The Effect And Mechanism Of Glycosides Of Cistanche On The Animal Model Of Alzheimer Disease Induced By Aβ_25-35

Objective:GCs (Glycosides of cistanche) was extracted from Xinjiang's Cistanche salsa. To establish animal model and cell model of Alzheimer disease (AD), explore the effects and mechanisms of GCs on model of AD.Methods:1. AD animal model was established by a single intracerebroventricular injection of micromolar dose of Aβ25-35 in mice. Experimental animals were randomly divided into six groups:sham-operated group, model group, VitE group and GCs groups[large (250mg·kg-1), middle (125mg·kg-1) and small doseage (62.5mg·kg-1)] treated groups. The model group and sham group were given double distilled water for 17days. VitE group and GCs groups were treated with VitE and three dosage of GCs (ig) for 17days.Before established AD mice model, the mices were given medicine-(GCs) for 7days.10 days after operation, a step-though type passive avoidance training was performed on the mice for one time, followed by a ability assessment of learning and memory; preparation heterogeneity of brain tissue, superoxidase dismutase (SOD) activites, gultathionine peroxides (GSH-Px) activites and malonidialdehyde (MDA) contents in mice brain were also detected; TUNEL method was used to observe the apoptosis of cells; the immunnohistochemical SABC method was used to determine expression of Bcl-2 and Bax.2. Dorsal hippocampus microinjection of aggregated Aβ25-35 was performed by stereotaxic technique to establish the AD rat model. Experimental animals were randomly divided into six groups:sham-operated group, model group, VitE group and GCs groups[large (160mg·kg-1), middle (80mg·kg-1) and small doseage (40mg·kg-1)] treated groups.4 days Before established AD rat model, the rats were given medicine. The model group and sham group were given double distilled water for 14days. VitE group and GCs groups were treated with VitE and three dosage of GCs (ig) for 14days.10 days after operation, learning and mermorizing ability of the rats were measured through a step-through type passive adoidance training and electrical maze test; preparation heterogeneity of brain tissue, SOD activites, GSH-Px activites, MDA contents and AchE activites were also assessed in rat brain; the ultrastructures changes and intracellular Ca2+ in rat's hippocampus neurons were observed under electronic microscope; pathological changes in rat's hippocampal neurons were observed by HE stain; Bieschowsky silver stain and the immunohistochemistry; the expression of phosphorylating of Ser 199/202-tau site and Ser396-tau site, total tau (Tau5) and phosphorylating GSK-3βactivity were observed by immunehistochemical and Westen blotting detection.3. We studied the neuroprotective effects of GCs on apoptosis of PC 12 cells induced by aggregated Aβ25-35 and set up AD cell model, PC12 cells culture in vitro.They divided into six groups:Control group, model group, VitE group and GCs groups[large (100μg·mL-1), middle (50μg·mL-1) and small dosage (25μg·mL-1)] treated groups. The PC12 cells were examined for livability by MTT, measured lactate dehydrogenase (LDH) leakage by the spectrophotometry. The apoptosis rate was detected by AnnexinV-FITC and activity of caspase-3 by enzyme-linked immunosorbent assay.Results:1. Effect of GCs on learing and memory in Alzheimer's disease mice induced by aggregated Aβ25-35:1.1 GCs markedly enhanced the ability of learing and memory which induced by AP25-35.In step-down test showed that erroneous numbers were more in the model group than that in the sham group (P<0.01),but less in GCs (middle and high) groups (P< 0.01). compared with sham group, the model group were obviously lower on SOD, GSH-Px and higher on MDA; compared with model group, the middle dose and large dose group of GCs were significantly higher on SOD, GSH-Px and lower on MDA (P< 0.01).1.2 10 days after intracerebroventricular injection of micromolar dose of Aβ25-35, under the optical microscope, many apopotosis cell in mice brain of the model group. Bal-2 expression was low and Bax expression was high. Compared with model group, little apopotosis cell which in the mice brain of the middle dose and large dose group of GCs, the Bax expression decreased and the Bcl-2 expression increased.2. Study on protective effect of GCs on impairmed hippocampal CA1 regions neurons of Alzheimer's rat model induced by aggregated Aβ25-352.1 In step-down test showed that the erroneous numbers and latency escape were significantly longer in the model group than that in the sham group (P<0.01). In the electrical maze test, in the model group, the erroneous numbers were more in the model group than that in the sham group, but less in GCs groups and VitE group (P<0.01). The model group were obviously lower on SOD, GSH-Px and higher on MDA, AchE (P< 0.01), compared with model group, GCs groups and VitE group were significantly higher on SOD, GSH-Px and lower on MDA and AchE (P<0.01).2.2 Compared with the sham group, Ca2+ were elevated in model rats brain hippocampus CA1 regions neurons, and Ca2+ were decreased in GCs groups and VitE group rat brain hippocampus CA1 regions neurons.2.3 Ultrastruture changes in rat's hippcampus CA1 regions2.3.1 HE stain:under the microscope, there were a lot of scattered apoptosis cells in the model group, it showed that cytoplasm concentrate, the colour was pink, appear some karyorrhexis, deeply stained karyopyknosis and the disappearance of the phenomenon of nucleolar; nerve fibers were thick, swelled just like broadband or cord-like structure. GCs groups and VitE group showed that form of neuron is normal.2.3.2 Bielschowski's sliver staining:the neurofibrils were disorded, thick, swelled and fused into broadband, and the axons were deeply stained, the neuron were obviously damaged and many neurons disappeared in the group of aggregated Aβ25-35. GCs groups and VitE group had evidently protective effect on the hippocampal neurons.2.3.3 Ultrastruture changes in rat's hippcampus CA1 regions under electronic mictoscope:neucleus of hippocampus neuron were obviously damaged, uneven matrix, Mitochondrial fracture, Vacuolation, abnormality, Synaptic structures had been obscure and synapses were reduced, nerve fiber was disordered, GCs groups and VitE group had evidently protective effect on the hippocampal neurons CA1 regions.2.4 The model group, the expression of phosphorylating tau with Ser 199/202 site, Ser396 site, and total tau in hippocampal neuons (P<0.01) were significantly increased compared with the sham group (P<0.01). And the expression of phosphorylating GSK-3βwere obviously increased too (P<0.01); GCs groups and VitE group's expression of phosphorylating tau with Ser 199/202 site, Ser396 site, and total tau in hippocampal neuons were significantly decreased compared with the model group. And the expression of phosphorylating GSK-3βwere obviously decreased too.3. The neuroprotective effects of GCs on apoptosis of PC12 cells induced by aggregated Aβ25-35.Comparing with normal group, the model group induced a notable decrease in viability of PC 12 cells; induced an significant increase in LDH leakage of PC 12 cells at 48h; increased in the cell apoptotic ratio of PC 12 cells; meanwhile enhanced caspase-3 activity (P<0.01, P<0.05). GCs groups and VitE group could raised significantly in viability of PC 12 cells; decreased the cell apoptotic ratio of PC 12 cells; reduced caspase-3 activity. GCs could inhibit the cell apoptosis and protect the neuron cell. GCs groups were near to VitE group.Conclusion:1. In A(325-35-induced AD animal model, the ability of learning and memory decreased, free radical scavenging enzyme decreased, enhanced lipid peroxidation, apoptosis, the activity of GSK-3β-P enhanced, the expression of Phosphorylating level with Ser199/202, Ser396 of total tau in hippocampal neuons were significantly increased, caused pathological changes in Bielschowski's Silver staining and ultrastructure in rat's hippocampus, all above indicated that the animal model was successful.2. GCs increased the ability of learning and memory in Aβ25-35-induced AD like animal model, improved the cognitive disorder, reduce the pathological changes of impaired cell and hippocampal neuron impairment, its possible mechanism might involve: enhancement of free radical scavenging enzyme, inhibition of lipid peroxidtion, protection of neurilemma, cutting down the acvivity of AchE, improving cholinergic nerve function, ameliorating the ability of learing and memory dysfunction, lowering the expression of bax, enhancing the expression of Bcl-2, inhibiting apoptosis, reducing the impairment of AP25-35 and protect the neuron.3. GCs suppressed the activity of GSK-3β-P, decreased the expression of Phosphorylating tau with Ser199/202 site, Ser396 site and total tau in hippocampal neurons, kept the normal structure of brain canaliculus, inhibited the formation of NFT, GCs had obviously protective function to rat hippocampal neuron in Aβ25-35-induced AD like rat model, In the AD cell model, GCs decreased the the activity of caspase-3, blocked the apoptosis road, protected the cell induced by Aβ25-35. GCs inhibited the activity of GSK-3β-P and caspase-3.It was the new target point to resist the AD and might have latent theoretical value in prohibiting and therapying AD.