Figure 4

MGDG (monogalactosyldiacylglycerol) inhibits NF-kB (Nuclear Factor-kappaB) involved in IL-6 (Interleukin-6) and IL-8 (Interleukin-8) production. A) Inhibition of IL-6 and IL-8 synthesis by the NF-kB inhibitor BAY117082. Human chondrocytes were treated with 5 μM BAY117082 for two hours before being supplemented with 100 U/ml IL-1α for 20 hours in the presence of BAY117082. Conditioned media were subjected to immunoblot analysis using IL-6 and IL-8 polyclonal antibodies; B-C) Quantitation of the inhibition by BAY117082 of the IL-6 and IL-8 synthesis. The average of the densitometric analysis of four Western blots performed on four independent experiments on two different primary cultures is presented. To show the repression by BAY117082 the % value referred to the value in IL-1α induced cells (100%) is calculated. D) NF-kB activity inhibition by MGDG. Human chondrocytes were pretreated overnight with 25 μM MGDG and stimulated with 100 U/ml IL-1α in serum free conditions for 24 hours. 5 μg of whole cell lysates were tested for binding of the activated p65 NF-kB subunit to a NF-kB consensus sequence using the Trans-Am NF-kB ELISA kit. Results are expressed as specific binding. Two experiments on two different primary cultures were performed in triplicate dishes, each one assayed in triplicate. One representative experiment is shown; E) To show the repression by MGDG the % value referred to the value in IL-1α induced cells (100%) is calculated. Each value was subtracted of the basal value. The average of the two experiments performed in triplicate and assayed in triplicate is shown. 15ΔPGJ2, 15-deoxy-Δ^12,14-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.