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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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AEA protected HT22 cells exposed to H2O2 via CB1. (a) CB1 antagonist AM251 reversed AEA-induced protection on cell metabolic activity (n = 8). (b) AM251 reversed AEA-induced protection on LDH release (n = 6). (c) AM251 reversed AEA-induced reduction of cleaved caspase-3 expression (n = 4). (d)–(h) Apoptotic rates assessed by flow cytometry. (d) Control cells cultured in drug-free medium. (e) Cells exposed to 200 μM H2O2 for 3 h. (f) Cells exposed to 10 μM AEA plus 200 μM H2O2 for 3 h. (g) Cells exposed to 10 μM AEA plus 10 μM AM251 in the presence of 200 μM H2O2 for 3 h. (h) Cells exposed to CB1 antagonist AM251 of 10 μM plus 200 μM H2O2 for 3 h. (i) Statistical results of (c)–(g). Results are expressed as means ± SD (n = 4). *P < 0.05 versus the 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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fig4: AEA protected HT22 cells exposed to H2O2 via CB1. (a) CB1 antagonist AM251 reversed AEA-induced protection on cell metabolic activity (n = 8). (b) AM251 reversed AEA-induced protection on LDH release (n = 6). (c) AM251 reversed AEA-induced reduction of cleaved caspase-3 expression (n = 4). (d)–(h) Apoptotic rates assessed by flow cytometry. (d) Control cells cultured in drug-free medium. (e) Cells exposed to 200 μM H2O2 for 3 h. (f) Cells exposed to 10 μM AEA plus 200 μM H2O2 for 3 h. (g) Cells exposed to 10 μM AEA plus 10 μM AM251 in the presence of 200 μM H2O2 for 3 h. (h) Cells exposed to CB1 antagonist AM251 of 10 μM plus 200 μM H2O2 for 3 h. (i) Statistical results of (c)–(g). Results are expressed as means ± SD (n = 4). *P < 0.05 versus the 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: In the absence of AEA, AM251 did not affect the cytotoxic impact of H2O2 (Figure 4(a)); however AM251 abolished the AEA-induced protection of HT22 cells, reducing the cell metabolic activity from 66.9 ± 2.4% to 49.5 ± 7.1% (P < 0.05). AM251 also reversed the influence of AEA on LDH release, increasing the LDH release from 29.1 ± 7.6 U/L to 51.2 ± 7.9 U/L (P < 0.05) (Figure 4(b)). We also evaluated cleaved caspase-3 expression and apoptotic rate by western blotting (Figure 4(c)) and flow cytometry (Figures 4(d)–4(i)), respectively, to assess the apoptosis of HT22 cells. AEA significantly decreased the expression of cleaved caspase-3 and the apoptotic rate of HT22 cells in response to H2O2 (P < 0.05). And AM251 abolished these effects caused by AEA. In addition, AEA ameliorated the changes in cellular morphology elicited by H2O2 and maintained the integrity of HT22 cells, and AM251 reversed this effect (Figure 5). These results indicated AEA protected HT22 cells from the damage caused by H2O2, and AM251 reversed this protection, suggesting that the protective effects of AEA may be mediated via CB1.


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 protected HT22 cells exposed to H2O2 via CB1. (a) CB1 antagonist AM251 reversed AEA-induced protection on cell metabolic activity (n = 8). (b) AM251 reversed AEA-induced protection on LDH release (n = 6). (c) AM251 reversed AEA-induced reduction of cleaved caspase-3 expression (n = 4). (d)–(h) Apoptotic rates assessed by flow cytometry. (d) Control cells cultured in drug-free medium. (e) Cells exposed to 200 μM H2O2 for 3 h. (f) Cells exposed to 10 μM AEA plus 200 μM H2O2 for 3 h. (g) Cells exposed to 10 μM AEA plus 10 μM AM251 in the presence of 200 μM H2O2 for 3 h. (h) Cells exposed to CB1 antagonist AM251 of 10 μM plus 200 μM H2O2 for 3 h. (i) Statistical results of (c)–(g). Results are expressed as means ± SD (n = 4). *P < 0.05 versus the 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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fig4: AEA protected HT22 cells exposed to H2O2 via CB1. (a) CB1 antagonist AM251 reversed AEA-induced protection on cell metabolic activity (n = 8). (b) AM251 reversed AEA-induced protection on LDH release (n = 6). (c) AM251 reversed AEA-induced reduction of cleaved caspase-3 expression (n = 4). (d)–(h) Apoptotic rates assessed by flow cytometry. (d) Control cells cultured in drug-free medium. (e) Cells exposed to 200 μM H2O2 for 3 h. (f) Cells exposed to 10 μM AEA plus 200 μM H2O2 for 3 h. (g) Cells exposed to 10 μM AEA plus 10 μM AM251 in the presence of 200 μM H2O2 for 3 h. (h) Cells exposed to CB1 antagonist AM251 of 10 μM plus 200 μM H2O2 for 3 h. (i) Statistical results of (c)–(g). Results are expressed as means ± SD (n = 4). *P < 0.05 versus the 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: In the absence of AEA, AM251 did not affect the cytotoxic impact of H2O2 (Figure 4(a)); however AM251 abolished the AEA-induced protection of HT22 cells, reducing the cell metabolic activity from 66.9 ± 2.4% to 49.5 ± 7.1% (P < 0.05). AM251 also reversed the influence of AEA on LDH release, increasing the LDH release from 29.1 ± 7.6 U/L to 51.2 ± 7.9 U/L (P < 0.05) (Figure 4(b)). We also evaluated cleaved caspase-3 expression and apoptotic rate by western blotting (Figure 4(c)) and flow cytometry (Figures 4(d)–4(i)), respectively, to assess the apoptosis of HT22 cells. AEA significantly decreased the expression of cleaved caspase-3 and the apoptotic rate of HT22 cells in response to H2O2 (P < 0.05). And AM251 abolished these effects caused by AEA. In addition, AEA ameliorated the changes in cellular morphology elicited by H2O2 and maintained the integrity of HT22 cells, and AM251 reversed this effect (Figure 5). These results indicated AEA protected HT22 cells from the damage caused by H2O2, and AM251 reversed this protection, suggesting that the protective effects of AEA may be mediated via CB1.

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