Study On The Protection And Mechanism Of Biochanin A Against Aβ_25-35 Induced Neurotoxicity

Objective:Alzheimer’s disease（AD）,named after its discoverer Alois Alzheimer,is characterized by progressive deterioration of memory,cognition and behavior.It is the leading cause of dementia worldwide,affecting around 24 million demented individuals across allnations.It has been estimated that 115.4 million people will suffer from AD by 2050.Mitochondrial damage,Oxidative stress and Ca²⁺ overload is actived by β-amyloid which is considered extremely critical for AD development.There is accumulating evidence that Aβ accuses the release of cytochrome C and the activation of cysteamine acid and aspartic acid protease,then neuron apoptosis process is started.Biochanin A,an O-methylated natural isoflavonoid classified as phytoestrogen.Compared with estrogen,phytoestrogens have estrogen-like effect and do not have the side effects of estrogen.Accumulated evidences have suggested that Biochanin A has been reported to show anti-tumorigenesis,anti-oxidation,and anti-inflammatory properties.Biochanin A has been investigated to explore its protective mechanism on dopaminergic neurons of the unilateral lipopolysaccharide（LPS）-injected rat.And Biochanin A has been shown to have neuroprotective effects in cerebral ischemia/reperfusion.However,little is known about the effects of biochanin A on the protection and mechanism of Biochanin A against Aβ_25-35 induced neurotoxicity.In this study,the effects of Biochanin A will be detected on AD cell model and AD rat model which are establed with Aβ_25-35.To further explore the function and mechanism of BiochaninA against the neurotoxicity of Aβ_25-35,aim at providing a new method for the prevention and treatment of AD.Methods:1 Primary culture of rat cortical neuronsThe cortex part were isolated from postnatal day 0（P0）Sprague Dawley rat,which were washed with D.Hank’Swashing liquid and minced,then subsequently digested for 15 min at 37?C water bath.Completely medium DMEM containing 10% FBS was added to terminate the digestion.After blowing into the cell suspension in completely medium,the cell suspension was seeded on the culture plates precoated with poly-L-lysine in the DMEM containing 10% fetal bovine serum at the density of 1.0×109·L^-1.The neurobasal medium was changed 4 hours after culture.48 h later,cytarabine was added to inhibite the growth of astrocytes.After 48 h the medium were completely changed.From then on,half of the culture medium was changed every other day.The neurons cultured for 7 days were used for experiment.2 Preparation of the AD rat model induced byAβ_25-35Rat was placed in the brain stereotaxic instrument after abdominal cavity anesthesia with 2% pentobarbital sodium.Cut along the midline skull skin,Five microliters aggregated Aβ_25-35 or vehicle were injected slowly over 10 min into bilateral CA1 hippocampus with a 10 μL microsyringe followed by 10 min remaining to allow the diffusion of injection content.Slowly withdraw the needle and sew up a wound.3 Preparation of Aβ peptidesAβ_25-35 was initially dissolved in sterile distilled H₂O（2 mM）,vortexed and stored at-80℃.Then peptides were aggregated at 2 mmol/L in sterile distilled H2 O at 37℃ for 72 h prior to use.Dilutions were further performed in free serum culture medium to obtain appropriate concentrations.4 Group of Experiments and Drug treatment4.1 Effects of Aβ_25-35 on neuronal survivalCortical neurons,prepared from postnatal day 0（P0）Sprague Dawley rat,were treated with Aβ_25-35.The cultures were treated with or without Aβ_25-35（0,10 μM,20 μM,or 40 μM）,used to the research of MTT.4.2 Effects of Biochanin A on neuronal survivalCortical neurons,prepared from postnatal day 0（P0）Sprague Dawley rat,were treated with Aβ_25-35.The cultures were treated with or without Biochanin A（0,0.5 μM,1 μM,2.5 μM,5 μM,10 μM,20 μM）,used to the research of MTT.4.3 Effect of Biochanin A on neuronal viability after Aβ_25-35 treatmentBiochanin A（0.2 μM,1 μM,5 μM,10 μM）was subjected to cells 6 h prior to addition of Aβ_25-35 at 20 μM for next 24 h,used to the research of MTT.4.4 Effects of Aβ_25-35 on the spatial learning and memory of ratsThirty SD rats were divided into three groups(sham group,vehicle group,and Aβ_25-35 group)randomly with ten in each.Six-day Morris water maze behavioral task was employed to test the spatial learning and memory of the animals since the seventh day after surgery.4.5 Effect of Biochanin A on behavioral in Aβ_25-35 treated ratsSeventy SD rats were divided into seven groups: sham group,Aβ_25-35 group,vehicle group（DMSO）,BL group(Aβ_25-35+Biochanin A 10 mg/kg weight),BM group(Aβ_25-35+Biochanin A 20 mg/kg weight),BH group(Aβ_25-35+Biochanin A 40 mg/kg weight),randomly with ten in each.Six-day Morris water maze behavioral task was employed to test the spatial learning and memory of the animals since the fourteenth day after surgery.4.6 Effect of estrogen receptor inhibitors on neuroprotection of Biochanin ACerebrocortical neurons from new born Sprague-Dawley rat pups（less than 24 h）were cultured as previously described.Cells were divided into seven groups: control group,Aβ_25-35 group（20 μM）,Biochanin A group(20 μM Aβ_25-35+1 mΜ Biochanin A),estrogen receptor inhibitors+Biochanin A group(20 μM Aβ_25-35+1 μM Biochanin A+10 μM ICI182780),E2 group(20 μM Aβ_25-35+100 nM E2)estrogen receptor inhibitors group+E2 group(20 μM Aβ_25-35+100 nM E2+10 μM ICI182780).Western blot was used to detect caspase-3.4.7 Effect of Biochanin A on MAPK in Aβ_25-35 treated neuronsCerebrocortical neurons from newborn Sprague-Dawley rat pups（less than 24 h）were cultured as previously described.Cells were divided into three groups: control group,Aβ_25-35 group（20 μM）,Biochanin A group(20 μM Aβ_25-35+1 μM BiochaninA).Western blot was used to detect P-JNK,P-ERK,P-p38.4.8 Effect of Biochanin A and p38 MAPK inhibitors SB202190 on caspase-3,Bcl-2,Bax,grp78 and p38 signaling pathway.Cerebrocortical neurons from newborn Sprague-Dawley rat pups（less than 24 h）were cultured as previously described.Cells were divided into three groups: control group,Aβ_25-35 group（20 μM）,Biochanin A group(20 μM Aβ_25-35+1 μM Biochanin A),p38 MAPK inhibitors+ Aβ_25-35 group(20 μM Aβ_25-35+10 μM SB202190),p38 MAPK inhibitors group（10 μM SB202190）,E2 group(20 μM Aβ_25-35+100 nM E2)Western blot was used to detect caspase-3,Bcl-2,Bax,P-p38,ATF2,grp78.5 Cell viability assayCell viability was assessed by MTT [3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2H-tetrazolium bromide] assay.Briefly neuronal cells were cultured on 24-well plates.Cultured neurons were treated with Aβ_25–35 and/or genistein.Afterwards,20 μL of MTT solution（5 mg/mL）was added to each well and incubated for 4 h at 37℃.The MTT solution was removed gently and 200 μL of DMSO was added to each well for 15 min.Detection and quantification of the formazan crystals are carried out with a microplate reader（Molecular Device Corporation,Sunnyvale,CA,USA）at 570 nm.The absorbance of the formazan formed in control cells was taken as 100 % viability.6 Western blot analysisNeuronal cells cultured on a 6-well plate were treated as previously described.After treatments,cells were washed twice by using ice-cold PBS.Then cells were collected and centrifuged at 12,000×g for 10 min at 4℃.Total cellular proteins were electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel,and then transferred to PVDF membranes.Membranes were blocked with 5% nonfat milk in Tris-buffered saline for 1 h at room temperature and incubated with primary antibody at 4℃ overnight.After washing with TBST（15min）,membranes were incubated with secondary antibody for 1 h at room temperature.Blots were developed with an ECL Western blot detection kit,integrated densities of bands were measured by Image J.7 Morris water maze testAnimals were placed in the test room 1 h early to adapt to the environment.Behavioral tests began at every day 9:30（room temperature: 25℃,water temperature : 20℃-22℃）.Animal test sequence remains the same.Rats were trained four trials each day,for five consecutive days.Recorded the time that from rats into the water to find the hidden platform,escape latency.On the sixth day,the platform was removed,the number of times the animal crossed the platform（number of platform crossings）and time ratio in platform quadrant（Ⅰtime ratio）were used as index of spatial memory retention.8 Statistical AnalysisAll data obtained were expressed as mean±SD,Statistical significance was determined by one-way analysis of variance（ANOVA）and student’ s t test followed by the post hoc Duncan test with spss13.0 software.P<0.05 was considered statistical significance.Results: 1 Biochanin A reduced Aβ_25–35-induced neuronal death 1.1 Effects of Aβ_25-35 on neuronal survivalCells treated with 20 μM Aβ_25–35 showed a nearly 40%（P< 0.05）reduction of viability,so 20 μM Aβ_25–35 was selected to be used to establish AD cell model in next experiments.1.2 Effects of Biochanin A on neuronal survivalCultured neurons were treated with Biochanin A at various doses for 24 h.Compared with control cells,cells with0.5 μM,1 μM,2.5 μM,5 μM,10 μM,20 μM didn’t be affected.1.3 Effect of Biochanin A on neuronal viability after Aβ_25–35 treatmentDifferent concentrations of Biochanin A（0.2 μM,1 μM,5 μM,10 μM）were subjected to cells 6 h prior to addition of Aβ_25–35 at 20 μM for next 24 h.Pretreatment of 1 μM Biochanin A significantly increased cell viability compared with that in the Aβ_25–35 group（P<0.05）and 1 μM Biochanin A was used in the subsequent experiments.2 Effects of Biochanin A on behavioral in Aβ_25-35 treated rats 2.1 Effects of Aβ_25-35 on the spatial learning and memory of ratsThe Morris water maze test results show that swimming speed no difference between groups.The mean escape latency in the Morris water maze behavioral task was significantly（P<0.05）higher in Aβ_25-35 group（59.5±0.8,54.3±1.7,49.4±2.3,44.3±2.1,40.7±1.2;Day 1 to Day 5,respectively.）compared with those of the vehicle group（59.3±0.9,49.8±1.6,45.3±1.2,29.1±2.0,19.9±2.1;Day 1 to Day 5,respectively.）The Morris water maze test results show that compared with vehicle group the number of platform crossings of Aβ_25-35 group significantly decreased.And the staying timer at ioinplatform quadrant of Aβ_25-35 group in the probe test were considerably（P<0.05）lower compared with those of vehicle group.2.2 Effect of Biochanin A on behavioral in Aβ_25-35 treated ratsThe Morris water maze test results show that swimming speed no difference between groups.The mean escape latency of BL,BM and BH groups were significantly（P<0.05）lower compared with Aβ_25-35 group.The number of platform crossings of the BL,BM and BH group was significantly（P<0.05）increased compared with those of Aβ_25-35 group.Meanwhile,the Ⅰ time ratio of BL,BM and BH group was significantly（P<0.05）increased compared with those of Aβ_25-35 group.3 Effect of estrogen receptor inhibitors on neuroprotection of Biochanin ACompared with the control group,Aβ_25–35 group showed significantly increased expression of Cleaved Caspase-3,which attenuated by pretreatment of Biochanin A（1 μM）and 17β-estradiol（100 nM）.When ICI182,780,Biochanin A,and Aβ_25-35 were added together to the culture medium,the level of Cleaved Caspase-3 significantly increased（P<0.05）.Likewise,the level of Cleaved Caspase-3 significantly increased when ICI182780 was added to cells treated with17 β-estradiol and Aβ_25-35（P<0.05）.4 Effect of Biochanin A on MAPK in Aβ25-35 treated neuronsThe expression of P-JNK, P-ERK and P-p38 were examined by using Western blot. After being exposed to 20 μM Aβ_25–35 for 24 h, the expression of P-JNK and P-p38 significantly increased, and the P-ERK level reduced. Biochanin A had no effect on Aβ-induced increase of P-JNK or decrease of P-ERK except increase of P-p38. 5 Effect of Biochanin A and p38 MAPK inhibitors SB202190 on caspase-3, Bcl-2 and p38 signaling pathwayAβ_25–35 group showed significantly increase expression of Cleaved Caspase-3 which attenuated by pretreatment of Biochanin A, SB202190 and 17 β-estradiol. Western blot results confirmed that the p38 inhibitor SB202190 can blocks Aβ-induced p38 phosphorylation. Biochanin A can functionally mimick SB202190 to partly inhibit Aβ25-35-induced phosphorylation of the p38. The results showed that Biochanin A attenuated Aβ_25–35-induced neurotocity at least inpart through inhibiting Aβ_25–35-induced p38 activation.Compared with the control group, Aβ_25–35 group showed significantly increased expression of ATF2 which attenuated by pretreatment of Biochanin A, SB202190 and 17β-estradiol. The expression of Bcl-2 was markedly decreased by Aβ_25–35 compared with the control group, while such decrease was obviously attenuated by pretreatment of Biochanin A, SB202190 and 17β-estradiol（P<0.05）.Conclusions:1 Biochanin A can attenuate neuronal apoptosis induced by Aβ_25–35,improve the spatial learning and memory in Aβ25-35 treated rats2 It is possible that the neuroprotective effect of Biochanin A against Aβ25-35 neurotoxicity depends on ERs.3 It is possible that the neuroprotective effect of Biochanin A against Aβ25-35 neurotoxicity depends on mitochondrial apoptotic pathways and p38 MAPK signaling pathway.And Biochanin A against Aβ25-35 induced neurotoxicity through inhibition of endoplasmic reticulum stress independent on p38 MAPK signaling pathway.