| Alzheimer’s disease(AD) is one of the most common neurodegenerative diseases, whose typical symptoms include progressive learning and cognitive dysfunctions. It is reported that Aβ neurotoxicity is the most vital cause for cognitive dysfunction. Aβ neurotoxicity is manifested as neuronal damage and microglial activation. Therefore, reducing Aβ neurotoxicity is regarded as an effective therapy for AD. Some epidemiological studies showed that the incidence of AD in smokers is lower than that of non-smokers, and the effect is believed to be induced mainly by nicotine. Some latest investigations reported that nicotine could ameliorate cognitive dysfunction in AD mice, the mechanism, however, is still elusive.Activation of cannabinoid system induces neuroprotection, activation of cannabinoid CB1 receptor protects neurons, and activation of CB2 receptor reduces microglial activation. In addition, cannabinoid receptor antagonist could be used in treating nicotine addiction, and cannabis tetrahydrocannabinol(THC) can also attenuate the withdrawal symptoms of nicotine. These findings indicate that partial bioactivities of nicotine may be mediated via cannabinoid receptors.In this study, we used Aβ1-42 to induce cell injury in HT22 cells, and investigate the mechanism of nicotine-induced protections. We also used Aβ1-42 to activate N9 microglial cells, and explore nicotine-induced inhibition of microglial activation. Furthermore, we investigated the role of cannabinoid receptor and PKC in nicotine-induced neuroprotection. We found that nicotine protects HT22 cells via CB1 receptor and its downstream signal PKC. In addition, nicotine shifts N9 microglial from M1 state to M2 state to reduce the activation of N9 cells, which is mediated by CB2 receptor and PKC.At present, nicotine’s protective effects have been confirmed. Further, low-dose products of nicotine(including E-cigarretes, nicotine patch et al.) have been proved to show low toxicity and low dependence and now are widely used for smoking cessation. Our findings offered new evidences for broadening the clinical indications of nicotine, and possibly proposed new therapies for AD.Part One: Nicotine Attenuates Aβ Neurotoxicity in HT22 Neuronal Cells via Cannabinoid CB1 ReceptorExperiment One: Nicotine Attenuates Aβ Neurotoxicity in HT22 Neuronal CellsObjective: To find a suitable Aβ concentration for the cell injury model and an effective nicotine concentration for the protective mechanism investigation.Methods: The HT22 cells were planted into a 96-well cell culture plate, and then the cells were cultured in drug-free medium(Control) or the medium containing various concentrations of Aβ1-42(1, 5 and 10 μM), after incubation for 24 h, cell viability was assessed via MTT method. And then, the cells were divided into 6 groups, including control and the cells treated with different concentrations of nicotine(0、1、10、100 and 500 μM) plus 5 μM Aβ1-42, after 24-h incubation, MTT was used to evaluate cell viability.Results: We observed 5 μM Aβ1-42 could reduce the cell viability to ~60 % of the control, and we used 5 μM Aβ1-42 in the subsequent experiments. In addition, we also noticed that 100 μM nicotine enhanced cell viability of the HT22 cells exposed to 5 μM Aβ1-42(P<0.001).Conclusions: We used 5 μM Aβ1-42 and 100 μM nicotine to investigate the nicotine-induced protections in the subsequent experiments.Experiment Two: Nicotine Protects HT22 Cells via Cannabinoid CB1 ReceptorObjective: To study the role of CB1 receptor in nicotine-induced protection against Aβ1-42 in HT22 cells.Methods: We divided the HT22 cells into 5 groups, including: Control(cultured in drug-free medium), Aβ1-42(exposed to 5 μM Aβ1-42), Nico+Aβ1-42(exposed to 5 μM Aβ1-42 plus 100μM Nico), CB1 antagonist AM251+Nico+Aβ1-42(exposed to 5 μM Aβ1-42 plus 100μM Nico plus 10μM AM251) and AM251(exposed to 10 μM AM251) groups. After 24-h incubation, we evaluated cell viability, LDH release, apoptotic rate and the protein expression of CB1, Bcl2 and Bax.Results: Compared with group Aβ1-42, we noticed increased cell viability, reduced LDH release, lower apoptotic rate, up-regulated expression of CB1, Bcl2 and down-regulated expression of Bax in group Nico+Aβ1-42(P<0.05). And the effects were significantly reversed by CB1 antagonist AM251(P<0.05).Conclusions: Nicotine protects HT22 cells against Aβ1-42 via cannabinoid CB1 receptor.Experiment Three: The Role of PKC in Nicotine-Induced Protection in HT22 Cells Objective: To study the role of PKC in nicotine-induced protection against Aβ1-42 in HT22 cells.Methods: The HT22 cells were divided into 5 groups, including: Control, Aβ1-42, Nico+Aβ1-42, AM251+Nico+Aβ1-42 and AM251 groups. The ratio of memberane PKC to cytoplasm PKC expression was calculated to assess PKC activation. And then,we divided the HT22 cells into 5 groups, including: Control(cultured in drug-free medium), Aβ1-42(exposed to 5 μM Aβ1-42), Nico+Aβ1-42(exposed to 5 μM Aβ1-42 plus 100 μM Nico), PKC inhibitor Che+Nico+Aβ1-42(exposed to 5 μM Aβ1-42 plus 100μM Nico plus 10 μM Che), and Che+Aβ1-42(exposed to 5 μM Aβ1-42 plus 10 μM Che) groups. After 24-h incubation, we evaluated cell viability and LDH release, and observed cell morphology.Results: Compared with group Aβ1-42, we observed enhanced PKC activation in group Nico+Aβ1-42(P<0.05), and the effect was reversed by CB1 antagonist AM251(P<0.05). Further, we noticed inceased cell viability, lower LDH release and improved cell morphology in group Nico+Aβ1-42 compared to group Aβ1-42(P<0.05), and PKC inhibitor Che significantly reversed these effects(P<0.05).Conclusions: As the downstream signal of CB1, PKC mediated the nicotine-induced protection against Aβ1-42 in HT22 neuronal cells.Part Two: Nicotine Attenuated Aβ1-42-Treated Microglial Activation via Cannabinoid CB2 ReceptorExperiment One: Nicotine Attenuated Aβ1-42-Treated Microglial ActivationObjective: To find a suitable Aβ1-42 concentration to activate N9 microglial cells, and search for an effective nicotine concentration for the mechanism investigation.Methods: The N9 cells were divided into 5 groups, treated with different concentration of Aβ1-42(0, 1, 2.5, 5 and 10 μM), after 24-h incubation, we used western blot to evaluate the i NOS expression. Then, the cells were divided into 6 groups, including Control group(cultured in drug-free medium), and 5 μM Aβ1-42 plus different concentrations of nicotine(0, 1, 10, 100 and 500 μM respectively), after the treatments, ELISA was used to evaluate TNF-α and IL-6 concentrations in the supernatants.Results: Compared with the control, 5μM Aβ1-42 up-regulated the i NOS protein expression in N9 cells(P<0.05). And 100 μM nicotine markedly decreased TNF-α and IL-6 release from N9 microglial cells(P<0.05).Conclusions: 5 μM Aβ1-42 can activate N9 microglial cells, and 100 μM nicotine could inhibit the activation of N9 microglial cells.Experiment Two: The Mechanism of Nicotine-Induced Attenuation of Microglial ActivationObjective: To explore the mechanism of nicotine in the inhibition of microglial activation induced by Aβ1-42.Methods: The N9 cells were divided into 5 groups, comprised of Control, Aβ1-42, Nico+Aβ1-42, CB2 antagonist AM630+Nico+Aβ1-42 and AM630+Aβ1-42 groups. After 24-h incubation, we used western blot, immunocytochemistry to evaluate the expression of i NOS and Arg-1. The concentrations of TNF-α, IL-10, IL-6 and BDNF in the supernatants were also determined by ELISA. Then the N9 cells were divided into 5 groups: Control, Aβ1-42, Nico+Aβ1-42, PKC inhibitor Che+Nico+Aβ1-42 and Che+Aβ1-42 groups. After the treatments, we used ELISA to measure the concentrations of TNF-α, IL-10, IL-6 and BDNF in the supernatants.Results: Compared with group Aβ1-42, i NOS expression was decreased while Arg-1 was inceased in group Nico+Aβ1-42(P<0.05). And CB2 antagonist AM630 reversed these effects(P<0.05). We also noticed that the concentrations of TNF-α, IL-6 were lower while the concentrations of BDNF was higher in group Nico+Aβ1-42 compared to group Aβ1-42(P<0.05). And the effects could be reversed by AM630 or Che(P<0.05). Conclusions: Nicotine attenuated Aβ1-42-induced microglial activation by transforming N9 cells from M1 state to M2 state. And cannabinoid CB2 receptor and PKC mediated the effects. |
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