Phytochemical mediated modulation of COX-3 and NFκB for the management and treatment of arthritis

Abstract

In this study, we investigated whether zerumbone (ZBN), ellagic acid (ELA) and quercetin (QCT), the plant-derived components, can modulate the role of COX-3 or cytokines liable in arthritic disorder. Initially, the effect of ZBN, ELA, and QCT on inflammatory process was investigated using in-vitro models. In-silico docking and molecular dynamics study of these molecules with respective targets also corroborate with in-vitro studies. Further, the in-vivo anti-arthritic potential of these molecules in Complete Freund's adjuvant (CFA)-induced arthritic rats was confirmed. CFA increases in TNF-α and IL-1β levels in the arthritic control animals were significantly (***p < 0.001) attenuated in the ZBN- and ELA-treated animals. CFA-induced attenuation in IL-10 levels recovered under treatment. Moreover, ELA attenuated CFA-induced upregulation of COX-3 and ZBN downregulated CFA-triggered NFκB expression in arthritic animals. The bonding patterns of zerumbone in the catalytic sites of targets provide a useful hint in designing and developing suitable derivatives that can be used as a potential drug. To our best knowledge, the first time we are reporting the role of COX-3 in the treatment of arthritic disorders which could provide a novel therapeutic approach for the treatment of inflammatory disorders.

Figure 1

Effect of zerumbone (ZBN), ellagic acid (ELA), quercetin (QCT) and standard diclofenac sodium (DfS) on in-vitro anti-inflammatory assays. (A) Inhibition of albumin denaturation, (B) heat-induced hemolysis, (C) protease inhibition assay. The average values of the raw data are expressed as the mean ± standard error mean (SEM), n = 3.

Figure 2

The structural stability of the protein–ligand complex monitored by the molecular dynamic simulations (30-ns). (a) COX-3 model bound to quercetin, ellagic acid, and zerumbone. Ellagic acid exhibits a greater RMSD value as the time progresses, while quercetin and zerumbone exhibit an almost constant stability pattern after 12 ns, except at approximately 22 ns. (b) TNF-α bound to quercetin, ellagic acid, and zerumbone. Quercetin exhibits a greater RMSD value as time progresses, while ellagic acid and zerumbone exhibit an almost constant stability pattern after 12 ns. (c) Interleukin (IL-10) bound to quercetin, ellagic acid, and zerumbone. Quercetin, ellagic acid and zerumbone exhibit almost constant RMSD values as time progresses. All the compounds (except ellagic acid) showed optimal stability at 27–30 ns.

Figure 3

The 3D target-ligand hydrogen bond interaction of IL-10 with (a) quercetin (b) ellagic acid and (c) zerumbone; TNF-α with (a) quercetin, (b) ellagic acid and (c) zerumbone; and COX-3 with (a) quercetin, (b) ellagic acid and (c) zerumbone.

Figure 4

Partial least squares simulation graph at 30 ns with amino acids of IL-10 interacting with (a) quercetin (b) ellagic acid and (c) zerumbone; of TNF-α with (a) quercetin, (b) ellagic acid and (c) zerumbone; and of COX-3 with (a) quercetin, (b) ellagic acid and (c) zerumbone. The scale on the right side of the graph indicates no contacts between IL-10 and ellagic acid, which indicates morePLS simulation, while the lack of contact points between IL-10 and zerumbone may be due to no hydrogen bond interactions.

Figure 5

Effect of zerumbone (ZBN), ellagic acid (ELA), quercetin (QCT) and standard diclofenac sodium (DfS) on in-vivo anti-arthritic bioassay. (A) Hind paw edema of healthy (NC) and CFA-induced arthritic (AC) control rats. (B) Pain intensity in the knee joint of healthy (NC) and CFA-induced arthritic (AC) rats. (C) Levels of TNF-α insera and knee joint tissues from and AC rats. (D) Levels of IL-10 insera and knee joint tissues from NC and AC rats. (E) Levels of IL-1β insera and knee joint tissues from NC and AC rats. [Average values of the raw data were expressed as the mean ± SEM, n = 5. For numerical results, one-way analysis of variance (ANOVA) with Tukey–Kramer Multiple Comparisons post-tests was performed using GraphPad InStat Version 3 (GraphPad Software). The minimum value of p < 0.05 was considered significant. *^Cp < 0.05, **^Cp < 0.01, and ***^Cp < 0.001 indicate significant differences of CFA-induced arthritic control group comparisons to the healthy control group; *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences of test groups comparisons to the CFA-induced arthritic control group] (F) CFA-induced increases in COX-3 and NFκB expression in knee joint tissues from AC rats. A group of NC rats was used as a reference control (western blotting analysis). [Average values of the raw data were expressed as the mean ± SEM, n = 3] (G). Zerumbone (ZBN) inhibited CFA-induced overexpression of COX-3 and NFκB. However, ellagic acid (ELA) and quercetin (QCT) inhibited CFA-induced overexpression of COX-3 but not CFA-induced overexpression of NFκB expression. CFA-induced arthritic rats were treated with test compounds at a dose of 50 mg/kg/day for 7 days. Knee joint tissue extracts were prepared in RIPA buffer containing aprotease inhibitor and subjected to western blotting.