Part 2 Effects Of Cistanche Deserticola Phenylethanoside On The Expression Of β-amyloid Protein in Hippocampus Of APP / PS1 Transgenic Mice

Part 2 The effective ingredients of Cistanche: the therapeutic effect of total phenylethanoid glycosides and Acteosides on Alzheimer's Disease

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4 . Conclusion

Alzheimer's disease mainly occurs in the central nervous system and is induced by multiple factors. So far, its pathogenesis is still unclear, but the core of the research is still around Aβ. As the main peptides secreted by APP in the brain, the abnormal deposition of Aβ1-42 and Aβ1-40 cannot directly cause neuronal degeneration. From the perspective of etiology, it is the soluble oligomer ADDLs produced by Aβ1-42 that cause the brain The real cause of neuronal degeneration. However, the investigation of the expression of these two proteins can also reflect the extent of AD brain lesions in real-time, indirectly show the expression of soluble oligomer ADDLs, and then dynamically monitor the neuronal damage in the brain from the source, providing a reference for disease prevention and treatment. In view of this, Aβ1-42 and Aβ1-40 have become key targets in the study of the pathogenesis of AD. The hippocampus is the main part of the brain to evaluate the ability of learning and memory. The damage of its neurons is the leading cause of cognitive dysfunction in AD. The CA1 area of the hippocampus is one of the main parts of AD pathological damage. This part of the experiment uses HE The staining method showed that the neurons in the CA1 area of the hippocampus of the normal group were arranged neatly and tightly, distributed more evenly, and had clear layers. In the model group, the hippocampal CA1 area of the mouse showed degenerative changes such as neuronal cell apoptosis due to injury. After the intervention of different doses of Cistanche phenethyl alcohol glycoside groups, the cell morphology of the hippocampal CA1 area

All have different degrees of improvement, suggesting that Cistanche phenylethanoid glycosides may have the effect of protecting neurons and inhibiting their apoptosis, but because HE staining is not specific for cell apoptosis and neuron loss, it is still necessary to target apoptosis-specific molecules in the later stage. Relevant detection of markers.

The pretreatment of the tissue to be tested is directly related to the staining result. In the experiment, the author found that because the hippocampus of the transgenic mice is located in the deep inner part of the temporal lobe of the telencephalon, it is small in size and difficult to separate the tissue. A little carelessness will cause mechanical damage to it so that the pathological changes cannot be effectively observed. At the same time, because genetically modified mice are expensive and the experimental dosing cycle is long, the loss of samples may affect the overall progress of the research. Therefore, when the mouse completes cardiac perfusion, the whole brain is taken out and fixed with paraformaldehyde, the scalpel blade is used to remove the olfactory bulb, and the rest of the brain tissue is dehydrated and embedded. The slice thickness is controlled at 3 to 5 μm to ensure that the hippocampus is intact. Under the premise of optimizing the operation process, the research efficiency is improved. In the Western-blot detection, the author found that due to the small molecular weight of Aβ1-42 and Aβ1-40 proteins, the expression bands are not easy to blot, so it is necessary to screen their SDS-PAGE gel concentration, electrophoresis time, and transfer time. If the concentration of the separating gel is too small, the electrophoresis lanes will migrate and the protein activation will be incomplete; too long the electrophoresis time will cause the band to shift out of the gel layer; the corresponding transfer time will make the western blot blurred, thereby affecting the primary antibody incubation Combine with secondary antibodies. In view of this, this study was through preliminary experiments, and finally selected 15% separation gel, electrophoresed, and transferred membrane for 2 hours to perform Western blotting on Aβ1-42 and Aβ1-40 to investigate their expression.

Among the technical methods currently used for protein regulation, the Western-blot method is mainly used for quantification to investigate the specific expression of related proteins. The immunohistochemistry method starts from a qualitative point of view. On the one hand, it can more intuitively show the degree of specific binding of protein and antibody, and on the other hand, it can support the quantitative results. Because immunohistochemistry is to detect protein expression by binding antibodies to antigenic sites on histopathological slices, the preparation of histopathological slices is particularly important. The section is not easy to be too thick. After the section is completed, care should be taken to save it to avoid external damage to the tissue section; adequate deparaffinization should be carried out before processing, and the time should not be less than 2 hours; choose a citric acid buffer with mild conditions and less incidental damage to the antigen Perform repair, remove catalase, fully expose the antigenic site, and make the primary antibody fit the site completely. The above two methods, as commonly used detection methods in molecular biology, complement each other and provide technical support for protein regulation research.

This study used HE and other methods to investigate the hippocampal lesions and the expression of Aβ-related proteins in the brain. The results showed that after the intervention of Cistanche phenylethanoid , the cell morphology of the hippocampal CA1 area in the brain of AD model mice improved to varying degrees. The expression levels of Aβ1-42 and Aβ1-40 were significantly down-regulated. In addition, it was found that the protective neuron effect of the verbascoside intervention group was better than that of the total phenylethanoid glycosides group. The total glycosides of Cistanche phenylethanoid glycosides have great potential to improve the pathological changes in the brain area of AD model mice and then play an antagonistic effect on AD. The specific mechanism of action needs to be further studied in the future. This study completes the research data on the mechanism of Cistanche antagonizing AD from the perspective of Aβ, and at the same time lays the research foundation for further elucidating the mechanism of the effective components or active monomers of Cistanche against AD in the later period.