The Effect And Mechanism Of TFSC On The Model Of AD Mice With Endocrine Recession

Alzheimer’s disease is a progressive disease of neurons system and also as known as dementia, the main clinical manifestation include of cognitive function obstacle. The epidemiological survey knows that the prevalence rate of AD increased when age was increased. The probability of the elderly with dementia is higher and higher and produce lots of matters for family and society. This disease happened hidden, the specific medicines have not been existence domestic and overseas to cure the alzheimer’s disease currently. So we have no time to delay to further study the pathogenesis of AD and the mechanism of action of drugs to treat AD. Cuscuta chinensis Lam belongs to convolvulaceae Cuscuta spp, is a Chinese traditional medicine. The main constituents include of TFSC have a great pharmacological effects on Endocrine recession, and less reports of the effects on females’ AD with endocrine recession. This research is main to study the pharmacological effects and mechanism of TFSC on female’s AD with endocrine recession.Objective:To study the effects and menchanism of TFSC on learning-memory ability and neurons apoptosis from the model of AD with endocrine recession.Methods:Fifty Kunming mice were randomly divided into five groups though the experiment includes:model group, control group with estrogen (0.3mg·kg-1), includes of low-dose experimental group (35.0mg·kg-1), middle-dose experimental group(70.0mg·kg-1), high-dose experimental group(140.0mg·kg-1) of TFSC treatment, and ten mice of each group. These mice were operated from backside to removing the bilateral ovaries, take another ten mice to operation just with skin and suture as a control group in order to built the model of AD with Endocrine recession. Four days after operation, the groups of model and sham operation were gave with NS. The rest four groups were gave with TFSC or estrogen as long as three months. The Vaginal epithelial cells smear was used to check the model had been successed or not. The abilities of learning-memory from these mice were checked after Morris water maze behavioral test; The weight of uterus from each group mice were checked by analytical balance; The levels of estrogen in blood from each group were checked by radioimmnunoassay; The change of neuronal pathological morphology from mice were checked after use the methods of hematoxylin-eosin staining(HE) and Nissl. The cells apoptosis rate from neurons in hippocampal were checked by flow cytometry; Used the method of western blotting to check the expression of apoptosis-related proteins. Used the method of reverse transcription-polymerase chain reaction(RT-PCR) to check the change of the expression of apoptosis-related genes from mice brain.Results:1.The effects of TFSC on mice uterine weight:Compared with the sham-operation group and Estrogen group, the weight of uterine from model group mice was dropped significantly(P<0.05); Compared with model group, the uterine weight were increased statistically significant from the groups mice of TFSC (P<0.05,P<0.01,P<0.01).2. The form of vaginal epithelial cells by eosin staining:compared with control group and estrogen group, there is no keratinization cell in model group, cells are round dot.Compared with model group, there have no round dot cells in each dose group of total flavonoids from semen cuscutae.3.The abilities of learning-memory check though Morris water maze experiment: compared with the sham-operated group and estrogen group, the model group mice’s escape latency and swimming distance were increased significantly, the times of cross platform were significantly reduced, the difference is significantly statistic (P<0.01); Three month later after Intragastric injection administration, compared with the mice from model group, each dose group of TFSC mice’s escape latency and swimming distance were significant reduced and times of cross platform obviously increased, the difference is significantly statistic(P<0.05, P<0.01, P<0.01).4.The experimental results about the level of estrogen of serum:compared with the sham-operated group and estrogen group, model group mice’s serum estrogen levels significantly decreased, the difference is significantly statistic(P<0.01); Compared with model group, the each dose group mice of TFSC increased estrogen levels in the serum significantly, but there are still a certainly difference compared with the normal group, the difference is significantly statistic (P<0.05, P<0.01, P<0.01).5. The pathological of neurons were checked by HE staining and Nissl staining: HE staining:In sham-operation group, the cells in hippocampal CA1region were closely packed neatly from, neuron assumes the circular or conical, the nucleoplasm ratio is larger, the nucleus is circular, nucleolus is obvious; Model group mice hippocampal CA1area cells loose and disorderly, more neuronal cells body were contraction and karyopyknosis, cytoplasm bubble becomes more large, the numbers of neuron less than normal group; The cells in hippocampal CA1region were closely packed neatly from Estrogen group.compared to model group mice,the cytoplasm bubble comes smaller, the number of neurons were increased; The cells in hippocampal CA1region were closely packed neatly from high dose group of TFSC. neuronal cytoplasm is full, the nucleus is circular, compared with model group the cytoplasm bubble become significantly smaller, the neurons were increased significantly well; The cells in hippocampal CA1region were closely packed neatly from middle dose group of TFSC. cytoplasm bubble become smaller and karyopyknosis. compared with model group,numbers of neurons were increased, the cells in CA1region in hippocampal were loose of low dose group of TFSC. Neuronal cell body contraction and nuclear pyknosis. cytoplasm bubble was same as model group and the numbers of neuron increased a little. Nissl staining:the neuron cells in hippocampal were closely packed neatly from normal control group, staining uniformity, Nissl is rich; The neuron cells in hippocampal of model group were disorder. Nissl are not clear and staining asymmetry, there have been many cell swelling, more dead cells. The numbers of neurons in mice were increased, rich,order dense, abundant cytoplasm, form a complete in estrogen group. The neuron cells were rules and rich in high dose group of TFSC.Nissl are rich and completely formed. The neurons in hippocampal from mice were general regular, Some cells slightly swollen and staining general uniform.The neuron cells in hippocampal from low dose group of TFSC decreased and irregularity, Nissl are not clear, Some cells swelling, and more dead cells.6.The cells apoptosis rate were checked thoughs flow cytometry experiment instrument:compared with number control group and estrogen group, model group mice brain neurons cells’ apoptosis rate significantly increased, the difference is significantly statistic(P<0.01); intragastric administration after three months, compared with model group, the apoptosis rate of each dose group of TFSC decreased obviously, the difference is significantly statistic (P<0.05, P<0.01, P<0.01).7.The expression of neuron cell apoptosis related proteins were checked by WB: compared with sham operated group and estrogen group, the expression of Cyt c and Bax protein in hippocampus significantly increased, the protein of Bel-2were significantly reduced from model group, the difference is significantly statistic(P<0.01); Compared with the mice from model group, the expression of proteins with Bax and Cyt-c were significantly decreased though continuous intragastric administration after3months later of each dose group with TFSC, the difference is significantly statistic (P<0.05, P<0.01, P<0.01).8.The expression of neuron cell apoptosis related protein’s gene’s were detected by RT-PCR:compared with normal control group and estrogen group, the expression of the mRNA of Bax, Cyt-c, brightness increased in model group mice’s brain, the expression had a significant rised, the Bcl-2protein mRNA stripe intensity is reduced, the difference is significantly statistic(P<0.01); Intragastric administration after3months, compared with model group, the expression of protein mRNA of Cyt c, in each dose group of TFSC in the hippocampus brightness were decreased, and the brightness of the protein of Bcl-2mRNA were increased, and the Bcl-2protein expression were raised, the difference is significantly statistic(P<0.05, P<0.01, P<0.01).Conclusions:1.After both ovaries were cut, the cutinization of vaginal epithelium cells from model group’s mice were disappeared and the level of estrogen from serum were decreased, the function of learning-memory abilities lysis. The results could be as preliminary evidence that endocrine recession type dementia model successed. 2.TFSC maybe can enhance the function of the hypothalamus-pituitary-gonad axis and the estrogen levels in mice’s serum were increased can improve the abilities of learning and memory in mice. Its the proof about the protective effects on hippocampal neuron cells.3.TFSC can be adjusted the expression of the protein of Bcl-2、Bax’s mRNA and the protein of Bax、Bax mRNA able to make the outer leakage of mitochondrial inner membrane closed and reduce the Cyt-c released into the cytoplasm and inhibit the Caspase-3and Caspase-9cascade reaction, and reduce the neurons apoptosis from the endogenous mitochondrial apoptosis.