An in vitro screening assay for inhibitors of proinflammatory mediators in herbal extracts using human synoviocyte cultures.
Tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase (COX)-2, and prostaglandin (PG)E-2 play a critical role in the pathophysiology of arthritis. Tumor necrosis factor-alpha 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 x 10(5) 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-alpha, 10 ng/ml interleukin-1beta, or control media alone at 5% carbon dioxide, 37 degrees C. Cell viability, TNF-alpha, COX-2, PGE-2, nuclear factor kappaB (NF-kappaB), and inhibitory subunit I kappa B-alpha (IkappaB-alpha) expression were analyzed by reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, and Western blots. Ginger extract-HAPC (100 microg/ml) significantly inhibited the activation of TNF-alpha and COX-2 expression in human synoviocytes as well as suppressed production of TNF-alpha and PGE-2. Inhibition of TNF-alpha and COX-2 activation was accompanied by suppression of NF-kappaB and IkappaB-alpha 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.
Frondoza
Carmelita G
CG
Division of Arthritis Surgery, Department of Orthopaedic Surgery, Johns Hopkins University, Baltimore, Maryland 21205, USA. cgfrondo@jhmi.edu
Sohrabi
Afshin
A
Polotsky
Anna
A
Phan
Phong V
PV
Hungerford
David S
DS
Lindmark
Lars
L
D015153:Blotting, Western; D002470:Cell Survival; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D015232:Dinoprostone / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D020939:Zingiber officinale; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D018836:Inflammation Mediators / Q000037:antagonists & inhibitors*; D007375:Interleukin-1 / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000032:analysis / Q000378:metabolism; D010003:Osteoarthritis / Q000473:pathology*; D010936:Plant Extracts / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000494:pharmacology
D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D020672:I-kappa B Proteins; D018836:Inflammation Mediators; D007375:Interleukin-1; D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D010936:Plant Extracts; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone