Regulation of interleukin‐1β–induced chemokine production and matrix metalloproteinase 2 activation by epigallocatechin‐3‐gallate in rheumatoid arthritis synovial fibroblasts

Objective

To evaluate the efficacy of epigallocatechin‐3‐gallate (EGCG), a potent antiinflammatory molecule, in regulating interleukin‐1β (IL‐1β)–induced production of the chemokines RANTES (CCL5), monocyte chemoattractant protein 1 (MCP‐1/CCL2), epithelial neutrophil–activating peptide 78 (ENA‐78/CXCL5), growth‐regulated oncogene α (GROα/CXCL1), and matrix metalloproteinase 2 (MMP‐2) activity in rheumatoid arthritis (RA) synovial fibroblasts.

Methods

Fibroblasts obtained from RA synovium were grown, and conditioned medium was obtained. Cell viability was determined by MTT assay. RANTES, MCP‐1, ENA‐78, and GROα produced in culture supernatants were measured by enzyme‐linked immunosorbent assay. MMP‐2 activity was analyzed by gelatin zymography. Western blotting was used to study the phosphorylation of protein kinase C (PKC) isoforms and nuclear translocation of NF‐κB.

Results

EGCG was nontoxic to RA synovial fibroblasts. Treatment with EGCG at 10 μM or 20 μM significantly inhibited IL‐1β–induced ENA‐78, RANTES, and GROα, but not MCP‐1 production in a concentration‐dependent manner. EGCG at 50 μM caused a complete block of IL‐1β–induced production of RANTES, ENA‐78, and GROα, and reduced production of MCP‐1 by 48% (P < 0.05). Zymography showed that EGCG blocked constitutive, IL‐1β–induced, and chemokine‐mediated MMP‐2 activity. Evaluation of signaling events revealed that EGCG preferentially blocked the phosphorylation of PKCδ and inhibited the activation and nuclear translocation of NF‐κB in IL‐1β–treated RA synovial fibroblasts.

Conclusion

These results suggest that EGCG may be of potential therapeutic value in inhibiting joint destruction in RA.

     

Combination treatment of db/db mice with exendin‐4 and gastrin preserves β‐cell mass by stimulating β‐cell growth and differentiation

Aim/Introduction: Preservation of β‐cell mass is crucial for maintaining long‐term glucose homeostasis. Therapies based on incretin and its mimetics are expected to achieve this goal through various biological functions, particularly the restoration of β‐cell mass. Here we tested the effects of gastrin and exendin‐4 in type 2 diabetic animals. Materials and Methods: The effects of exendin‐4 and gastrin on β‐cell function and mass were examined in 8‐week‐old db/db mice. INS‐1 beta cells and AR42J cells were used to determine the molecular mechanism underlying the effects of the two agents. Immunohistochemistry, western blotting and RT‐PCR assays were used to assess the biological effects of the two agents. Results: Two weeks of combination administration of exendin‐4 plus gastrin resulted in a significant improvement of glucose tolerance associated with a marked preservation of β‐cell mass in db/db mice. Immunohistochemical analysis showed that such treatment resulted in the appearance of numerous irregularly‐shaped small islets and single insulin‐positive cells. While gastrin had little biological effect on INS‐1 β‐cells consistent with low expression of its intrinsic receptor on these cells, it caused differentiation of AR42J cells into insulin‐producing cells. Co‐stimulation with exendin‐4 significantly enhanced gastrin‐induced endocrine differentiation of AR42J precursor cells. These findings were further supported by enhanced expression of key genes involved in β‐cell differentiation and maturation, such as neurogenin3 (Ngn3) and MafA. Conclusions: These results suggest that combination treatment of db/db mice with exendin‐4 and gastrin preserves β‐cell mass by stimulating β‐cell growth and differentiation. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.00044.x, 2010)