TNF-α and IL-6, but not IL-1β, formed a positive-feedback circuit with ROS generation upon PQ stimulus.

<p>(A) TNF-α, IL-1β, and IL-6 production levels were remarkably promoted by PQ and significantly decreased by DPI. (B) PQ-induced ROS generation was decreased by preincubation with TNF-α or IL-6 antibody respectively, but not by IL-1β, which indicated a positive-feedback circuit between the regulation of ROS generation and TNF-α and IL-6 production under PQ stimulus. ROS contents were normalized to control. *<i>P</i><0.05.</p

ROS were involved in PQ-induced neutrophil apoptosis delay.

<p>(A) ROS generation was assessed with DCFH-DA fluorescence intensity and was remarkably increased by all PQ concentration (5, 50, and 100 μM) at all time points (6, 12, 18, and 24 h) in a concentration-independent manner. (B) DPI or apocynin pretreatment suppressed PQ-induced ROS generation. (C) PQ-induced neutrophil apoptosis was also attenuated by DPI or apocynin pretreatment. ROS contents were normalized to control. *<i>P</i><0.05.</p

p38 MAPK and NF-κB signaling pathways, but not Akt, were involved in PQ-induced ROS generation and reduced neutrophil apoptosis.

<p>(A, B) p-p38 MAPK and p-Mcl-1 production were predominantly induced by PQ and suppressed by DPI co-treatment. The specific p38 MAPK inhibitor SB203580 blocked both p-p38MAPK and p-Mcl-1 productions, whereas SC200137 suppressed p-Mcl-1 but not p-p38MAPK. (C) The Akt pathway was not affected by PQ. (D, E) Both p-IκBα and p-P65 were activated by PQ pretreatment and decreased by DPI. (F) PQ-mediated reduction in neutrophil apoptosis could be rescued by SB203580, SC200137, and PDTC but not SC221226, which indicated that p38MAPK, Mcl-1, and NF-κB pathways were involved in the PQ-regulated decrease in neutrophil apoptosis. *<i>P</i><0.05.</p

PQ delayed neutrophil apoptosis in a concentration-independent manner.

<p>Neutrophil apoptosis was assessed with annexin V-FITC and PI staining followed by flow cytometry. Compared with the control, the doses of PQ (5, 50, or 100 μM) significantly attenuated neutrophil apoptosis at all time points after treatment (6, 12, 18, and 24 h). No difference was found between different concentration groups. *<i>P</i><0.05 compared with control at the same time point.</p

Repressive effects of TET on the expression of COX-1 and COX-2 in LPS-induced hyperalgesia.

<p>(A) Western blotting of COX-1 and COX-2 in brain tissues and the quantified comparison of relative densities are shown. (B) COX-1 and COX-2 mRNA expression were tested by qRT-PCR in brain tissues. (C) COX-1 and COX-2 mRNA expression were tested by qRT-PCR in cultured astroglia. Values are shown as M±SD, and normalized to the NS groups. *, <i>P</i><0.05.</p

Signaling targets of LPS and TET.

<p>Small RNA-interfering (si) experiments with siIKKα, siIKKβ, and control siRNA were performed to investigate the specific targets of LPS and TET in astroglia. (A) Both LPS and TET initially target the phosphorylation of IKKβ, but not that of IKKα. (B) Both siIKKβ and TET decrease COX-2 generation, but not siIKKα. Values are shown as M±SD, and normalized to the LPS(-)siRNA(-)TET(-) groups. *, <i>P</i><0.05.</p

Repressive effects of TET on PGE₂ levels in sera, brain tissues, and cultured astroglia.

<p>(A, B) PGE₂ levels in the sera and brain tissues, respectively, were significantly increased after LPS stimulation and markedly suppressed by TET. (C) MTT analysis of astroglia viability <i>in vitro</i> in the presence of different concentrations of TET. (D) PGE₂ levels in LPS-treated astroglia were suppressed by TET in a dose-dependent manner. Values are shown as M±SD. *, <i>P</i><0.05.</p

Induction of hyperalgesia by LPS in BALB/C mice.

<p>(A) Molecular structure of TET. (B) Latency time of hind-paw licking in the hot-plate test in mice that were treated with LPS at different concentrations and time points. (C) Writhing counts obtained from the acetic acid-induced abdominal constriction test in mice that were treated with LPS at different concentrations and time points. (D and E) Percentages of protection by TET (15, 30, 45 mg/kg) at 6 h after LPS (100 μg/kg) stimulus, as indicated by the hot-plate test (D) or acetic acid-induced abdominal constriction test (E). Indomethacin (5 mg/kg) and morphine (10 mg/kg) were applied as the positive controls. Values are shown as M±SD. *, <i>P</i><0.05.</p

Regulatory effects of TET on the NF-κB pathway in LPS-induced cultured astroglia.

<p>(A) The expression of P65, pP65, IκBα, and pIκBα in the presence or absence of TET and LPS was assessed by western blotting, and the quantified comparisons of relative densities are shown. (B) The protein expression of IKKs in the presence or absence of TET and LPS was assessed by western blotting, and the quantified relative densities. Values are shown as M±SD, and normalized to the NS groups. *, <i>P</i><0.05.</p