Epigallocatechin-3-gallate (EGCG) is a bioactive polyphenol of green tea and exerts potent anti-inflammatory effects by inhibiting signaling events and gene expression. Interleukin-1beta (IL-1β) is the principal cytokine linked to cartilage degradation in osteoarthritis (OA). The objective of this study was to evaluate the global effect of EGCG on IL-1β-induced expression of proteins associated with OA pathogenesis in human chondrocytes.
Primary OA chondrocytes were pretreated with EGCG (10 to 100 uM) and then stimulated with IL-1β (5 ng/ml) for 24 hours. Culture supernatants were incubated with cytokine antibody arrays and immunoreactive proteins (80 proteins) were visualized by enhanced chemiluminiscence. Effect of EGCG on IL-1β-induced expression of 18 selected genes was verified by Real time-PCR and effect on IL-6, IL-8 and tumor necrosis factor-alpha (TNF-α) production was determined using specific ELISAs. Western immunoblotting was used to analyze the effect of EGCG on the interleukin-1 receptor-associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF-6) proteins in IL-1β-stimulated chondrocytes. The role of nuclear factor kappa-B (NF-κB) and mitogen activated protein kinases (MAPKs) in the regulation of selected genes and the mechanism involved in EGCG mediated modulation of these genes was determined by using specific inhibitors for NF- κB (MG132) and MAPKs (p38-MAPK, SB202190; JNK-MAPK, SP600125, ERK-MAPK, PD98059).
Out of 80 proteins present on the array, constitutive expression of 14% proteins was altered by EGCG treatment. No significant stimulatory effect was observed on the proteins associated with cartilage anabolic response. Stimulation with IL-1β enhanced the expression of 29 proteins. Expression of all 29 proteins up-regulated by IL-1β was found to be suppressed by EGCG. EGCG also inhibited the expression of the signaling intermediate TRAF-6 at 50 and 100 uM concentrations (P < 0.05). Our results identified several new targets of EGCG, including epithelial neutrophil activating peptide-78 (ENA-78), granulocyte macrophage colony stimulation factor (GM-CSF), growth- related oncogene (GRO), GRO-α, IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), MCP-3, macrophage inflammatory protein-1beta (MIP-1β), granulocyte chemotactic protein-2 (GCP-2), MIP-3alpha, interferon-gamma-inducible protein-10 (IP-10), nucleosome assembly protein-2 (NAP-2) and leukemia inhibitory factor (LIF). The inhibitory effects of EGCG were mainly mediated by inhibiting the activation of NF-κB and c-Jun N-terminal Kinase (JNK)-MAPK in human chondrocytes.
Our results suggest that the potential of EGCG in OA treatment/prevention may be related to its ability to globally suppress the inflammatory response in human chondrocytes. These results identify additional new targets of EGCG and advocate that EGCG may be a potent chondroprotective agent in OA.