The Neuroprotective Effects Of Gossypium Herbaceam L. Extracts On AD Rat Induced By Aβ And The Study Of Inflammatory Mechanisms Involved In AD Rat Model

Alzheimer's disease(AD),one of the most prominent neurodegenerative diseases,is characterized by progressive loss of cognitive abilities.The neuropathological hallmarks of the disease are senile plaque(SP),neurofibrillary tangles(NFT) and loss of neurons. There have been a lot of studies providing some hypotheses on the pathophysiology of AD,but the mechanisms of AD remain unclear.Most of the studies suggested that AD was resulted from a lot of different etiopathogenisis and their interactions.And the formation,metabolism and toxicity ofβ-amyloid protein(Aβ) have been suggested to play a central role in the pathogenesis of AD.Some causes,such as gene defect,might induce abnormal metabolism of Aβprecursor protein(APP),which can contribute to Aβaccumulation.As a consequence,Aβwould induce the damage of neurons through several different pathways,including excitatory toxicity,dysfunctions of mitochondria, abnormal energy metabolism,disequilibrium of calcium homeostasis,apoptosis, oxidative stress,neuroinflammation process,and so on.At the present,there is no definitive treatment or cure for AD.A large segment of the public finds solace in herbs,in part believing that herbs are natural and hence safer than synthetic drugs,and that a complex mixture of herbs can effectively treat complex diseases.In addition,the metabolite profile of herbal medicine is important for screening its active constituents,thus providing a valuable contribution to the drug discovery process and elucidation of the underlying mechanism of action.Gossypium herbaceam L. extracts(GHE) is an active standardized extract obtained from Gossypium herbaceam L., an ethical herb used by Uygur people in Xinjiang,China,to treat mental retardation.In the present study,the neuroprotective effect of GHE against Aβ_25-35 induced learning and memory impairment was observed in vivo.We used GHE to treat the rats injected with Aβ_25-35,and used donepezil as the positive control.The results suggested GHE could be considered as a potential agent for preventing or retarding the development or progression of AD.Additionally,recent evidences showed that inflammatory disorders were involved in the onset and progression of AD.Glial cells(microglia and astrocytes) played a major role in the neuroinflammatory process in AD.The activation of microglia,in response to Aβaccumulation,leaded to enhancements in the production of inflammatory mediators. As a consequence,inducible nitric oxide synthase(iNOS) was activated,which produced nitric oxide(NO),and reactive nitrogen specie(RNS).Moreover,toxic reactive oxygen species(ROS) were produced,which could also react with RNS to form peroxynitrite that was highly toxic to cells and caused apoptosis.Additionally,interactions between microglia and astrocytes have been observed in the brain,which might further result in the increased production of pro-inflammatory cytokines,complement proteins,and oxidants.Both types of glial cells might contribute to neuronal damage and the accumulation of Aβ.Aβcould further induce a cascade of cellular mechanism,including activation of both glial cells to release inflammatory mediators.In contrast to the neurotoxic effects,neuroprotective effects of glial cells,especially astrocytes,in the process of neurodegeneration have been observed.Some investigations supported that both microglia and astrocytes,while responding to pro-inflammatory agents,could protect neuronal cells by producing neurotrophic factors(NTFs),and anti-inflammatory mediators(i.e.IL-4,IL-10).Thus,in the present study the chronic animal model of AD induced by Aβwas used to evaluate the relationship between neuroinflammatory process and neuroprotective effect resulted from glial cells activation.And it could provide some valuable evidences for the drug discovery process in the future.PartⅠ:The neuroprotective effects of Gossypium herbaceam L.extracts (GHE) on AD rat induced by Aβ1.There were many complex components in GHE.The retention times of main components were 18.36,18.46,18.56,19.51,20.46,20.79,21.99,22.26,and 23.37, respectively. 2.SD rats were randomly divided into 6 groups:rats treated with i.c.v,normal saline and i.g.distilled water were used as control group;rats treated with i.c.v.Aβ_25-35 and i.g. distilled water were used as model group;rats treated with i.c.v.Aβ_25-35 and i.g. donepezil were used as positive control group;rats treated with i.c.v.Aβ_25-35 and i.g. GHE 35 mg/kg were used as GHE low dose group;rats treated with i.c.v.Aβ_25-35 and i.g.GHE 70 mg/kg were used as GHE medium dose group;rats treated with i.c.v. Aβ_25-35 and i.g.GHE 140 mg/kg were used as GHE high dose group.3.Morris water maze data showed that Aβ_25-35-treated animals exhibited obviously longer escape latencies in probe test than that of the control(P＜0.01).The GHE (70mg/kg and 140mg/kg) significantly shortened escape latencies(P＜0.05),and ameliorated memory impairment due to Aβ_25-35,as did donepezil(P＜0.01).However, there was no dose dependence observed among each GHE treatment group.4.Step through test suggested that treatment with donepezil and GHE(70 mg/kg) effectively reduced Aβ-induced increase of numbers of errors in the passive performance test.GHE intragastricly administrated tended to increase the step-through latency,but the improvement did not achieve statistical significance.5.Nissl staining analysis indicated Aβ_25-35 significantly decreased the healthy cell density in CA1 region of hippocampus in rats,as compared to that in the control group(P＜0.05),and treatment with GHE-M(70 mg/kg) for following 14 days markedly reversed the Aβ_25-35-induced changes(P＜0.05) in the CA1 region.6.TUNEL results suggested apoptotic neurons in dentate gyrus in model rats were markedly increased compared to those in control rats,and positive cells were occasionally seen in dentate gyrus of rats treated with donepezil and GHE.7.The activity of GSH-Px and CAT in the hippocampus tissues of rats treated by Aβ_25-35 alone decreased in comparison with those in the control group.The most significant benefits exerted on improvement of GSH-Px and CAT activities in the hippocampus tissue were observed in GHE-L and GHE-M groups.Unfortunately,no significant differences existed in the activity of SOD and the level of MDA in hippocampus between the model group and each GHE group. 8.ELISA and western blotting results showed that Aβ_25-35 i.c.v,injection could significantly induce the enhancement of IL-Iβconcentration in hippocampus(P＜0.01),while to some extent,the IL-1RA expression showed a trend of increase as well. However,the ratio of IL-1RA/IL-1βobviously increased in hippocampus of rats treated with Aβ_25-35 alone(P＜0.01).Which suggested the activity of IL-1 system in model group is higher than that in the control group.Besides,GHE(70 mg/kg and 140 mg/kg) could reverse the changes induced by Aβ,which indicated that GHE could play an important role against the inflammation induced by IL-1 system.9.The nuclear translocation and activation of NF-κB were analyzed by western blot and EMSA,respectively.Western blot analysis proved that GHE,especially at the dose of 70mg/kg administration(P＜0.05),could effectively inhibit the Aβ_25-35 induced translocation of NF-κB into cell nucleus in hippocampus of rats,and EMSA result indicated that treatment of GHE could prevent the activation of NF-κB due to Aβ_25-35. Aβeffectively caused degradation of IκB-α(P＜0.05),which could be significantly inhibited by treatment with GHE at the doses of 70 and 140 mg/kg(P＜0.05). Immunohistochemistry assay data also showed that the levels of NF-κB in cell nucleus in CA1 and CA3 region of hippocampus were significantly increased in the presence of Aβ_25-35 after two weeks(P＜0.01).Interestingly,this increase in nucleus in CA1 region was prevented by treatment with donepezil,GHE-M and GHE-H(P＜0.05). Meanwhile,donepezil,GHE-L,GHE-M and GHE-H could also reduce the level of NF-κB in nucleus in CA3 region of hippocampus in rats,respectively(P＜0.01).PartⅡ:The study of inflammatory mechanisms involved in AD rat model induced by i.c.v,infusion of Aβ1.Long Evans rats were randomly divided into 2 groups:rats treated with i.c.v. continuous infusion of normal saline(NS) 5μl per day for 14 days were used as control group;rats treated with i.c.v,continuous infusion of Aβ_25-35 300 pmol per day for 14 days were used as model group.2.Morris water maze data showed that compared with the control group,the escape latency obviously prolonged due to Aβ_25-35 i.c.v,infusion(P＜0.01).3.Immunohistochemistry analysis results indicated that compared with the control group, the number of GFAP immunopositive cells was significantly increased in CA1,CA3 and DG regions in hippocampus of rats in Aβmodel group(P＜0.01),which suggested that GFAP expression enhanced and astrocytes activated;While the number of Ibal (one of the markers of microglia) immunopostive cells were obviously increased only in CA1 region of hippocampus in rats in Aβmodel group compared with those in the control group,which suggested that microglia could be activated by Aβi.c.v,infusion. In addition,real-time PCR results provided further supports that the transcriptions level of markers for astrocytes and microglia were significantly increased in Aβmodel group compared with those in the control group.4.Bio-plex cytokine assay showed that among the 9 cytokines which were detected, IL-1α(P＜0.01),IL-1β(P＜0.01) and TNF-α(P＜0.05) were obviously up-regulated by Aβi.c.v,infusion,while the anti-inflammatory cytokine IL-4 was down-regulated by Aβi.c.v,infusion.However the latter did not achieve significant change.5.Western blotting and ELISA data indicated that among some NTFs and their receptors, the BDNF(P＜0.01) and GDNF(P＜0.05) expressions were markedly higher in hippocampus of rats in Aβmodel group.While the NGF(P＜0.01) and p75^NTR(P＜0.05) expressions obviously decreased due to Aβi.c.v,infusion.Real-time PCR results showed that BDNF(P＜0.05) and Trk B(P＜0.05) transcription levels were significantly lower than those in the control group.6.The corticosterone levels in serum in rats were analyzed by EIA test.The data suggested that the corsticosterone level was obviously increased due to Aβi.c.v. infusion in this study(P＜0.05).7.The protein expression and the transcription level of APP in hippocampus of rats were evaluated by western blotting and real-time PCR analysis respectively.The results showed that to some extent,both of the protein expression and transcription level altered due to Aβi.c.v,infusion in this study,but the differences did not achieve statistical significance.