Figure 1.
Effect of various inhibitors on secreted embryonic alkaline phosphatase (SEAP) release in monophosphoryl lipid A (MPLA) - and oxidant-induced HEK-Blue mTLR4 activation.
Cells were pre-incubated with various inhibitors for 30 min followed by incubation with MPLA (57 nM), PPC (1 µM), PDO (50 µM) or PN (1 mM) for 16 h in continued presence of the inhibitors. SEAP release in the supernatant was determined using Quanti-Blue, and the absorbance read at 650 nm. The color-coded column in each panel (A to D) represents treatment with MPLA, PPC, PDO or PN alone, respectively, in the absence of TLR4 antagonists/inhibitors. The data represent 5 independent experiments; *p≤0.01 versus treatments with MPLA alone, or [TIRAPc+MPLA]; #p≤0.001 vs treatments with PPC alone, [LPS-RS +PPC], and [TIRAPc +PPC]; +p≤0.001 vs treatments with PDO alone, [LPS-RS +PDO], or [TIRAPc+PDO]; $p≤0.001 vs treatments with PN alone, [LPS-RS +PN], or [TIRAPc +PN].
Figure 2.
Effect of anti-TLR4 pAb on secreted embryonic alkaline phosphatase (SEAP) release in MPLA and pro-oxidant-stimulated HEK-Blue mTLR4 cells.
Cells were preincubated with anti-TLR4 pAb (5 µg/ml) for 2 h followed by stimulation overnight with either MPLA (57 nM) or PPC (1 µM). Preincubation with rat polyclonal IgG (10 µg/ml) was used as control. SEAP release in the supernatant was determined using Quanti-Blue with the absorbance read at 650 nm. The data represent 3 independent experiments. *p≤0.001 vs treatments with MPLA or PPC preincubated with anti-TLR4.
Figure 3.
Effect of anti-oxidants on secreted embryonic alkaline phosphatase (SEAP) release in pro-oxidant-stimulated HEK-Blue mTLR4 cells.
Cells were pre-treated with Ebselen (50 µM) or PBN (100 µM) followed by stimulation with MPLA (57 nM), PPC (1 µM), PDO (50 µM) or PN (1 mM) for 16 h in the continued presence of the anti-oxidants. SEAP release in the supernatant was determined using Quanti-Blue, and absorbance was read at 650 nm. The color-coded column in each panel (A to D) represents treatment with MPLA, PPC, PDO or PN alone, respectively, in the absence of the anti-oxidants. [The data represent n = mean ± SEM in 6 independent experiments; *p≤0.01 compared to MPLA treatment alone; **p≤0.001 compared with MPLA treatment alone; #p≤0.001 vs treatment with PPC alone; +p≤0.001 vs treatment with PDO alone; $p≤0.001 vs treatments with PN alone].
Figure 4.
Effect of MPLA and pro-oxidants on levels of SEAP release from HEK-Blue mTLR2 cells.
Cells were stimulated with 1 µg/ml of LPS from Porphyromonas gingivalis (LPS-PG) (1 µg/ml), MPLA (57 nM), PPC (1 µM), PDO (50 µM), or PN (1 mM) for 16 h. SEAP released into the supernatant was quantified using Quanti-Blue (Fig. 4A) [n = means ± SEM from 5 independent experiments; #p≤0.01 vs treatments with LPG-PG or PPC alone; +p≤0.01 versus treatment with LPS-PG alone]. Fig. 4B - A representative qualitative representation of LPS-PG concentration-dependent changes in purple/blue color development (absorbance) indicative of increased SEAP release (i.e., enhanced with increasing NF-κB) with increasing LPS-PG concentration after Quanti-Blue reaction. HEK-Blue mTLR2 cells incubated with different concentrations of LPS-PG at 0.1, 1.0 and 10 µg/ml for 16 h were compared to treatments with different prooxidants assumed to be potential TLR2 activators.
Figure 5.
Representative immunoblots of nitrated proteins following incubation of HEK-Blue mTLR4 and HEK-Blue mTLR2 cells with an equimolar concentration (1 mM) of either PN or SIN-1 for 3 h.
Cell lysates were subjected to immunoblot using anti-nitrotyrosine. BSA nitrated at the tyrosine residue(s) was used as positive marker for protein nitration.
Figure 6.
Effect of pro-oxidants on secreted embryonic alkaline phosphatase (SEAP) release in HEK-Blue null 1-v cells.
Cells were stimulated with MPLA (57 nM), PPC (1 µM), PDO (50 µM), or PN (1 mM) for 16 h. The SEAP levels in the cell supernatant were determined using Quanti-Blue with the absorbance was read at 650 nm. The data are presented as means ± SEM from at least 5 independent experiments.
Figure 7.
Percentage (%) responses in mechanical head withdrawal threshold (MHWT) to a von Frey filament following PPC-induced inflammation in the mouse masseter muscle at d0, d0.25, d1, d2 and d7.
[n = 5–9 mice/group; #p≤0.01 compared to all test groups; **p = 0.001 compared to [EUK-134 vehicle/NS] and [EUK-134 (10 nmol)/NS] groups; *p = ≤0.01 compare to [EUK-134 (10 nmol)/NS] on d1 and d7 [2-way ANOVA (group and time)/Fisher’s PLSD post hoc test].
Figure 8.
A representative masseter TLR4 expression levels in different treatment groups (A) at d7 after treatments with EUK-134 (10 nmol/mouse), NS and/or PPC (250 pmol/mouse).
The histograms (B) represent the optical density (OD) ratios of TLR4 immunoblot signals normalized to those of β-actin from the same test groups. The numbers in parenthesis in (B) are related to the immuno-blot signals in (A) [n = 5–6 mice/group; *p ≤ 0.01 compared with [EUK-134 (10 nmol)/NS]- and [EUK-134 vehicle/NS]-treated group; +p = 0.05 versus [EUK-134 vehicle/PPC] [1-way ANOVA/Fisher’s PLSD post hoc test].
Figure 9.
Masseter muscle tissue levels of (A) TNFα and (B) IL-1β at d7 following different treatments.
Values were normalized to total tissue protein levels. [n = 5–6 mice/group; *p≤0.01, **p≤0.001 compared with [EUK-134(10 nmol/mouse)/PPC]-treated group for each cytokine.