The Effect Of Nicotine On Cognitive Function Disorder And The Mechanism In AD Rats

Alzheimer's disease(AD) is a neurodegeneration disease of the nervous system.AD patients present with impairment in learning,memory and intelligence,and loss of the self-care ability at late stages.Due to aging of the population,AD has become the fourth cause of death of the elderly next to heart disease,tumor and stroke.The treatment and nursing of AD patients are costly,bringing heavy financial burdens to the family and the society as well.The hallmarks of AD pathology are senile plaques,neurofibrillary tangles, granulovacuolar degeneration of neurons and cerebrovascular amyloidosis.The precise etiology and pathogenesis of AD are unclear;therefore,an effective therapy of AD is not available yet.Recent studies have demonstrated that excessive,regional inflammatory reaction in the brain may cause the release of large amounts of free radicals and cytokines, resulting in the incidence and progression of AD.As with senile plaque(SP) and neurofibrillary tangles(NFTs),inflammatory reaction is also a pathologic hallmark of AD. In light of the pathologic hallmarks of AD,we consider blockage of inflammatory reaction as one of the therapeutic targets for AD.Aβand neurofibrils may activate microglial cells,leading to enhanced expression of cell surface MHC-Ⅱand increased secretion of inflammatory cytokines such as IL-1β,IL-6 and TNFα.IL-1β,in turn,activates astrocytes,and activated astrocytes secrete more inflammatory cytokines,resulting in Aβdeposition and neurofibrillary tangles;therefore,a vicious cycle forms,which causes irreversible damages to neurons and synapses as well as pathologic and clinical changes of the nervous system.Inflammatory cytokines resulting from brain inflammation are also trigger factors for brain neuroinflammation.Accordingly,non-steroid anti-inflammatory drugs(NSAIDs) are used to treat AD.It has been shown that the long-term use of NSAIDs may reduce the risk of AD and ameliorate the symptoms of AD patients.Therefor,anti-inflammation therapy can delay the onset of AD as well as improve cognitive function in the aymptoms of AD.Recent studies suggest that nicotine can inhibbite the inflammation.The rats are chronically administrated with nicotine.Results showed that nicotine can decrease the chemotaxis/chemokinesis of peripheral vlood mononuclear cells,impaire T-help cells antigen present,attenuate T-cell activation.Nicotine-treatment decrease T-cell proliferation, inhibite the migration of leukocytes,attenuates the plasma effusion induced inflammation.Smoking may also reduce the risk of AD.Treatment with nicotine transdermal patches may improve the intelligence of AD patients as revealed by the Coruner's continuous performance test(CPT).There is a cholinergic anti-inflammatory pathway in the body, through which acetylcholine or nicotine may reduce lipopolysaccharide-induced release of inflammatory cytokines such as TNF-α,IL-1βand IL-6 viaα7nAChR on macrophages and thus lessen lipopolysaccharide-induced inflammatory response.Since neurons,microglial cells and astrocytes expressα7nAChR in the central nervous system,is there a similar cholinergic anti-inflammatory pathway in the central nervous system?Acute treatment of ex vivo microglial cells with nicotine may reduce LPS-induced secretion of TNF-α.Nevertheless,the effect of nicotine on Aβactivated microglial cells, secretion of IL-1βand IL-6 has not been reported yet.In the study,we administered AD model rats with nicotine orally,and detected the changes in the contents ofα7nAChR,IL-1β,and IL-6,so as to observe the effect of nicotine on cognition and release of inflammatory cytokines.Nicotine and Aβwere added into the mixed nerve cell cultures,and the protective effect of nicotine on neurons was observed; meanwhile,the changes in IL-1 and IL-6 were detected,so as to further elucidate the anti-inflammatory effect of nicotine.Main contents and results:1.Effect of nicotine on Aβ-induced inflammatory reaction in the nervous systemS-D rats were orally administered with nicotine to perform nicotine pretreatment.Then, Aβ25-35 was injected into bilateral hippocampus to establish the nicotinic AD rat model and the AD control rat model.The differences in learning and memory among the AD nicotine rats,AD rats and normal rats were observed by the Morris water maze test.During the place navigation test,the latency was longer in the nicotine AD group than in the normal control group,and the percentages of swimming time and swimming distance at each quadrant were lower in the nicotine AD group than in the normal control group,but were still significantly higher than in the AD control group.In the AD control group,the latency tended to decrease,but was not stable,and the frequency of search at each quadrant was low and the purposiveness was not clear,and there were significant differences in the regards between the nicotine AD group and the normal control group.Compared to the normal control group,rats of the nicotine AD group presented with certain impairment of intelligence,but the impairment was significantly milder than that in the AD group. Variance analysis revealed significant differences among the three groups of animals.Western blot analysis ofα7nAChR protein in hippocampal tissue revealed thatα7nAChR protein content was decreased slightly in the nicotine AD group at days 1,7 and 15 after injection of Aβ(P＜0.05 at day 1),and there were no significant differences between the nicotine AD group and the normal control group.However,in the AD group,α7nAChR protein content was decreased at days 1,7 and 15 after injection of Aβand existed till day 15,and there were significant differences between the nicotine AD group and the normal control group.α7nA ChR immunoreactive cells:In the normal control group,cells were darkly stained and well-arranged in great density.In the nicotine AD group,cells were stained relatively darkly and arranged relatively well,just similar to cells of the normal control group.However,in the AD group,cells were faintly stained and arranged in mess.The levels of IL-1βand IL-6 in the hippocampal tissue:ELISA results indicated that IL-1βand IL-6 contents of hippocampal tissue were low in the normal control group and increased slightly in the nicotine AD group following injection of Aβ(the contents were the highest at day 1 and then decreased gradually),and there were no significant differences between the nicotine AD group and the normal control group.However,in the AD group, IL-1βand IL-6 contents increased drastically and reached a peak at day 7 and then decreased,but the contents were still significantly higher than those in the normal control group and the nicotine AD group at day 15.IL-1βimmunoreactive cells:In the hippocampus of rats of the normal control group and the nicotine AD group,IL-1βimmunoreactivity was weak and cells were faintly stained; however,in the AD group,IL-1βimmunoreactivity was strong and cells were darkly stained.IB4 immunoreactive microglial cells:In the normal control group:the number of IB4 immunoreactive hippocampal cells was small;cells were faintly stained,with flat and long body and short and thin processes.In the nicotine AD group:the number of IB4 immunoreactive hippocampal cells was increased compared to the normal group,and cells were faintly stained with similar morphology with cells of the normal group.In the AD group:The number of IB4 immunoreactive hippocampal cells was significantly increased, and cell body size was increased,with prolonged and thickened processes.Some of the IB4 immunoreactive cells were of shrub- or rod-shape.GFAP immunoreactive astrocytes:In the normal control group:the number of GFAP immunoreactive hippocampal cells was small,cells were faintly and evenly stained,cell body size was small,and nerve processes were thin and short.In the nicotine AD group:the number of GFAP immunoreactive hippocampal cells was increased,cells were darkly stained,the body size of some cells was increased,and nerve processes were prolonged.In the AD group:The number of GFAP immunoreactive hippocampal cells was significantly increased,the size of cell body was increased,and nerve processes were thickened.2.Counteraction of nicotine against Aβ_25-35 neurotoxicityThe mixed hippocampal cells of 2d-old Sprague-Dawley rats were cultured.There were four groups:the normal control group,Aβ(2μM) group,the nicotine(10μM) +Aβ(2μM) group and the nicotine(20μM)+Aβ(2μM) group.Hippocampal tissue was isolated under sterile condition,and mixed hippocampal nerve cells were cultured.At day 5 of culture,cells were observed under phase-contrast microscope.Cells grew well,cell body was flat,with marked halos around,body size was increased,and nerve processes were long and extended gradually,forming a network.The hippocampal cultures incubation with differentia concentration nicotine for 1h prior to treatment with Aβ_25-3.After 24h incubation of cells with various concentrations of nicotine or nicotine +Aβ,IL-1βand IL-6 contents in culture supernatants were determined by ELISA and neurons were subjected to fluorescence staining,followed by determination of neuron body diameter and process length as well as cell viability assessment by the MTT assay.IL-1βand IL-6 contents in culture supernatants:In the nicotine Aβgroup,IL-1βand IL-6 contents were increased mildly after addition of Aβ;the differences in IL-1βand IL-6 contents were not significant between the nicotine Aβgroup and the normal control group, but were significant between the nicotine Aβgroup and the Aβgroup,suggesting that nicotine has inhibitory effect on Aβ-induced release of IL-1βand IL-6.Provided with a constant Aβconcentration,the inhibitory effect was related to the nicotine concentration.In the Aβgroup,IL-1βand IL-6 contents in culture supernatants were significantly higher than those in the nicotine Aβgroup and the normal control group,confirming that Aβmay promote nerve cells to secrete excessive IL-1βand IL-6.Effect of nicotine on hippocampal cultures counteracting Aβneurotoxicity:In the normal control group,fluorescence stains of the neuron were even,neuronal body was full, cell contour was smooth and round,and nerve processes formed a network.In the nicotine AD group,fluorescence stains of the neuron were similar to those of normal neurons, neuron body was full,cell contour was smooth and round,nerve processes grew like normal ones,neuron body diameter and process length were near to those of normal neurons.In the Aβgroup,the number of neurons was small,some cells detached from the bottom wall and floated in the medium,damaged neurons degenerated gradually,cell body was swollen,and there were sediments,ruptured nerve processes,and disintegrated neurons.Moreover, neuron body diameter was increased,while nerve processes shortened.MTT assay of cell proliferation and viability:Cell viability was 54.7%and 62.7%in the nicotine(10uM) +Aβgroup and the nicotine(20uM) +Aβgroup respectively,but only 29.57%in the Aβgroup,suggesting that nicotine may counteract the neurotoxicity of Aβ, increase cell viability,and protect neurons.ConclusionsOral administration of nicotine dissolved in drinking water simulated spontaneous smoking well in terms of nicotine absorption and drug action.The nicotine AD rat models were established by injecting Aβ_25-35 in bilateral hippocampus after certain periods of oral administration of nicotine.During the Morris water maze test,the latency in the place navigation test was prolonged in the nicotine AD group as compared to the normal control group,but was significantly shortened compared to the AD group.In the spatial exploration test,the percentages of swimming time and distance at each quadrant were significantly increased in the nicotine AD group than in the AD group.These findings suggest that nicotine may prevent and treat cognitive disorder of AD rats.It was found that oral nicotine may increase expression ofα7nAChR,attenuate Aβ-reduced reduction ofα7nAChR and suppress Aβ_25-35-induced activation of microglial cells and astrocytes,thus reducing secretion of IL-1βand IL-6 by glial cells.Nicotine, mediated by microglial cellα7nAChR,suppresses secretion of IL-1βand IL-6,and thus exerts its anti-inflammatory effect.As revealed in the mixed hippocampal cultures,nicotine may counteract Aβ_25-35 cytotoxicity.Following nicotine pretreatment,the secretion of IL-1βand IL-6 by glial cells was suppressed,the neurotoxicity of Aβ_25-35 to neurons was decreased,resulting higher cell viability;moreover.