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Punicalagin Induces Nrf2/HO-1 Expression via Upregulation of PI3K/AKT Pathway and Inhibits LPS-Induced Oxidative Stress in RAW264.7 Macrophages.

Xu X, Li H, Hou X, Li D, He S, Wan C, Yin P, Liu M, Liu F, Xu J - Mediators Inflamm. (2015)

Bottom Line: However, the potential antioxidant effects of PUN in macrophages remain unclear.Here, LY294002, a specific PI3K/Akt inhibitor, suppressed PUN-induced HO-1 expression and led to ROS accumulation in macrophages.Furthermore, PUN inhibited LPS-induced oxidative stress in macrophages by reducing ROS and NO generation and increasing superoxide dismutase (SOD) 1 mRNA expression.

View Article: PubMed Central - PubMed

Affiliation: CAU-BUA TCVM Teaching and Researching Team, College of Veterinary Medicine, China Agricultural University (CAU), No. 2 West Yuanmingyuan Road, Beijing 100193, China.

ABSTRACT
Reactive oxygen species (ROS) and oxidative stress are thought to play a central role in potentiating macrophage activation, causing excessive inflammation, tissue damage, and sepsis. Recently, we have shown that punicalagin (PUN) exhibits anti-inflammatory activity in LPS-stimulated macrophages. However, the potential antioxidant effects of PUN in macrophages remain unclear. Revealing these effects will help understand the mechanism underlying its ability to inhibit excessive macrophage activation. Hemeoxygenase-1 (HO-1) exhibits antioxidant activity in macrophages. Therefore, we hypothesized that HO-1 is a potential target of PUN and tried to reveal its antioxidant mechanism. Here, PUN treatment increased HO-1 expression together with its upstream mediator nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). However, specific inhibition of Nrf2 by brusatol (a specific Nrf2 inhibitor) dramatically blocked PUN-induced HO-1 expression. Previous research has demonstrated that the PI3K/Akt pathway plays a critical role in modulating Nrf2/HO-1 protein expression as an upstream signaling molecule. Here, LY294002, a specific PI3K/Akt inhibitor, suppressed PUN-induced HO-1 expression and led to ROS accumulation in macrophages. Furthermore, PUN inhibited LPS-induced oxidative stress in macrophages by reducing ROS and NO generation and increasing superoxide dismutase (SOD) 1 mRNA expression. These findings provide new perspectives for novel therapeutic approaches using antioxidant medicines and compounds against oxidative stress and excessive inflammatory diseases including tissue damage, sepsis, and endotoxemic shock.

No MeSH data available.


Related in: MedlinePlus

PUN induced HO-1 protein expression via PI3K/Akt pathway. Cells were incubated at 37°C in a humidified incubator with 5% CO2. (a) RAW264.7 cells were treated with 100 μM PUN for indicated durations (0, 2, 4, 6, 8, and 12 h). The Akt protein and Akt phosphorylation level were analyzed by Western blotting. (b) RAW264.7 cells were treated for 8 h with 100 μM PUN in the presence or absence of 20 μM LY294002. The HO-1 and Nrf2 proteins expression were analyzed by Western blotting. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ∗∗P < 0.01 and ##P < 0.01 indicate significant differences compared with the PUN-treated group of indicated proteins, respectively.
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fig3: PUN induced HO-1 protein expression via PI3K/Akt pathway. Cells were incubated at 37°C in a humidified incubator with 5% CO2. (a) RAW264.7 cells were treated with 100 μM PUN for indicated durations (0, 2, 4, 6, 8, and 12 h). The Akt protein and Akt phosphorylation level were analyzed by Western blotting. (b) RAW264.7 cells were treated for 8 h with 100 μM PUN in the presence or absence of 20 μM LY294002. The HO-1 and Nrf2 proteins expression were analyzed by Western blotting. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ∗∗P < 0.01 and ##P < 0.01 indicate significant differences compared with the PUN-treated group of indicated proteins, respectively.

Mentions: Recent studies have demonstrated that the PI3K/Akt pathway acts as an important upstream regulator of HO-1 expression [25]; thus we investigated whether the PI3K/Akt pathway also plays a central role in the PUN-induced HO-1 expression. Western blot analysis showed that PUN treatment notably enhanced Akt phosphorylation after 2 h of treatment in a time-dependent manner, but not the total Akt protein level, suggesting that the PUN-induced HO-1 expression may be associated with the PI3K/Akt signaling pathway (Figure 3(a)). To further reveal the mechanism, we used LY294002, a specific PI3K/Akt inhibitor. The results showed that PUN treatment significantly increased HO-1 expression; however, inhibiting Akt phosphorylation by LY294002 significantly attenuated the PUN-induced HO-1 activation (Figure 3(b)). Therefore, we concluded that PI3K/Akt is essential for the PUN antioxidant activity by regulating PUN-induced HO-1 expression. LPS treatment triggers ROS generation in macrophages, so we used LPS to determine whether PI3K/Akt signaling regulates the ROS scavenging property of PUN. As expected, both fluorescence staining and assay values demonstrated that the PI3K/Akt pathway regulates the ability of PUN to scavenge ROS (Figure 4), which is in accordance with its effect on HO-1 expression.


Punicalagin Induces Nrf2/HO-1 Expression via Upregulation of PI3K/AKT Pathway and Inhibits LPS-Induced Oxidative Stress in RAW264.7 Macrophages.

Xu X, Li H, Hou X, Li D, He S, Wan C, Yin P, Liu M, Liu F, Xu J - Mediators Inflamm. (2015)

PUN induced HO-1 protein expression via PI3K/Akt pathway. Cells were incubated at 37°C in a humidified incubator with 5% CO2. (a) RAW264.7 cells were treated with 100 μM PUN for indicated durations (0, 2, 4, 6, 8, and 12 h). The Akt protein and Akt phosphorylation level were analyzed by Western blotting. (b) RAW264.7 cells were treated for 8 h with 100 μM PUN in the presence or absence of 20 μM LY294002. The HO-1 and Nrf2 proteins expression were analyzed by Western blotting. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ∗∗P < 0.01 and ##P < 0.01 indicate significant differences compared with the PUN-treated group of indicated proteins, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4417599&req=5

fig3: PUN induced HO-1 protein expression via PI3K/Akt pathway. Cells were incubated at 37°C in a humidified incubator with 5% CO2. (a) RAW264.7 cells were treated with 100 μM PUN for indicated durations (0, 2, 4, 6, 8, and 12 h). The Akt protein and Akt phosphorylation level were analyzed by Western blotting. (b) RAW264.7 cells were treated for 8 h with 100 μM PUN in the presence or absence of 20 μM LY294002. The HO-1 and Nrf2 proteins expression were analyzed by Western blotting. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. ∗∗P < 0.01 and ##P < 0.01 indicate significant differences compared with the PUN-treated group of indicated proteins, respectively.
Mentions: Recent studies have demonstrated that the PI3K/Akt pathway acts as an important upstream regulator of HO-1 expression [25]; thus we investigated whether the PI3K/Akt pathway also plays a central role in the PUN-induced HO-1 expression. Western blot analysis showed that PUN treatment notably enhanced Akt phosphorylation after 2 h of treatment in a time-dependent manner, but not the total Akt protein level, suggesting that the PUN-induced HO-1 expression may be associated with the PI3K/Akt signaling pathway (Figure 3(a)). To further reveal the mechanism, we used LY294002, a specific PI3K/Akt inhibitor. The results showed that PUN treatment significantly increased HO-1 expression; however, inhibiting Akt phosphorylation by LY294002 significantly attenuated the PUN-induced HO-1 activation (Figure 3(b)). Therefore, we concluded that PI3K/Akt is essential for the PUN antioxidant activity by regulating PUN-induced HO-1 expression. LPS treatment triggers ROS generation in macrophages, so we used LPS to determine whether PI3K/Akt signaling regulates the ROS scavenging property of PUN. As expected, both fluorescence staining and assay values demonstrated that the PI3K/Akt pathway regulates the ability of PUN to scavenge ROS (Figure 4), which is in accordance with its effect on HO-1 expression.

Bottom Line: However, the potential antioxidant effects of PUN in macrophages remain unclear.Here, LY294002, a specific PI3K/Akt inhibitor, suppressed PUN-induced HO-1 expression and led to ROS accumulation in macrophages.Furthermore, PUN inhibited LPS-induced oxidative stress in macrophages by reducing ROS and NO generation and increasing superoxide dismutase (SOD) 1 mRNA expression.

View Article: PubMed Central - PubMed

Affiliation: CAU-BUA TCVM Teaching and Researching Team, College of Veterinary Medicine, China Agricultural University (CAU), No. 2 West Yuanmingyuan Road, Beijing 100193, China.

ABSTRACT
Reactive oxygen species (ROS) and oxidative stress are thought to play a central role in potentiating macrophage activation, causing excessive inflammation, tissue damage, and sepsis. Recently, we have shown that punicalagin (PUN) exhibits anti-inflammatory activity in LPS-stimulated macrophages. However, the potential antioxidant effects of PUN in macrophages remain unclear. Revealing these effects will help understand the mechanism underlying its ability to inhibit excessive macrophage activation. Hemeoxygenase-1 (HO-1) exhibits antioxidant activity in macrophages. Therefore, we hypothesized that HO-1 is a potential target of PUN and tried to reveal its antioxidant mechanism. Here, PUN treatment increased HO-1 expression together with its upstream mediator nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). However, specific inhibition of Nrf2 by brusatol (a specific Nrf2 inhibitor) dramatically blocked PUN-induced HO-1 expression. Previous research has demonstrated that the PI3K/Akt pathway plays a critical role in modulating Nrf2/HO-1 protein expression as an upstream signaling molecule. Here, LY294002, a specific PI3K/Akt inhibitor, suppressed PUN-induced HO-1 expression and led to ROS accumulation in macrophages. Furthermore, PUN inhibited LPS-induced oxidative stress in macrophages by reducing ROS and NO generation and increasing superoxide dismutase (SOD) 1 mRNA expression. These findings provide new perspectives for novel therapeutic approaches using antioxidant medicines and compounds against oxidative stress and excessive inflammatory diseases including tissue damage, sepsis, and endotoxemic shock.

No MeSH data available.


Related in: MedlinePlus