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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 increased intracellular SOD and ameliorated GSH/GSSG ratio. The cells were divided into six groups, Control: cells cultured in drug-free medium; AEA: cells exposed to 10 μM AEA for 3 h; 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. The intracellular SOD, GSH, and GSSG levels were assessed by the corresponding reagent kit, and the GSH/GSSG ratio was calculated according to the GSH and GSSG levels. (a) Intracellular SOD level. (b) Intracellular GSH level. (c) Intracellular GSSG level. (d) Intracellular GSH/GSSG ratio. Results are expressed as means ± SD (n = 6). *P < 0.05 versus control (no H2O2, no AEA, and no AM251), #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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fig7: AEA increased intracellular SOD and ameliorated GSH/GSSG ratio. The cells were divided into six groups, Control: cells cultured in drug-free medium; AEA: cells exposed to 10 μM AEA for 3 h; 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. The intracellular SOD, GSH, and GSSG levels were assessed by the corresponding reagent kit, and the GSH/GSSG ratio was calculated according to the GSH and GSSG levels. (a) Intracellular SOD level. (b) Intracellular GSH level. (c) Intracellular GSSG level. (d) Intracellular GSH/GSSG ratio. Results are expressed as means ± SD (n = 6). *P < 0.05 versus control (no H2O2, no AEA, and no AM251), #P < 0.05 versus the cells exposed to H2O2 alone, and ∧P < 0.05 versus the cells exposed to AEA plus H2O2.

Mentions: Exposure of HT22 cells to H2O2 led to accumulation of intracellular ROS, and simultaneous treatment with AEA markedly reduced the generation of ROS (Figure 6). SOD plays a vital role in protecting cells against oxidative injury. H2O2 treatment sharply decreased SOD activity in HT22 cells to 32.2 ± 5.0% (Figure 7(a)), and AEA restored SOD activity to 70.3 ± 4.0% of baseline (P < 0.05). GSH is also an important cellular antioxidant. H2O2 treatment sharply decreased GSH activity in HT22 cells from 18.8 ± 2.7 μM to 6.1 ± 1.0 μM (Figure 7(b)). Simultaneous application of AEA partially restored GSH levels to 12.1 ± 1.4 μM (P < 0.05). GSSG levels were increased in response to H2O2 treatment from 1.5 ± 0.3 μM to 2.9 ± 0.3 μM (Figure 7(c)), and this effect was almost entirely abolished by AEA, which reduced GSSH levels to 1.6 ± 0.4 μM (P < 0.05). The GSH/GSSG ratio was reduced from 12.3 ± 1.1 to 2.2 ± 0.3 by H2O2 treatment, and AEA partially restored this balance, increasing the ratio to 7.3 ± 0.7 (Figure 7(d)). The influences of AEA on intracellular ROS, SOD, GSH, GSSG, and GSH/GSSG ratio were abolished by the CB1 antagonist AM251, indicating that the antioxidative effects of AEA may be mediated via CB1 of HT22 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)

AEA increased intracellular SOD and ameliorated GSH/GSSG ratio. The cells were divided into six groups, Control: cells cultured in drug-free medium; AEA: cells exposed to 10 μM AEA for 3 h; 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. The intracellular SOD, GSH, and GSSG levels were assessed by the corresponding reagent kit, and the GSH/GSSG ratio was calculated according to the GSH and GSSG levels. (a) Intracellular SOD level. (b) Intracellular GSH level. (c) Intracellular GSSG level. (d) Intracellular GSH/GSSG ratio. Results are expressed as means ± SD (n = 6). *P < 0.05 versus control (no H2O2, no AEA, and no AM251), #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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fig7: AEA increased intracellular SOD and ameliorated GSH/GSSG ratio. The cells were divided into six groups, Control: cells cultured in drug-free medium; AEA: cells exposed to 10 μM AEA for 3 h; 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. The intracellular SOD, GSH, and GSSG levels were assessed by the corresponding reagent kit, and the GSH/GSSG ratio was calculated according to the GSH and GSSG levels. (a) Intracellular SOD level. (b) Intracellular GSH level. (c) Intracellular GSSG level. (d) Intracellular GSH/GSSG ratio. Results are expressed as means ± SD (n = 6). *P < 0.05 versus control (no H2O2, no AEA, and no AM251), #P < 0.05 versus the cells exposed to H2O2 alone, and ∧P < 0.05 versus the cells exposed to AEA plus H2O2.
Mentions: Exposure of HT22 cells to H2O2 led to accumulation of intracellular ROS, and simultaneous treatment with AEA markedly reduced the generation of ROS (Figure 6). SOD plays a vital role in protecting cells against oxidative injury. H2O2 treatment sharply decreased SOD activity in HT22 cells to 32.2 ± 5.0% (Figure 7(a)), and AEA restored SOD activity to 70.3 ± 4.0% of baseline (P < 0.05). GSH is also an important cellular antioxidant. H2O2 treatment sharply decreased GSH activity in HT22 cells from 18.8 ± 2.7 μM to 6.1 ± 1.0 μM (Figure 7(b)). Simultaneous application of AEA partially restored GSH levels to 12.1 ± 1.4 μM (P < 0.05). GSSG levels were increased in response to H2O2 treatment from 1.5 ± 0.3 μM to 2.9 ± 0.3 μM (Figure 7(c)), and this effect was almost entirely abolished by AEA, which reduced GSSH levels to 1.6 ± 0.4 μM (P < 0.05). The GSH/GSSG ratio was reduced from 12.3 ± 1.1 to 2.2 ± 0.3 by H2O2 treatment, and AEA partially restored this balance, increasing the ratio to 7.3 ± 0.7 (Figure 7(d)). The influences of AEA on intracellular ROS, SOD, GSH, GSSG, and GSH/GSSG ratio were abolished by the CB1 antagonist AM251, indicating that the antioxidative effects of AEA may be mediated via CB1 of HT22 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