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. 2015 Feb;35(2):325-32.
doi: 10.3892/ijmm.2014.2014. Epub 2014 Nov 27.

The thymoquinone-induced production of reactive oxygen species promotes dedifferentiation through the ERK pathway and inflammation through the p38 and PI3K pathways in rabbit articular chondrocytes

Seon-Mi Yu  1 , Song-Ja Kim  1
Affiliations

VSports - The thymoquinone-induced production of reactive oxygen species promotes dedifferentiation through the ERK pathway and inflammation through the p38 and PI3K pathways in rabbit articular chondrocytes

Seon-Mi Yu et al. Int J Mol Med. 2015 Feb.

VSports - Abstract

Dedifferentiation and inflammation are major features of cartilage degeneration during the pathogenesis of osteoarthritis (OA) VSports手机版. Thymoquinone (TQ) is the major compound of black seed oil isolated from Nigella sativa with various beneficial or harmful effects on several diseases; however, its effects on the dedifferentiation and inflammation of chondrocytes have not yet been characterized. In the present study, we investigated whether TQ regulates the dedifferentiation and inflammation of rabbit articular chondrocytes, focusing on the production of reactive oxygen species (ROS) in rabbit articular chondrocytes. TQ induced the generation of ROS in a dose-dependent manner, as shown by staining with the fluorescent probe, 2'-7'-dichlorofluorescein diacetate. We confirmed that TQ induced dedifferentiation by measuring the loss of type II collagen and the reduction in chondroitin sulfate proteoglycan levels. TQ also caused inflammation by inducing the expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). The antioxidant, N-acetyl cysteine (NAC), prevented the dedifferentiation and inflammation which was generated by the TQ-induced production of ROS. Furthermore, TQ caused a dose-dependent increase in p38, phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphoinositide 3-kinase (PI3K) expression. NAC abrogated this effect and attenuated the dedifferentiation and inflammation which was generated by the TQ-induced production of ROS. To identify the ROS-regulated pathways, we treated the chondrocytes with the p38 inhibitor, SB203580, the MEK inhibitor, PD98059, and the PI3K inhibitor, LY294002. PD98059 inhibited the TQ-induced dedifferentiation and SB203580 and LY294002 prevented the TQ-induced inflammation. These findings suggest that the TQ-induced production of ROS causes dedifferentiation through the ERK pathway and inflammation through the PI3K and p38 pathways in rabbit articular chondrocytes. .

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Figures

Figure 1
Figure 1
Thymoquinone (TQ) induces the generation of intracellular reactive oxygen species (ROS) in primary rabbit articular chondrocytes. (A) Chondrocytes were exposed to TQ for 2 h. Dichlorofluorescein (DCF) fluorescence intensity was observed under an inverted fluorescence microscopy (left panel). DCF fluorescence intensity was quantified using ImageJ software (right panel). (B) Chondrocytes were exposed to TQ for 2 h. ROS fluorescence was measured using an Flx8000 Bio-Tek fluorometer (B). Data are presented as the means ± SD from 3 independent experiments performed in triplicate. *P<0.05, compared with the control group.
Figure 2
Figure 2
Thymoquinone (TQ) induces dedifferentiation in rabbit articular chondrocytes. (A) Chondrocytes were exposed to TQ for 24 h. (B) Cells were exposed to 20 μM TQ for 24 h. (A and B) The expression of type II collagen and actin was determined by western blot analysis with actin as a loading control. Chondrocytes were treated with TQ for 24 h. (C) The synthesis of sulfate proteoglycan was determined by Alcian blue staining. Articular chondrocytes were exposed to 20 μM TQ for 24 h. (D) The expression of type II collagen was determined by IF staining. Data are presented as the means ± SD from 3 independent experiments performed in triplicate. *P<0.05, compared with the control group.
Figure 3
Figure 3
Thymoquinone (TQ) increases cyclooxygenase-2 (COX-2) expression in rabbit articular chondrocytes. (A) Primary chondrocytes were exposed to TQ for 24 h. (B) Cells were exposed to 20 μM TQ for 24 h. (A and B) The expression of COX-2 and actin was determined by western blot analysis with actin as a loading control. (C) Rabbit chondrocytes were treated with TQ for 24 h. (C) Production of prostaglandin E2 (PGE2) was determined by PGE2 assay. (D) Chondrocytes were exposed to 20 μM TQ for 24 h. (D) The expression of COX-2 was determined by immunofluorescence staining. Data are presented as the means ± SD from 3 independent experiments performed in triplicate. *P<0.05, compared with the control group.
Figure 4
Figure 4
Thymoquinone (TQ)-induced dedifferentiation and cyclooxygenase-2 (COX-2) expression are blocked by an inhibitor of reactive oxygen species (ROS), N-acetyl cysteine (NAC). Chondrocytes were exposed to 20 μM TQ in the absence or presence of 5 mM NAC (A and B) for 2 h or (C and D) for 24 h. ROS production was determined by fluorescence microscopy (A; magnification, ×200). (B) Reactive oxygen species (ROS) fluorescence was measured using an Flx8000 fluorometer. (C) The expression of type II collagen and COX-2 was determined by western blot analysis with actin as a loading control. (D) The synthesis of sulfate proteoglycan was detected by Alcian blue staining. (E) The secretion of prostaglandin E2 (PGE2) was analyzed by PGE2 assay. Data are presented as the means ± SD from 3 independent experiments performed in triplicate. *P<0.01, compared with the control group.
Figure 5
Figure 5
Thymoquinone (TQ) activates the PI3K/Akt and the MAPK (p38, ERK and JNK) pathways in chondrocytes. (A) Rabbit chondrocytes were exposed to TQ for 24 h. (B) Articular chondrocytes were exposed to 20 μM TQ for the indicated periods of time. (A and B) The activation of p38, ERK, JNK and Akt was determined by western blot analysis with actin as a loading control. Data are presented as the means ± SD from 3 independent experiments performed in triplicate.
Figure 6
Figure 6
Thymoquinone (TQ)-induced reactive oxygen species (ROS) generation regulates dedifferentiation through p38 and cyclooxygenase-2 (COX-2) expression through PI3K/Akt and ERK. (A) Chondrocytes were exposed to 20 μM TQ in the absence or presence of 5 mM N-acetyl cysteine (NAC) for 24 h. (A) The expression of p-p38, p-ERK, p-JNK, p-Akt and actin was determined by western blot analysis with actin as a loading control. Primary chondrocytes were exposed to 20 μM TQ in the absence or presence of SB203580 (SB, PI3K inhibitor), PD98059 (PD, p38 inhibitor), LY294002 (LY, ERK inhibitor) or SP600125 (SP, JNK inhibitor) (B) for 2 h or (C–E) for 24 h. (B) ROS fluorescence was measured using an Flx 8000 fluorometer. (C) The expression of type II collagen, COX-2, p-Akt, p-p38, p-JNK and p-ERK was determined by western blot analysis with actin as a loading control. (D) The production of sulfate proteoglycan was determined by Alcian blue staining. (E) The synthesis of prostaglandin E2 (PGE2) was analyzed by PGE2 assay. Data are presented as the means ± SD from 3 independent experiments performed in triplicate. *P<0.01, compared with the control group.
Figure 7
Figure 7
4,4′-Diisothiocyano-2,2′-stilbenedisulphonic acid (DIDS) inhibits dedifferentiation and cyclooxygenase-2 (COX-2) expression which is generated by the thymoquinone (TQ)-induced production of reactive oxygen species (ROS). Chondrocytes were exposed to 20 μM TQ in the presence or absence of 200 μM DIDS for (A) 2 h or (B–D) 24 h. (A) ROS fluorescence was measured using an Flx8000 fluorometer. (B) The expression of type II collagen and COX-2 was determined by western blot analysis with actin as a loading control. (C) The production of sulfate proteoglycan was determined by Alcian blue staining. (E) The synthesis of prostaglandin E2 (PGE2) was analyzed by PGE2 assay. Data are presented as the means ± SD from 3 independent experiments performed in triplicate. *P<0.05, compared with the control group.
Figure 8
Figure 8
Schematic diagram of thymoquinone (TQ)-induced dedifferentiation and cyclooxygenase-2 (COX-2) expression. The TQ-induced production of reactive oxygen species (ROS) regulates dedifferentiation through the ERK pathway and modulates COX-2 expression through the PI3K and p38 pathways. These effects are blocked by treatment with N-acetyl cysteine (NAC) and 4,4′-diisothiocyano-2,2′-stilbenedisulphonic acid (DIDS).
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