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

Xing B, Xin T, Hunter RL, Bing G - J Neuroinflammation (2008)

Bottom Line: 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.Furthermore, wortmannin prevented the inhibitory effect of pioglitazone on the LPS-induced NO increase.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Neurobiology, 310 Davis Mills Building, University of Kentucky, Chandler Medical Center, 800 Rose Street, Lexington, KY 40536-0298, USA. bxing3@email.uky.edu

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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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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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).

Mentions: To determine if pioglitazone enhances PI3K/Akt expression and if its inhibition enhances LPS-induced NO generation, the levels of PI3K and Akt were determined. PPAR-γ, PI3K, and Akt phosphorylation were measured after LPS (1 μg/ml) exposure. As shown in Fig 5, PPAR-γ activation was observed in pioglitazone-treated cultures within 10 min after DMSO or LPS. PI3K and phosphorylated Akt were increased 60 min after LPS in the pioglitazone-treated cultures (Fig. 5, p < 0.05). Next, wortmannin (1 μM) was added 30 mins before pioglitazone (10 μM) treatment and the NO level was measured 48 h after LPS (1 μg/ml). The results showed that pretreatment with pioglitazone inhibited the LPS-induced NO increase (p < 0.01). However, when wortmannin was given 30 mins before pioglitazone, NO production was increased over LPS exposure (p < 0.05). Interestingly, administration of wortmannin (1 μM) 30 min before pioglitazone followed by LPS 1 hr later did not show the inhibitive effect of pioglitazone on NO level. Wortmannin alone, or together with pioglitazone, did not influence NO generation without LPS stimulation. Thus, pioglitazone prevents LPS-induced NO production, and pretreatment with wortmannin increases NO generation (Fig 6).


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

Xing B, Xin T, Hunter RL, Bing G - J Neuroinflammation (2008)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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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).
Mentions: To determine if pioglitazone enhances PI3K/Akt expression and if its inhibition enhances LPS-induced NO generation, the levels of PI3K and Akt were determined. PPAR-γ, PI3K, and Akt phosphorylation were measured after LPS (1 μg/ml) exposure. As shown in Fig 5, PPAR-γ activation was observed in pioglitazone-treated cultures within 10 min after DMSO or LPS. PI3K and phosphorylated Akt were increased 60 min after LPS in the pioglitazone-treated cultures (Fig. 5, p < 0.05). Next, wortmannin (1 μM) was added 30 mins before pioglitazone (10 μM) treatment and the NO level was measured 48 h after LPS (1 μg/ml). The results showed that pretreatment with pioglitazone inhibited the LPS-induced NO increase (p < 0.01). However, when wortmannin was given 30 mins before pioglitazone, NO production was increased over LPS exposure (p < 0.05). Interestingly, administration of wortmannin (1 μM) 30 min before pioglitazone followed by LPS 1 hr later did not show the inhibitive effect of pioglitazone on NO level. Wortmannin alone, or together with pioglitazone, did not influence NO generation without LPS stimulation. Thus, pioglitazone prevents LPS-induced NO production, and pretreatment with wortmannin increases NO generation (Fig 6).

Bottom Line: 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.Furthermore, wortmannin prevented the inhibitory effect of pioglitazone on the LPS-induced NO increase.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Neurobiology, 310 Davis Mills Building, University of Kentucky, Chandler Medical Center, 800 Rose Street, Lexington, KY 40536-0298, USA. bxing3@email.uky.edu

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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