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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

Experimental protocol diagram. (a) The HT22 cells were assigned into seven groups. The control cells were cultured in drug-free medium, and the other six groups were exposed to different concentrations of H2O2 for 3 h, ranging from 50 μM to 1000 μM. MTT assay was taken to determine the injury degree. (b) The cells were divided into six groups; except for control and H2O2 only groups, the other four groups were exposed to 200 μM H2O2 plus different concentrations of AEA for 3 h. MTT assay was taken to evaluate the injury degree. (c) The cells were assigned into six groups, including control, AEA, H2O2, AEA + H2O2, AM251 + AEA + H2O2, and AM251 + H2O2 groups. After an incubation of 3 h, MTT assay, LDH release, and western blotting were taken to determine the roles of CB1 and Nox2 in AEA-induced protection. (d) The cells were divided into three groups, including control, CB1-siRNA, and SC-siRNA groups; after an incubation of 5 h, western blotting was used to evaluate the silencing rate of CB1 protein expression. (e) Then, the cells were divided into five groups, including control, H2O2, AEA + H2O2, CB1-siRNA + AEA + H2O2, and SC-siRNA + AEA + H2O2; the cell injury was evaluated by MTT and LDH release at 3 h after incubation, and ROS generation was evaluated by measuring fluorescence intensity. (f) The cells were divided into five groups, including control, H2O2, AEA + H2O2, apocynin + AEA + H2O2, and apocynin + AEA + H2O2; western blotting, MTT assay, and LDH release were taken to measure Nox2 expression and cell injury.
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fig1: Experimental protocol diagram. (a) The HT22 cells were assigned into seven groups. The control cells were cultured in drug-free medium, and the other six groups were exposed to different concentrations of H2O2 for 3 h, ranging from 50 μM to 1000 μM. MTT assay was taken to determine the injury degree. (b) The cells were divided into six groups; except for control and H2O2 only groups, the other four groups were exposed to 200 μM H2O2 plus different concentrations of AEA for 3 h. MTT assay was taken to evaluate the injury degree. (c) The cells were assigned into six groups, including control, AEA, H2O2, AEA + H2O2, AM251 + AEA + H2O2, and AM251 + H2O2 groups. After an incubation of 3 h, MTT assay, LDH release, and western blotting were taken to determine the roles of CB1 and Nox2 in AEA-induced protection. (d) The cells were divided into three groups, including control, CB1-siRNA, and SC-siRNA groups; after an incubation of 5 h, western blotting was used to evaluate the silencing rate of CB1 protein expression. (e) Then, the cells were divided into five groups, including control, H2O2, AEA + H2O2, CB1-siRNA + AEA + H2O2, and SC-siRNA + AEA + H2O2; the cell injury was evaluated by MTT and LDH release at 3 h after incubation, and ROS generation was evaluated by measuring fluorescence intensity. (f) The cells were divided into five groups, including control, H2O2, AEA + H2O2, apocynin + AEA + H2O2, and apocynin + AEA + H2O2; western blotting, MTT assay, and LDH release were taken to measure Nox2 expression and cell injury.

Mentions: To find a suitable H2O2 concentration, the HT22 cells were assigned into seven groups (Figure 1(a)). Except for control group, the other six groups were exposed to different concentrations of H2O2 for 3 h, ranging from 50 μM to 1000 μM. Then MTT assay was taken to determine the injury degree of the cells. To find a suitable AEA concentration, the cells were divided into six groups. Except for control and H2O2 only groups, the other four groups were exposed to 200 μM H2O2 plus different concentrations of AEA (Figure 1(b)). Then MTT assay was taken to evaluate the injury degree of the cells.


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)

Experimental protocol diagram. (a) The HT22 cells were assigned into seven groups. The control cells were cultured in drug-free medium, and the other six groups were exposed to different concentrations of H2O2 for 3 h, ranging from 50 μM to 1000 μM. MTT assay was taken to determine the injury degree. (b) The cells were divided into six groups; except for control and H2O2 only groups, the other four groups were exposed to 200 μM H2O2 plus different concentrations of AEA for 3 h. MTT assay was taken to evaluate the injury degree. (c) The cells were assigned into six groups, including control, AEA, H2O2, AEA + H2O2, AM251 + AEA + H2O2, and AM251 + H2O2 groups. After an incubation of 3 h, MTT assay, LDH release, and western blotting were taken to determine the roles of CB1 and Nox2 in AEA-induced protection. (d) The cells were divided into three groups, including control, CB1-siRNA, and SC-siRNA groups; after an incubation of 5 h, western blotting was used to evaluate the silencing rate of CB1 protein expression. (e) Then, the cells were divided into five groups, including control, H2O2, AEA + H2O2, CB1-siRNA + AEA + H2O2, and SC-siRNA + AEA + H2O2; the cell injury was evaluated by MTT and LDH release at 3 h after incubation, and ROS generation was evaluated by measuring fluorescence intensity. (f) The cells were divided into five groups, including control, H2O2, AEA + H2O2, apocynin + AEA + H2O2, and apocynin + AEA + H2O2; western blotting, MTT assay, and LDH release were taken to measure Nox2 expression and cell injury.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Experimental protocol diagram. (a) The HT22 cells were assigned into seven groups. The control cells were cultured in drug-free medium, and the other six groups were exposed to different concentrations of H2O2 for 3 h, ranging from 50 μM to 1000 μM. MTT assay was taken to determine the injury degree. (b) The cells were divided into six groups; except for control and H2O2 only groups, the other four groups were exposed to 200 μM H2O2 plus different concentrations of AEA for 3 h. MTT assay was taken to evaluate the injury degree. (c) The cells were assigned into six groups, including control, AEA, H2O2, AEA + H2O2, AM251 + AEA + H2O2, and AM251 + H2O2 groups. After an incubation of 3 h, MTT assay, LDH release, and western blotting were taken to determine the roles of CB1 and Nox2 in AEA-induced protection. (d) The cells were divided into three groups, including control, CB1-siRNA, and SC-siRNA groups; after an incubation of 5 h, western blotting was used to evaluate the silencing rate of CB1 protein expression. (e) Then, the cells were divided into five groups, including control, H2O2, AEA + H2O2, CB1-siRNA + AEA + H2O2, and SC-siRNA + AEA + H2O2; the cell injury was evaluated by MTT and LDH release at 3 h after incubation, and ROS generation was evaluated by measuring fluorescence intensity. (f) The cells were divided into five groups, including control, H2O2, AEA + H2O2, apocynin + AEA + H2O2, and apocynin + AEA + H2O2; western blotting, MTT assay, and LDH release were taken to measure Nox2 expression and cell injury.
Mentions: To find a suitable H2O2 concentration, the HT22 cells were assigned into seven groups (Figure 1(a)). Except for control group, the other six groups were exposed to different concentrations of H2O2 for 3 h, ranging from 50 μM to 1000 μM. Then MTT assay was taken to determine the injury degree of the cells. To find a suitable AEA concentration, the cells were divided into six groups. Except for control and H2O2 only groups, the other four groups were exposed to 200 μM H2O2 plus different concentrations of AEA (Figure 1(b)). Then MTT assay was taken to evaluate the injury degree of the cells.

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