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Anandamide protects HT22 cells exposed to hydrogen peroxide by inhibiting CB1 receptor-mediated type 2 NADPH oxidase.

Jia J, Ma L, Wu M, Zhang L, Zhang X, Zhai Q, Jiang T, Wang Q, Xiong L - Oxid Med Cell Longev (2014)

Bottom Line: HT22 cells exposed to H2O2 demonstrated morphological changes, decreased LDH release, reduced metabolic activity, increased levels of intracellular ROS and oxidized glutathione (GSSG), reduced levels of superoxide dismutase (SOD), and reduced glutathione (GSH) and increased expression of Nox2.AEA prevented these effects, a property abolished by simultaneous administration of CB1 antagonist AM251 or CB1-siRNA.Nox2 inhibition is involved in AEA-induced cytoprotection against oxidative stress through CB1 activation in HT22 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.

ABSTRACT

Background: Endogenous cannabinoid anandamide (AEA) protects neurons from oxidative injury in rodent models; however the mechanism of AEA-induced neuroprotection remains to be determined. Activation of neuronal NADPH oxidase 2 (Nox2) contributes to oxidative damage of the brain, and inhibition of Nox2 can attenuate cerebral oxidative stress. We aimed to determine whether the neuronal Nox2 was involved in protection mediated by AEA.

Methods: The mouse hippocampal neuron cell line HT22 was exposed to hydrogen peroxide (H2O2) to mimic oxidative injury of neurons. The protective effect of AEA was assessed by measuring cell metabolic activity, apoptosis, lactate dehydrogenase (LDH) release, cellular morphology, intracellular reactive oxygen species (ROS), and antioxidant and oxidant levels and Nox2 expression.

Results: HT22 cells exposed to H2O2 demonstrated morphological changes, decreased LDH release, reduced metabolic activity, increased levels of intracellular ROS and oxidized glutathione (GSSG), reduced levels of superoxide dismutase (SOD), and reduced glutathione (GSH) and increased expression of Nox2. AEA prevented these effects, a property abolished by simultaneous administration of CB1 antagonist AM251 or CB1-siRNA.

Conclusion: Nox2 inhibition is involved in AEA-induced cytoprotection against oxidative stress through CB1 activation in HT22 cells.

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Related in: MedlinePlus

Nox2 expression was inhibited in the presence of AEA via CB1. (a) Nox2 expression was increased in HT22 cells exposed to H2O2 in a time-dependent manner. Then the cells were divided into five groups, Control: cells cultured in drug-free medium; H2O2: cells exposed to 200 μM H2O2 for 3 h; AEA + H2O2: cells exposed to 10 μM AEA plus 200 μM H2O2 for 3 h; AM251 + AEA + H2O2: cells exposed to 10 μM AEA plus 10 μM CB1 antagonist AM251 in the presence of 200 μM H2O2 for 3 h; AM251 + H2O2: cells exposed to 10 μM AM251 plus 200 μM H2O2 for 3 h. Nox2 protein expression (b) and mRNA transcription (c) were evaluated by western blotting and real-time PCR, respectively. (d) Incubation with CB1-siRNA for 5 h abolished the AEA-induced inhibition of Nox2 mRNA transcription. Results are expressed as means ± SD (n = 4). *P < 0.05 versus the control (no H2O2, no AEA, and no AM251 or siRNA), #P < 0.05 versus the cells exposed to H2O2 alone, and ∧P < 0.05 versus the cells exposed to AEA plus H2O2.
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fig9: Nox2 expression was inhibited in the presence of AEA via CB1. (a) Nox2 expression was increased in HT22 cells exposed to H2O2 in a time-dependent manner. Then the cells were divided into five groups, Control: cells cultured in drug-free medium; H2O2: cells exposed to 200 μM H2O2 for 3 h; AEA + H2O2: cells exposed to 10 μM AEA plus 200 μM H2O2 for 3 h; AM251 + AEA + H2O2: cells exposed to 10 μM AEA plus 10 μM CB1 antagonist AM251 in the presence of 200 μM H2O2 for 3 h; AM251 + H2O2: cells exposed to 10 μM AM251 plus 200 μM H2O2 for 3 h. Nox2 protein expression (b) and mRNA transcription (c) were evaluated by western blotting and real-time PCR, respectively. (d) Incubation with CB1-siRNA for 5 h abolished the AEA-induced inhibition of Nox2 mRNA transcription. Results are expressed as means ± SD (n = 4). *P < 0.05 versus the control (no H2O2, no AEA, and no AM251 or siRNA), #P < 0.05 versus the cells exposed to H2O2 alone, and ∧P < 0.05 versus the cells exposed to AEA plus H2O2.

Mentions: The expression of Nox2 in HT22 cells was upregulated in the presence of 200 μM H2O2 in a time-dependent manner (Figure 9(a)). AEA decreased the Nox2 protein expression (Figure 9(b)) and mRNA transcription (Figure 9(c)). However, CB1 antagonist AM251 or CB1-siRNA (Figure 9(d)) abolished AEA-induced influence on Nox2 protein expression and mRNA transcription, suggesting that the Nox2 may be involved in AEA-induced cytoprotection against H2O2 via CB1.


Anandamide protects HT22 cells exposed to hydrogen peroxide by inhibiting CB1 receptor-mediated type 2 NADPH oxidase.

Jia J, Ma L, Wu M, Zhang L, Zhang X, Zhai Q, Jiang T, Wang Q, Xiong L - Oxid Med Cell Longev (2014)

Nox2 expression was inhibited in the presence of AEA via CB1. (a) Nox2 expression was increased in HT22 cells exposed to H2O2 in a time-dependent manner. Then the cells were divided into five groups, Control: cells cultured in drug-free medium; H2O2: cells exposed to 200 μM H2O2 for 3 h; AEA + H2O2: cells exposed to 10 μM AEA plus 200 μM H2O2 for 3 h; AM251 + AEA + H2O2: cells exposed to 10 μM AEA plus 10 μM CB1 antagonist AM251 in the presence of 200 μM H2O2 for 3 h; AM251 + H2O2: cells exposed to 10 μM AM251 plus 200 μM H2O2 for 3 h. Nox2 protein expression (b) and mRNA transcription (c) were evaluated by western blotting and real-time PCR, respectively. (d) Incubation with CB1-siRNA for 5 h abolished the AEA-induced inhibition of Nox2 mRNA transcription. Results are expressed as means ± SD (n = 4). *P < 0.05 versus the control (no H2O2, no AEA, and no AM251 or siRNA), #P < 0.05 versus the cells exposed to H2O2 alone, and ∧P < 0.05 versus the cells exposed to AEA plus H2O2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig9: Nox2 expression was inhibited in the presence of AEA via CB1. (a) Nox2 expression was increased in HT22 cells exposed to H2O2 in a time-dependent manner. Then the cells were divided into five groups, Control: cells cultured in drug-free medium; H2O2: cells exposed to 200 μM H2O2 for 3 h; AEA + H2O2: cells exposed to 10 μM AEA plus 200 μM H2O2 for 3 h; AM251 + AEA + H2O2: cells exposed to 10 μM AEA plus 10 μM CB1 antagonist AM251 in the presence of 200 μM H2O2 for 3 h; AM251 + H2O2: cells exposed to 10 μM AM251 plus 200 μM H2O2 for 3 h. Nox2 protein expression (b) and mRNA transcription (c) were evaluated by western blotting and real-time PCR, respectively. (d) Incubation with CB1-siRNA for 5 h abolished the AEA-induced inhibition of Nox2 mRNA transcription. Results are expressed as means ± SD (n = 4). *P < 0.05 versus the control (no H2O2, no AEA, and no AM251 or siRNA), #P < 0.05 versus the cells exposed to H2O2 alone, and ∧P < 0.05 versus the cells exposed to AEA plus H2O2.
Mentions: The expression of Nox2 in HT22 cells was upregulated in the presence of 200 μM H2O2 in a time-dependent manner (Figure 9(a)). AEA decreased the Nox2 protein expression (Figure 9(b)) and mRNA transcription (Figure 9(c)). However, CB1 antagonist AM251 or CB1-siRNA (Figure 9(d)) abolished AEA-induced influence on Nox2 protein expression and mRNA transcription, suggesting that the Nox2 may be involved in AEA-induced cytoprotection against H2O2 via CB1.

Bottom Line: HT22 cells exposed to H2O2 demonstrated morphological changes, decreased LDH release, reduced metabolic activity, increased levels of intracellular ROS and oxidized glutathione (GSSG), reduced levels of superoxide dismutase (SOD), and reduced glutathione (GSH) and increased expression of Nox2.AEA prevented these effects, a property abolished by simultaneous administration of CB1 antagonist AM251 or CB1-siRNA.Nox2 inhibition is involved in AEA-induced cytoprotection against oxidative stress through CB1 activation in HT22 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.

ABSTRACT

Background: Endogenous cannabinoid anandamide (AEA) protects neurons from oxidative injury in rodent models; however the mechanism of AEA-induced neuroprotection remains to be determined. Activation of neuronal NADPH oxidase 2 (Nox2) contributes to oxidative damage of the brain, and inhibition of Nox2 can attenuate cerebral oxidative stress. We aimed to determine whether the neuronal Nox2 was involved in protection mediated by AEA.

Methods: The mouse hippocampal neuron cell line HT22 was exposed to hydrogen peroxide (H2O2) to mimic oxidative injury of neurons. The protective effect of AEA was assessed by measuring cell metabolic activity, apoptosis, lactate dehydrogenase (LDH) release, cellular morphology, intracellular reactive oxygen species (ROS), and antioxidant and oxidant levels and Nox2 expression.

Results: HT22 cells exposed to H2O2 demonstrated morphological changes, decreased LDH release, reduced metabolic activity, increased levels of intracellular ROS and oxidized glutathione (GSSG), reduced levels of superoxide dismutase (SOD), and reduced glutathione (GSH) and increased expression of Nox2. AEA prevented these effects, a property abolished by simultaneous administration of CB1 antagonist AM251 or CB1-siRNA.

Conclusion: Nox2 inhibition is involved in AEA-induced cytoprotection against oxidative stress through CB1 activation in HT22 cells.

Show MeSH
Related in: MedlinePlus