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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

CB1-siRNA reversed AEA-induced protection against oxidative stress. 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; CB1-siRNA + AEA + H2O2: cells incubated with CB1-siRNA for 5 h and then exposed to 10 μM AEA plus 200 μM H2O2 for 3 h; scrambled siRNA (SC-siRNA) + AEA + H2O2: cells incubated with SC-siRNA for 5 h and then exposed to 10 μM AEA plus 200 μM H2O2 for 3 h. CB1-siRNA abolished the AEA-induced protection against 200 μM H2O2 in HT22 cells; SC-siRNA did not affect the protection. (a) CB1-siRNA significantly downregulated the expression of CB1, assessed by western blotting. (b) Cell metabolic activity, assessed by MTT (n = 8). (c) LDH release, assessed by reagent kit and spectrophotometry (n = 6). (d)–(h) The fluorescence intensity of ROS. (i) Statistical results of (d)–(h) (n = 6). Results are expressed as means ± SD, *P < 0.05 versus the control (no H2O2, no AEA, and no 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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fig8: CB1-siRNA reversed AEA-induced protection against oxidative stress. 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; CB1-siRNA + AEA + H2O2: cells incubated with CB1-siRNA for 5 h and then exposed to 10 μM AEA plus 200 μM H2O2 for 3 h; scrambled siRNA (SC-siRNA) + AEA + H2O2: cells incubated with SC-siRNA for 5 h and then exposed to 10 μM AEA plus 200 μM H2O2 for 3 h. CB1-siRNA abolished the AEA-induced protection against 200 μM H2O2 in HT22 cells; SC-siRNA did not affect the protection. (a) CB1-siRNA significantly downregulated the expression of CB1, assessed by western blotting. (b) Cell metabolic activity, assessed by MTT (n = 8). (c) LDH release, assessed by reagent kit and spectrophotometry (n = 6). (d)–(h) The fluorescence intensity of ROS. (i) Statistical results of (d)–(h) (n = 6). Results are expressed as means ± SD, *P < 0.05 versus the control (no H2O2, no AEA, and no siRNA), #P < 0.05 versus the cells exposed to H2O2 alone, and ∧P < 0.05 versus the cells exposed to AEA plus H2O2.

Mentions: To further determine whether AEA-induced antioxidative ability was mediated by CB1 in HT22 cells, we used CB1-siRNA to knock down the expression of CB1. CB1-siRNA was effective in reducing the expression of CB1 (Figure 8(a)) and reversed AEA-induced cytoprotection, leading to a significant reduction of cell metabolic activity (Figure 8(b)), an increase of LDH release (Figure 8(c)), and a rise of intracellular ROS level (Figures 8(d)–8(i)).


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)

CB1-siRNA reversed AEA-induced protection against oxidative stress. 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; CB1-siRNA + AEA + H2O2: cells incubated with CB1-siRNA for 5 h and then exposed to 10 μM AEA plus 200 μM H2O2 for 3 h; scrambled siRNA (SC-siRNA) + AEA + H2O2: cells incubated with SC-siRNA for 5 h and then exposed to 10 μM AEA plus 200 μM H2O2 for 3 h. CB1-siRNA abolished the AEA-induced protection against 200 μM H2O2 in HT22 cells; SC-siRNA did not affect the protection. (a) CB1-siRNA significantly downregulated the expression of CB1, assessed by western blotting. (b) Cell metabolic activity, assessed by MTT (n = 8). (c) LDH release, assessed by reagent kit and spectrophotometry (n = 6). (d)–(h) The fluorescence intensity of ROS. (i) Statistical results of (d)–(h) (n = 6). Results are expressed as means ± SD, *P < 0.05 versus the control (no H2O2, no AEA, and no 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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Related In: Results  -  Collection

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fig8: CB1-siRNA reversed AEA-induced protection against oxidative stress. 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; CB1-siRNA + AEA + H2O2: cells incubated with CB1-siRNA for 5 h and then exposed to 10 μM AEA plus 200 μM H2O2 for 3 h; scrambled siRNA (SC-siRNA) + AEA + H2O2: cells incubated with SC-siRNA for 5 h and then exposed to 10 μM AEA plus 200 μM H2O2 for 3 h. CB1-siRNA abolished the AEA-induced protection against 200 μM H2O2 in HT22 cells; SC-siRNA did not affect the protection. (a) CB1-siRNA significantly downregulated the expression of CB1, assessed by western blotting. (b) Cell metabolic activity, assessed by MTT (n = 8). (c) LDH release, assessed by reagent kit and spectrophotometry (n = 6). (d)–(h) The fluorescence intensity of ROS. (i) Statistical results of (d)–(h) (n = 6). Results are expressed as means ± SD, *P < 0.05 versus the control (no H2O2, no AEA, and no siRNA), #P < 0.05 versus the cells exposed to H2O2 alone, and ∧P < 0.05 versus the cells exposed to AEA plus H2O2.
Mentions: To further determine whether AEA-induced antioxidative ability was mediated by CB1 in HT22 cells, we used CB1-siRNA to knock down the expression of CB1. CB1-siRNA was effective in reducing the expression of CB1 (Figure 8(a)) and reversed AEA-induced cytoprotection, leading to a significant reduction of cell metabolic activity (Figure 8(b)), an increase of LDH release (Figure 8(c)), and a rise of intracellular ROS level (Figures 8(d)–8(i)).

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