Study On The Regulatory Mechanism Of High Fat High Meat Protein Diet On Hippocampal Amyloid Precursor Protein(APP)

Alzheimer’s disease（AD）is a neurodegenerative disease,and the main pathogenesis is that the amyloid precursor protein（APP）abnormally cleaved byβ-secretase（mainly BACE1）to insoluble Aβ₄₂ aggregates and produce neurotoxicity.The main feature of AD is severe memory and cognitive impairment,and may be accompanied by a series of complications such as personality abnormalities,digestive system and gastrointestinal tract dysfunction,which brings mental and financial burden to patients.There are about 10million AD patients in China until 2019,and the AD patients will continue to rise due to population ageing.AD is incurability at present in the clinic,which caused by genetic and environmental factors represented by diet.Studies have found that Western diet patterns characterized by high fat and high protein diets can lead to the occurrence of AD.There are a wide range of dietary protein sources,and their effects on human health are different.However,little is known about the effects of different dietary proteins on high fat diets induced-AD.Therefore,changing the type and content of protein may become one of the methods to prevent/delay the onset of AD.This study used animal and cell models to investigated the effects of different types（casein,chicken and pork protein）and content（20%and 40%）of protein on APP expression and cleavage in hippocampus under high fat diet.The specific research is as follows:1.The effect of high fat high meat protein diet on the expression of APP in hippocampus To investigated the effect of different type and content protein on the expression of hippocampal APP under high fat diet,we used high fat combined with different type（casein,chicken and pork protein）and content（20%and 40%）protein to fed Wistar rat for 12 weeks,collecting hippocampus.We measured the volume of the hippocampus,the content of inflammatory factors（IL-6,IL-1β,TNF-αand LPS）,neurotransmitter（GABA）and metabolites（glutamate,creatine,inositol,N-acetylaspartic acid and choline）,the m RNA level of APP,BACE1,astrocyte protein（SCNA,P₂X₇,GFAP and Caspase3）,glutamatergic system（GLT-1,GLAST and NMDA）,neurotransmitter（BDNF）.The results showed that under the high fat diet,the high protein group significantly up-regulated the m RNA levels of GLT-1 and GLAST in the hippocampus（P<0.05）,and down-regulated metabolites,BDNF and GABA content（P<0.05）.In the high protein group,compared with casein chicken protein diet significantly increased the level of inflammation in the hippocampus and the m RNA levels of APP and BACE1（P<0.05）.Therefore,excessive intake of chicken protein up-regulated hippocampal APP expression,promoted a series of neuron changes related to AD caused by high fat diet.2.The effect of high fat high meat protein diet on the enteric nervous system and APP expression in N2a cells We used high fat combined with different type（casein,chicken and pork protein）and content（20%and 40%）protein to fed Wistar rat for 12 weeks,collecting serum and duodenum.The content of inflammatory factors（IL-6,IL-1β,TNF-αand LPS）in serum were measured.In duodenum,the content of inflammation and neurotransmitters（acetylcholine,glutamate and GABA）,the m RNA levels of nutrient transporters（CD36,FATP4 and B0AT1）,enteric nerve cell protein（GFAP and S100β）expression and intestinal epithelial morphology were measured.At the same time,the dietary protein,collagen and myosin digest were intervened on nerve cell N2a,and the protein expression of APP,BACE1 and GLT-1 in nerve cells were investigated.The results showed that high protein diet significantly increased the m RNA levels of FAPT4 and B0AT1（P<0.05）,and down-regulated the m RNA level of VIP（P<0.05）under high fat diet.In the high protein group,compared with casein,chicken protein diet significantly increased serum and duodenal inflammation levels（P<0.05）,inhibited the vitality of enteric nerve cells,and down-regulated the content of acetylcholine（P<0.05）.The expression of APP and BACE1 in N2a cells were positively correlated with protein concentration in chicken protein digest,while the casein and pork protein groups showed the opposite trend,which may be related to the amino acid composition of the digest.Therefore,the enteric nervous system of Wistar rats was inhibited when excessive intake of chicken protein under high fat diet,and the"gut-brain axis"may be an important way for diet to up-regulated APP expression.3.The effect of high fat high chicken protein diet on the cleavage of APP in the hippocampus of APP/PS1 mice To further explore the effect of chicken protein diet on APP cleavage under high fat diet,we used AD model APP/PS1 double transgenic mice and wild type WT mice in this part of study,which were fed with high fat high chicken protein diet and high fat low chicken protein diet for 12 weeks,and the hippocampus and serum were collected.The Morris water maze was used to explore the behavior of APP/PS1 mice,the m RNA levels of hippocampal APP cleavage(APP,BACE1,ADAM10 and Aβ₄₂),glutamatergic system（glutamate,GABA,GLT-1,GLAST and NMDA）,nerve synapse protein（SCNA and PSD95）and serum metabolomics were investigated.The results showed that under high fat diet,high levels of chicken protein aggravated the memory impairment of APP/PS1 mice,and up-regulated the m RNA level of BACE1 and the content of Aβ₄₂（P<0.05）,while there were no significant effect in WT mice（P>0.05）.In APP/PS1 mice,high fat high chicken protein diet significantly down-regulated the mRNA levels of SCNA,GLT-1,GLAST and NMDA（P<0.05）,and up-regulated the m RNA levels of p38 MAPK,MKK and MLK3（P<0.05）.By comparing the serum metabolomics of APP/PS1 mice with high fat high protein and high fat low protein diets,48 different metabolites were identified,mainly enriched on organic acids and their derivatives,lipids and lipid molecules,organic oxygen compounds,nucleosides,nucleotides and analogs,organic heterocyclic compounds.Among them,the concentration of phospholipid substances decreased in the serum of APP/PS1 mice fed with high fat high protein diet.Therefore,under high fat diet,high chicken protein promoted the APP cleavage,inhibited the activity of neurons and glutamatergic system,activated the p38MAPK pathway in the hippocampus,and aggravated dyslipidemia and lipid metabolism disorders in APP/PS1 mice.4.The effect of high fat high chicken diet on the enteric nervous system of APP/PS1mice We used APP/PS1 double transgenic mice and wild type WT mice in this part of study,which were fed high fat high chicken protein diet and high fat low chicken protein diet for 12 weeks,and the duodenum was collected.The enteric nerve cell protein（GFAP and S100β）,neurotransmitter（acetylcholine and VIP）and amino acid levels in duodenal tissue and chyme were measured.The results showed that under high fat diet,high chicken protein down-regulated the content of duodenal glutamate,citrulline,ornithine,arginine and aspartic acid in APP/PS1 and WT mice（P<0.05）.At the same time,high chicken protein significantly down-regulated the m RNA levels of duodenal VIP and the content of acetylcholine in APP/PS1 mice,and up-regulated the contents of GABA and choline（P<0.05）.Compared with the low protein group,the duodenum of APP/PS1 mice and WT mice in the high chicken protein group had obvious villi rupture.Therefore,under high fat diet,high chicken protein inhibited duodenal glutamate metabolism and enteric nervous system function in APP/PS1 mice,and may participated in the hippocampal process of aggravating the cleavage of APP through the gut-brain axis.5.The effect of chicken protein digest on p38 MAPK-mediated APP cleavage Taking the nerve cell N2a which stably transfected with APP as the experimental object,treated cells with different concentrations（0.1,0.3,0.5 mg/mL）of chicken protein digest for 24 hours,and the protein expression of APP,BACE1,GLT-1 and p38 MAPK were measured by Western blotting.The results showed that 0.5 mg/m L of chicken protein digest could up-regulated the expression of p38 MAPK,BACE1 and APP in APP-transfected N2a cells（P<0.05）,and down-regulated the expression of GLT-1（P<0.05）.After activating p38 MAPK in APP transfected N2a cells,the expression of BACE1was significantly up-regulated,and the expression of GLT-1 was significantly down-regulated（P<0.05）.When chicken protein digest interfered with cells while inhibiting the expression of p38 MAPK,the expression of BACE1 and GLT-1 had no significant changes in APP transfected N2a cells（P>0.05）.Therefore,in APP-transfected nerve cells,p38 MAPK activated was a way to mediated the cleavage of APP and chicken protein digest.