Figure 6.

Group V secretory phospholipase A₂ (sPLA₂V) promotes the aggressive properties of rheumatoid synovial fibroblasts (RASFs) via endothelial protein C receptor (EPCR). RASFs were transfected with control or sPLA₂V small interfering RNA (siRNA). (A) RASF viability after siRNA transfection for 72 hours, detected by colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and confirmed by trypan blue exclusion assay. (B) Cartilage degradation by RASFs. After siRNA transfection for 24 hours, medium was replaced with Dulbecco’s modified Eagle’s medium (DMEM) without fetal bovine serum (FBS), and osteoarthritis (OA) cartilage explants were incubated with RASFs for a further 24 hours. Media were collected for detection of sulphated glycosaminoglycans (sGAGs) by 1,9-dimethylmethylene blue (DMMB) assay. (C) Interleukin-1-beta (IL-1β) protein in the culture media of RASFs transfected with control or sPLA₂V siRNA. After siRNA transfection for 24 hours, medium was replaced with DMEM without FBS, RASFs were incubated for a further 24 hours, and media were collected for enzyme-linked immunosorbent assay (ELISA). (D) Cartilage degradation ability of RASFs in response to recombinant sPLA₂V (50, 100, and 500 ng/mL) for 24 hours, measured by DMMB assay. (E) Cartilage-degradative ability of RASFs transfected with EPCR siRNA for 48 hours and recombinant sPLA₂V (100 ng/mL) for 24 hours, measured by DMMB assay. (F) NF-κB activation in whole cell lysates of RASFs transfected with EPCR siRNA for 48 hours and recombinant sPLA₂V (100 ng/mL) for 24 hours and semi-quantified by image analysis software in comparison with β-actin. The images represent one of three experiments. Data on graph are shown as mean ± standard deviation (SD) of three or four different RASF cell lines. Data were analyzed by Student t test (A-C) by one-way analysis of variance (D-F), followed by Tukey’s HSD post hoc test. *P <0.05, **P <0.01 when compared with relevant control.