Figure 1.
Inhibition of TNF-α-induced IL-6 expression by 5-LOX inhibitors in human synovial fibroblasts.
Human synovial fibroblasts were incubated with TNF-α (10 ng/ml) for indicated time intervals. mRNA and released IL-6 were determined by QPCR and ELISA respectively. Treatment with TNF-α enhanced the IL-6 mRNA (A) expression and cytokine release (B) in a time-dependent manner. Pretreatment with two 5-LOX inhibitors, NDGA (5 or 10 µM) or MK-886 (5 µM) for 1 hr significantly inhibited the TNF-α-induced mRNA (C) or protein levels (D) of IL-6. Data are presented as mean ± SEM. *, p<0.05 compared with vehicle control (con). #, p<0.05 compared with TNF-α treatment alone.
Figure 2.
Attenuation of TNFα-induced MCP-1/CCL-2 expression by 5-LOX inhibitors in human synovial fibroblasts.
Human synovial fibroblasts were pretreated with NDGA (10 µM) or MK-886 (5 µM) for 1 hr, and then treated with TNF-α for another 6 hr. Cell lysate was extracted for real-time PCR evaluation (A) and conditioned medium was collected for ELISA measurement of MCP-1 (B). Note that 5-LOX inhibitors NDGA and MK-886 antagonized TNF-α-induced mRNA and protein levels of MCP-1/CCL-2. Data are presented as mean ± SEM. *, p<0.05 compared with control (con). #, p<0.05 compared with TNF-α-treatment alone.
Figure 3.
Involvement of leukotriene B4 in TNF-α-induced cytokine expression in human synovial fibroblasts.
Human synovial fibroblasts were pretreated with LTB4 receptor antagonist LY29311 (1 µM) for 1 hr, and then treated with TNF-α for another 6 hr. TNF-α-induced up-regulation of IL-6 and MCP-1 were antagonized by LY29311 (A, B). RASFs were treated with TNF-α (10 ng/ml) for 4 and 12 hr, and the conditioned medium was collected for ELISA measurement of LTB4. Data show that LTB4 was increased at 4 hr (C). (D) Human synovial fibroblasts were treated with leukotriene B4 for 6 hrs at different concentrations, and conditioned medium was collected for ELISA assay. ELISA analysis showed that treatment with leukotriene B4 enhanced IL-6 release in a concentration-dependent manner. Data are presented as mean ± SEM. *, p<0.05 compared with control (con). #, p<0.05 compared with TNF-α-treatment alone.
Figure 4.
Knockdown of 5-LOX inhibits TNF-α-induced IL-6 and MCP-1 release in human synovial fibroblasts.
Total protein extracts from RASFs transfected with empty vector or 5-LOX shRNA were used for Western blotting. (A) Immunoblotting showed that cells transfected with 5-LOX shRNA of clone No.3 and No.7 markedly decreased 5-LOX expression. Human synovial fibroblasts transfected with empty vector or 5-LOX shRNA were treated with TNF-α for 6 hr. IL-6 and MCP-1 protein release in the conditioned medium was evaluated by ELISA. Note that knockdown of 5-LOX decreased IL-6 (B) and MCP-1 (C) release following TNF-α treatment for 6 hr. Data are presented as mean ± SEM. *, p<0.05 compared with empty vector control (con).
Figure 5.
5-LOX inhibitors antagonize TNF-α-induced IKKα/β activation, IκBα phosphorylation, IκBα degradation and NF-κB nuclear translocation in human synovial fibroblasts.
(A) Immunoblotting showed that treatment of TNF-α (10 ng/ml) enhanced the phosphorylation of IκBα time-dependently in human synovial fibroblasts. (B) Human synovial fibroblasts were pre-incubated with 5-LOX inhibitors NDGA (10 µM) or MK-886 (5 µM) for 1 hr and then exposed to TNF-α for another 10 min. Note that pretreatment with NDGA or MK-886 could antagonize TNF-α-induced effects. (C) Human synovial fibroblasts were pretreated with NDGA (10 µM) or MK-886 (5 µM) for 1 hr. TNF-α (10 ng/ml) was then added for another 30 min. Cytosolic and nuclear extracts were separated by NE-PER kit. Note that NDGA or MK-886 significantly antagonized the nuclear translocation of NF-κB subunits of p65 and p50. C23 was used as nucleus marker.
Figure 6.
5-LOX inhibitors antagonize TNF-α-induced nuclear translocation of p65.
Immunofluorescent staining showed that p65 translocated into nucleus after treatment of TNF-α (25 ng/ml) for 30 min. Pretreatment of 5-LOX inhibitors NDGA (10 µM) or MK-886 (5 µM) antagonized the nuclear translocation of p65. DAPI staining was used to indicate the location of nucleus. Scale: 5 µm.
Figure 7.
Antagonism by 5-LOX inhibitors on TNF-α-induced up-regulation of serum monocyte chemo-attractant protein-1 (MCP-1) level and paw edema in mice.
(A) Male C57BL/6 mice (8 weeks-old) were injected with TNF-α (40 µg/kg in 0.2 ml saline) into femoral vein and 5-LOX inhibitor NDGA (10 mg/kg) was co-treated in another cohort. After 6 hr, mice were sacrificed and serum was collected for ELISA analysis. Note that mice injected with TNF-α markedly increased MCP-1 levels in serum, which was inhibited by co-treatment with 5-LOX inhibitor NDGA (10 mg/kg) (n = 4–5). (B) TNF-α was intraplantarly injected onto the paw of male C57BL/6 mice (8 weeks-old). 5-LOX inhibitors, NDGA (10 µM) or MK-886 (10 µM) was co-injected with TNF-α (10 µl). It was found that TNF-α induced acute paw edema and increased paw thickness during 4–6 hr. Note that co-administration of NDGA or MK-886 significantly attenuated TNF-α-induced paw edema (n = 5). Data are presented as mean ± SEM. *, p<0.05 compared with vehicle control. #, p<0.05 compared with TNF-α-treatment alone.
Figure 8.
Schematic diagram of the involvement of 5-LOX in TNF-α-induced cytokine/chemokine release.
TNF-α increased the release of IL-6 and MCP-1 via the activation of NF-κB signaling in RASFs. 5-LOX inhibitors, NDGA or MK886 inhibited TNF-α-induced activation of IKK and IκBα degradation. 5-LOX inhibitors also inhibited the translocation of NF-κB subunits of p50 and p65 into nucleus (response element) and then decreased TNF-α-induced IL-6 and MCP-1 release. Knockdown of 5-LOX by RNAi exerted similar inhibitory effects.