Tiku ML, Shah R and Allison GT: Evidencelinking chondrocyte lipid peroxidation to cartilage matrix proteindegradation. Possible role in cartilage aging and the pathogenesisof osteoarthritis. J Biol Chem. 275:20069–20076. 2000. : :
Our findings also suggested that TQ increased theexpression and production of the pro-inflammatory mediators, COX-2and PGE ().Dedifferentiation and inflammation are supported by anintracellular signaling network involving the PI3K/Akt and MAPKspathways (). Phosphorated Akttranslocates to the nucleus and phosphorylates numerous targetmolecules to mediate signals (). MAPKs are a family of proteinspromoting a phosphorylative signaling cascade, leading to theactivation of transcription factors involved either in cellulardedifferentiation and inflammation (). It has also been reported that ROSinduces dedifferentiation, inflammation and proliferation in avariety of cell types through the temporal activation of the PI3Kand MAPKs pathways (,). In addition, several studies havelinked dedifferentiation and COX-2 expression with MAPKs, p38,ERK-1/2 and JNK and PI3K/Akt. (,,).
Reactive oxygen species (ROS), such as hydrogenperoxide (HO), superoxide anion(O). and hydroxyl radical (OH)are generally believed to be harmful to cells and tissues (). ROS are generated by a variety ofendogenous and exogenous processes through several pathways and themitochondria are the major source of intracellular ROS (,).ROS are destructive to DNA and proteins (). ROS are involved in the regulationof the production of biochemical factors involved in cartilagedegradation. They may cause damage to all matrix components, eitherdirectly or indirectly by reducing matrix component synthesis().
Thymoquinone (TQ) inducesdedifferentiation in rabbit articular chondrocytes. (A)Chondrocytes were exposed to TQ for 24 h. (B) Cells were exposed to20 μM TQ for 24 h. (A and B) The expression of type IIcollagen and actin was determined by western blot analysis withactin as a loading control. Chondrocytes were treated with TQ for24 h. (C) The synthesis of sulfate proteoglycan was determined byAlcian blue staining. Articular chondrocytes were exposed to 20μM TQ for 24 h. (D) The expression of type II collagen wasdetermined by IF staining. Data are presented as the means ± SDfrom 3 independent experiments performed in triplicate.*P
Thymoquinone (TQ)-induceddedifferentiation and cyclooxygenase-2 (COX-2) expression areblocked by an inhibitor of reactive oxygen species (ROS), N-acetylcysteine (NAC). Chondrocytes were exposed to 20 μM TQ in theabsence or presence of 5 mM NAC (A and B) for 2 h or (C and D) for24 h. ROS production was determined by fluorescence microscopy (A;magnification, ×200). (B) Reactive oxygen species (ROS)fluorescence was measured using an Flx8000 fluorometer. (C) Theexpression of type II collagen and COX-2 was determined by westernblot analysis with actin as a loading control. (D) The synthesis ofsulfate proteoglycan was detected by Alcian blue staining. (E) Thesecretion of prostaglandin E2 (PGE2) wasanalyzed by PGE2 assay. Data are presented as the means± SD from 3 independent experiments performed in triplicate.*P
Prostaglandins are produced by cyclooxygenases (COX)from arachidonic acid and are induced in arthritic joints (). COX has three forms: COX-1, COX-2 andCOX-3. Whereas COX-1 is constituvely expressed in various celltypes to maintain homeostasis, COX-2 is the inducible form of COX,implicated in prostaglandin synthesis in the inflammatory responseand has been associated with osteoarthritic cartilage (). COX-3 is a recently described variantof COX-1 and is also known as COX-1 VI. However, to date, there isnot much conclusive evidence available regarding the existence ofCOX-3 protein ().
In order to gain further insight into the molecularmechanisms underlying the induction of differentiation and COX-2expression in chondrocytes, we investigated the activation of theMAPK and PI3K pathways ().Our results revealed that TQ induced a dose-dependent increase inthe expression of the MAPKs, p-p38, p-ERK, p-JNK and PI3K/pAkt(). The TQ-inducedphosphorylation of MAPKs and PI3K was long-lasting and reachedmaximum levels after 10 min of treatment for p-p38, p-ERK and p-JNKand 30 min for p-Akt; the levels decreased thereafter (). We then determined whether theTQ-induced activation of MAPKs and PI3K is blocked by NAC (). NAC inhibited the TQ-inducedphosphorylation of MAPKs and PI3K (). To determine the associationbetween the TQ-induced generation of ROS, dedifferentiation andCOX-2 expression and the activation of MAPKs and PI3K, we inhibitedthe phosphorylation of MAPKs and PI3K using specific inhibitors(SB203580 for p38, PD98059 for ERK, LY294002 for PI3K/Akt andSP600125 for JNK) prior to treatment with TQ (). None of these inhibitors blockedthe TQ-induced generation of ROS, but some slightly inhibited theTQ-induced dedifferentiation and the expression of COX-2 (). The inhibition of ERK byPD98059 attenuated the TQ-induced loss in type II collagenexpression and proteoglycan synthesis (). The inhibition of p38with SB203580 or PI3K/Akt with LY294002 blocked the TQ-inducedexpression of COX-2 and PGE synthesis ().
Osteoarthritis (OA), a degenerative joint disease,is a multifactorial process in which mechanical factors play acentral role and is characterized by alterations in the structureand function of the whole joint (). OA involves the entire joint organ,including the subchondral bone, meniscus, ligaments, periarticularmuscle, capsule and synovium, and is associated with risk factors,such as age, gender, obesity, prior joint injury, geneticpredisposition and mechanical factors, including malalignment andabnormal joint shape (). Duringskeletal development, chondrocytes differentiate from mesenchymalprogenitors to synthesize the cartilage (). Differentiated chondrocytes expresscartilage-specific collagens II, IX and XI. Under normalconditions, chondrocytes rest in a non-stimulated steady state andmaintain the synthesis of proteoglycans and other non-collagenmolecules ().
Thymoquinone (TQ)-induced reactiveoxygen species (ROS) generation regulates dedifferentiation throughp38 and cyclooxygenase-2 (COX-2) expression through PI3K/Akt andERK. (A) Chondrocytes were exposed to 20 μM TQ in theabsence 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 wasdetermined by western blot analysis with actin as a loadingcontrol. Primary chondrocytes were exposed to 20 μM TQ inthe 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 fluorescencewas measured using an Flx 8000 fluorometer. (C) The expression oftype II collagen, COX-2, p-Akt, p-p38, p-JNK and p-ERK wasdetermined by western blot analysis with actin as a loadingcontrol. (D) The production of sulfate proteoglycan was determinedby Alcian blue staining. (E) The synthesis of prostaglandinE2 (PGE2) was analyzed by PGE2assay. Data are presented as the means ± SD from 3 independentexperiments performed in triplicate. *P
A recent study demonstrated that DIDS, a selectiveinhibitor of mitochondrial electron transport, prevents theproduction of ROS (). In thepresent study, the cells treated with DIDS no longer produced ROSin response to TQ ().Treatment with DIDS restored type II collagen expression andsulfate proteoglycan synthesis and decreased the expression ofCOX-2 and PGE production in the TQ-treated chondrocytes(). These findingssuggest that the inhibition of ROS production from the mitochondriaby DIDS inhibits dedifferentiation and COX-2 expression and thatROS is the key source of cartilage destruction ().