| BackgroundAlzheimer’s disease（AD）is a progressive neurodegenerative disease with cognitive impairment and behavioral damage as the main clinical symptoms.It is mainly characterized by extracellular senile plaques formed by the deposition ofβ-amyloid（Aβ）in the brain,intracellular neurofibrillary tangles formed by hyperphosphorylation of tau protein,and neuronal loss.At present,the most recognized amyloid cascade hypothesis holds that Aβaggregation is the central trigger factor leading to a series of pathophysiological changes in AD,which can induce neuronal damage and loss,synaptic deletion,neuroinflammation,and tau protein hyperphosphorylation.These pathological processes in turn further promote Aβdeposition,thus forming a cascade amplification effect and eventually leading to cognitive dysfunction.According to the pathogenesis of AD and the amyloid hypothesis,targeting Aβis a promising therapeutic direction and also a hot research topic at present.AstragalosideⅣ（AS-Ⅳ）is the main active component of Astragalus membranaceus.The previous study of our group found that AS-Ⅳcan improve the learning and memory loss in a variety of dementia models,but its mechanism is not completely clear.Peroxisome proliferators-activated receptor gamma（PPARγ）is one of nuclear receptor superfamily members,which mainly regulates transcription of target genes and participates in many physiological reactions such as lipid metabolism,sugar homeostasis,inflammatory reaction,apoptosis and so on.PPARγexpression or activation plays an important protective role in neurodegenerative diseases.PPARγagonists can improve the memory and cognitive ability of AD patients,and PPARγmay be a potential target for AD therapy.Furthermore,BDNF has been proved to be closely related to the pathogenesis and progression of AD.Its expression is regulated by PPARγ.BDNF plays an important regulatory role in synaptic plasticity and learning and memory through Trk B receptor.In this study,we intend to further verify the neuroprotective effect of AS-Ⅳin vitro and in vivo model of AD-like induced by Aβ1-42,and further explore its potential mechanism.PartⅠEffect of AS-Ⅳon AD-like behavior induced by Aβ1-42 in mice and its mechanismObjectivesWe aim to study the effect of AS-Ⅳon AD-like behavior in mice and explore its mechanism via PPARγ-BDNF/Trk B signal pathway.MethodsMale C57BL/6 mice aged 5-6 weeks were randomly divided into sham group,Aβ1-42 injection group,AS-Ⅳ（10,20,40 mg/kg）,PPARγinhibitor group,and positive control group.Except for the sham group and the positive control group,the mice in the other groups were intragastrically administered with AS-Ⅳonce a day for one week,and then bilateral hippocampi were injected with Aβ1-42.Twenty-four hours after the injection,the positive control group was given donepezil 5 mg/kg by gavage;the PPARγinhibitor group was given AS-Ⅳ20 mg/kg by gavage and GW9662 1 mg/kg by intraperitoneal injection,respectively;the AS-Ⅳlow,medium and high dose groups were given AS-Ⅳ10 mg/kg,20 mg/kg and 40 mg/kg by gavage as before,respectively;the sham group and the Aβ1-42 injection group were given physiological saline of the same volume by gavage,once a day for four weeks.After the administration,the cognitive function and AD-like phenotypes of the mice were evaluated.Behavioral tests related to cognitive function in mice were:Morris water maze test to detect spatial learning and memory,new object recognition task to observe recognition memory,and conditioned fear test to evaluate fear memory.After the behavioral test,the hippocampal tissues were isolated and Nissl staining was used to observe the damage degree of hippocampal neurons.Enzyme Linked Immunoserbent Assay（ELISA）was used to detect endogenous Aβ1-42 level and the contents of IL-1β,IL-6 and TNF-α.Immunohistochemistry was used to detect the positive expression of BDNF and PPARγ.Golgi-Cox staining was used to measure the density of dendritic spines in hippocampal neurons.The ultrastructure of synapse was observed by transmission electron microscope.RT-q PCR was used to detect BDNF and PPARγm RNA levels;Immunofluorescence was used to detect the co-expression of PPARγand BDNF,SYN and MAP-2,PPARγand GFAP,and the positive expression of PSD95,SYN and GAP43.Western blotting was used to detect the expression of t-tau,p-tau,BDNF,t-Trk B,p-Trk B,PPARγ,GFAP,cleaved caspase-3 protein,and synaptic related proteins PSD95,SYN,GAP43 and Arc.TUNEL staining was used to detect apoptosis of hippocampal neurons.Results1.AS-Ⅳimproves Aβ1-42-induced cognitive impairment in miceThe results of morris water maze test showed that the spatial learning and memory of mice injected with Aβ1-42 decreased significantly.The results of the new object recognition experiment showed that the recognition memory of mice injected with Aβ1-42 decreased significantly.The results of conditioned fear memory test showed that the cued fear memory and contextual fear memory of mice injected with Aβ1-42 decreased significantly.After mice were given AS-Ⅳ,The results of the navigation experiment showed that AS-Ⅳ（10,20,and 40 mg/kg）could reverse the escape latency in Aβ1-42-injected mice in a concentration-dependent manner.In the space exploration experiment,AS-Ⅳ（10,20,and 40 mg/kg）could increase the detention time spent in the target quadrant and the swimming distance spent in the target quadrant in a concentration-dependent manner,and improve the spatial learning and memory in Aβ1-42-injected mice.The results of new object recognition showed that AS-Ⅳ（10,20,and 40 mg/kg）had no effect on Aβ1-42-impaired recognition memory in mice.The results of conditional fear memory test showed that AS-Ⅳ（10,20,and 40 mg/kg）could reverse the freezing time in a concentration-dependent manner,and restore the contextual fear memory and the cued fear memory in Aβ1-42-injected mice.2.AS-Ⅳattenuates Aβ1-42-induced pathological changes in hippocampusThe results of Nissl staining showed that compared with Aβ1-42 injection group,the number of hippocampal neurons in AS-Ⅳadministration group increased,with regular morphology and relatively neat arrangement,and the number of Nissl corpuscles increased significantly,and the degree of neuronal damage decreased.Aβ1-42 injection did not result in significant increase of endogenous Aβ1-42 content in hippocampus of mice,but could significantly increase the phosphorylation level of tau.However,AS-Ⅳcould significantly reverse the increase in tau phosphorylation level in mice induced by Aβ1-42 injection.3.AS-Ⅳsuppresses Aβ1-42-induced synaptic toxicity in hippocampusThe results of immunofluorescence and Western blotting showed that compared with the sham group,the expression of synaptic specific proteins,such as PSD95,SYN and GAP43 in hippocampus of mice injected with Aβ1-42 decreased significantly,while AS-Ⅳsignificantly increased the expression of PSD95,SYN and GAP43 in hippocampus of mice injected with Aβ1-42.However,there was no significant difference in Arc protein expression among the experimental groups.The results of MAP-2 and SYN by immunofluorescence co-labeling showed that compared with the sham group,the number of MAP-2 positive cells in hippocampal neurons of Aβ1-42-injected mice was obviously reduced,accompanied by the reduced positive expression of SYN.AS-Ⅳcould significantly increase the expression of MAP-2 in hippocampus of mice injected with Aβ1-42,meanwhile the expression of SYN was also significantly increased.The results of Golgi-Cox staining showed that the density of dendritic spines in hippocampal neurons of mice injected with Aβ1-42 decreased significantly.AS-Ⅳcould significantly increase the density of dendritic spines in hippocampal neurons of mice injected with Aβ1-42.The results of the transmission electron microscope showed that the synapse number of mice injected with Aβ1-42 was significantly reduced.While AS-Ⅳcould improve the damage of synaptic structure and alleviate synaptic toxicity in hippocampus of mice injected with Aβ1-42.4.AS-Ⅳinhibits Aβ1-42-induced BDNF reduction via promoting PPARγexpression in hippocampusThe results of RT-q PCR,Western blotting and immunohistochemistry showed that the levels of BDNF and PPARγm RNA and protein in hippocampus of mice injected with Aβ1-42 decreased significantly.AS-Ⅳcould significantly increase the expression of PPARγand BDNF m RNA and protein in hippocampus of mice injected with Aβ1-42,and up-regulate the phosphorylation level of Trk B protein.In addition,the results of PPARγand BDNF by immunofluorescence co-labeling showed that the expression of PPARγin hippocampus of mice injected with Aβ1-42 decreased,meanwhile the expression of BDNF also decreased significantly.AS-Ⅳcould significantly increase PPARγexpression in hippocampus of mice injected with Aβ1-42,accompanied by increased BDNF expression.Furthermore,the co-administration of PPARγinhibitor,GW9662 and AS-Ⅳnot only blocked the effect of AS-Ⅳon PPARγexpression,but also blocked the effect of AS-Ⅳon BDNF expression in mice injected with Aβ1-42.The above results indicated that AS-Ⅳinhibited the decline of BDNF level induced by Aβ1-42,and triggered the BDNF/Trk B signal pathway by promoting PPARγexpression in hippocampal neurons.5.AS-Ⅳinhibits Aβ1-42-induced neuroinflammation via promoting PPARγexpression in hippocampusThe results of ELISA showed that the contents of IL-1β,IL-6 and TNF-αin hippocampus of mice injected with Aβ1-42 increased significantly.AS-Ⅳcould significantly inhibit the increase of IL-1β,IL-6 and TNF-αin hippocampus of mice induced by Aβ1-42.The results of immunofluorescence co-labeling of PPARγand GFAP and western blotting showed that the expression of PPARγin hippocampus of mice injected with Aβ1-42 decreased significantly,meanwhile the expression of GFAP was increased significantly.AS-Ⅳupregulated the expression of PPARγin hippocampus of mice induced by Aβ1-42,accompanied by the significant decrease in GFAP expression.While the co-admistration of PPARγinhibitor,GW9662 and AS-Ⅳblocked the effect of AS-Ⅳon the expression of IL-1β,IL-6,TNF-α,PPARγand GFAP induced by Aβ1-42in hippocampus of mice.The above results showed that AS-Ⅳalleviated the neuroinflammation induced by Aβ1-42 via promoting the expression of PPARγin hippocampus,inhibiting the proliferation of glial cells,and reducing the expression of pro-inflammatory factors.6.AS-Ⅳinhibits Aβ1-42-induced apoptosis in hippocampusThe results of TUNEL staining,transmission electron microscopy and western blotting showed that the apoptosis morphology of hippocampal neurons in mice injected with Aβ1-42 were obvious,and the expression of cleaved caspase-3 protein increased significantly.AS-Ⅳcould significantly downregulate the expression of cleaved caspase-3 protein in hippocampal neurons of mice induced by Aβ1-42 and inhibited neuronal apoptosis.SummaryAS-Ⅳpromotes PPARγexpression,and then up-regulates the BDNF level and activates BDNF/Trk B signaling pathway,repairs synaptic damage,inhibits the activation of glial cells in hippocampus,decreases the expression of pro-inflammatory factors,reduces the neuroinflammatory response,prevents hippocampal neurons apoptosis,and improve cognitive dysfunction induced by Aβ1-42 in mice.PartⅡProtective effect of AS-Ⅳon HT-22 cells induced by Aβ1-42 and its mechanismObjectivesWe aim to study the effect of AS-Ⅳon HT-22 cells induced by Aβ1-42 and its mechanism.MethodsThe AD-like cell model was established after exposure to 10μM Aβ1-42for 48 h in HT-22 cells.MTT assay was used to detect cell viability.LDH leakage was used to evaluate the degree of cell injury.Trypan blue dye exclusion test was used to detect cell death rate.RT-q PCR was used to detect BDNF and PPARγm RNA levels.PPARγRNAi test and treatment of PPARγspecific inhibitor（GW9662）were used to block PPARγexpression.Western blotting was used to detect the expression of BDNF,PPARγ,t-Trk B,p-Trk B and cleaved caspase-3 proteins.The contents of IL-1β,IL-6and TNF-αwere detected by ELISA.Flow cytometry was used to detect the level of intracellular reactive oxygen species.DAPI staining and Annexin V-FITC/PI double staining were used to detect the degree of apoptosis.Results1.AS-Ⅳsuppresses Aβ1-42-induced cytotoxicity in HT-22 cellsThe results of MTT assay showed that AS-Ⅳcould significantly increase the cell viability induced by Aβ1-42 in a concentration-dependent manner within the concentration range of 5-100μM.When the concentration of AS-Ⅳreached 500μM,there was no significant difference in cell viability compared with the Aβ1-42 treatment group.The results of LDH test showed that the cell injury degree increased significantly in HT-22 cells treated with Aβ1-42.The damage degree of cells pretreated with AS-Ⅳ（25,50,and 100μM）was significantly reduced.The results of trypan blue staining showed that the cell death rate decreased significantly in HT-22 cells treated with Aβ1-42.AS-Ⅳcould significantly inhibit the cell injury and death induced by Aβ1-42.2.AS-Ⅳsuppresses Aβ1-42-induced BDNF reduction in HT-22 cellsThe results of RT-q PCR and Western blotting showed that the expression of BDNF m RNA and protein and the phosphorylation level of Trk B were significantly decreased in HT-22 cells treated with 10μM Aβ1-42.Consistent with the effect of exogenous BDNF,AS-Ⅳsignificantly upregulated the expression of BDNF m RNA and protein induced by Aβ1-42 and the phosphorylation level of Trk B in a concentration-dependent manner.In addition,AS-Ⅳalone could also significantly promote the expression of BDNF protein in normal HT-22 cells(without Aβ1-42).The above results showed that AS-Ⅳinhibited the decline of BDNF level induced by Aβ1-42 and activated the BDNF/Trk B signal pathway.3.AS-Ⅳsuppresses Aβ1-42-induced decline of BDNF level via promoting PPARγexpression in HT-22 cellsThe results of Western blotting showed that BDNF protein level also decreased significantly after interfering PPARγ,blocking its expression in HT-22 cells.In HT-22cells induced by Aβ1-42,interference with PPARγor blocking its expression with GW9662 could significantly reverse the effect of AS-Ⅳon BDNF protein expression.In addition,PPARγm RNA and protein levels decreased significantly in HT-22 cells injured by Aβ1-42.AS-Ⅳcould significantly upregulate PPARγm RNA and protein levels induced by Aβ1-42.In addition,AS-Ⅳalone could also promote the expression of PPARγprotein in normal HT-22 cells(without Aβ1-42).The above results showed that AS-Ⅳinhibited the decline of BDNF level induced by Aβ1-42 via promoting PPARγexpression.4.AS-Ⅳinhibits Aβ1-42-induced neuroinflammattion via promoting PPARγexpression in HT-22 cellsThe results of ELISA showed that the contents of IL-1β,IL-6 and TNF-αincreased significantly in HT-22 cells treated with 10μM Aβ1-42.AS-Ⅳcould significantly reduce the content of IL-1β,IL-6 and TNF-αinduced by Aβ1-42 in a concentration-dependent manner.PPARγinhibitor,GW9662 could significantly reverse the effect of AS-Ⅳon Aβ1-42-treated HT-22 cells.The above results showed that AS-Ⅳreduced pro-inflammatory factor expression and inhibited the neuroinflammattion induced by Aβ1-42via promoting PPARγexpression.5.AS-Ⅳsuppresses Aβ1-42-induced oxidative damage and apoptosis in HT-22 cellsThe results of flow cytometry showed that Aβ1-42 induced the significant increase in the level of reactive oxygen species in HT-22 cells,consistent with the effect of NAC,AS-Ⅳcould significantly inhibit the oxidative damage induced by Aβ1-42 in HT-22 cells.Additionally,exogenous BDNF had the same effect as AS-Ⅳand could significantly reduce the neurotoxicity induced by Aβ1-42.While PPARγinhibitor,GW9662significantly reversed the enhancement effect of AS-Ⅳon the cell viability induced by Aβ1-42 in HT-22 cells.The results of DAPI staining,Annexin V-FITC/PI double staining and Western blotting further showed that Aβ1-42 significantly induced apoptosis in HT-22 cells.Consistent with the effect of exogenous BDNF,AS-Ⅳcould significantly down-regulate the expression of cleaved caspase-3 protein induced by Aβ1-42 and inhibit cell apoptosis.While PPARγinhibitor,GW9662 significantly reversed the anti-apoptotic effect of AS-Ⅳ.The above results showed that AS-Ⅳinhibited apoptosis induced by Aβ1-42 in HT-22 cells via promoting PPARγexpression.SummaryAS-Ⅳcould significantly inhibit the neuronal damage,neuroinflammation and apoptosis in HT-22 cells induced by Aβ1-42,which may be related to its promotion of PPARγexpression,further up-regulation of BDNF level and activation of BDNF/Trk B signaling pathway.ConclusionsAS-Ⅳup-regulated BDNF level and activated the BDNF/Trk B signaling pathway via promoting PPARγexpression,subsequently repaired synaptic damage,reduced neuroinflammation,inhibited hippocampal neuron injury and apoptosis,thereby improved cognitive dysfunction,and exerted neuroprotective effects in Aβ1-42-treated HT-22 cells and C57BL/6 mice. |
