The Role Of REST In Stress And The Mechanism Of LW-AFC Improving Learning And Memory Impairment Induced By Stress

Chronic stress can cause excessive psychological and physiological demage to the body,affecting mental and physical health as well as daily life.There are mounting evidences that chronic stress can reduce the number and function of neurons in hippocampus,amygdala and prefrontal cortex,even impair cognitive function,emotional regulation and autonomous behavior.morever,these impairments are more serious in elderly or diseased individuals.NRSF/REST(neuron-restrictive silencer factor,NRSF;also known as RE-1 silencing transcription factor,REST)is a critical negative transcription regulator in the nervous system,which participates in neuronal differentiation,axon growth,vesicle transport release,and ion conduction.REST,increased with age,is highly expressed in neurons in the healthy aged.However,its decreased expression and nuclear deletion play a key role in the pathogenesis of Alzheimer's disease(AD).REST in hippocampus was positively correlated with learning and memory.These findings strongly suggest that REST is not only a classical inhibitor to maintain normal neurogenesis,but also a potential site to protect neurodegeneration.REST is an effective therapeutic target for learning and memory function.The experimental model is a critical tool to study diseases.REST has not yet been reported in cells and animal of AD models.RSET has multiple protective effects on different stresses.REST can maintain neuronal activity through antioxidant stress.The most direct response caused by stress is the hyperfunction of the hypothalamicpituitary-adrenal axis(HPA or HTPA axis).REST can improve stress-induced disorders by directly regulating the expression of CRH [4] [5,6] and indirectly regulating the production of corticosterone.In the course of aging,the sensitivity of negative feedback of HPA axis system is reduced,and the abnormality of glucocorticoid reccptors(GRs)will lead to cognitive disfunction because of the excessive secretion of corticosterone caused by stress.In the aging state,whether the body can quickly regulate the learning and memory impairment caused by HPA axis disorder through REST is worth further investigation.Traditional Chinese medicine Liuwei Dihuang Decoction(LW)has obvious regulative effect on endocrine and immune imbalance caused by various types of stress.Liuwei Dihuang Glycoside Tablets(LW-AFC)with controllable quality and good safety were separated from LW.Previous studies in our laboratory confirmed that LW-AFC has multiple effects on restore HPA axis disorder,improve cognition.LW-AFC can also improve learning and memory impairment caused by multiple acute stress [13] and single factor stress.These studies suggest that LW-AFC can improve cognitive impairment induced by stress.However,the mechanism of LWAFC in improving learning and memory impairment induced by chronic stress still remains unclear.REST can improve stress-induced cognitive impairment by regulating HPA axis,NMDAR and synaptic plasticity.Whether LW-AFC can alleviate stress-induced learning and memory impairment by regulating REST calls for further exploration.In this study,both human serum and human induced pluripotent stem cells(IPSC)were used to study the expression of REST in human aging process.Then,the AD animal model of senescence accelerated mouse(SAM)was used to study the dynamic expression of REST in central and peripheral serum during senescence of rapidly aging mice.The effects of chronic mild stress(CMS)were observed on cognitive function,corticosterone concentration,REST content and synaptic plasticity-related signaling pathways of SAMP8,besides,the effects of chronic stress on stress tolerance of aging brain were also studied.Finally,the effects of LW-AFC on stress tolerance were observed,we intend to explore whether LW-AFC can play an anti-stress role by regulating the content of REST.?.expression of REST in human peripheral blood samples and human IPSC1.The expression of REST in peripheral blood of the cognitively normal elderly,Mild cognitive impairment(MCI)patients and AD patientsELISA was used to detect the REST content in serum of the elderly with normal cognition,mild cognitive impairment(MCI)and alzheimer's disease(AD).The results of correlation analysis showed that REST expression in serum had no significant correlation with the MMSE.2.Expression of REST in IPSC and its induced differentiation of nerve cellsIPSC were induced to differentiate into neural stem cells,microglia cells,and neurons using IPSCs that were cognitively derived from young,elderly,and AD patients.Western Blot(WB)was used to detect REST expression.The results showed that REST protein expression in IPSC of cognitively normal elderly was significantly higher than that of young people,and REST protein expression in IPSC of AD patients was significantly lower than that of cognitively normal elderly people.No differences in REST protein expression were observed in neural stem cells,neurons and microglia induced by IPSC in young adults,cognitively normal elderly and AD patients.? REST expression in SAMR1/P8 and the influence of chronic stress on REST expression1.Dynamic expression of REST in the aging process of SAMR1/P8Senescence Accelerated Mouse P8(SAMP8)aged with 2,6 and 12 months were selected as the model group,and the same aged Senescence Accelerated Mouse Resistant 1(SAMR1)was taken as the control group.WB was used to detect the REST expression in the hippocampus during the aging process of SAMR1 and SAMP8.The results showed that REST was most highly expressed in the hippocampus of SAMR1 at 2 months of age,but no significant difference was found between 2,6 and 12 months.The expression of REST was the lowest in the hippocampus of 2-month old SAMP8,and the expression of REST was gradually increased in the hippocampus of 6-month and 12-month SAMP8.The expression of REST was significantly higher in the hippocampus of 12-month SAMP8 than in the hippocampus of 2-month SAMP8,but there was no significant difference between 12-month and 6-month.Compared with SAMR1 at the same age,REST expression in the hippocampal tissue of SAMP8 mice at 6 months was significantly increased.2.Effects of chronic stress on learning,memory and REST expression in rapidly aging miceTaking SAMR1 as the control,the nesting ability of 8-month old of SAMP8 was tested after 28 days of chronic mild stress.LTP study was performed for 28 days after stress to observe the effects of CMS on learning and memory in mice.The results showed that SAMP8 could improve its nesting ability after stress.Compared with the model group,the object recognition ability of the stress group was significantly improved,and LTP was enhanced.The plasma cortical ketone level was determined by ELISA after 28 days of stress,and the content of the hippocampal REST protein,brain derived neurotrophic factor(BDNF)and NMDAR subunit NMDAR2A/2B in mice was determined by WB.The effects of CMS on the restful NMDAR axis in SAMP8 brain were observed.The level of corticosterone in the stress group was higher than that in the model group.In addition,CYP11B1 expression was significantly decreased in the SAMR1 stress group compared with the SAMR1 control group.NMDAR1,REST and BDNF increased significantly in the SAMP8 stress group compared with the SAMP8 control group.There was a significant decrease in CYP11B1.The results showed that moderate stress could improve the learning and memory ability of aging mice.The improvement of SAMP8 learning and memory by moderate stress may be related to the increase of NMDAR1,REST and BDNF content.?.effect of LW-AFC on improving learning and memory impairment caused by chronic stress8 weeks of Balb/c was used to give chronic mild stress.After 7 days of stress,compared with the control group,the nesting ability of the stress group was significantly reduced,and the expression of NMDAR2 A and BDNF protein in the hippocampus was increased.Significantly increased serum corticosterone,LW-AFC can significantly improve the ability to nest after stress damage.After 14 days of stress,compared with the control group,the nesting ability of the stress group was decreased,and the serum corticosterone was significantly increased,and the expression of NMDAR2A/2B and BDNF protein in the hippocampus was increased.After lw-afc administration,NMDAR2A/2B protein expression increased compared with the stress group.Significantly increased REST content.After 21 days of stress,serum corticosterone was significantly increased in the stress group compared with the control group,the 1-hour identification index in object recognition ability was decreased,and LTP was significantly reduced.After lw-afc administration,compared with the stress group,LTP was significantly increased,and NMDAR2 A,REST and BDNF protein expressions were increased.28 days of stress,stress group compared with control group,significantly lower nesting ability,significantly elevated serum corticosterone,1 hour identification index is significantly lower,LTP significantly lower,after giving LW-an AFC,compared with stress group,serum corticosterone significantly lower,1 hour identification index increased significantly,has obvious effect of improving LTP,NMDAR2 A and rise of the REST.REST content increased significantly.After 35 days of stress,stress group compared with control group,significantly lower nesting ability,1 hour identification index decreased,but no significant difference,24 hours of the identification index is significantly lower,LTP extremely significantly lower,after giving LW-an AFC,compared with stress group,nesting ability significantly increased,identification index 1 hour and 24 hour identification index were significantly increased,significantly increase the LTP.Based on the above studies,the following conclusions are drawn in this study:(1)there was no significant difference in the content of REST protein in the serum of the olded,MCI and AD patients;REST was most highly expressed in IPSC cells of cognitively normal elderly people,and the expression of REST in IPSC cells of patients with AD was significantly lower than that of the IPSC cells of patients with AD.However,no difference in REST expression was observed in IPSC-induced differentiation of NSC,neurons and microglia cells.(2)in the hippocampus of SAMP8,REST protein expression increased with the increase of the age of months,among which the content of SAMP8 was the lowest at 2 months and the highest at 12 months.mild chronic stress can improve the learning and memory ability of 8-month-old SAMP8 mice,and its mechanism may be related to the increase of REST content to increase the switch of NMDAR2 B to 2A and inhibit the generation of corticosterone.(3)LW-AFC can improve learning and memory impairment caused by chronic multiple stress for 4 weeks,and its mechanism may be related to melioration HPA axis disorder,increasing REST content,and regulating the balance of NMDAR2A/2B,suggesting that LW-AFC may play an anti-stress role by affecting REST signaling pathway,and LW-AFC is expected to develop into an anti-stress drug.