Study On The Relationship Between Short-term High-fat Diet And Alzheimer's Disease And Related Mechanism

Background:Alzheimer’s disease（AD）is a serious neurodegenerative disease,and progressive cognitive impairment is its main clinical manifestation.Its neuropathological characteristics mainly include senile plaque（SP）,neurofibrillary tangles（NFTs）,neuronal loss and associated glial cell proliferation caused by the deposition of beta-amyloid protein（Aβ）.Traditional Chinese medicine holds that the abundant vitality and vigorous marrow are the material basis of brain function,while the deficiency of vital energy,the loss of nutrients in marrow and the absence of vital energy are the pathological basis of brain-related diseases.In addition to adipose tissue,the distribution of fat in the central nervous system（CNS）is the highest.In CNS,fat forms the myelin sheath in the form of fatty acids,which then surrounds the axons in the brain and spinal cord,rather than being stored in adipose tissue.CNS-derived adipokines can be produced by non-adipocyte types and nuclei in different brain regions,such as the hypothalamic arcuate nucleus,which regulates diet.Previous studies on AD suggest that adipokines may accelerate the progression of neurodegenerative diseases and vascular diseases by aggravating inflammation in multiple brain regions and nuclei.Previous studies have shown that high-fat diet can aggravate the pathogenesis of AD,and adipokines may aggravate inflammation by activating glial cells in the course of neurodegenerative diseases,thus aggravating the process of neurodegenerative diseases.In recent years,some studies have shown that high-fat diet has no effect on the progression of AD.The Lancet recently reported a 10-year epidemiological study involving more than 135,000 subjects.It was pointed out that,compared with people with low fat intake and energy mainly obtained through carbohydrates,proper increase in fat intake not only did not increase the incidence of cardiovascular diseases,but also reduced the overall mortality rate,overturning the previous high-fat diet on cardiovascular diseases.The conclusion of disease damage suggests whether high-fat diet really does damage the brain and nervous system.Based on these,this study is to explore the relationship between high-fat diet and AD and the related mechanisms,in order to provide the corresponding theoretical and experimental basis for the daily prevention and adjuvant therapy of Alzheimer’s disease.Section Ⅰ:Effects of short-term high-fat diet on AD animal models and related mechanismsOBJECTIVE:To explore the effect of high-fat diet on brain cognitive impairment induced by Okadaic acid in a short period of time and to detect its possible biological mechanism,so as to provide theoretical and experimental basis for the daily prevention and adjuvant treatment of Alzheimer’s disease.Methods:To observe the effect of short-term high-fat diet on cognitive impairment in Alzheimer’s disease model mice and its related mechanism.Two months later,a model of Alzheimer’s disease was established and fed with a high-fat diet.Morris water maze（MWM）was used to test the learning and memory abilities of experimental animals.Nissl staining was used to detect neuronal damage in hippocampal slices.Immunohistochemical staining was used to detect the deposition of Abeta and the content of adiponectin.Western blot was used to detect the expression of Tau and pTau.The expression of NLRP3（NOD-like receptor family,pyrin domain containing 3）and caspase-1 pathway were also measured.RESULTS:The results of water maze showed that short-term high-fat diet could alleviate the cognitive impairment caused by Okadaic acid,and the behavioral manifestation was the impairment of spatial learning and memory impairment caused by OKA.The results of Nissl staining also indicated that neuronal damage was significantly alleviated in the high-fat diet group.Western blot results showed that highfat diet reduced the abnormal phosphorylation of Tau protein.Immunohistochemical staining showed that high-fat diet reduced the deposition of A beta.At the same time,immunohistochemical detection of adiponectin content indicated that short-term highfat diet may reduce adiponectin content by increasing Okadaic acid.Western blot results showed that high-fat diet may regulate NLRP3 and caspase-1 signaling.Signal pathway alleviates neuronal damage and plays a neuroprotective role.CONCLUSION:Short-term high-fat diet may play a protective role in the nervous system to some extent,and its possible mechanisms are speculated.Firstly,high-fat diet reduces the expression of inflammatory factors by mediating NLRP3/caspase 1 pathway.Secondly,it may be that the central nervous system is the second highest fat content in the body,second only to adipose tissue.Short-term high-fat diet may have certain nutritional effects on the brain nerve,which can alleviate the neurological damage caused by OKA and the decline of adiponectin level.This provides a new way of thinking and theoretical basis for the research on the treatment of Alzheimer’s disease.Section Ⅱ Preparation of Lentivirus Packaging SystemOBJECTIVE:The diameter of recombinant virus vector is mostly in the range of 20100 nm,which plays an important role in life-related scientific research and clinical drug development.Viral vectors can be used as a tool to mediate the transmission of RNAi or the expression of related genes.This not only helps people understand the function of genes,but also can be used to develop new therapies（such as gene therapy and stem cell therapy）.Because the target gene can not be directly integrated into most eukaryotic cells,in order to better achieve the desired experimental results,the commonly used method in the experiment is to package the target gene into a virus to infect cells,so as to obtain expression to meet the experimental needs.Compared with other classical viral vectors such as adenovirus,adeno-associated virus and retrovirus,lentivirus has the advantages of high infection spectrum,low immunogenicity and long expression time.Therefore,this experiment carried out a preliminary study on the operation of the lentivirus packaging system,in order to provide feasibility for the establishment of animal models and related gene determination in subsequent experiments.METHODS:293T cells are human renal epithelial cell lines expressing large T antigen of SV40.They are derived from 293 cells and are often used for instantaneous transfection and packaging of viruses.After starvation treatment,293T cells in logarithmic phase were transfected with three plasmids（transfer plasmid,packaging plasmid and envelope plasmid）packaging system.After transfection,the transfection solution was withdrawn to collect,concentrate,purify,freeze-store the viral solution and determine the expression effect at cell level and in animal brain.The expression levels of lentiviruses obtained from two different transfer plasmids pLJMl-EGFP（Plasmid₁9319）and PLKO.3G（Plasmid₁4748）,24 hours and 48 hours of collection,the difference of infection effect before and after concentration,and the effect of PLL encapsulation on the expression of lentiviruses in 10 cm dishes at different conditions during virus collection were determined.The expression of lentiviruses in the substantia nigra of rats was detected by localized intracerebral injection.Results:（1）The expression of lentiviruses collected in the three-plasmid packaging system consisting of PLKO.3G（Plasmid₁4748）was better than that in the pLJM1EGFP（Plasmid₁939）packaging system.（2）In the same plasmid packaging system,the infection effect of lentiviruses collected 24 hours was stronger than that of lentiviruses collected 48 hours.（3）Compared with the same plasmid,the expression level of concentrated lentivirus solution in cells was higher.（4）During the collection of lentiviruses,the infection effect of 10 cm cell culture dish was better than that of 10 cm cell culture dish.（5）The green fluorescence expression of lentivirus was detected in substantia nigra,lateral ventricle,striatum and substantia nigra of rat brain after localized injection of lentivirus.CONCLUSION:（1）The expression of lentiviruses in PLKO.3G（Plasmid₁4748）packaging system is better than that in pLJMl-EGFP（Plasmid₁939）packaging system,and the expression effect of lentiviruses collected 24 hours after transfection is higher than that of lentiviruses collected 48 hours after transfection.（2）During the collection of lentiviruses,10 cm dishes of cell culture should be coated with PLL.The mechanism may be that PLL coating is beneficial to the adherence growth of cells and the collection of viral solution.（3）After concentration of lentivirus solution,the infection effect is improved,which can provide guidance for the follow-up experiment.（4）The green fluorescence expression of lentiviruses was observed in frozen sections of the striatum,substantia nigra and lateral ventricles,which indicated that the lentiviruses obtained from the packaging system could be expressed in the brain of animals.Section Ⅲ Identification of Transgenic Mice GenotypesObjective:The genes of a species store and regulate all the information about its race and its own growth and reproduction.The essence of genes is DNA molecular fragments with genetic effects.By analyzing DNA,we can understand the basic genetic information of organisms.Specific genotypes of mice can be used to determine the relationship between specific gene fragments and related diseases and to facilitate the establishment of disease animal models.Methods:after about 3-4 weeks of birth,the rats were harvested with a scissors scissors,and about 1-2mm of the rat tail was harvested for tissue DNA extraction and purification.Polymerase chain reaction（PCR）was used for agarose gel electrophoresis.APP and PSEN1 are commonly used gene fragments related to AD modeling in current research.APP/PSEN1 double-transgenic mice can express mutant human presenilin（DeltaE9）and mouse amyloid precursor protein（APPswe）fusion,which can be used to model AD animal models.RESULTS:Genetically engineered mice expressing both APP and PSEN1 were detected.In addition to the AD model induced by Okadaic acid,more aspects of the establishment of AD-related mechanisms.It is convenient for the improvement of animal models related to high-fat diet.CONCLUSION:APP/PSEN1 double transgenic mice model has been successfully identified.