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-Δ^12,14-prostaglandin J_2,; 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.