| Summary: | 博士 === 國防醫學院 === 醫學科學研究所 === 99 === Introduction
Accumulation of advanced glycation end products (AGEs) in joints contributes to the pathogenesis of cartilage damage in osteoarthritis (OA). We first explore the potential chondroprotective effects of resveratrol on AGEs-stimulated porcine chondrocytes and cartilage explants. In addition, by using tumor necrosis factor-alpha (TNF-α)-activated porcine chondrocytes as a screening tool, we further studied and identified small-molecule inhibitors preserving immunomodulatory effects as therapeutics for OA.
Methods
Chondrocytes were isolated from pig joints. A minilibrary of 300 benzamide-linked small molecules was established. Activation of the IκB kinase (IKK)-IκBα-nuclear factor-kappaB (NF-κB) and c-Jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)-activator protein-1 (AP-1) pathways was assessed by electrophoretic mobility shift assay (EMSA), Western blot and transfection assay. The levels of inducible nitric oxide synthase (iNOS)-NO and cyclooxygenase-2 (COX-2)-prostaglandin E2 (PGE2) were measured by Western blot, Griess reaction or ELISA. Zymography and real-time RT-PCR were used to determine enzyme activity and expression of matrix metalloproteinases (MMPs) and aggrecanases, respectively. Proteoglycan degradation in cartilage explants was determined by histochemistry analysis.
Results
We show that AGEs-induced expression of iNOS and COX-2 and production of NO and PGE2 were suppressed by resveratrol. Such effects of resveratrol were likely mediated through inhibiting IKK-IκBα-NF-κB and JNK/ERK-AP-1 signaling pathways induced by AGEs. By targeting these critical signaling pathways, resveratrol decreased AGEs-stimulated expression and activity of MMP-13 and prevented AGEs-mediated destruction of collagen II. Histochemistry analysis further confirms that resveratrol could prevent AGEs-induced degradation of proteoglycan and aggrecan in cartilage explants. Future studies on the small molecules are therefore recommended. Bioassay screening of benzamide-linked small molecules revealed that 2-hydroxy-N-[3-(trifluoromethyl)phenyl]benzamide (HS-Cf) was a potent inhibitor of NO production and iNOS expression in TNF-α-stimulated porcine chondrocytes. HS-Cf suppressed TNF-α-induced activity of MMP-13 and expressions of several aggrecanases and prevented TNF-α-mediated reduction of collagen II. Histochemistry analysis confirmed that HS-Cf could prevent TNF-α-induced degradation and release of proteoglycan/aggrecan in cartilage explants. Such effects by HS-Cf were likely through suppressing TNF-α-induced interferon regulatory factor-1 (IRF-1) but not nuclear factor kappaB (NF-κB) signaling. The significance of IRF-1 was further confirmed by short hairpin knock-down studies.
Conclusions
The present study reveals not only the effects and mechanisms regarding how resveratrol may protect cartilage from AGEs-mediated damage but also the potential therapeutic benefit of resveratrol in the treatment of OA. In a minilibrary containing 300 small molecules, we identified a benzamide-linked small molecule, 2-hydroxy-N-[3-(trifluoromethyl)phenyl]benzamide (HS-Cf), that through down-regulating TNF-α-induced IRF-1 activity suppressed chondrocyte activation and prevented cartilage destruction. Both resveratrol and HS-Cf might be a potential disease-modifying drug for OA therapeutics.
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