Effect And Mechanism Of The Cattle Encephalon Glycoside And Ignotin Injection On The Expression Of Amyloid β-peptide And Some Proteins Relating To Neuroprotection In The Brain Of APPswe/PS1dE9 Double Transgenic Mice

The first part:the therapeutic with CEGI effect of APPswe/PSlDe double transgenic mice Aβ42、GFAP in brain cortex and hippocampusObjective As the most common type of senile dementia, Alzheimer’s disease (AD) has a high incidence. The main clinical manifestation of AD is a progressive cognitive dysfunction, then it may lead to dementia and eventually a complete loss of daily living ability. The main pathological changes of AD are the appearance of senile plaque (SP) and neurofibrillary tangles(NFTs), which are accompanied by synaptic and neuronal losses. Amyloid protein (Aβ) cascade hypothesis attractes widespread attention among the pathogenesis of AD. Astrocytes are one kind of glial cells, which have the most widely distribution and the largest volume. Astrocytes have multiple effects on neuron support, protection and restoration, nutrition and neurotransmitter regulation and immunity. Glial fibrillary acidic protein (GFAP) is a unique marker of astrocytes. The changes of its content or structure not only reflect the morphological changes of astrocytes, but also better reflect the degree and range of neuronal damage. Cattle Encephalon Glycoside and Ignotin Injection (CEGI) is small molecule peptide amino acids and contains a variety of ganglioside components, which play an important role for promoting nerve regeneration and restoring nerve function. Based on APP/PS1 double transgenic AD mice model, this study discusses CEGI therapeutic effect for AD by Morris water maze behavioral measurement and observe the changes of β-amyloid (Aβ42) and GFAP, aiming to providing theoretical basises and experimental evidences for clinical application of CEGI in the treatment of AD.Methods The 90 mice of APP/PS1 double transgenic for 5 months were randomly divided into 6 groups,15 of each:the model group(Tg), the low-dose group of CEGI (Tg+CEGI-L), the mid-one (Tg+CEGI-M), the high-one (Tg+CEGI-H), Tg+Donelize group (Tg+Donelize), and the combination one of the mid-dose of CEGI and Tg+Donelize (Tg+CEGI+Donelize). The 10 non-transgenic mice (C57BL/6) for the same age and background were as the normal control group (nTg). The 7 groups of animals were given different doses of saline, CEGI, Donepezil 30 days, then were made a six-day Morris water maze behavioral tests. By (Hematoxylin-eosin)HE, immunohistochemistry(IHC), Thioflavin staining-S(Thioflavin-S) and western blot, the different expressions of Aβ42 and GFAP in hippocampus and cortex were detected.Results There were no statistically significant (P> 0.05) of the body weight of mice between before and after treatment in the same group; In behavioral trials, compared with the ones of Tg group, the landing station numbers of the Tg+CEGI-M, nTg and Tg+CEGI+Donepezil groups significantly increased at 60 seconds (P＜0.05); The differences about the counts and area of Aβ42 senile plaques in hippocampus, were statistically significant (P<0.001), in Tg+Donepezil and Tg+CEGI+Donepezil groups, respectively compared with the Tg group; There were all significantly statistical differences in Tg+CEGI-M, Tg+CEGI-H, Tg+Donepezil and Tg+CEGI+Donepezil groups, when respectively compared with Tg group (P<0.005), about the counts and area in cortical AP42 senile plaques; The quantities of GFAP in the hippocampal CA1and CA3 area, were less in Tg+CEGI-M, Tg+CEGI-H, Tg+Donepezil and Tg+CEGI+Donepezil groups than Tg group.Conclusion The treatment of CEGI significantly improved APPswe/PS1dE9 double transgenic mice spatial learning and memory; The treatment of CEGI could significantly reduce the generation and expression of Aβ42 in APP/PS1 double transgenic mice brain, and the mid-dose treatment of CEGI was more effective; CEGI therapy could reduce the expression of GFAP of astrocytes. The study results showed that:CEGI by inhibiting Aβ42 generation, accumulation and expression in APPswe/PS1dE9 double transgenic mouse brain, could reduce GFAP expression in astrocytes, which improved APPswe/PS1dE9 double transgenic mice learning and memory ability. These has a clear therapeutic effect on AD.The second part:the CEGI on the expression effects of the calcium-binding protein-D28K and vitamin D receptor in the hippocampus of APPswe/PS1dE9 double transgenic miceObjective AD pathogenesis includes Aβ damage theory, Tau protein aberrant modification, calcium homeostasis, inflammation, cholinergic and gene mutations, etc. It is now widely recognized that Aβ is the initiating factor of AD pathogenesis. The neurotoxic effects of Aβ affect calcium signaling pathway of neurons, leading to calcium overload and neurotoxicity, thereby affecting learning and memory mechanisms and inducing neuronal cell death. Calbindin D28K is mainly present in the cytoplasm of neurons, axons and dendrites of some brain regions, which can maintain neuronal calcium homeostasis and reduce the toxicity of neurons due to high concentrations of calcium ions. The combination of vitamin D receptor (VDR) and vitamin D can regulate calcium ion channels, affect calcium levels, maintain calcium homeostasis and reduce the neurotoxicity. Based on APP/PS1 double transgenic AD mice model, this study discuss CEGI therapeutic effect for AD by observing the changes in expression of neuron protective proteins and CB and VDR having the ability of buffering Calcium.Methods The 90 mice of APP/PS1 double transgenic for 5 months were randomly divided into 6 groups,15 of each:the model group(Tg), the low-dose group of CEGI (Tg+CEGI-L), the mid-one (Tg+CEGI-M), the high-one (Tg+CEGI-H), Tg+Donelize group (Tg+Donelize), and the combination one of the mid-dose of CEGI and Tg+Donelize (Tg+CEGI+Donelize). The 10 non-transgenic mice (C57BL/6) for the same age and background were as the normal control group (nTg). The 7 groups of animals were given different doses of saline, CEGI, Donepezil 30 days, then were made a six-day Morris water maze behavioral tests. By immunohistochemistry(IHC), RT-PCR and western blot, the different expressions of CB and VDR in hippocampus CA1 and CA3 were detected.Results In the hippocampal CA1 and CA3 areas, the expression of CB was lower in Tg group than other six groups. The difference was statistically significant (P<0.05); The expression results of CB in mRNA and Western Blot were consistent with immunohistochemical one.; The expression of VDR in the hippocampal CA1 and CA3 areas, was lower in Tg group than nTg and Tg+CEGI+Donepezil. The difference was statistically significant (P<0.05);Conclusion The treatment of CEGI significantly improves the expression of hippocampal CB and VDR in the APPswe/PS1dE9 double transgenic mouse model. Thereby this enhances the calcium chelating in neurons, buffers neurotoxicity caused by high calcium concentration, and has a neuroprotective effect.