AN IN VITRO SCREENING ASSAY FOR INHIBITORS OF PROINFLAMMATORY MEDIATORS IN HERBAL EXTRACTS USING HUMAN SYNOVIOCYTE CULTURES

• Published in: In vitro cellular & developmental biology. Animal Vol. 40; no. 3; pp. 95 - 101
• Main Authors: FRONDOZA, CARMELITA G, SOHRABI, AFSHIN, POLOTSKY, ANNA, PHAN, PHONG V, HUNGERFORD, DAVID S, LINDMARK, LARS
• Format: Journal Article
• Language: English
• Published: Germany Society for In Vitro Biology 01.03.2004
• Subjects: Antiinflammatories; Arthritis; Blotting, Western; CELL AND TISSUE MODELS; Cell Survival; Cells, Cultured; Chondrocytes; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors - pharmacology; Cytokines; Dinoprostone - metabolism; Drug Evaluation, Preclinical; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation - drug effects; Ginger; ginger extract; Humans; I-kappa B Proteins - metabolism; Inflammation; Inflammation Mediators - antagonists & inhibitors; Interleukin-1 - antagonists & inhibitors; Interleukin-1 - pharmacology; Isoenzymes - metabolism; Membrane Proteins; Necrosis; NF-kappa B - analysis; NF-kappa B - metabolism; NF-KappaB Inhibitor alpha; Osteoarthritis; Osteoarthritis - pathology; Plant Extracts - pharmacology; Prostaglandin-Endoperoxide Synthases - metabolism; prostaglandins; Reverse Transcriptase Polymerase Chain Reaction; Synovial Membrane - cytology; Synovial Membrane - drug effects; Synovial Membrane - metabolism; synoviocytes; TNF-α Tumor Necrosis Factor-alpha - antagonists & inhibitors; Tumor Necrosis Factor-alpha - pharmacology; Tumors; Zingiber officinale
• ISSN: 1071-2690; 1543-706X; 1543-706X
• DOI: 10.1290/1543-706X(2004)040<0095:AIVSAF>2.0.CO;2

Tumor necrosis factor–α (TNF-α), cyclooxygenase (COX)-2, and prostaglandin (PG)E-2 play a critical role in the pathophysiology of arthritis. Tumor necrosis factor–α mediates induction of other cytokines, COX-2, PGs, and metalloproteinases, which leads to cartilage degradation. We developed an in vitro human synoviocyte assay system for screening inhibitors of proinflammatory mediators in herbal extracts. Synoviocytes (5 × 105 cells/well) obtained during primary knee replacement from osteoarthritic patients were incubated with: control media alone or ginger extract (hydroxy-methoxy-phenyl compounds [HAPC]: EV.EXT®77), 1 h before activation with 1 ng/ml TNF-α, 10 ng/ml interleukin-1β, or control media alone at 5% carbon dioxide, 37° C. Cell viability, TNF-α, COX-2, PGE-2, nuclear factor κB (NF-κB), and inhibitory subunit I kappa B-alpha (IκB-α) expression were analyzed by reverse transcriptase–polymerase chain reaction, enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, and Western blots. Ginger extract-HAPC (100 μg/ml) significantly inhibited the activation of TNF-α and COX-2 expression in human synoviocytes as well as suppressed production of TNF-α and PGE-2. Inhibition of TNF-α and COX-2 activation was accompanied by suppression of NF-κB and IκB-α induction. Using our in vitro assay, we discovered that the ginger extract blocks activation of proinflammatory mediators and its transcriptional regulator suggesting its mode of action. These observations indicate that ginger extract-HAPC offers a complementary and alternative approach to modulate the inflammatory process involved in arthritis.