A?_25-35Oligomers Induced Insulin Signaling Impairment And The Underlying Mechanisms In Intestinal Macrophages

Background:Alzheimer's disease(AD)is the most common neurodegenerative disorder of the central nervous system,occurring of the senile dementia patients.The core pathological features of AD are the tangles of the senile plaques formed by the extracellular beta amyloid proteins(amyloid-?,A?)and the intracellular hyperphosphorylated Tau protein.The abnormal deposition of A? in the brain can lead to the degeneration of synapses and the neuronal apoptosis.AD has been a major global public health problem.Until now,AD has no effective intervention because the underlying pathogenesis is still not clear and has no targeted treatment strategy.This is the fundamental problem to be solved urgently.The investigation of the AD patient registry(Mayo Clinic)suggested that almost 80%of patients with AD exhibited glucose intolerance or diabetes.Epidemiologic studies suggested that diabetes increased the risk for progressive AD up to 50-100%.Since the original Rotterdam study,the epidemiological or clinical observations had accumulated mounting evidences showing that diabetic patients were significantly more likely to develop cognitive deterioration and to exhibit increased susceptibility to dementia,particularly AD.Indeed,studies by Ott and colleagues had shown that dementia occurrence rate were twice as frequent in diabetic patients as in normal subjects.It is now thought that even prolonged pre-diabetes can significantly increase the risk of dementia.AD and type 2 diabetes mellitus(T2D)have a lot of common pathologic characteristics,including an abnormal protein processing,dysregulated insulin signal pathway,disorder of glucose metabolism,oxidative stress,advanced glycosylation end products formation and activated inflammatory pathways.Insulin deficiency and insulin resistance as well as the interference of insulin and insulin-like growth factor represent the early abnormal and progressing pathological processes which can explain the most lesions of AD from molecular,biochemical and histopathological aspects.Therefore,AD had been regarded as an special selectived cerebral diabetes which was named "type 3 diabetes".The AD brain showed signs of insulin-signaling defects and changes in the insulin signaling pathway and/or abnormal activation,and most importantly,decreased responsiveness to insulin.Insulin resistance in AD is the critical role in the pathological mechanism which is related to A? production and aggregation,neurofibrillary tangles formation,neuronal degeneration and synaptic transmission disorder.Neuroinflammation is an early and persisitent pathological feature in neurodegenerative disease,while peroxidative stress was proved to be a factor.The JNK signaling pathway is associated with oxidative stress and insulin resistance.At the same time,JNK also activates gamma-secretase,which affects the formation of A?.A? was found that not only deposited in the central nervous system but also in skin,subcutaneous tissue and intestinal tract and so on.The intestinal tract was referred to as the "second brain" of the body.There was a complex interaction between intestines and the brain through neural,endocrine,immune,and humoral links.The complexity of these interactions was enrolled in the denomination of"gut brain axis(GBA)".The GBA not only ensures the proper maintenance of gastrointestinal,homeostasis,but is likely to have important effects on affection,motivation,and advanced cognitive function.Clinical and laboratory evidence indicated that gut microbiota has important influence to the GBA,and gut microbiota was confirmed to be connected with metabolic diseases The change of the microbial communities can activate inflammatory signaling pathways,leading to impaired insulin signaling pathway.Intestinal macrophages are the body's largest resident macrophages groups which distributing along the intestinal tract.The macrophage has close links between the nervous system through the ?2-AR and its ligands.Macrophages have important effects on intestinal flora and GBA.Therefore,our study will focus on the insulin signaling pathway and the undelying mechanism of the intestinal macrophages reduced by A(3.Aims:1.To study the insulin signaling in the gut of transgenic mice reduced by A? and the underlying mechanisms.2.To study the insulin signaling of intestinal macrophages reduced by A? and the underlying mechanisms.Methods1.Animals and CellsAnimal models:APPswe/PSEN1dD9(APP/PS1)transgenic mouse was purchased from the Jackson Laboratory.It is the most widely used animal model in the study of AD.And WT mice was purchased from Guangdong medical laboratory animal center.20 animals were fed seperately in cages(4-5 cages).The cage temperature was controled in 20?24 ?,with light and dark period of 12 h,free drinking water and feeding.We selected healthy mice of 12 months of age to carry out relevant experiments.Cell culture:The mouse intestinal macrophages(RAW264.7)cells were purchased from the Shanghai institute of biology.Dulbecco's modified Eagle's medium was used to culture the cells which was supplemented with 10%fetal bovine serum and added in 100 u/ml penicillin and 100 ug/ml streptomycin.The humidified atmosphere was kept at 37? and containied 5%C02.According to cell growth,the cell generation and processing were carried out.2.ImmunohistochemistryWe took the ileum of about 20 cm from pyloric after the mice was put to death and removed of residual waste.After rinsing with the saline solution,the ileum was put into 4%polyformaldehyde fixation,and then were fixed and embedded in paraffin blocks.The primary antibodies concentration were following like these:lR 1:1000?IRS 1 1:1000?A? 1:200?Tuj 1:500?KIBRA 1:200?32AR 1:400?eNOS 1:400.DAB system was applied for displaying color at room temprature.All images were analyzed by the QUANTITY ONE gel electrophoresis image analyzer.Protein expression levels were marked with the protein beta-actin.3.A?25-35 oligomers preparationA? 25-35 peptides 1 mg was dissolved in 0.33 ml double evaporate water and then the concentration was adjusted of 1 nmol/uL.After sealed with sealing membrane,A? 25-35 peptides was incubated in 37 ? water bath for 96 h until it was fully assembled and "aging".4.MTT assayAfter the end of drug treatment,MTT assay was used to asses the cell viability.5.ELISA assayThe logarithmic growth of RAW264.7 was inoculated in 96-orifice plate and then was treated with a group of drugs.The concentration of IL-6 and TNF-a was checked using ELISA assay.6.Western BlotProteins in macrophages homogenate were extracted with RIPA buffer.The expression of proteins in insulin signaling pathway(IR-??IRS1?p-IRS1?AKT?p-AKT?JNK?p-JNK)was detected by blot western.QUANTITY ONE gel electrophoresis image analyzer was used to analysis the results.7.Statistic AnalysisStatistical analysis was performed by SPSS version 19.0(IBM,Armonk,NY,USA).The one-way ANOVA detection method was adopted in the multi-group data comparison,and LSD method was used in the comparison between the two groups.The results of all the analyses were indicated by means±SD,And the difference was p<0.05,which was statistically significant.Results:1.The expression of IRS1,A?,Tuj,KIBRA,?2AR,and eNOS in AD group were increased,but IR not.Compared with WT group,levels of IRS1,A?,Tuj,KIBRA,P2AR,and eNOS were all increased,with statistical difference(P<0.05).There was no significant difference in IR in the two groups of mice.2.The proliferation of macrophages RAW264.7 was increased induced by A?25-35.Compared with the blank group,the proliferation of RAW264.7 cells showed a statistically significant difference(P<0.01),and this effect was dose dependent with A?25-35.After insulin treatment,the increase rate of the cell was significantly lower than that of A?25-35 group(P<0.05).1.The expression of TNF-a and IL-6 secreted by macrophages RAW264.7 was increased induced by A?25-35.Compared with the blank group,the expression of TNF-a and IL-6 showed a statistically significant difference(P<0.01),and this effect was dose dependent with A?25-35.After insulin treatment,the expression of TNF-a and IL-6 was significantly reduced and had statistically significant difference(P<0.05)compared to non-insulin group.2.The expression of IR-? and p-AKT/AKT in macrophages RAW264.7 was increased and the expression of p-IRS1/IRS1 and p-JNK/JNK was decreased induced by A?25-35.AP25-35 can significantly reduce the content of the macrophage IR-? protein(P<0.01),while the insulin treatment can significantly improve the amount of IR-?(P<0.05).In addition,A?25-35 significantly reduced the ratio of p-AKT/AKT of macrophages(p<0.01),while insulin treatment could significantly improve the ratio(p<0.05).AP25-35 can significantly increase the ratio of p-IRS 1/IRS1 and p-JNK/JNK in macrophages(p<0.01),and the two ratios tend to be equal with blank(p<0.01)after insulin treatment.Conclusions:1.The APP/PS1 mice showed abnormal deposition of the A? and the highly expression of IRS 1 suggesting the insulin resistance,and A? induced it.2.The APP/PS1 mice showed highly expression of the intestinal ?2AR,KIBRA and Tuj sugesting that the sympathetic activity increased,and the function of macrophage was activated.3.A?25-35 induced the proliferation of macrophages and the function was significantly enhanced.4.A?25-35 impaired the insulin signaling pathway of macrophages,and the underlying mechanism was related with JNK pathway.