| Alzheimer’s disease (AD), characterized clinically by progressivelycognitive impairment and pathologically by the appearance of senile plaquesand neurofibrillary tangles, is the most prevalent senile dementia. Amyloid β(Aβ), a β-sheet peptide fragment produced by the proteolytic cleavage ofamyloid precursor protein (APP) by γ-and β-secretases, is the majorcomponent of senile plaques. Both in vivo and in vitro studies have shown thataccumulation of Aβ fibrils trigger neurodegeneration, supporting the view thatAβ aggregation is of importance in AD. Aβ causes oxidative damage,inflammatory responses, and memory impairment, all of which may lead tothe neuronal dysfunction and degeneration responsible for the cognitivedeficits observed in AD.Steroids hormones and their metabolites within the central nervoussystem (CNS) are commonly defined as neuroactive steroids or neurosteroids.They can be either synthesized de novo in the CNS by glial cells and neuronsfrom cholesterol or synthesized in the periphery by the adrenals and gonads.The concentration of neurosteroid was higher in CNS than in periphery.Neurosteroids mainly include pregnenolone (PREG), dehydroepiandrosterone(DHEA), their sulfate derivatives pregnenolone sulfate (PREGS) anddehydroepiandrosterone sulfate (DHEAS), and progesterone (PROG),5α-dihydroprogesterone (5α-DHP),3α,5α-tetrahydroprogesterone(allopregnanolone/ALLO), deoxycorticosterone (DOC),tetrahydrodeoxycorticosterone (THDOC), estradiol (E2), etc. Neurosteroidsplay an important role as endogenous modulators in altering neuronalexcitability rapidly; however, no specific receptor has been reported to datefor neurosteroids. Most of their actions in the nervous tissue were reported tomodulate membrane neurotransmitter receptors, such as GABAA, NMDA, and sigma1receptors, and thus affect neuronal plasticity, anxiety, responses tostressful stimuli, and neuropsychiatric symptoms represented during AD.Some neurosteroids were reported to improve learning and memory abilityand protect against Aβ peptide-induced neurotoxicity, thus exerting neuronalprotection. However, changes in the metabolic pathway of neurosteroidsduring AD and their role in Aβ-mediated impairment remain relativelyelusive given the limitations in sample sizes and analysis methods.Objective: We have reported previously that the concentration ofprogesterone decreased in the media of Aβ25-35-induced cortical neurons, andprogesterone treatment inhibited the declination of cell viability as well as theapoptosis induced by Aβ25-35. The present study was designed to investigatethe levels of neurosteroids in encephalic regions related to learning andmemory (the prefrontal cortex and hippocampus) of Sprague-Dawley (SD)rats injected with Aβ25-35as well as the expression of neurosteroidogenesisenzymes. Furthermore, the effect of progesterone administration againstAβ25-35induced impairment was investigated in vivo and in vitro. The presentstudy provided a potential therapeutic strategy for disorders with learning andmemory impairment, such as AD.Material and Methods:1Effect of Aβ micro-injection on the spatial learning and memory of ratsTo investigate the effect of Aβ25-35administration on spatial learning andmemory of rats, a total of thirty SD rats were divided into three groups (shamgroup, vehicle group, and Aβ group) randomly with ten in each. Afteranesthetized with2%pentobarbital sodium (40mg/kg weight, intraperitonealinjection), the animals were placed in stereotaxic apparatus (Jiangwai typeâ… ).Five microliters aggregated Aβ25-35or vehicle (sterile distilled water, forvehicle group) were injected slowly over10min into bilateral CA1hippocampus (anterior/posterior:-3.5mm, media/lateral:±2.0mm, anddorsal/ventral:-2.7mm ventral to the skull surface) with a10μLmicrosyringe, followed by10min remaining to allow the diffusion ofinjection content. The rectal temperature was maintained at36℃to37℃for all animals throughout the surgery. Six-day Morris water maze behavioraltask was employed to test the spatial learning and memory of the animalssince the seventh day after surgery. After the behavioral test, all animals wereanesthetized with pentobarbital sodium and perfused through the ascendingaorta with normal saline followed by4%paraformaldehyde. The brain tissueswere then dissected and fixed for histological examination.Behavioral Tests: The Morris water maze (JLBehv-MWM, Shanghai,China) consisted of a black pool (180cm diameter,60cm high) filled withwater (40cm,23±1℃), and was divided into four quadrants with fourequidistant release points around the edge, with a circular black platform (10cm diameter) submerging1cm below the water surface and remaining thesame position of the target quadrant. The behavior of rats in the pool wastracked with a camera which allowing us to measure the swim distance andtime to find the platform. Rats were trained four trials each day, with aninterval of20min at least, for five consecutive days. The starting quadrantwas randomized every day with all animals according to the same order. Theanimals were faced towards the pool wall before being released, and allowedto stay on the platform for10s after finding the platform within60s, orguided to the platform to stay for10s if failed to reach the platform in60s.The time needed to reach the platform (escape latency) was analyzed asindices of spatial learning. The probe trial was conducted on the sixth day,when each subject allowed90s to search the water from which the platformhad been removed. The number of times the animal crossed the platform(number of platform crossings) and time ratio in platform quadrant (â…£ timeratio) were used as index of spatial memory retention. A camera was used totrack the time to find the platform as well as to measure the swim speed anddistance travelled.Histological Examination: The brain tissues were fixation in4%paraformaldehyde in phosphate buffer (pH7.4), dehydration in a series ofethanol, embedded in wax and sectioned into5-micrometer-thick sections.Tissue sections were then deparaffinized, dehydrated, and stained with thionin, followed by differentiation, dehydration and clearance before they weremounted. The number of intact pyramidal cells (per1mm linear length of thesame section of CA1hippocampus) was then counted by three experimentersand analyzed.2The metabolism alteration of neurosteroids in the prefrontal cortex andhippocampus of Aβ treated ratsTo investigate the effect of Aβ25-35on neurosteroids level in brain, a totalof ninety SD rats were divided into three groups (sham group, vehicle group(sterile distilled water), and Aβ group) randomly with thirty in each group andtreated the same as above. Ten rats in each group were sacrificed (viadecapitation) during16:00-18:00on the seventh day and the twelfth dayafter Aβ injection respectively, with the prefrontal cortex and hippocampusremoving (on ice) and stored in-70℃until neurosteroids detectionthroughhigh performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. On the twelfth day after Aβ injection, five rats in eachgroup were sacrificed (via decapitation) with hippocampus removed (on ice)and stored at-70℃for steroidogenesis enzymes analysis through RT-PCR,Western blot, and immunohistochemisty. The rests were anesthetized withpentobarbital sodium and perfused through the ascending aorta with normalsaline followed by4%paraformaldehyde. The brain tissues were thendissected and fixed for histological examination.Sample Preparation and HPLC-MS/MS Analysis: Samples (about100mg) were added with10μL30ng/mL MT (internal standards), homogenizedin1mL of phosphate buffered saline (PBS) using an ultrasonic homogenizer(Heidolph, Germany), mixed with2mL ethyl acetate/n-hexane (9:1, v/v)and centrifuged at12000rpm for10min. The pellet was extracted three timeswith2mL ethyl acetate/n-hexane (9:1, v/v), and the organic phases werecombined and dried with a gentle stream of nitrogen in a50℃water bath.The samples were derivatized with dansyl chloride in a60℃water bath for40min, concentrated at12000rpm for10min and then transferred inautosampler vials before the high performance liquid chromatography- tandem mass spectrometry (HPLC-MS/MS) analysis.The HPLC-MS/MS system (Thermo Fisher Scientific, Waltham, MA,USA) consisted of Surveyor MS Pump Plus, Surveyor AS Plus, TSQQuantum Access triple quadrupole mass spectrometer and Xcalibur DataSystems (Thermo Fisher Scientific). Separation was achieved on a XDB C18analytical column (4.6mm×50mm, Agilent, Palo Alto, CA, USA) fittedwith a XDB C18guard column (4.6mm×12mm, Agilent). The HPLC mobilephases were (A) H2O/0.1%formic acid and (B) MeOH/0.1%formic acid,and the flow rate was0.5mL/min. The gradient was as follows:0-6.5min，36%A;6.5-6.6min,30%A;6.6-17min,10%A;17-18min,36%A.The column temperature was40℃and the injection volume was30μL. AMSD quadrupole mass spectrometer equipped with atmospheric pressurechemical ionization (APCI) source (Thermo Fisher Scientific) was used forthe detection of analytes in the positive ion mode. The optimized conditionswere as follows: the spray voltage,4500V; the vaporizer temperature,400℃; the sheath gas,30L/min; the aux gas,5L/min; the capillary temperature,270℃. The quantification was performed using multiple-reaction monitoring(MRM) method with the transitions of (m/z)299.03â†'(m/z)158.86and280.9for PREG,(m/z)315.03â†'(m/z)97and109.01for PROG,(m/z)301â†'(m/z)188.9and282.85for ALLO,(m/z)254.97â†'(m/z)132.9and158.9for17β-E2,(m/z)303.1â†'(m/z)97.04and109.06for MT, respectively.RT–PCR: Total RNA was isolated from cells using TRIzol Reagent(Invitrogen, CA, USA) and equal amounts were reverse transcribed intocDNA using the oligo dT primer, then the cDNAs were used as DNAtemplates for PCR. GAPDH was used to ensure equal loading. The PCRproducts were separated on1.5%agarose gel and visualized by ethidiumbromide.Western Blot: Lysates from brain tissue were prepared with lysis buffer(1%Triton X-100,150mM NaCl,10mM Tris-HCl, pH7.4,1mM EDTA,pH8.0,0.2mM Na3VO4,0.2mM phenylmethylsulfonyl fluoride, and0.5%NP-40). Equal amounts of protein (40μg) were separated by10%SDS- PAGE, and electrotransferred to a PVDF membrane (Millipore, Billerica, MA,USA). Membranes were blocked with5%BSA for2h at room temperature,and incubated with the primary antibodies,1:1000mouse anti-TNF-α(Abcam),1:500rabbit anti-IL-1β (Abcam),1:500rabbit anti-CYP11A1(anbobio,),1:500rabbit anti-3β-HSD (Epitomics),1:500rabbit anti-aromatase (Abcam), and1:5000rabbit anti-β-actin (Santa Cruz) overnight,respectively, and then with the respective secondary antibody for2h. Proteinswere detected using the Chemiluminescence Plus Western blot analysis kit(Santa Cruz). The experiments were replicated three times.Immunohistochemistry: Five-micrometer-thick sections weredeparaffinized, dehydrated, followed by Antigen retrieval (microwave). Thensections were in turn incubated with3%H2O2for10min, with primaryantibody overnight at4℃, with diluted biotinylated IgG secondary antibody,with DAB, followed by being differentiated, dehydrated, cleared, andmounted.3The effect of progesterone on behavioral and cellular impairment in Aβtreated ratsTo investigate the effect of PROG on cognition impairment induced byAβ25-35CA1hippocampus injection, a total of fifty SD rats were dividedrandomly into five groups: vehicle group (sterile distilled water), Aβ group, PLgroup (Aβ+progesterone4mg/kg weight), PMgroup (Aβ+progesterone8mg/kg weight), and PHgroup (Aβ+progesterone16mg/kg weight). During6-12days after Aβ injection described above, animals were injected withdifferent dosages of progesterone (PL,PM,and PHgroup) or vehicle (sesame oilfor vehicle group and Aβ group) daily at13:30. Behavioral tests wereconducted during7-12days after Aβ injection beginning at14:30.Immediately after behavior tests, five rats in each group were sacrificed (viadecapitation) with hippocampus removed (on ice) and stored at-70℃forinflammatory factors analysis through RT–PCR and Western blot. The restanimals were anesthetized with pentobarbital sodium and perfused through theascending aorta with normal saline followed by4%paraformaldehyde. The brain tissues were dissected and fixed for histological examination.Behavioral tests, RT–PCR, Western blot and histological examinationwere conducted as mentioned above.4The effect of PROG on Aβ25-35induced cell impairment in vitroHippocampus cells of newborn SD rat were cultured and treated asdescribed. In brief, bilateral hippocampus newborn rat were dissociated with0.125%trypsin/HBSS for15min at37℃, followed by trypsin quenchingwith DMEM-HG containing10%FBS. Cell suspensions were centrifuged(5min,200×g), resuspended, dissociated by repeated passage through a fire-polished Pasteur pipette, and then filtered through a sterile cell strainer. Thedissociated hippocampus cells plated on poly-L-lysine (Sigma,50μg/mL)coated96-well plates at a density1×106cells/mL were grown in Neurobasalmedium-A supplemented with5units/mL penicillin,5mg/mL streptomycin,1%GlutaMAX I and2%B27neuromix at37℃in humidified5%CO2/95%air atmosphere for7days to9days before experimentation.The cultures were treated with or without Aβ (1μM,10μM, or50μM)for48h, or10μM Aβ for12h,24h, or48h. In the following experiment,cultures were pre-treated with or without PROG (0.1μM,0.5μM, or1μM)for60min followed by exposure to10μM aggregated Aβ for48h, or treatedsimultaneously with different dosages of PROG and Aβ for48h. Cellviability was assessed by MTT analysis. The results were presentedgraphically as a percentage of live cells in the control group.Assessment of Cell Viability: Loss of cell viability was measured by theMTT (methyl thiazolyl tetrazolium) assay. In brief,2×105cells per well wereseeded in triplicate onto96-well plates in Neurobasal-A medium and allowedto grow for7days. The cells were treated according to the experimentaldesign. After drug treatment, cells were incubated with5mg/mL MTT for2h,and subsequently solubilized in dimethyl sulfoxide (DMSO). The absorbencyat570nm was then measured using ELIASA (BMG Labtech, Germany). Theresults were expressed as the mean of the absorbance relative to the controlgroup. The experiments were replicated three times. 5Statistical AnalysisThe data were expressed as mean±standard error of the mean (SEM).The mean escape latency of the Morris water maze behavioral tests collectedduring the training days were analyzed by repeated-measure analysis ofvariance (ANOVA). Other data were statistically analyzed by one-wayANOVA, followed by between-group comparisons using LSD post hoc test.Statistical significance was concluded with a value of P <0.05for all analyses.Data analyses were performed with the SPSS software version13.0.Results:1Effect of Aβ micro-injection on the spatial learning and memory of ratsThe Morris water maze behavioral task was employed to examinehippocampus-dependent spatial learning and memory. The mean swimspeeds did not differ across the groups. The mean escape latency in the Morriswater maze behavioral task was significantly (P <0.01) higher in Aβ group(47.9±2.3,39.1±3.0,37.4±2.9,33.3±2.6, and29.4±3.0; Day1to Day5,respectively) compared with those of the vehicle group (47.3±1.3,28.6±2.7,21.1±2.7,15.8±1.9, and12.8±1.4; Day1to Day5, respectively.).Moreover, the number of platform crossings (48.7%) and the staying timeratio in platform quadrant (32.7%) of Aβ group in the probe test wereconsiderably (P <0.01) lower compared with those of vehicle group, thusconfirming spatial learning and memory impairment in Aβ25-35treated rats.Overall, no difference was observed between sham and vehicle group.Histological examination showed that Aβ25-35significantly (P <0.01)decreased (37.6%) the intact pyramidal cell number in the CA1hippocampuscompared with the vehicle group.2The metabolism alteration of neurosteroids in the prefrontal cortex andhippocampus of Aβ treated ratsHigh performance liquid chromatography-tandem mass spectrometrywas employed to simultaneously quantify the level of neurosteroids indifferent encephalic region. Levels of neurosteroids were altered in theprefrontal cortex and hippocampus of Aβ treated rats7and12days after surgery. Levels of PREG in the hippocampus of Aβ group were significantly(P <0.05) lower (66.1%and70.8%)7days and12days after surgerycompared with those of vehicle group, while levels of PREG in the prefrontalcortex did not differ compared with those of vehicle group on either timepoint. Furthermore, levels of PROG were significantly (P <0.01) reduced inboth the prefrontal cortex and hippocampus (63.8%and61.8%for7daysand12days after surgery in the prefrontal cortex;44.3%and42.2%for7days and12days after surgery in hippocampus, respectively) compared withthose of vehicle group. However, the hippocampus of Aβ treated ratscontained significantly (P <0.05) elevated (146.2%and137.9%) levels of17β-E27days and12days after surgery compared with those of vehiclegroup, while the prefrontal cortex of Aβ treated rats contained significantly (P<0.05) higher (171.8%) levels of17β-E2only on7days after surgerycompared with those of vehicle group. The level of ALLO did not differ ineither brain regions on both time points. The level of PREG, PROG and17β-E2did not differ between sham and vehicle group in either brain region onboth time points.The expression of neurosteroidogenesis enzymes was altered inhippocampus of Aβ treated rats. RT-PCR showed that gene expression ofCYP11A1,3β-HSD, and aromatase in hippocampus were significantly (P <0.01) higher in Aβ group (149.0%,247.6%, and159.5%) compared withthose of vehicle group. The up-regulation of CYP11A1,3β-HSD, andaromatase were confirmed when the protein level of CYP11A1,3β-HSD,and aromatase were significantly (P <0.01) increased in Aβ group (142.3%,219.9%, and145.0%) compared with those of vehicle group, showed byWestern blot. Results of immunohistochemisty analysis confirmed the up-regulation of aromatase in hippocampus of Aβ treated rats (113.6%, P <0.01,compared with vehicle group).3The protective effect of progesterone against the behavioral and cellularimpairment in Aβ treated ratsAdministration of PROG enhanced the cognitive performance of Aβ treated rats in a dose-dependent manner (4mg/kg,8mg/kg,16mg/kg for PL,PM, PHgroups, respectively). The mean swim speeds did not differ across thegroups. The mean escape latency of PL, PMand PHgroups were significantly(P <0.05for PL; P <0.01for PMand PH) lower compared with Aβ group. Therestoration of cognitive impairment was confirmed when the number ofplatform crossings of the PMand PHgroup was significantly increased by170.6%(P <0.05) and217.7%(P <0.01), compared with those of Aβ group.Meanwhile, the â…£ time ratio of PL, PM, and PHgroup was significantly (P <0.01) increased by170.6%,190.13%, and217.7%, compared with Aβgroup.PROG reversed Aβ25-35-induced cell loss in CA1hippocampus dosedependently. Compared with vehicle group, the intact pyramidal cell numberof Aβ group significantly (P <0.01) decreased by35.9%. Compared with Aβgroup, the intact pyramidal cell number of PL, PM, and PHgroups weresignificantly (P <0.01) increased by130.9%,190.1%, and241.3%,respectively.On the cellular level, PROG reversed Aβ25-35injection induced up-regulation of TNF-α and IL-1β. RT-PCR showed that that gene expressionof TNF-α and IL-1β in hippocampus were significantly (P <0.01) higher inAβ group (176.4%and176.4%) compared with those of vehicle group. Theup-regulation of TNF-α and IL-1β were confirmed when the protein levelof TNF-α and IL-1β were significantly (P <P <0.01) increased in Aβgroup (162.7%and158.3%) compared with those of vehicle group, showedby Western blot. The gene expression of TNF–α (83.6%,71.9%, and55.5%for PL, PM, and PHgroups, P <0.01, respectively) and IL-1β (90.9%,80.2%, and67.5%for PL(P <0.05), PM(P <0.01), and PHgroups (P <0.01),respectively) in hippocampus were significantly lower in the PROG-treatedgroup compared with Aβ group. The down-regulation of TNF-α and IL-1βby PROG were confirmed when the protein level of TNF–α (86.4%,73.8%,and58.1%for PL, PM, and PHgroups, P <0.01, respectively) and IL-1β(91.9%,81.8%, and69.6%for PL(P <0.05), PM(P <0.01), and PHgroups (P <0.01), respectively) were significantly decreased in the PROG-treatedgroup compared with Aβ group, showed by Western blot.4The protective effect of progesterone against Aβ25-35-induced culturedhippocampus cells viability decrease in vitroThe MTT assay was used to evaluate the viability of culturedhippocampus cells. Aβ25-35decreased cell viability dose and time dependently.Compared with vehicle group, the cell viability of Aβ treated group (48h)significantly (P <0.05for Aβ10μM, and P <0.01for Aβ50μM) decreasedby66.6%and55.4%. Compared with vehicle group, the cell viability of Aβtreated group (10μM) significantly (P <0.05for Aβ24h, and P <0.01forAβ48h) decreased by68.2%and66.9%. Meanwhile, PROG protectedagainst Aβ25-35-induced impairment dose dependently. PROG (1μM)treatment simultaneously with Aβ25-35significantly (P <0.05) reversed Aβ25-35-induced cell viability decrease. Moreover, PROG treatment1h prior toAβ25-35incubation significantly (P <0.05for PROG0.5μΜ, and P <0.01forPROG1μM) reversed Aβ25-35-induced cell viability decrease, confirming theprotection of PROG against Aβ25-35-induced cultured hippocampus cellsviability decrease in vitro.Conclusions:1Injection of Aβ25-35into the CA1hippocampus sub-region impairedthe spatial learning and memory of rats. Aβ-induced learning and memeoryimpairment model was successfully established.2The present study showed, for the first time, that injection of Aβ25-35into the CA1hippocampus sub-region of rats decreased levels of PREG andPROG and increased the level of17β-E2in the prefrontal cortex andhippocampus simultaneously quantified by HPLC-MS/MS. Moreover, theexpression of neurosteroidogenesis enzymes CYP11A1,3β-HSD, andaromatase was up-regulated in Aβ treated rats. Increased expression of3β-HSD and aromatase accelerated the metabolism of PREG and PROG thusreduced the levels of PREG and PROG and increased the level of E2.Meanwhile, the expression of CYP11A1was up-regulated to compensate the decreased level of PREG and PROG.3Administration of PROG reversed the up-regulation of pro-inflammatory cytokines TNF-α and IL-1β, as well as the behavioral andcellular impairment of Aβ treated rats, thus exerting neuronal protection.4PROG protectived against Aβ25-35-induced cells viability decrease ofcultured hippocampus neurons in vitro, comfirming the protection of PROGagainst Aβ25-35-induced neural impaiment. |
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