Figure 5
Effect of MGDG (monogalactosyldiacylglycerol) treatment on pathways of prostaglandins production in cultured articular chondrocytes. A) Human chondrocytes were pretreated overnight with 25 μM MGDG before treatment with 100 U/ml IL-1α for 24 hours. Upper panel: cell lysates were collected and analyzed by Western blot with a COX-2 polyclonal antibody. Middle panel: Western blot of cell lysates using a mPGES polyclonal antibody. Actin was blotted as an internal control; B) Quantitation of COX-2 expression in IL-1α + MGDG treated cells calculated as fold increase related to IL-1α only treatment. The average of three Western blots from three different primary cultures is shown; C) Quantitation of the inhibition of mPGES expression by the MGDG treatment. To show the repression by MGDG the % value referred to the value in IL-1α induced cells (100%) is calculated. The average of two Western blots from two different primary cultures is shown; D) PGE2 production. Conditioned media collected from human chondrocytes were analyzed for prostaglandin E2 content by a competitive immunoassay using the prostaglandin E2 EIA kit Monoclonal. Two experiments were performed in triplicate dishes, each one assayed in triplicate. One representative experiment is shown. Concentrations are expressed in pg/ml. E) 15ΔPGJ2 quantitation. 15ΔPGJ2 was measured in cell serum-free media using a 15ΔPGJ2 -specific competitive EIA Kit. Two experiments were performed, each one assayed in triplicate. One representative experiment is shown. Concentrations are expressed in pg/ml. 15ΔPGJ2, 15-deoxy-Δ12,14-prostaglandin J2,; COX-2, cyclooxygenase-2; DGDG, digalactosyldiacylglycerol; IL-1, interleukin-1; IL-6, interleukin-6; IL-8, interleukin-8; MGDG, monogalactosyldiacylglycerol; mPGES, microsomal PGE synthase; NF-kB, nuclear factor-kappaB; P1, cell passage number1; p38, p38 mitogen activated protein kinase; PGE2, prostaglandin E2; TNFα, tumor necrosis factor alpha.