Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation

Abstract

Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1-5μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p<0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p<0.05; n=4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p<0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA.

Keywords: ASK1; MAP kinase; Rheumatoid arthritis; Synovial fibroblasts; TNF-α; Thymoquinone.

Fig. 1. Effect of Thymoquinone on human RA-FLS viability

RA-FLS were treated with (A) Thymoquinone (TQ) (0.1–5 μM) for 24 hrs; (B) TQ (1–5 μM) with or without TNF-α (20 ng/ml).

Fig. 2. Effect of TQ on IL-6 and IL-8 production and ICAM-1/VCAM-1 and Cad-11 expression in RA-FLS

(A and B) RA-FLS were pre-treated with TQ (1–5 μM) for 2 hrs, followed by TNF-α (20 ng/ml) stimulation for 24 hrs. IL-6 and IL-8 production was determined in the conditioned media using commercially available ELISA kits. (C) Expression of adhesion molecule (ICAM-1, VCAM-1 and Cad-11) were analyzed in cell lysates using Western blotting. Densitometric analysis was performed and values are presented as mean ± SEM of n=4 experiments using different donors. ^## p<0.01 for NS vs TNF-α; *p<0.05 **p<0.01 for TNF-α vs TQ.

Fig. 3. TQ inhibits TNF-α-induced activation of p38 and JNK, but not ERK or IκBα, in RAFLS

RA-FLS were pre-treated with TQ (1–5 μM) for 2 hrs, followed by TNF-α (20 ng/ml) stimulation for 30 min. Cell lysates were evaluated for the total and phosphorylated form of p38, JNK, ERK, IκB-α and β-actin by Western blotting (A). Densitometric analysis were done by Image lab 5.1 and values are represented as mean ±SEM of n=4 experiments using different donors (B). ^## p<0.01 for NS vs TNF-α; **p<0.01 for TNF-α vs TQ.

Fig. 4. Involvement of ASK1 in TNF-α stimulated RA fibroblast and their abrogation by TQ

RA-FLS were treated with TQ (1–5 μM) for 2 hrs, followed by stimulation with TNF-α (20 ng/ml) for 30 min were lysed and probed for the expression of phospho-ASK1 (Thr845), ASK1, and phospho-c-jun (A). Densitometric analysis normalized with β-actin was done by Image Lab 5.1 and values are the mean ± SEM of experiments performed in four different RA-FLS donors (B). ^# p<0.05, ^## p<0.01 for NS vs TNF-α; *p<0.05, **p<0.01 for TNF-α vs TQ.

Fig. 5. Effect of TQ on TRAF-2 and ASK1 association in RA-FLS

RA-FLS were treated with TQ (1–5 μM) for 2 hrs, followed by stimulation with TNF-α (20 ng/ml) for 30 min were lysed and IP ASK1 followed by Western blotting for the detection of TRAF-2 (A). RA-FLS were grown to >80% confluence in 8 chamber slides for pre-treatment with TQ (5 μM) for 2 hrs, followed by TNF-α (20 ng/ml) stimulation for 30 min. Cells were fixed and stained with mouse monoclonal TRAF-2 or rabbit polyclonal ASK1 antibody (B), followed by Alexa staining. Images were acquired using 40X magnification.

Fig. 6. Inhibition of TNF-α-induced expression of adhesion molecules (ICAM-1, VCAM-1, and Cad-11) by ASK1 inhibitor in RA-FLS

RA-FLS were pre-treated with TC-ASK 10 (ASK1 inhibitor; 12.5–25 μM) for 2 hrs, followed by stimulation with TNF-α (20 ng/ml) for 24 hrs. Cells were lysed and probed for the expression of ICAM-1, VCAM-1 and Cad-11 (A). Densitometric analysis normalized with β-actin was done by Image Lab 5.1 and values are represented as mean ±SEM of 4 independent experiments using different donors (B). ^# p<0.05, ^## p<0.01 for NS vs TNF-α; *p<0.05, **p<0.01 for TNF-α vs ASK inhibitor.