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. 2008 Jan 18:5:4.
doi: 10.1186/1742-2094-5-4.

Pioglitazone inhibition of lipopolysaccharide-induced nitric oxide synthase is associated with altered activity of p38 MAP kinase and PI3K/Akt

Bin Xing  1 , Tao Xin, Randy Lee Hunter, Guoying Bing
Affiliations

Pioglitazone inhibition of lipopolysaccharide-induced nitric oxide synthase is associated with altered activity of p38 MAP kinase and PI3K/Akt

Bin Xing et al. J Neuroinflammation. .

Abstract

Background: Previous studies have suggested that peroxisome proliferator activated receptor-gamma (PPAR-gamma)-mediated neuroprotection involves inhibition of microglial activation and decreased expression and activity of inducible nitric oxide synthase (iNOS); however, the underlying molecular mechanisms have not yet been well established. In the present study we explored: (1) the effect of the PPAR-gamma agonist pioglitazone on lipopolysaccharide (LPS)-induced iNOS activity and nitric oxide (NO) generation by microglia; (2) the differential role of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun NH(2)-terminal kinase (JNK), and phosphoinositide 3-kinase (PI3K) on LPS-induced NO generation; and (3) the regulation of p38 MAPK, JNK, and PI3K by pioglitazone.

Methods: Mesencephalic neuron-microglia mixed cultures, and microglia-enriched cultures were treated with pioglitazone and/or LPS. The protein levels of iNOS, p38 MAPK, JNK, PPAR-gamma, PI3K, and protein kinase B (Akt) were measured by western blot. Different specific inhibitors of iNOS, p38MAPK, JNK, PI3K, and Akt were used in our experiment, and NO generation was measured using a nitrite oxide assay kit. Tyrosine hydroxylase (TH)-positive neurons were counted in mesencephalic neuron-microglia mixed cultures.

Results: Our results showed that pioglitazone inhibits LPS-induced iNOS expression and NO generation, and inhibition of iNOS is sufficient to protect dopaminergic neurons against LPS insult. In addition, inhibition of p38 MAPK, but not JNK, prevented LPS-induced NO generation. Further, and of interest, pioglitazone inhibited LPS-induced phosphorylation of p38 MAPK. Wortmannin, a specific PI3K inhibitor, enhanced p38 MAPK phosphorylation upon LPS stimulation of microglia. Elevations of phosphorylated PPAR-gamma, PI3K, and Akt levels were observed with pioglitazone treatment, and inhibition of PI3K activity enhanced LPS-induced NO production. Furthermore, wortmannin prevented the inhibitory effect of pioglitazone on the LPS-induced NO increase.

Conclusion: We demonstrate that pioglitazone protects dopaminergic neurons against LPS insult at least via inhibiting iNOS expression and NO generation, which is potentially mediated via inhibition of p38 MAPK activity. In addition, the PI3K pathway actively participates in the negative regulation of LPS-induced NO production. Our findings suggest that PPAR-gamma activation may involve differential regulation of p38 MAPK and of the PI3K/Akt pathway in the regulation of the inflammatory process.

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Figures

Figure 1
Figure 1
Pioglitazone inhibits LPS-induced NO production in microglia-enriched cultures. Microglia cultures were treated with pioglitazone (1 μM and 10 μM) 1 hr before LPS treatment, and 48 hrs later NO levels were measured. LPS significantly induced NO generation, and pretreatment with pioglitazone inhibited this LPS-induced NO production in a dose-dependent manner. Data presented are representative of three independent experiments (n = 3). (***p < 0.001 vs. control ###p < 0.001 vs. LPS)
Figure 2
Figure 2
Pioglitazone inhibits LPS-induced iNOS expression, and iNOS inhibition protects dopaminergic neurons from LPS insults. Rat mesencephalic mixed cultures were treated with 1 μg/ml LPS for 48 hours. A: LPS treatment upregulated the expression of iNOS, and pretreatment with pioglitazone (10 μM), 1 hr before LPS, prevents its expression. B: Rat mesencephalic mixed cultures were treated with the selective iNOS inhibitor 1400 W, with different doses from 1 ng/ml to 10 μM/ml, 1 hr before a 72 hr LPS exposure. The number of TH-positive neurons was determined by immunocytochemistry. Data presented are representative of three independent experiments (n = 3). (**p < 0.01 vs. control, ***p < 0.001 vs. control, #p < 0.05 vs. LPS, ##p < 0.01 vs. LPS, ###p < 0.001 vs. LPS).
Figure 3
Figure 3
Inhibition of NO by pioglitazone is related to inhibition of p38 MAPK activity. A: A selective p38 MAPK inhibitor (SB203580), or a selective JNK inhibitor (SP600125), was added to microglia-enriched cultures 1 hr before LPS (1 μg/ml) exposure and, after 24 hrs, NO levels were measured. Only the p38 MAPK inhibitor prevented NO production. B: Pretreatment with pioglitazone inhibited LPS-induced phosphorylation of p38 MAPK in mesencephalic neuronal-microglia mixed cultures. Pioglitazone was added 1 hr before LPS treatment (1 μg/ml) and, after 30 mins, p38 MAPK was immunobloted. As shown in 3B, LPS increased phosphorylation of p38 MAPK, and pretreatment with pioglitazone inhibited this expression. Data presented are representative of three independent experiments (n = 3). (*p < 0.05 vs. control, ***p < 0.001 vs. control, #p < 0.05 vs. LPS).
Figure 4
Figure 4
Inhibition of PI3K activity increases LPS-induced p38 MAPK activity. Wortmannin (1 μM and 10 μM) was administered to mesencephalic neuronal-microglia mixed cultures before LPS (1 μg/ml) was added and, after 30 mins, p38 MAPK was immunobloted. As shown in 4A, wortmannin enhances the phosphorylation of p38 MAPK under LPS stimulation in a dose-dependent manner (Fig 4A and 4B. p < 0.05), however, wortmannin did not increase p38 phosphorylation without LPS stimulation (Fig 4D). In contrast, inhibition of PI3K activity by wortmannin did not change JNK expression (Fig 4C). Data presented are representative of three independent experiments (n = 3). (*p < 0.05 vs. wortmannin 1 μM + LPS).
Figure 5
Figure 5
Pioglitazone activates PPAR-γ and enhances PI3K/Akt activity. Rat mesencephalic cultures (2 × 106 cells/well) were treated with pioglitazone (10 μM) only, or 1 hr before LPS (1 μg/ml) exposure. PPAR-γ activation was assessed after 10 min, and P13K and Akt were assessed after 60 min. PPAR-γ activation, PI3K and Akt expression were observed in the pioglitazone-treated cultures, compared to control and LPS-only groups. Data presented are representative of three independent experiments (n = 3). (*p < 0.05 vs. control, #p < 0.05 vs. LPS, ##p < 0.01 vs. LPS).
Figure 6
Figure 6
PI3K negatively regulates the LPS-induced increase in NO production. The specific PI3K inhibitor wortmannin (1 μM) was administered individually 90 mins before LPS treatment (1 μg/ml), or 30 mins before pioglitazone followed by LPS 60 mins later in microglia-enriched culture, and after 48 hrs NO levels were measured. The results show that the LPS-induced NO level was significantly higher than control (p < 0.01), and that pretreatment with pioglitazone inhibits LPS-induced NO (p < 0.01). In contrast, pretreatment with wortmannin enhanced the LPS-induced increase in NO generation (p < 0.05), and this pretreatment prevented the inhibitory effect of pioglitazone on LPS-induced NO generation. Data presented are representative of three independent experiments (n = 3).). (**p < 0.01 vs. control, #p < 0.05 vs. LPS, ##p < 0.01 vs. LPS, &&p < 0.01 vs. Piog plus LPS).
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