Intervention And Mechanism Of Salidroside On Alzheimer’s Disease Models Induced By β-amyloid

Alzheimer’s disease (AD) is a insidious progressive neurodegenerativedisease. In clinnical，it is characterised by progressive loss of memory,cognitive dysfunction, agnosia，apraxia，aphasia，execution dysfunction, visionspace damages and personality behavior changes which generalized dementia.The pathogenesis of AD is multitude and complex and not fully understoodyet. Researchers proposed various hypotheses with respect to etiology of AD,such as the cholinergic hypothesis, amyloid hypothesis, the free radicals andapoptosis hypothesis, the tau proteins hypothesis, dysmetabolism hypothesis,the excitatory amino acids hypothesis, the genic mutation hypothesis, et al.However, none of them can fully explain the pathogenesis of AD. With thedevelopment of research, people have come to realize that the oxidative stress,apoptosis and Mitochondrial dysfunction which are general reactions in thebody have inseparable connection with AD.Plenty of evidence show that abnormal metabolism of β-amyloid (Aβ)peptides and accumulation of excessive Aβ play critical roles in progress ofAD. The stimulation by Aβ persistently can activate microglia, generatereactive oxygen species (ROS), then produce harmful free radicals andactivate apoptosis molecules, These reactions exert direct or indirect injuriesof nervous system. Increasing evidence demonstrate that Aβ deposition causeda series of neurotoxic effect which cause neuron disfunction, death and furtherlead to dementia. Oxidative stress in the brain and apoptosis played significantroles in this course. The therapy from anti-oxidative and anti-apoptosis hasbecome one of the most important strategies for the prevention of AD.In recent years, many studies have confirmed that the incidence of AD isclosely related to tissue and cell apoptosis. P53is a key protein in theapoptosis pathway, the normal and basal expression of P53is closely related to the stability of the genome and cell cycle. Under the normal conditions, italso plays an important role in the body’s internal environment of stability andreconstruction process of the organization, the overexpression andaccumulation of P53will lead to a series of pathophysiological changes. Theimmunohistochemical studies have confirmed that the plaques andneurofibrillary tangles in the brain of AD contain a lot of P53protein. So, withthe course of the disease, its expression in the central nervous system of ADwhether reinforcing or not, whether there is a potential link between increasedP53and cognitive abilities in patients with AD or not, and what factorspromote its overexpression, this article will discuss one by one.Plateau rhodiola is a perennial plant of Rhodiola family. It was atraditional famous Tibetan medicine. Researches show that rhodiola havespecial effects in preventing oxidative damage, scavenging free radicals,improving cell metabolism and enhancing cell vitality. In the past few years,growing attention has been received on its benefits of enhancing the brainfunction and improving memory. Animal experiments also showed thatrhodiola may have good prospects in the treatment of dementia and relatedfields. However, salidroside as the main effective component of rhodiola, itseffect and mechanism on againsting damages of axoneures by Aβ, improvingcognitive deficit of Alzheimer’s disease is still not clear and need systematicand profound researches.The rat received hippocampal injection of Aβ and the SH-SY5Y cellswere stinulated by Aβ are mature models for AD. These models is closer tothe real pathological processes of AD than the others, because of its fulllyresponsed for the courses of Aβ deposition in the brain. This study establishedAβ induced rats and cell models of AD. Systematical investigations were firstundertaken from the aspects of behavior cognitive change, generation ofphysiopathologic metabolism product, enzymology changes in activity,protein expression, transcriptional regulation and so on. The aim to explore thesignal transduction mechanisms and the effects of salidroside on cognitivedysfunction in Alzheimer’s disease model rats induced by Aβ1-40. Part1The effects of salidroside on cognitive dysfunction of AD modelratsObjective: To establish Aβ1-40induced rat models of AD and observe theeffects of salidroside on cognitive dysfunction of this model.Methods: Aβ1-40was injected into bilateral hippocampus to create ADmodel. Rats were administered by gavage with salidroside in settled dose anddensity (50mg·kg-1·d-1) everyday for21days. Morris water maze testingsystem was undertaken since17th day to observe the change of learning andmemory abilities in rats，and continuous5days. Data were presented asˉx±sand analyzed with multi-variate test of repetitive measure ANOVA usingSPSS16.0statistical program. Enumeration data were analyzed with Rank sumtest. A level of P<0.05was considered statistically significant.Results: The analysis of the place navigation trial showed that the escapelatency decreased from Day1to Day5in all groups. The AD model ratsdisplayed longer escape latencies than the rats of sham control group(P＜0.05).The animals which were treated with salidroside displayed significantly lowerescape latencies than those in AD model group(P＜0.05). Representativenavigation paths at day5of training demonstrated that spatial learningacquisition was impaired in the animals of AD model group relative toanimals of salidroside-treated group. In the spatial probe trial, The AD modelrats spent significantly less time in the quadrant where the platform washidden than animals in sham control group(P＜0.05). The number of crossingsto the previous location of the platform was decreased in AD model grouprelative to animals in negtive control group(P＜0.05). Animals in salidroside(50mg·kg-1·d-1) group spent more time in the target quadrant and showedstatistically more platform-passing times than animals in AD model group(P＜0.05).Conclusions: In rats with bilateral hippocampal injections by Aβ1-40, thememory, learning ability are significant declined, the therapy with quantitativesalidroside intervention of AD rats and the declined ability of learning and memory reversed.The behavioural data obtained in the Morris water maze testdemonstrate that salidroside is able to protect animals from the memoryimpairments induced by hippocampal injection of Aβ1-40.Part2The effects of salidroside on anti-oxidative activities of AD modelratsObjective: Oxidative stress can be seen as a neuronal cell injury inducedby different stimuli and a variety of degenerative disorders of central nervousfunction common pathways. In a variety of ways, Aβ1-40can stimulateintracellular oxidative stress in brain tissue reaction, and a large number ofROS produce consequently. The aim of this part is to observe the effects ofsalidroside on the generation of total ROS, the superoxide dismutase (SOD)activity, the malondialdehyde (MDA) level and the in serum and hippocampusof AD model rats to discuss the effect of salidroside on ROS in AD models,then the apoptosis rate was checked.Methods: Aβ1-40was injected into bilateral hippocampus to create ADmodel. Rats were administered by gavage with salidroside in settled dose anddensity (50mg·kg-1·d-1) everyday for21days. The generation of total ROS inhippocampus was determined by flow cytomertry technology usingDCFH-DA, the superoxide dismutase (SOD) activity and the malondialdehyde(MDA) level in serum and hippocampus were determined by separatebiochemical kit, last the apoptosis rate was checked by TUNEL. Data werepresented as xˉ±s and analyzed with ANOVA and LSD using SPSS16.0statistical program. A level of (P＜0.05) was considered statisticallysignificant.Results:(1) This experimental study examined the total ROS productsin the hippocampus cellular of AD model rats. To detect ROS levels in eachgroup of cells by flow cytometry (FCM). Compared with the sham controlgroup, the ROS levels of hippocampus intracellular in AD model groupincreased significantly (P<0.05). AD model rats were treated with salidroside （50mg·kg-1·d-1）for21days, the ROS levels were significantly inhibited(P<0.05), although not back to normal levels, compared with negative controlgroup (P>0.05).(2) According to the experimental requests, to determineSOD activity (content) in serum of AD model rats. Compared with thecontrol group, the serum SOD activity in AD model group decreasedsignificantly (P<0.05), giving Salidroside (50mg·kg-1·d-1) for21days, serumSOD activity was significantly higher compared with the AD model group(P<0.05), although not back to normal levels, but compared with the negativecontrol group (P>0.05), SOD activity in the hippocampus cells the same as theserological results.(3) Compared with the negative control group, serumMDA in AD model group was significantly higher (P<0.05), givingsalidroside (50mg·kg-1·d-1) for21days, serum MDA content decreasedsignificantly compared with the AD model group (P<0.05), although not backto the normal level, but compared with the sham control group (P>0.05),MDA content in hippocampus cells have the same trend with the serologicalresults.(4) To determine apoptosis in hippocampus cells of AD model rats.Compared with the sham control group, the apoptosis rate in hippocampalcells of AD model rats were significantly higher (P<0.05), giving salidroside(50mg·kg-1·d-1) for21days, the apoptosis in hippocampus cells decreasedsignificantly compared with the AD model group (P<0.05), although not backto normal levels, but compared with the sham control group (P>0.05).Conclusion: Salidroside can effectively inhibit ROS generate inhippocampal tissue cells of AD model rats,while increase SODactivity(content) in serum and hippocampal tissue cells, and to reduce theMDA levels in serum and hippocampus tissue cells, thus, it was validatedfrom local and global mutual that salidroside can increase the ability ofoxidative stress in AD model rats and reduce damage to nerve cells caused byoxidative stress. Part3The impacts of salidroside on NADPH oxidase-ROS pathway inhippocampus of AD model rats induce by Aβ1-40Objective: NADPH oxidase is a major source of ROS products in cells.The aim of this part is to discuss the underlying mechanisms that whethersalidroside inhibited ROS upstream passage or not by observing the subunitexpression and activation in NADPH oxidase family members.Methods: Aβ1-40was injected into hippocampus to create AD model.Rats were administered by gavage with salidroside in settled dose and density(50mg·kg-1·d-1) everyday for21days. After establishing a successful model,using RT-PCR and Western blot to detect the expressions of gp91phox andother subunits reside in hippocampal tissues in the mRNA and protein Level.Data were presented asˉx±s and analyzed with multi-variate test of repetitivemeasure ANOVA using SPSS16.0statistical program. Enumeration data wereanalyzed with Rank sum test. A level of P<0.05was considered statisticallysignificant.Results:(1) Western Blot showed：Aβ1-40may induce p22phox, p67phoxgp9lphox and p47phox protein expression was significantly increased inhippocampus cells of AD model rats. Protein content above-mentioned in ADmodel group were2.16,2.57,3.02and2.71times than the sham control group(P<0.05). But after given quantitative salidroside, the protein expression ofp22phox, p67phox gp9lphox p47phox in hippocampal tissue were lower by24.1%,26.3%,31.1%and34.2%than the AD model group(P<0.05).(2)RT-PCR testing showed that: compared with the sham control group, theexpression of p22phox, p67phox gp9lphox and p47phox mRNA significantlyincreased in AD model rats hippocampus cells. The mRNA levels in ADmodel group were increased by84.1%,96.3%,101.1%and104.2%than thenegative control group (P<0.05). But after given quantitative salidroside, itcan significantly inhibit the effect induced by Aβ1-40. Salidroside interventiongroup decreased by34.3%,36.1%,33.4%and30.1%than AD model group(P<0.01).Conclusion: Salidroside inhibits NADPH oxidase family membersthereby inhibiting ROS production, this process may be caused by inhibitingthe expression and activation of family members subunit, or inhibiting NADPH oxidase activation by mutually synergy. Salidroside throughsuppressed NADPH oxidase-ROS pathway effectively, reduced oxidativestress response significantly in brain cells of AD rat model induced by Aβ1-40.Part4The impacts of salidroside on ROS-P53mitochondrial apoptoticpathway in hippocampus of AD model rats induce by Aβ1-40Objective: To further search the specific mechanism of apoptosis rateafter ROS, we examined the expression of apoptosis-related protein P53afterROS generation. Also verify the impact salidroside on Bcl-2, Bax andmitochondrial apoptotic pathway based on P53gene.Methods: Aβ1-40was injected into hippocampus to create AD model.Rats were administered by gavage with salidroside in settled dose and density(50mg·kg-1·d-1) everyday for21days. After establishing the successful models,Western-blot was used to detect the expressions of apoptosis-related proteinsP53. Also verify that the expression salidroside on Bcl-2, Bax andmitochondrial apoptotic pathway basised on P53gene. Data were presented asxˉ±s and analyzed with multi-variate test of repetitive measure ANOVA usingSPSS16.0statistical program. Enumeration data were analyzed with Rank sumtest. A level of P<0.05was considered statistically significant.Results:(1) Western Blot showed: Aβ1-40can cause nucleus P53expression in hippocampus cells of AD model rats, and it was significantlyhigher expression. In AD model group, the highest in the nucleus of P53protein up to5-6times than the sham control group(P<0.05). After givenquantitative salidroside, the nuclear P53protein expression in hippocampus ofAD model rats decreased nearly50%compared with the AD model group(P<0.05).(2)Compared with the sham control group, the Bax expression inhippocampal cells of AD model rats significantly increased; while Bcl-2expression in hippocampal tissue cells of AD model rats was significantlyreduced. The Bax increased151%than the sham control group (P<0.05); TheBcl-2in AD model rats down nearly70%than the sham control group (P<0.05). After given quantitative salidroside, it can significantly inhibiteffects induced by Aβ1-40. Bax and Bcl-2in the salidroside intervention groupdecreased and increased by40%and55%than AD model group (P<0.05).(3)Compared with the sham control group, the caspase-9and caspase-3expression was significantly increased in hippocampus cells of AD model rats;The caspase-9and caspase-3in AD model group increased by151%and124%than the sham control group (P<0.05). After given the quantitativesalidroside, it can significantly inhibit the effects induced by Aβ1-40. caspase-9and caspase-3in the intervention group with salidroside decreased by40%and55%compared with AD model group (P<0.05).Conclusion: The salidroside can inhibit NADPH oxidase to generateROS, thereby inhibiting P53tranfer into the nucleus to activation, thusinhibiting the expression of caspase-9and caspase-3, it indicated thatsalidroside can inhibit the NADPH oxidase-ROS-P53-mitochondrial apoptosispathway, reducing the oxidative stress and apoptosis in AD brain tissueinduced by Aβ1-40.Part5Salidroside reduces p53during Aβ1-40-induced neurotoxicity byinduction of heme oxygenase-1through PI3K/Akt/Nrf-2signal pathwaysObjective: The objective of this study was to investigate how salidrosideprotects neurocytes from Aβ1-40. Animal models of AD induced by injection ofAβ1-40into hippocampus provide numberous evidences for cellular apoptosis.Thus, SH-SY5Y cells stimulated by Aβ1-40and rat received hippocampalinjection of Aβ1-40AD models were used for research in vitro and vivo,respectively. We found that salidroside in vitro experiment could preventAβ1-40-induced apoptosis of SH-SY5Y cells and protect brain in vivo fromoxidative damage and also reduce p53nucleus expression in betaamyloid-induced neurocytes injury through upregulation of HO-1.Methods: Total protein (30μg) for each sample was separated by10%SDS-PAGE and transferred overnight to PVDF membranes, and the nonspecific binding of antibodies was blocked by5%nonfat dried milk orBSA in phosphate-buffered saline. Membranes were then probed with HO-1,p53, t-Akt, p-Akt, Bax, Bcl-2, cleaved caspase-9/3or Nrf-2antibody (1:1000)overnight at4C. After three washes with phosphate-buffered saline(0.1%Tween-20PH7.4). The protein bands were visualized using an enhancedchemiluminescence Western blotting detection kit and the results wereanalyzed using imaging densitometer. ARE-luciferase activity assay:SH-SY5Y cells were plated in24well plates at a density of1×104/cells andincubated overnight.2μg of the luciferase reporter plasmid with AREpromoter and the β-galactosidase vector plasmid were co-transfected to eachsample, using transfection reagents at the proportion of2μg DNA per10μl. Tocorrect for the transfection efficiency, phRL-SV40-β-galactosidase was used.2hours later, the luciferase activity was detected according to the methodprovided by the manufacturer. In brief, cells were washed with PBS in4C andharvested with lysis buffer then centrifugated, supernatant (20μl) was used forthe detection of the luciferase activity, which was measured by a luminometer.TUNEL staining: The terminal deoxynucleotidyl transferase dUTP-mediatednicked end labeling (TUNEL) assay in SH-SY5Y cells was used to assess celldeath by examining DNA fragmentation following treatment of cells. Briefly,cells were grown on6well cell culture plates and treated as described above.After treatment, cells were washed with phosphate-buffered saline (PBS), theslides were immersed in4%formaldehyde in PBS at4C and then in3%H2O2and in0.2%Triton X-100for5-10min at room temperature. Equilibrationbuffer (100μl) was added, and the slides were incubated at room temperaturefor10min. TdT reaction mix (50μl) was then added, and the cells wereincubated for60min at37C. The slides were then immersed in2×SSC for15min. Propidium iodide (PI) was added and incubated for15min to stain allcells. The localized green fluorescence of apoptotic cells was detected againsta red background by fluorescence microscopy. For the quantification ofTUNEL-positive (apoptotic) cells, a minimum of200cells was counted pergroup, and the percentage of the positively labeled cells was calculated. All analyses were performed using the SPSS16.0for Windows, statisticaldifferences were checked by ANOVAs correction or Student’s t-tests formultiple comparisons. P﹤0.05were set as statistically significant. All datawere checked for normality and homogeneity of variance previously and wereexpressed as mean±standard error.Results:(1) Aβ1-40can inhibit the cell viability,and salidroside can induceHO-1protein expression through PI3K/Akt signals involvement in inductionof HO-1,(2)The effect of salidroside can be reversed by ZnPPIX,(3)Salidroside induces HO-1by Nrf-2activation,Salidroside inhibits oxidativestress-induced apoptosis,Salidroside attenuates cognitive deficits and inhibitsp53nucleus translocation induced by Aβ1-40, P53activator-RITA inducesapoptosisConclusion: In summary, we demonstrated that salidroside protects braincells from Aβ1-40via induction of HO-1. The expression of HO-1bysalidroside was dependent on PI3K/Akt pathway and Nrf-2translocation.Salidroside also attenuated cognitive deficits, LDH release, and P53release inAD rat brain by ZnPPIX-sensitive manner (Fig.7). Thus, salidroside may bean important novel therapeutic agent for treatment of AD.