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Anesthetic propofol reduces endotoxic inflammation by inhibiting reactive oxygen species-regulated Akt/IKKβ/NF-κB signaling.

Hsing CH, Lin MC, Choi PC, Huang WC, Kai JI, Tsai CC, Cheng YL, Hsieh CY, Wang CY, Chang YP, Chen YH, Chen CL, Lin CF - PLoS ONE (2011)

Bottom Line: Western blot analysis showed propofol inhibited LPS-induced activation and phosphorylation of IKKβ (Ser180) and nuclear factor (NF)-κB (Ser536); the subsequent nuclear translocation of NF-κB p65 was also reduced.Additionally, propofol inhibited LPS-induced Akt activation and phosphorylation (Ser473) partly by reducing reactive oxygen species (ROS) generation; inter-regulation that ROS regulated Akt followed by NF-κB activation was found to be crucial for LPS-induced inflammatory responses in macrophages.These results suggest that propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting the interconnected ROS/Akt/IKKβ/NF-κB signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Chi Mei Medical Center, Tainan, Taiwan. hsing@mail.chimei.org.tw

ABSTRACT

Background: Anesthetic propofol has immunomodulatory effects, particularly in the area of anti-inflammation. Bacterial endotoxin lipopolysaccharide (LPS) induces inflammation through toll-like receptor (TLR) 4 signaling. We investigated the molecular actions of propofol against LPS/TLR4-induced inflammatory activation in murine RAW264.7 macrophages.

Methodology/principal findings: Non-cytotoxic levels of propofol reduced LPS-induced inducible nitric oxide synthase (iNOS) and NO as determined by western blotting and the Griess reaction, respectively. Propofol also reduced the production of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 as detected by enzyme-linked immunosorbent assays. Western blot analysis showed propofol inhibited LPS-induced activation and phosphorylation of IKKβ (Ser180) and nuclear factor (NF)-κB (Ser536); the subsequent nuclear translocation of NF-κB p65 was also reduced. Additionally, propofol inhibited LPS-induced Akt activation and phosphorylation (Ser473) partly by reducing reactive oxygen species (ROS) generation; inter-regulation that ROS regulated Akt followed by NF-κB activation was found to be crucial for LPS-induced inflammatory responses in macrophages. An in vivo study using C57BL/6 mice also demonstrated the anti-inflammatory properties against LPS in peritoneal macrophages.

Conclusions/significance: These results suggest that propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting the interconnected ROS/Akt/IKKβ/NF-κB signaling pathways.

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Related in: MedlinePlus

Non-cytotoxic levels of propofol inhibit LPS-induced Akt activation, which Akt signaling is required for LPS-induced NF-κB activation as well as NO generation.RAW264.7 cells (1×106 cells/well in 6-well culture plates or 5×104 cells/well in 96-well culture plates) were treated with propofol or vehicle for 0.5 h. Next, cells were stimulated with LPS (2 µg/ml) for 6 or 24 h. (A) Western blot analysis was used to determine the phosphorylation of Akt (Ser473), p38 MAPK (Thr180/Tyr182), JNK (Thr183/Tyr185), and ERK1/2 (Thr185/Tyr187). β-actin was the internal control. The ratio of pAkt to Akt is shown. Data are representative of three individual experiments. (B) RAW264.7 cells (1×106 cells/well in 6-well culture plates) were treated with LPS (2 µg/ml) for the indicated time periods with or without LY294002 (100 µM) pre-treatment for 0.5 h. Western blot analysis was used to determine the phosphorylation of IKKβ (Ser180). β-actin was the internal control. The ratio of pIKKβ to IKKβ is shown. Data are representative of three individual experiments. (C) Meanwhile, Griess reagent was used to detect the generation of nitrite. Data, obtained from triplicate cultures, are means ± SD. One of representative data obtained from three individual experiments is shown. *p<0.05 compared to the LPS group.
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pone-0017598-g003: Non-cytotoxic levels of propofol inhibit LPS-induced Akt activation, which Akt signaling is required for LPS-induced NF-κB activation as well as NO generation.RAW264.7 cells (1×106 cells/well in 6-well culture plates or 5×104 cells/well in 96-well culture plates) were treated with propofol or vehicle for 0.5 h. Next, cells were stimulated with LPS (2 µg/ml) for 6 or 24 h. (A) Western blot analysis was used to determine the phosphorylation of Akt (Ser473), p38 MAPK (Thr180/Tyr182), JNK (Thr183/Tyr185), and ERK1/2 (Thr185/Tyr187). β-actin was the internal control. The ratio of pAkt to Akt is shown. Data are representative of three individual experiments. (B) RAW264.7 cells (1×106 cells/well in 6-well culture plates) were treated with LPS (2 µg/ml) for the indicated time periods with or without LY294002 (100 µM) pre-treatment for 0.5 h. Western blot analysis was used to determine the phosphorylation of IKKβ (Ser180). β-actin was the internal control. The ratio of pIKKβ to IKKβ is shown. Data are representative of three individual experiments. (C) Meanwhile, Griess reagent was used to detect the generation of nitrite. Data, obtained from triplicate cultures, are means ± SD. One of representative data obtained from three individual experiments is shown. *p<0.05 compared to the LPS group.

Mentions: Activation of MAPKs and Akt may act upstream of NF-κB signaling [22], [23], [24], [25], [26]. We found that propofol treatment reduced LPS-induced phosphorylation of Akt (Ser473) (0.77 with LPS only vs. 0.06 with LPS + propofol) but not ERK1/2 (Thr185/Tyr187), p38 MAPK (Thr180/Tyr182), or JNK (Thr183/Tyr185) 0.25 h after LPS treatment (Figure 3A). To confirm the effect of Akt on NF-κB activation, we demonstrated that LY294002, a PI3K inhibitor, reduced LPS-induced phosphorylation of IKKβ (Ser180) 0.25 h after LPS treatment (1.14 with LPS only vs. 0.07 with LPS + LY294002, Figure 3B). We further found that pre-treatment with LY294002 significantly (p<0.05) reduced LPS-induced upregulation of nitrite in macrophages in vitro (37.8±4.9 with LPS only vs. 7.4±0.3 with LPS + propofol, Figure 3C). Overall, these results demonstrate that treatment with propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting Akt phosphorylation and Akt-regulated NF-κB activation.


Anesthetic propofol reduces endotoxic inflammation by inhibiting reactive oxygen species-regulated Akt/IKKβ/NF-κB signaling.

Hsing CH, Lin MC, Choi PC, Huang WC, Kai JI, Tsai CC, Cheng YL, Hsieh CY, Wang CY, Chang YP, Chen YH, Chen CL, Lin CF - PLoS ONE (2011)

Non-cytotoxic levels of propofol inhibit LPS-induced Akt activation, which Akt signaling is required for LPS-induced NF-κB activation as well as NO generation.RAW264.7 cells (1×106 cells/well in 6-well culture plates or 5×104 cells/well in 96-well culture plates) were treated with propofol or vehicle for 0.5 h. Next, cells were stimulated with LPS (2 µg/ml) for 6 or 24 h. (A) Western blot analysis was used to determine the phosphorylation of Akt (Ser473), p38 MAPK (Thr180/Tyr182), JNK (Thr183/Tyr185), and ERK1/2 (Thr185/Tyr187). β-actin was the internal control. The ratio of pAkt to Akt is shown. Data are representative of three individual experiments. (B) RAW264.7 cells (1×106 cells/well in 6-well culture plates) were treated with LPS (2 µg/ml) for the indicated time periods with or without LY294002 (100 µM) pre-treatment for 0.5 h. Western blot analysis was used to determine the phosphorylation of IKKβ (Ser180). β-actin was the internal control. The ratio of pIKKβ to IKKβ is shown. Data are representative of three individual experiments. (C) Meanwhile, Griess reagent was used to detect the generation of nitrite. Data, obtained from triplicate cultures, are means ± SD. One of representative data obtained from three individual experiments is shown. *p<0.05 compared to the LPS group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3050912&req=5

pone-0017598-g003: Non-cytotoxic levels of propofol inhibit LPS-induced Akt activation, which Akt signaling is required for LPS-induced NF-κB activation as well as NO generation.RAW264.7 cells (1×106 cells/well in 6-well culture plates or 5×104 cells/well in 96-well culture plates) were treated with propofol or vehicle for 0.5 h. Next, cells were stimulated with LPS (2 µg/ml) for 6 or 24 h. (A) Western blot analysis was used to determine the phosphorylation of Akt (Ser473), p38 MAPK (Thr180/Tyr182), JNK (Thr183/Tyr185), and ERK1/2 (Thr185/Tyr187). β-actin was the internal control. The ratio of pAkt to Akt is shown. Data are representative of three individual experiments. (B) RAW264.7 cells (1×106 cells/well in 6-well culture plates) were treated with LPS (2 µg/ml) for the indicated time periods with or without LY294002 (100 µM) pre-treatment for 0.5 h. Western blot analysis was used to determine the phosphorylation of IKKβ (Ser180). β-actin was the internal control. The ratio of pIKKβ to IKKβ is shown. Data are representative of three individual experiments. (C) Meanwhile, Griess reagent was used to detect the generation of nitrite. Data, obtained from triplicate cultures, are means ± SD. One of representative data obtained from three individual experiments is shown. *p<0.05 compared to the LPS group.
Mentions: Activation of MAPKs and Akt may act upstream of NF-κB signaling [22], [23], [24], [25], [26]. We found that propofol treatment reduced LPS-induced phosphorylation of Akt (Ser473) (0.77 with LPS only vs. 0.06 with LPS + propofol) but not ERK1/2 (Thr185/Tyr187), p38 MAPK (Thr180/Tyr182), or JNK (Thr183/Tyr185) 0.25 h after LPS treatment (Figure 3A). To confirm the effect of Akt on NF-κB activation, we demonstrated that LY294002, a PI3K inhibitor, reduced LPS-induced phosphorylation of IKKβ (Ser180) 0.25 h after LPS treatment (1.14 with LPS only vs. 0.07 with LPS + LY294002, Figure 3B). We further found that pre-treatment with LY294002 significantly (p<0.05) reduced LPS-induced upregulation of nitrite in macrophages in vitro (37.8±4.9 with LPS only vs. 7.4±0.3 with LPS + propofol, Figure 3C). Overall, these results demonstrate that treatment with propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting Akt phosphorylation and Akt-regulated NF-κB activation.

Bottom Line: Western blot analysis showed propofol inhibited LPS-induced activation and phosphorylation of IKKβ (Ser180) and nuclear factor (NF)-κB (Ser536); the subsequent nuclear translocation of NF-κB p65 was also reduced.Additionally, propofol inhibited LPS-induced Akt activation and phosphorylation (Ser473) partly by reducing reactive oxygen species (ROS) generation; inter-regulation that ROS regulated Akt followed by NF-κB activation was found to be crucial for LPS-induced inflammatory responses in macrophages.These results suggest that propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting the interconnected ROS/Akt/IKKβ/NF-κB signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Chi Mei Medical Center, Tainan, Taiwan. hsing@mail.chimei.org.tw

ABSTRACT

Background: Anesthetic propofol has immunomodulatory effects, particularly in the area of anti-inflammation. Bacterial endotoxin lipopolysaccharide (LPS) induces inflammation through toll-like receptor (TLR) 4 signaling. We investigated the molecular actions of propofol against LPS/TLR4-induced inflammatory activation in murine RAW264.7 macrophages.

Methodology/principal findings: Non-cytotoxic levels of propofol reduced LPS-induced inducible nitric oxide synthase (iNOS) and NO as determined by western blotting and the Griess reaction, respectively. Propofol also reduced the production of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 as detected by enzyme-linked immunosorbent assays. Western blot analysis showed propofol inhibited LPS-induced activation and phosphorylation of IKKβ (Ser180) and nuclear factor (NF)-κB (Ser536); the subsequent nuclear translocation of NF-κB p65 was also reduced. Additionally, propofol inhibited LPS-induced Akt activation and phosphorylation (Ser473) partly by reducing reactive oxygen species (ROS) generation; inter-regulation that ROS regulated Akt followed by NF-κB activation was found to be crucial for LPS-induced inflammatory responses in macrophages. An in vivo study using C57BL/6 mice also demonstrated the anti-inflammatory properties against LPS in peritoneal macrophages.

Conclusions/significance: These results suggest that propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting the interconnected ROS/Akt/IKKβ/NF-κB signaling pathways.

Show MeSH
Related in: MedlinePlus