Figure 5.

Effect of AVX001 and AVX002 on IL-1β-stimulated sPLA₂ protein (A), mRNA expression (B) and promoter activity (C) in mesangial cells. Quiescent cells were stimulated with either DMEM (-), or IL-1β (1 nM) in the absence (-) or presence of the indicated concentrations of AVX001, AVX002, AACOCF₃ and DHA (all inhibitors were added as a pretreatment for 90 min before stimulation). (A) Supernatants were taken for protein precipitation, separated by SDS-PAGE, transfered to membranes and subjected to Western blot analysis using a monoclonal antibody against rat sPLA₂. Corresponding cell lysates were stained for GAPDH and used for normalization. Bands were densitometrically evaluated and the ratio between secreted sPLA₂ and cytosolic GAPDH was calculated. Results are expressed as % of IL-1β stimulation. Data are means ± SD (n = 3–5). The inset shows representative Western blots of sPLA₂ (upper panels) from supernatants (SN) and GAPDH (lower panels) from cell lysates. (B) Cells were taken for RNA extraction and subjected to quantitative PCR analysis of rat IIA-sPLA₂ and 18S RNA. ΔΔCt values were calculated and results are expressed as % of maximal IL-1β-stimulated response and are means ± SD (n = 3–5). (C) Cells were transfected with the sPLA₂ promoter construct plus a plasmid coding for Renilla luciferase. After transfection, cells were stimulated for 24 h with vehicle (-), IL-1β (1 nM), or IL-1β in the absence or presence of AVX001 or AVX002 (both at 20 μM). Both inhibitors were added as a pretreatment 90 min before stimulation. sPLA₂ promoter activity was calculated and results are expressed as relative luciferase units (RLU) and are means ± SD (n = 3). **P < 0.01, ***P < 0.001 considered statistically significant when compared with the control values; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 when compared with the IL-1β-stimulated values.