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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

AEA upregulated the expression of CB1 in HT22 cells. Immunofluorescence staining and western blotting were used to investigate the AEA-induced effect on CB1 protein expression in HT22 cells. 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 at the presence of 200 μM H2O2 for 3 h; AM251 + H2O2: cells exposed to 10 μM AM251 plus 200 μM H2O2 for 3 h. CB1 protein (red) was expressed in HT22 cells. AEA upregulated the expression of CB1 receptor; however CB1 antagonist AM251 reversed the CB1 upregulation in HT22 cells. Nuclei were counter-stained with DAPI (blue). Results are expressed as means ± SD (n = 4). *P < 0.05 versus control (no H2O2, no AEA, and no AM251), ∧P < 0.05 versus the cells exposed to AEA plus H2O2. Bar = 20 μm.
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fig3: AEA upregulated the expression of CB1 in HT22 cells. Immunofluorescence staining and western blotting were used to investigate the AEA-induced effect on CB1 protein expression in HT22 cells. 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 at the presence of 200 μM H2O2 for 3 h; AM251 + H2O2: cells exposed to 10 μM AM251 plus 200 μM H2O2 for 3 h. CB1 protein (red) was expressed in HT22 cells. AEA upregulated the expression of CB1 receptor; however CB1 antagonist AM251 reversed the CB1 upregulation in HT22 cells. Nuclei were counter-stained with DAPI (blue). Results are expressed as means ± SD (n = 4). *P < 0.05 versus control (no H2O2, no AEA, and no AM251), ∧P < 0.05 versus the cells exposed to AEA plus H2O2. Bar = 20 μm.

Mentions: We used immunofluorescence and western blotting to assess whether AEA could up-regulate CB1 expression in HT22 cells. We observed CB1 staining in the cell membrane and cytoplasm of HT22 cells, consistent with a previous study [11]. Treatment with 10 μM AEA induced a significant up-regulation of CB1 expression (P < 0.05), and the selective CB1 antagonist AM251 reversed the AEA-induced up-regulation of CB1 expression (Figure 3).


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)

AEA upregulated the expression of CB1 in HT22 cells. Immunofluorescence staining and western blotting were used to investigate the AEA-induced effect on CB1 protein expression in HT22 cells. 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 at the presence of 200 μM H2O2 for 3 h; AM251 + H2O2: cells exposed to 10 μM AM251 plus 200 μM H2O2 for 3 h. CB1 protein (red) was expressed in HT22 cells. AEA upregulated the expression of CB1 receptor; however CB1 antagonist AM251 reversed the CB1 upregulation in HT22 cells. Nuclei were counter-stained with DAPI (blue). Results are expressed as means ± SD (n = 4). *P < 0.05 versus control (no H2O2, no AEA, and no AM251), ∧P < 0.05 versus the cells exposed to AEA plus H2O2. Bar = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: AEA upregulated the expression of CB1 in HT22 cells. Immunofluorescence staining and western blotting were used to investigate the AEA-induced effect on CB1 protein expression in HT22 cells. 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 at the presence of 200 μM H2O2 for 3 h; AM251 + H2O2: cells exposed to 10 μM AM251 plus 200 μM H2O2 for 3 h. CB1 protein (red) was expressed in HT22 cells. AEA upregulated the expression of CB1 receptor; however CB1 antagonist AM251 reversed the CB1 upregulation in HT22 cells. Nuclei were counter-stained with DAPI (blue). Results are expressed as means ± SD (n = 4). *P < 0.05 versus control (no H2O2, no AEA, and no AM251), ∧P < 0.05 versus the cells exposed to AEA plus H2O2. Bar = 20 μm.
Mentions: We used immunofluorescence and western blotting to assess whether AEA could up-regulate CB1 expression in HT22 cells. We observed CB1 staining in the cell membrane and cytoplasm of HT22 cells, consistent with a previous study [11]. Treatment with 10 μM AEA induced a significant up-regulation of CB1 expression (P < 0.05), and the selective CB1 antagonist AM251 reversed the AEA-induced up-regulation of CB1 expression (Figure 3).

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