Novel Mechanisms Of Neuronal Redox Homeostasis In Reducing Oxidative Stress Injury

Neuronal redox homeostasis is an intracellular state, reflecting the balance of oxidants and anti-oxidants in cells. An over-production of neuronal oxidants induces a large consumption of anti-oxidants in neurons, causing a breakage of neuronal redox homeostasis. Then, the oxidative damage and apoptosis may happen. For this reason, maintaining neuronal redox homeostasis is critical in reducing oxidative stress injury in CNS.Oxidative stress injury induces a variety of neurological conditions, including Alzhimer’s disease(AD), Parkinson’s disease(PD) and ischemic stroke. Oxidative stress leads to neuronal apoptosis and microglial activation, resulting in CNS damage ultimately. Therefore, administration of anti-oxidant is believed to be effective in treating the diseases induced by oxidative stress. The Chinese use natural anti-oxidants traditionally. More than 2000 years ago, the Chinese started drinking green tea, which contains natural anti-oxidant tea polyphenols. However, unfortunately, several large scale clinical trials indicated that some commonly used antioxidants, including vitamin C, vitamin D and β-carotene, did not induce significant anti-oxidative effects. The mechanism of anti-oxidative injury is not clear, which is one of the most important reasons for the failure of anti-oxidative therapies.It is reported that cannabis tetrahydrocannabinol(THC) and synthetic cannabinoid WIN55212-2 could attenuate oxidative stress injury and induce neuroprotections. Because THC brings about addiction, and WIN55212-2 induces mental symptoms, which limit the clinical use of the two drugs greatly. Anandmide(AEA) is an endocannabinoid, which could induce neuroprotective effects by activating cannabinoid receptors. However, whether AEA could reduce oxidative stress injury in neurons is still unknown.Amifostine is a common anti-radiative medicine, used in protecting normal tissue against X-ray and chemotherapeutic drugs in the patients with malignant tumors. In peripheral tissue, amifostine was found to be anti-oxidative. However, whether amifostine could induce anti-oxidative actions in neurons is still elusive.The aim of this study is to explore the neuroprotective effects of AEA and amifostine, and investigate anti-oxidative actions of the the two drugs in maintaining neuronal redox homeostasis.Part 1 Cannabinoid AEA-Induced Neuroprotective and Anti-oxidative MechanismsExperiment 1 Searching for a Suitable AEA Concentration and Observing CB1 Receptor Expression in the Presence of AEA in HT22 CellsObjective: To find a suitable AEA concentration and observe CB1 receptor expression in HT22 cells exposed to AEA.Methods: The HT22 cells were exposed to H2O2 to mimic oxidative stress injury. To find a suitable AEA concentration, the cells were divided into six groups. Except for control and H2O2 only groups, the other four groups were exposed to 200 μM H2O2 plus differentconcentrations of AEA(ranging from 1 μM to 20 μM). After 3-h incubation, according to the MTT result, we determined AEA concentration for the subsequent experiments. Then, the cells were assigned into five groups, including control, H2O2, AEA+H2O2, CB1 receptor antagonist AM251+AEA+H2O2 and AM251+H2O2 groups. After an incubation of 3 h, Western blot and immunocytochemistry were taken to observe CB1 receptor expression.Results: Compared with the H2O2 group, 10 μM and 20 μM of AEA increased the cell viability significantly(P < 0.05). Moreover, AEA markedly up-regulated CB1 receptor expression in HT22 cells(P < 0.05), co-administatrion of CB1 receptor antagonist AM251, however, partially reversed AEA-induced effects on CB1 expression(P < 0.05).Conclusions: We used 10 μM AEA in the subsequent experiments, and observed that AEA could up-regulate CB1 expression in HT22 cells.Experiment 2 CB1 Receptor Is Involved in AEA-Induced Maintenance of Neuronal Redox HomeostasisObjective: To explore the role of CB1 receptor in AEA-induced maintenance of neuronal redox momeostasis in HT22 cells.Methods: 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 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. We used MTT assay, LDH release, cell morphology and apoptotic rate to evaluate cell injury degree. Meanwhile, we tested the intracellular reactive oxygen species(ROS), superoxide dismutase(SOD), reduced glutathione(GSH) and oxidized glutathione(GSSG) to assess anti-oxidative ability of the cells. In addition, we used CB1-si RNA to further study the role of CB1 in AEA-induced neuroprotection. Moreover, we tested AEA-induced neuroprotection in a rat middle cerebral artery occlusion(MCAO) model.Results: AEA attenuated cell injury, decreased apoptotic rate, increased intracellular SOD and GSH levels, and down-regulated ROS and GSSG accumulations. The AEA-induced anti-oxidative injury actions were significantly reversed by CB1 antagonist AM251 or CB1-si RNA. And AEA-induced anti-ischemic ability was also abolished by administration of CB1 antagonist AM251.Conclusions: CB1 receptor is involved in AEA-induced neuroprotection by maintaining neuronal redox homeostasis.Experiment 3 Exploring the Role of NADPH Oxidase 2 in AEA-Induced Anti-oxidative EffectsObjective: To explore the role of NADPH oxidase 2(Nox2) in AEA-induced anti-oxidative ability in HT22 cells.Methods: We used Western blot and Real-Time PCR to evaluate AEA-induced effects on Nox2 protein expression and m RNA transcription. And then, we divided the cells 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; Nox inhibitor Apocynin+AEA+H2O2: cells exposed to 10 μM AEA plus 50 μM apocynin in the presence of 200 μM H2O2 for 3 h; Apocynin+H2O2: cells exposed to 50 μM apocynin plus 200 μM H2O2 for 3 h. We used Western blot to evaluate Nox2 protein expression, and took MTT assay and LDH release to test cell injury degree.Results: Compared with the control group, H2O2 exposure markedly up-regulated Nox2 protein expression and m RNA transcription(P < 0.05), and co-administration of AEA down-regulated Nox2 protein expression and m RNA transcription in the presence of H2O2(P < 0.05), CB1 receptor antagonist AM251 or CB1-si RNA, however, significantly abolished the AEA-induced effects on Nox2(P < 0.05). In addition, we noticed Nox inhibitor apocynin did not cause obvious effects on the AEA-induced cytoprotection and Nox2 protein expression(P > 0.05).Conclusions: Nox2 mediates AEA-induced anti-oxidative ability in HT22 cells.Part 2 Cytoprotectant Amifostine Induces Neuroprotective and Anti-oxidative Effects by Maintaining Redox Homeostasis in PC12 cellsObjective: To explore cytoprotectant amifostine(Ami)-induced anti-oxidative and neuroprotective effects.Methods: PC12 cells were exposed to a high concentration of glutamate(Glu) to mimic chronic oxidative stress injury in neurons. We divided PC12 cells into six groups, control: cells cultured in drug-free medium; Ami: cells exposed to 500 μM amifostine for 24 h; Glu: cells exposed to 15 m M glutamate for 24 h; Ami+Glu: cells exposed to 500 μM amifostine plus 15 m M glutamate for 24 h; SOD2-si RNA+Ami+Glu: cellular SOD2 protein was down-regulated by SOD2-si RNA, and then exposed to 500 μM amifostine plus 15 m M glutamate for 24 h; SC-si RNA+Ami+Glu: cells were treated with scrambled si RNA, and then exposed to 500 μM amifostine plus 15 m M glutamate for 24 h. We used immunocytochemistry and Western blot to observe SOD2 expression level, and took MTT assay and LDH release to evaluate cell injury degree. Meanwhile, we tested mitochondrial superoxide product, intracellular ROS, GSH and catalase(CAT) levels.Results: Compared with control, glutamate exposure down-regulated SOD2 protein expression, injured PC12 cells, increased mitochondrial superoxide product and intracellular ROS accumulations, and decreased GSH and CAT levels(P < 0.05). Amifostine up-regulated SOD2 expression, attenuated cell injury degree, decreased mitochondrial superoxide product and intracellular ROS accumulations, and increased GSH and CAT levels(P < 0.05) in PC12 cells. Administration of SOD2-si RNA obviously abolished the amifostine-induced neuroprotective and anti-oxidative effects(P < 0.05), in contrast, SC-si RNA did not induce significant effects on the amifostine-induced anti-oxidative actions(P > 0.05).Conclusions: SOD2 mediates amifostine-induced neuroprotective and anti-oxidative actions by maintaining redox homeostasis.Summary: Nox2 is involved in AEA-induced neuroprotection via CB1 receptor by maintaining neuronal redox homeostasis. And SOD2 mediates amifostine-induced neuroprotective and anti-oxidative effects by maintaining redox homeostasis in PC12 cells.Oxidative stress-associated CNS conditions bring great harm to public health, effective anti-oxidants, however, are very limited. Moreover, the limited anti-oxidants are not suitable for long-term use. Our findings offer new choices for the sufferers of oxidative stress-associated CNS conditions, and also provide novel mechanisms in exploring pharmacological actions of cannabinoid AEA and cytoprotectant amifostine.