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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

Nox inhibitor did not induce a more significant reduction of Nox2 expression than AEA alone. 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; Apocynin + AEA + H2O2: cells exposed to 10 μM AEA plus 50 μM Nox inhibitor AM251 in the presence of 200 μM H2O2 for 3 h; Apocynin + AEA + H2O2: cells exposed to 50 μM apocynin plus 10 μM AEA in the presence of 200 μM H2O2 for 3 h. Nox2 protein expression (a) was evaluated by western blotting (n = 4). (b) Cell metabolic activity and (c) LDH release were determined by MTT (n = 8) and reagent kit (n = 6), respectively. Results are expressed as means ± S.D. *P < 0.05 versus the control (no H2O2, no AEA, and no apocynin), #P < 0.05 versus the cells exposed to H2O2 alone, n.s.: no significance.
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fig10: Nox inhibitor did not induce a more significant reduction of Nox2 expression than AEA alone. 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; Apocynin + AEA + H2O2: cells exposed to 10 μM AEA plus 50 μM Nox inhibitor AM251 in the presence of 200 μM H2O2 for 3 h; Apocynin + AEA + H2O2: cells exposed to 50 μM apocynin plus 10 μM AEA in the presence of 200 μM H2O2 for 3 h. Nox2 protein expression (a) was evaluated by western blotting (n = 4). (b) Cell metabolic activity and (c) LDH release were determined by MTT (n = 8) and reagent kit (n = 6), respectively. Results are expressed as means ± S.D. *P < 0.05 versus the control (no H2O2, no AEA, and no apocynin), #P < 0.05 versus the cells exposed to H2O2 alone, n.s.: no significance.

Mentions: To further investigate the role of Nox2 in AEA-induced protection in HT22 cells exposed to H2O2, we used apocynin, a specific Nox inhibitor. We found that the presence of 50 μM apocynin decreased the expression of Nox2 protein significantly (Figure 10(a)), and there was no significance between AEA and apocynin alone on Nox2 expression in H2O2-treated HT22 cells. In addition, a combination of AEA and apocynin did not cause a more significant reduction of Nox2 expression than either AEA or apocynin used alone (P > 0.05). Similarly, we noticed a combination of AEA and apocynin did not induce a more significant increase of cell metabolic activity (Figure 10(b)) and reduction of LDH release (Figure 10(c)) than either AEA or apocynin alone (P > 0.05), indicating that Nox2 inhibition may be involved in AEA-induced cytoprotection against H2O2.


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)

Nox inhibitor did not induce a more significant reduction of Nox2 expression than AEA alone. 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; Apocynin + AEA + H2O2: cells exposed to 10 μM AEA plus 50 μM Nox inhibitor AM251 in the presence of 200 μM H2O2 for 3 h; Apocynin + AEA + H2O2: cells exposed to 50 μM apocynin plus 10 μM AEA in the presence of 200 μM H2O2 for 3 h. Nox2 protein expression (a) was evaluated by western blotting (n = 4). (b) Cell metabolic activity and (c) LDH release were determined by MTT (n = 8) and reagent kit (n = 6), respectively. Results are expressed as means ± S.D. *P < 0.05 versus the control (no H2O2, no AEA, and no apocynin), #P < 0.05 versus the cells exposed to H2O2 alone, n.s.: no significance.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig10: Nox inhibitor did not induce a more significant reduction of Nox2 expression than AEA alone. 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; Apocynin + AEA + H2O2: cells exposed to 10 μM AEA plus 50 μM Nox inhibitor AM251 in the presence of 200 μM H2O2 for 3 h; Apocynin + AEA + H2O2: cells exposed to 50 μM apocynin plus 10 μM AEA in the presence of 200 μM H2O2 for 3 h. Nox2 protein expression (a) was evaluated by western blotting (n = 4). (b) Cell metabolic activity and (c) LDH release were determined by MTT (n = 8) and reagent kit (n = 6), respectively. Results are expressed as means ± S.D. *P < 0.05 versus the control (no H2O2, no AEA, and no apocynin), #P < 0.05 versus the cells exposed to H2O2 alone, n.s.: no significance.
Mentions: To further investigate the role of Nox2 in AEA-induced protection in HT22 cells exposed to H2O2, we used apocynin, a specific Nox inhibitor. We found that the presence of 50 μM apocynin decreased the expression of Nox2 protein significantly (Figure 10(a)), and there was no significance between AEA and apocynin alone on Nox2 expression in H2O2-treated HT22 cells. In addition, a combination of AEA and apocynin did not cause a more significant reduction of Nox2 expression than either AEA or apocynin used alone (P > 0.05). Similarly, we noticed a combination of AEA and apocynin did not induce a more significant increase of cell metabolic activity (Figure 10(b)) and reduction of LDH release (Figure 10(c)) than either AEA or apocynin alone (P > 0.05), indicating that Nox2 inhibition may be involved in AEA-induced cytoprotection against H2O2.

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