilage 17, 8390.CrossRefGoogle ScholarPubMed
13. Duan, C, Guo, X, Zhang, XD, et al. (2010) Comparative analysis of gene expression profiles between primary knee osteoarthritis and an osteoarthritis endemic to Northwestern China, Kashin-Beck disease. Arthritis Rheum 62, 771780.CrossRefGoogle Scholar
14. Han, J, Guo, X, Tan, W, et al.. (2013) The expression of p-ATF2 involved in the chondeocytes apoptosis of an endemic osteoarthritis, Kashin-Beck disease. BMC Musculoskelet Disord 14, 209.CrossRefGoogle ScholarPubMed
15. Wang, W, Wei, S, Luo, M, et al. (2013) Oxidative stress and status of antioxidant enzymes in children with Kashin-Beck disease. Osteoarthritis Cartilage 21, 17811789.CrossRefGoogle ScholarPubMed
16. Davis, RJ (2000) Signal transduction by the JNK group of MAP kinases. Cell 103, 239252.CrossRefGoogle ScholarPubMed
17. Dhanasekaran, DN & Reddy, EP (2008) JNK signaling in apoptosis. Oncogene 27, 62456251.CrossRefGoogle ScholarPubMed
18. Liu, J & Lin, A (2005) Role of JNK activation in apoptosis: a double-edged sword. Cell Res 15, 3642.CrossRefGoogle ScholarPubMed
19. Pulverer, BJ, Kyriakis, JM, Avruch, J, et al. (1991) Phosphorylation of c-jun mediated by MAP kinases. Nature 353, 670674.CrossRefGoogle ScholarPubMed
20. Aigner, T, Kim, HA & Roach, HI (2004) Apoptosis in osteoarthritis. Rheum Dis Clin North Am 30, 639653 xi.CrossRefGoogle ScholarPubMed
21. Han, Z, Boyle, DL, Chang, L, et al. (2001) c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest 108, 7381.CrossRefGoogle ScholarPubMed
22. Fan, Z, Soder, S, Oehler, S, et al. (2007) Activation of interleukin-1 signaling cascades in normal and osteoarthritic articular cartilage. Am J Pathol 171, 938946.CrossRefGoogle ScholarPubMed
23. Chun, JS (2004) Expression, activity, and regulation of MAP kinases in cultured chondrocytes. Methods Mol Med 100, 291306.Google ScholarPubMed
24. Mengshol, JA, Vincenti, MP, Coon, CI, et al. (2000) Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappa B: differential regulation of collagenase 1 and collagenase 3. Arthritis Rheum 43, 801811.3.0.CO;2-4>CrossRefGoogle ScholarPubMed
25. Han, Z, Boyle, DL, Aupperle, KR, et al. (1999) Jun N-terminal kinase in rheumatoid arthritis. J Pharmacol Exp Ther 291, 124130.Google ScholarPubMed
26. Sundarrajan, M, Boyle, DL, Chabaud-Riou, M, et al. (2003) Expression of the MAPK kinases MKK-4 and MKK-7 in rheumatoid arthritis and their role as key regulators of JNK. Arthritis Rheum 48, 24502460.CrossRefGoogle ScholarPubMed
27. Schett, G, Tohidast-Akrad, M, Smolen, JS, et al. (2000) Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis. Arthritis Rheum 43, 25012512.3.0.CO;2-K>CrossRefGoogle ScholarPubMed
28. Yang, JB, Wang, ZW & Liu, JX (1994) Diagnostic criteria of Kashin-Beck disease. Chin J Endemiol 13, 2432.Google Scholar
29. Wang, H & Joseph, JA (1999) Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med 27, 612616.CrossRefGoogle ScholarPubMed
30. Hafeman, DG, Sunde, RA & Hoekstra, WG (1974) Effect of dietary selenium on erythrocyte and liver glutathione peroxidase in the rat. J Nutr 104, 580587.Google ScholarPubMed
31. Karin, M & Gallagher, E (2005) From JNK to pay dirt: jun kinases, their biochemistry, physiology and clinical importance. IUBMB Life 57, 283295.CrossRefGoogle ScholarPubMed
32. Bogoyevitch, MA & Kobe, B (2006) Uses for JNK: the many and varied substrates of the c-Jun N-terminal kinases. Microbiol Mol Biol Rev 70, 10611095.CrossRefGoogle ScholarPubMed
33. Guma, M, Ronacher, L, Firestein, GS, et al. (2011) JNK1 deficiency limits macrophage mediated antigen-induced arthritis. Arthritis Rheum 63, 16031612.CrossRefGoogle ScholarPubMed
34. Guma, M, Kashiwakura, J, Crain, B, et al. (2010) JNK1 controls mast cell degranulation and IL-1{beta} production in inflammatory arthritis. Proc Natl Acad Sci U S A 107, 2212222127.CrossRefGoogle ScholarPubMed
35. Kallunki, T, Deng, T, Hibi, M, et al. (1996) c-Jun can recruit JNK to phosphorylate dimerization partners via specific docking interactions. Cell 87, 929939.CrossRefGoogle ScholarPubMed
36. Chu, CR, Kaplan, LD, Fu, FH, et al. (2004) Recovery of articular cartilage metabolism following thermal stress is facilitated by IGF-1 and JNK inhibitor. Am J Sports Med 32, 191196.CrossRefGoogle ScholarPubMed
37. de Launay, D, van de Sande, MG, de Hair, MJ, et al. (2012) Selective involvement of ERK and JNK mitogen-activated protein kinases in early rheumatoid arthritis (1987 ACR criteria compared to 2010 ACR/EULAR criteria): a prospective study aimed at identification of diagnostic and prognostic biomarkers as well as therapeutic targets. Ann Rheum Dis 71, 415423.CrossRefGoogle ScholarPubMed
38. Manning, AM & Davis, RJ (2003) Targeting JNK for therapeutic benefit: from junk to gold? Nat Rev Drug Discov 2, 554565.CrossRefGoogle Scholar
39. Arkin, MR & Whitty, A (2009) The road less traveled: modulating signal transduction enzymes by inhibiting their protein-protein interactions. Curr Opin Chem Biol 13, 284290.CrossRefGoogle ScholarPubMed
40. Wang, SJ, Guo, X, Zuo, H, et al. (2006) Chondrocyte apoptosis and expression of Bcl-2, Bax, Fas, and NOS in articular cartilage in patients with Kashin-Beck disease. J Rheumatol 33, 615619.Google Scholar
41. Lange-Carter, CA, Pleiman, CM, Gardner, AM, et al. (1993) A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science 260, 315319.CrossRefGoogle ScholarPubMed
42. Yujiri, T, Sather, S, Fanger, GR, et al. (1998) Role of MEKK1 in cell survival and activation of JNK and ERK pathways defined by targeted gene disruption. Science 282, 19111914.CrossRefGoogle ScholarPubMed
43. Yujiri, T, Ware, M, Widmann, C, et al. (1999) MEK kinase 1 (MEKK1) transduces c-Jun NH2-terminal kinase activation in response to changes in the microtubule cytoskeleton. J Biol Chem 274, 1260512610.CrossRefGoogle ScholarPubMed
44. Zhang, L, Deng, M, Kao, CW, et al. (2003) MEK kinase 1 regulates c-Jun phosphorylation in the control of corneal morphogenesis. Mol Vis 9, 584593.Google ScholarPubMed
45. Xia, Y, Makris, C, Su, B, et al. (2000) MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration. Proc Natl Acad Sci U S A 97, 52435248.CrossRefGoogle ScholarPubMed
46. Berry, MJ, Tujebajeva, RM, Copeland, PR, et al. (2001) Selenocysteine incorporation directed from the 3′UTR: characterization of eukaryotic EFsec and mechanistic implications. Biofactors 14, 1724.CrossRefGoogle ScholarPubMed
47. Steinbrenner, H & Sies, H (2009) Protection against reactive oxygen species by selenoproteins. Biochim Biophys Acta 1790, 14781485.CrossRefGoogle ScholarPubMed
48. Bellinger, FP, Raman, AV, Reeves, MA, et al. (2009) Regulation and function of selenoproteins in human disease. Biochem J 422, 1122.CrossRefGoogle ScholarPubMed
49. Ge, K & Yang, G (1993) The epidemiology of selenium deficiency in the etiological study of endemic diseases in China. Am J Clin Nutr 57, 259S263S.Google Scholar
Figure 0

Fig. 1 Increased protein expression of phosphorylated c-Jun N-terminal kinase (p-JNK) in whole blood of Kashin–Beck disease (KBD) patients compared with healthy control subjects. (a) Protein extracts from patient and healthy control whole blood samples were prepared and analysed by immunoblotting with JNK, p-JNK and α-tubulin antibodies. (b) Signal intensity was then quantified, and the results of the densitometric analysis are shown as mean values and standard deviations represented by vertical bars for p-JNK expression relative to α-tubulin (right panel) and mean values and standard deviations represented by vertical bars for JNK expression relative to α-tubulin (left panel). One experiment is representative of three independent experiments. p-JNK protein expression levels in the KBD group were significantly higher than that in the control group (P<0·05). K, samples from KBD patients; C, samples from control subjects. * P<0·05.

Figure 1

Fig. 2 Effects of different concentrations of tert-butyl hydroperoxide (tBHP) on the cellular viability of chondrocytes were estimated by methylthiazolyl tetrazolium (MTT) reduction. (a) Cells were incubated in absence or presence of several tBHP concentrations for different time periods (6–24 h: , 6 h; , 12 h; , 18 h; , 24 h). (b) Chondrocytes were treated with various doses of tBHF for 24 h and viability was determined by MTT. Values are means and standard deviations represented by vertical bars. One experiment is representative of four independent experiments. Mean value was significantly different from that of the control group: * P<0·05, ** P<0·01 (chondrocytes untreated with tBHP).

Figure 2

Fig. 3 Protection against tert-butyl hydroperoxide (tBHP)-induced oxidative stress by Na2SeO3. Cell viability was determined by the methylthiazolyl tetrazolium (MTT) assay 24 h after exposure to tBHP, following a 24 h pre-treatment with Na2SeO3. Treatment with tBHP alone was seen to significantly decrease cell viability. Na2SeO3 showed protection against tBHP-induced cell toxicity. Values are means and standard deviations represented by vertical bars. One experiment is representative of four independent experiments. C, control group; O, tBHP injury group (tBHP300 mmol/l); OS1, low Se pre-protection group (0·05 mg/ml Na2SeO3+300 mmol/l tBHP); OS2, middle Se pre-protection group (0·1 mg/ml Na2SeO3+300 mmol/l tBHP); OS3, high Se pre-protection group (0·15 mg/ml Na2SeO3+300 mmol/l tBHP). ** P<0·01.

Figure 3

Fig. 4 The apoptosis of C28/I2 human chondrocytes detected by Hoechst 33342 staining and the protective effects of different Na2SeO3 concentrations on C28/I2 cells. Tert-butyl hydroperoxide (tBHP) injury obviously increased apoptosis of C28/I2 chondrocytes, whereas pre-treatment with Na2SeO3 reduced apoptosis. One experiment is representative of three independent experiments. C, control group; O, tBHP injury group (tBHP 300 mmol/l); OS1, low Se pre-protection group (0·05 mg/ml Na2SeO3+300 mmol/l tBHP); OS2, middle Se pre-protection group (0·1 mg/ml Na2SeO3+300 mmol/l tBHP); OS3, high Se pre-protection group (0·15 mg/ml Na2SeO3+300 mmol/l tBHP).

Figure 4

Fig. 5 The expressions of apoptosis-related proteins in C28/I2 cells using Western blots. (a) Protein extracts were prepared and analysed by immunoblotting with antibodies recognising phosphorylated (p)-c-Jun and β-actin. Signal intensity was then quantified and the results of the densitometric analysis are shown as mean values and standard deviations represented by vertical bars for p-c-Jun expression relative to β-actin (right panel). (b) Protein extracts were prepared and analysed by immunoblotting with c-Jun and actin antibodies. Signal intensity was then quantified and the results of the densitometric analysis are shown as mean values and standard deviations represented by vertical bars for c-Jun expression relative to β-actin (right panel). (c) Protein extracts were analysed by immunoblotting with activating transcription factor 2 (ATF2) and β-actin antibodies. The results of the densitometric analysis are shown as mean values and standard deviations represented by vertical bars for ATF2 expression relative to β-actin (right panel). (d) The results of immunoblotting with antibodies recognising B-cell lymphoma 2 (Bcl-2) and β-actin and densitometric analysis are shown as mean values and standard deviations represented by vertical bars for Bcl-2 expression relative to β-actin (right panel). For all, one experiment is representative of four independent experiments. C, control group; O, tBHP injury group; OS1, low Se pre-protection group; OS2, middle Se pre-protection group; OS3, high Se pre-protection group. * P<0·05, ** P<0·01.

Figure 5

Fig. 6 Intracellular reactive oxygen species (ROS) levels and activity of glutathione peroxidase (GPX). (a) ROS levels after pre-treatment with various concentrations of Na2SeO3 for 24 h followed by treatment with tert-butyl hydroperoxide (tBHP) (300 mmol/l) for 24 h. Treatment with tBHP significantly increased the ROS levels. Pre-treatment with Na2SeO3 significantly decreased the ROS levels. Values are means and standard deviations represented by vertical bars. One experiment is representative of four independent experiments. * P<0·05, ** P<0·01. (b) Activity of GPX. Values are means and standard deviations represented by vertical bars. *P<0·05. DCF, 5-(and-6)-chloromethyl-2-,7-dichlorofluorescin.

Figure 6

Fig. 7 Effect of Na2SeO3 on the c-Jun N-terminal kinase (JNK) signalling pathway. (a–c) Western blotting and densitometric analysis results of mitogen-activated protein kinase kinase kinase 1 (MEKK1), JNK and phosphorylated JNK (p-JNK). It showed that tert-butyl hydroperoxide (tBPH) injury for 24 h increased MEKK1 and p-JNK levels but not JNK expression in chondrocytes. Pre-treatment with various concentrations of Na2SeO3 for 24 h significantly decreased the expression levels of MEKK1 and p-JNK. One experiment is representative of four independent experiments. Values are means and standard deviations represented by vertical bars. C, control group; O, tBHP injury group; OS1, low Se pre-protection group; OS2, middle Se pre-protection group; OS3, high Se pre-protection group. Values are statistically significant at * P<0·05 and ** P<0·01.

You have Access
15
Cited by

Save article to Kindle

To save this article to your Kindle, first ensure coreplatform@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about saving to your Kindle.

Note you can select to save to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be saved to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

Find out more about the Kindle Personal Document Service.

Effects of sodium selenite on c-Jun N-terminal kinase signalling pathway induced by oxidative stress in human chondrocytes and c-Jun N-terminal kinase expression in patients with Kashin–Beck disease, an endemic osteoarthritis
Available formats
×

Save article to Dropbox

To save this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you used this feature, you will be asked to authorise Cambridge Core to connect with your Dropbox account. Find out more about saving content to Dropbox.

Effects of sodium selenite on c-Jun N-terminal kinase signalling pathway induced by oxidative stress in human chondrocytes and c-Jun N-terminal kinase expression in patients with Kashin–Beck disease, an endemic osteoarthritis
Available formats
×

Save article to Google Drive

To save this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you used this feature, you will be asked to authorise Cambridge Core to connect with your Google Drive account. Find out more about saving content to Google Drive.

Effects of sodium selenite on c-Jun N-terminal kinase signalling pathway induced by oxidative stress in human chondrocytes and c-Jun N-terminal kinase expression in patients with Kashin–Beck disease, an endemic osteoarthritis
Available formats
×
×

Reply to: Submit a response

Please enter your response.

Your details

Please enter a valid email address.

Conflicting interests

Do you have any conflicting interests? *