Effect And Mechanism Of Salidroside On Cognitive Deficits Of Alzheimer's Disease Model Rats Induced By β-amyloid

Alzheimer's disease (AD) is a chronic progressive neurodegenerativedisorder. It is characterised clinically by progressive loss of memory,cognitive dysfunction and personality changes as the three primary groups ofsymptoms. Pathogenesis of AD is complex and not fully understood yet.People proposed various hypotheses with respect to etiology of AD,such asthe amyloid hypothesis, the tau proteins hypothesis, the cholinergic hypothesis,the dysmetabolism hypothesis, the free radicals and apoptosis hypothesis, theexcitatory 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 realized that the oxidativestress, inflammation which are widespread reactions in the body haveinseparable connection with AD.Plenty of evidence shows that abnormal metabolism of β-amyloid (Aβ)peptides and accumulation of excessive Aβ plays a critical role in progress ofAD. Abnormal deposition of Aβ can activate microglia secreteproinflammatory molecules and cellular factors, induce nicotinamide adeninedinucleotide phosphate oxidase (NADPH oxidase) to generate reactive oxygenspecies (ROS), activate nuclear factor-κB (NF-κB), induce the expression oftumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS).These reactions exert direct or indirect injuries of nervous system. Increasingevidence demonstrates that Aβ caused a series of neurotoxic effect whichcause neuron disfunction, death and further lead to dementia. Chronicinflammation in the brain and oxidative stress played significant roles in thiscourse. Anti-inflammatory and anti-oxidative therapy has become one of themost important strategies for the prevention and treatment of AD.Plateau rhodiola is a perennial plant of Rhodiola family. It was a traditional famous Tibetan medicine. Researches show that rhodiola havespecial effects in preventing oxidative damage, scavenging free radicals,improving cell metabolism and enhancing cell vitality. Growing attention hasbeen received on it's benefits of enhancing the brain function and improvingmemory, in the past few years. Animal experiments also showed that rhodiolamay have good prospects in the treatment of dementia and related fields.However, salidroside as the main effective component of rhodiola, its effectand mechanism on cognitive deficit of Alzheimer's disease is still not clearand need systematic and profound researches.The rat received intrahippocampal injection of Aβ is a mature model forAD. This model is closer to the real pathological processes of AD than othermodels, because of its good simulation of Aβ deposition in the brain. Aim toexplore the potential effects of salidroside on cognitive impairment of AD andthe related molecular mechanism, this study established Aβ1～40induced ratsmodel of AD. Systematical investigations were first undertaken from theaspects of behavior changes, generation of metabolism products, enzymologychanges in activity, protein expression, transcriptional regulation and so on.1The effects of salidroside on cognitive deficits of AD model rats.Objective: To establish Aβ1～40induced rat models of AD and observethe effects of salidroside on cognitive deficits of these models.Methods: Male SD rats were randomly divided into6groups: shamcontrol group, AD model group, salidroside in small, medium and large dose(25mg·kg^-1,50mg·kg^-1,75mg·kg^-1) groups and huperzine A group(0.05mg·kg^-1). Aβ1～40was injected into bilateral hippocampus to create AD model.Rats were administered by gavage with salidroside for3weeks to determinethe protective and therapeutic effects on treatment rats. Morris water mazetesting system was undertaken to observe the changes of learning and memoryabilities in rats. Data were presented as xˉ±s and analyzed with multi-variatetest of repetitive measure ANOVA using SPSS statistical program.Enumeration data were analyzed with Rank sum test. A level of P<0.05wasconsidered statistically significant. Results:(1) The analysis of the place navigation trial showed that theescape latencies decreased from Day1to Day5in all groups. The AD modelrats displayed longer escape latencies than the rats of sham control group(P＜0.01). The animals that treated with salidroside displayed significantlylower escape latencies than those in AD model group (P＜0.01). But therewas no significant difference between salidroside(25mg·kg^-1)-treated groupand AD model group at the day1,2and5(P＞0.05). The animals inhuperzine A group displayed significantly lower escape latencies than those inAD model group (P＜0.05). Representative navigation paths at day5oftraining demonstrated that spatial learning acquisition was impaired in theanimals of AD model group relative to animals of salidroside-treated groupand huperzine A group.(2) In the spatial probe trial, The AD model rats spentsignificantly less time in the quadrant where the platform was hidden thananimals in sham control group (P＜0.01). The number of crossings to theprevious location of the platform was decreased in AD model group relative toanimals in sham control group (P＜0.01). Animals treated with salidroside(25mg·kg^-1) spent more time in the target quadrant and showed moreplatform-passing times than animals in AD model group, but there was nosignificant difference between them (P＞0.05). Animals in salidroside (50mg·kg^-1,75mg·kg^-1) groups and huperzine A group spent more time in thetarget quadrant and showed statistically more platform-passing times thananimals in AD model group (P＜0.01).Conclusions: The behavioural data obtained in the Morris water mazetest demonstrate that salidroside is able to protect animals from the memoryimpairments induced by intrahippocampal injection of Aβ1～40.2The effects of salidroside on anti-oxidative activities of AD modelrats.Objective: To observe the effects of salidroside on the generation of totalROS, the superoxide dismutase (SOD) activity, the malondialdehyde (MDA)level and the acetylcholinesterase (AChE) activity in serum and hippocampusof AD model rats.Methods: Male SD rats were randomly divided into4groups: sham control group, AD model group, salidroside (50mg·kg^-1) group and huperzineA group (0.05mg·kg^-1). Aβ1～40was injected into bilateral hippocampus tocreate AD model. Rats were administered by gavage with salidroside for3weeks. The generation of total ROS in hippocampus was determined by flowcytomertry technology, the superoxide dismutase (SOD) activity and themalondialdehyde (MDA) level in serum and hippocampus as well as theacetylcholinesterase (AChE) activity in serum were determined by separatebiochemical kit. Data were presented as xˉ±s and analyzed with ANOVA andLSD using SPSS statistical program. A level of P＜0.05was consideredstatistically significant.Results:(1) The results showed animals received intrahippocampalinjection of Aβ1～40displayed a significantly enhancement of ROS productioncompared with the sham control group (P <0.01). The level of ROS wassignificantly inhibited after treated with salidroside or huperzine A incomparison with the AD model group (P <0.01, P <0.01). The inhibition ofROS by huperzine A was weaker than that by salidroside.(2) Animalsreceived intrahippocampal injection of Aβ1～40displayed a significantlydecrease of the SOD activity in serum and hippocampus compared with thesham control group (P <0.01). The SOD activity was significantly increasedafter treated with salidroside or huperzine A in comparison with the AD modelgroup (P <0.01, P <0.01). The increase of SOD activity by huperzine A wasweaker than that by salidroside.(3) Intrahippocampal injection of Aβ1～40induced significant increase of the MDA level in the serum and hippocampusof AD model group (P <0.01). On the other hand, we observed a significanttrend for decrease of the MDA level after salidroside or huperzine A treatmentin comparison with the AD model groups (P <0.01, P <0.05). The decrease ofthe MDA level by huperzine A was weaker than that by salidroside.(4) ADmodel group showed a significantly increase of the AChE activity inhippocampus compared with the sham control group (P <0.01). The AChEactivity was significantly inhibited after treated with salidroside or huperzineA in comparison with the AD model group (P <0.05, P <0.01). The inhibition of AChE activity by salidroside was weaker than that by huperzine A.Conclusions: Salidroside could significantly decrease the generation oftotal ROS in hippocampus, increase the SOD activity in serum andhippocampus, decrease the malondialdehyde (MDA) level in serum andhippocampus and inhibit the AChE activity in hippocampus of AD model rats.These effects enhanced the anti-oxidative activities and prevented the neuronaldamage from oxidative stress. There are different mechanisms and advantagesbetween salidroside and huperzine A on the effects of preventing oxidativedamage and suppressing AchE activity.3The effects of salidroside on NADPH oxidase-ROS signalpathway in hippocampus of AD model rats.Objective: NADPH oxidase is the main source of ROS in cells. In thepresent study, the expression and activation of NADPH oxidase inhippocampus of AD model rats were observed in order to elucidate the signalmechanism of salidroside on the suppressing generation of ROS.Methods: Male SD rats were randomly divided into3groups: shamcontrol group, AD model group, salidroside (50mg·kg^-1) group. Aβ1～40wasinjected into bilateral hippocampus to create AD model. Rats wereadministered by gavage with salidroside for3weeks. The expression level ofgp91phoxand p47phoxprotein in hippocampus of AD model rats weredetermined by Westernblot. The expression level of gp91phoxand p47phoxmRNA were assayed by means of RT-PCR. The phosphorylation level ofp47phoxprotein was detected by immuno precipitation analysis. Data werepresented as xˉ±s and analyzed with ANOVA and LSD using SPSS statisticalprogram. A level of P＜0.05was considered statistically significant.Results:(1) Western blot analysis showed that the expression level ofgp91phoxand p47phoxprotein in hippocampus of AD model rats weresignificantly increased by2.39times and2.71times, compared with shamcontrol group (P＜0.01). After treated with salidroside the expression levelof gp91phoxand p47phoxprotein in hippocampus of salidroside group weresignificantly inhibited by32.6%and46.1%respectively, in comparison with the AD model group (P＜0.01).(2) RT-PCR analysis showed that theexpression level of gp91phoxand p47phoxmRNA in hippocampus of AD modelrats were significantly rosed by1.99times and2.12times, compared withsham control group (P＜0.01). After treated with salidroside the expressionlevel of gp91phoxand p47phoxmRNA in hippocampus of salidroside group weresignificantly reduced by36.5%and26.8%respectively, in comparison withthe AD model group (P＜0.01).(3) IP-Western Bolt analysis showed that thephosphorylation level of p47phoxprotein in hippocampus of AD model ratswere significantly increased by5.13times, compared with sham control group(P＜0.01). After treated with salidroside the phosphorylation level of p47phoxprotein in hippocampus of salidroside group were significantly inhibited by44.5%respectively, in comparison with the AD model group (P＜0.01).Conclusions: Salidroside could significantly decrease the genetranscription and protein expression as well as the subunit activation ofNADPH oxidase. The findings indicate that both the effects of suppressingsubunits expression and inhabiting subunit activation on NADPH oxidase,decreased the level of ROS in hippocampus, further reduced the oxidativedamage.4The effects of salidroside on ROS-NF-κB signal pathway inhippocampus of AD model rats.Objective: It has been demonstrated that activated microglia produceROS, which may induce or exacerbate neurotoxicity by causing oxidativestress to neurons. Studies also showed that ROS production was attributable tothe activation of NF-κB and consequent pro-inflammatory cytokines.Aβ-ROS-NF-κB signal pathway has been shown to play an important role inchronic inflammation during the onset and progression of AD. In the presentstudy, the activation of ROS-NF-κB signal pathway in hippocampus of ADmodel rats was observed in order to elucidate the signal mechanism ofsalidroside on the attenuation of inflammatory damage in the brain.Methods: Male SD rats were randomly divided into3groups: shamcontrol group, AD model group, salidroside (50mg·kg^-1) group. Aβ1～40was injected into bilateral hippocampus to create AD model. Rats wereadministered by gavage with salidroside for3weeks. The expression level ofNF-κB and IκBα protein in hippocampus of AD model rats were determinedby Westernblot and Immunohistochemical analysis. The expression level ofNF-κB and IκBα mRNA were assayed by means of RT-PCR. Thephosphorylation level of NF-κB protein was detected by Westernblot. Thephosphorylation level of IκBα protein was detected by immuno precipitationanalysis. Data were presented asˉx±s and analyzed with ANOVA and LSDusing SPSS statistical program. A level of P＜0.05was consideredstatistically significant.Results:(1) Western blot analysis showed that the expression level ofNF-κB protein in hippocampus of AD model rats were significantly increasedby2.43times, compared with sham control group (P＜0.01). After treatedwith salidroside the expression level of NF-κB protein in hippocampus ofsalidroside group were significantly inhibited by32.9%respectively, incomparison with the AD model group (P＜0.01). Immunohistochemicalanalysis showed the same trend as shown in Western blot. The NF-κB positivecells were increased in AD Model group and decreased in salidroside group.(2) RT-PCR analysis showed that the expression level of NF-κB mRNA inhippocampus of AD model rats were significantly rosed by2.07timescompared with sham control group (P＜0.01). After treated with salidrosidethe expression level of NF-κB mRNA in hippocampus of salidroside groupwere significantly reduced by35.9%respectively, in comparison with the ADmodel group (P＜0.01).(3) Western Bolt analysis showed that thephosphorylation level of NF-κB protein in hippocampus of AD model ratswere significantly increased by4.91times, compared with sham control group(P＜0.01). After treated with salidroside the phosphorylation level of NF-κBprotein in hippocampus of salidroside group were significantly inhibited by53.5%respectively, in comparison with the AD model group (P＜0.01).(4)Western blot analysis showed that the expression level of IκBα protein inhippocampus of AD model rats were significantly decreased by58.7%, compared with sham control group (P＜0.01). After treated with salidrosidethe expression level of IκBα protein in hippocampus of salidroside group weresignificantly rosed by99.2%respectively, in comparison with the AD modelgroup (P＜0.01). Immunohistochemical analysis showed the same trend asshown in Western blot. The IκBα positive cells were decreased in AD Modelgroup and rosed in salidroside group.(5) RT-PCR analysis showed that theexpression level of IκBα mRNA in hippocampus of AD model rats weresignificantly reduced by62.7%compared with sham control group (P＜0.01).After treated with salidroside the expression level of IκBα mRNA inhippocampus of salidroside group were significantly increased by92.2%respectively, in comparison with the AD model group (P＜0.01).(6)IP-Western Bolt analysis showed that the phosphorylation level of IκBαprotein in hippocampus of AD model rats were significantly increased by2.98times, compared with sham control group (P＜0.01). After treated withsalidroside the phosphorylation level of IκBα protein in hippocampus ofsalidroside group were significantly inhibited by33.4%respectively, incomparison with the AD model group (P＜0.01).Conclusions: On the one hand salidroside could inhibit the ROS-NF-κBsignal pathway by reducing the NF-κB protein expression, on the other handsalidroside could inhibit the ROS-NF-κB signal pathway by reducing thephosphorylation level of IκBα protein and enhancing IκBα protein expression,both of which can inhibit the NF-κB activation. These effects have greatsignificance for attenuating Aβ-mediated inflammation in the brain.5The effects of salidroside on iNOS and RAGE expression inhippocampus of AD model rats.Objective: Excessive expression of iNOS and RAGE can be induced byNF-κB. These productions can not only mediate the neurotoxicity of Aβderectly by themselves, but also activate NF-κB again, which will amplify theinflammatory effects and resulted in vicious circles. In the present study, theexpression of iNOS and RAGE in hippocampus of AD model rats wereobserved in order to further elucidate the comprehensive intervention of salidroside on the oxidative stress and inflammation in the brain.Methods: Male SD rats were randomly divided into3groups: shamcontrol group, AD model group, salidroside (50mg·kg^-1) group. Aβ1～40wasinjected into bilateral hippocampus to create AD model. Rats wereadministered by gavage with salidroside for3weeks. The expression level ofiNOS and RAGE protein in hippocampus of AD model rats were determinedby Westernblot and Immunohistochemical analysis. The expression level ofiNOS and RAGE mRNA were assayed by means of RT-PCR. Data werepresented as xˉ±s and analyzed with ANOVA and LSD using SPSS statisticalprogram. A level of P＜0.05was considered statistically significant.Results:(1) Western blot analysis showed that both the expression levelof iNOS and RAGE protein in hippocampus of AD model rats weresignificantly increased by3.72times and1.86times, compared with shamcontrol group (P＜0.01). After treated with salidroside the expression levelof iNOS and RAGE protein in hippocampus of salidroside group weresignificantly inhibited by47.4%and31.4%respectively, in comparison withthe AD model group (P＜0.01). Immunohistochemical analysis showed thesame trend as shown in Western blot. Both of the iNOS and RAGE positivecells were increased in AD Model group and decreased in salidroside group.(2) RT-PCR analysis showed that the expression level of iNOS and RAGEmRNA in hippocampus of AD model rats were significantly rosed by2.08times and1.65times compared with sham control group (P＜0.01). Aftertreated with salidroside the expression level of iNOS and RAGE mRNA inhippocampus of salidroside group were significantly reduced by37.0%and23.8%respectively, in comparison with the AD model group (P＜0.01).Conclusions: Salidroside could inhibit the activation of NF-κB-iNOSand NF-κB-RAGE signal pathways and their feedback loop in hippocampus ofAD model rats. All of these effects might suppress the interaction and thevicious cycle from mutual promotion between oxidative stress andinflammatory damage, that would give a comprehensive protection fornervous tissue.