Study On Important Amino Acids And Biological Effects In Cognitive Impairment Induced By Maternal Separation Stress

Mother separation(Maternal Separation,MS)is a classic early stress model describing adverse stress in early life of mammals,which can lead to cognitive and emotional disorders in adulthood.Amino acids are important bioactive substances.Studies have shown that maternal and infant separation stress can lead to metabolic shocks,in which the changes of amino acids and their metabolism may be closely related to cognitive impairment,but the results are still inconclusive.In recent years,metabonomic techniques have been used to analyze and screen metabolites with potential biological characteristics in patients with different diseases,providing interventions for disease research,diagnosis and treatment.As an essential amino acid,leucine can quickly enter the central nervous system through the blood-brain barrier and play an important role in the survival regulation of neurons,but the specific mechanism is not clear.In this study,metabonomic techniques were used to analyze and screen the amino acids that may be involved in cognitive regulation in serum and cerebrospinal fluid of rats with maternal and infant separation stress,and to explore the biological role of key amino acids(leucine)in cognitive impairment.From the point of view of neuronal autophagy,the regulatory mechanism of leucine deficiency on neuronal autophagy was revealed.To provide a new theoretical basis for elucidating the molecular mechanism of cognitive impairment caused by maternal and infant separation stress,and to provide new therapeutic ideas for improving the mental health status of this kind of stress population.Ⅰ.Amino acid study on cognitive impairment induced by maternal and infant separation stress in ratsIn order to observe the effects of MS stress on the cognitive function of female and male rats,the differences of amino acids in peripheral serum and central cerebrospinal fluid under MS stress were explored from the perspective of metabonomics,and the amino acids with certain biological significance were screened out and verified by molecular biology experiments.The early stress models of female and male rats were established by MS method.Open-field open field test was used to evaluate the ability of rats to explore unfamiliar environment independently,object recognition test was used to evaluate the memory ability of new and old objects,Morris water maze test was used to evaluate the spatial learning and memory ability.Amino acids in serum and cerebrospinal fluid were detected and screened by ultra high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)and metabonomics.LDH rapid detection kit and flow cytometry were used to detect the LDH release and apoptosis rate of neuronal injury model induced by amino acid intervention of GC.The results showed that the spatial self-exploration ability,memory ability and spatial learning and memory function of MS-stressed female and male rats were significantly lower than those of CON-stressed female rats(P<0.05).The cognitive ability score of MS-stressed female rats was slightly higher than that of male rats.Nissl staining showed that the number of neurons in CA1 and CA3 regions of hippocampus in male MS rats and in hippocampal CA3 regions in female MS rats were significantly lower than those in CON group(P<0.05).The arrangement of nerve cells was loose and stained lighter,but there was no significant difference in the number and arrangement of neurons in other regions.The contents of GC and NE in serum and cerebrospinal fluid were detected by Elisa method,and the content of GC increased significantly,but there was no difference in the level of NE.Absolute quantification of amino acids in serum and cerebrospinal fluid,and a mathematical model was established according to the quantitative results.The results of PLS-DA and OPLS-DA showed that there were significant differences in amino acid profiles in serum and cerebrospinal fluid between male and female rats under MS stress.The projection importance of variables(VIP>1.0),the reliability of metabolites(Corr.Coeffs)and the one-dimensional test analysis of |log2F| > 0 of the comprehensive quantitative data in the whole data model showed that canine in serum and threonine in cerebrospinal fluid of female rats were amino acids with potential biological characteristics.The sequence of VIP and Corr.Coeffs showed that lysine,leucine,5-hydroxylysine and aspartic acid in cerebrospinal fluid of female and male MS rats had high coincidence in multiple groups.Cytological experiment confirmed that 100 μmol/L GC was the experimental concentration of stress injury model of neurons in vitro,and superimposed different concentration gradients of four kinds of amino acids.It was found that when leucine was more than 10 μmol/L and the other three amino acids were more than 100 μmol/L,the release of LDH and the rate of apoptosis were significantly lower than those in 100 μmol/L GC group(P<0.05).Ⅱ.Biological role of leucine in cognitive impairment induced by maternal and fetal separation stress in rats to observe the regulation of different contents of leucine diet on cognitive function in rats with MS stress.To explore the main biological effects of leucine in cognitive impairment induced by MS stress.Female and male rats were divided into four groups: control normal diet group(CON+N),control low leucine diet group(CON+L-LEU),MS stress normal diet group(MS+N),MS stress low leucine diet group(MS+L-LEU),MS stress high leucine diet group(MS+H-LEU).The leucine level in cerebrospinal fluid was detected by enzyme labeled immunosorbent assay(ELISA).The autophagosomes in the hippocampus of rats were detected by transmission electron microscope.Apoptosis in hippocampus of rats was detected by Tunel immunofluorescence staining.The expressions of autophagy and apoptosis-related proteins Beclin-1,Bcl-2 and Bax were detected by automatic Western blotting quantitative analysis system(WES).The results showed that the leucine content in cerebrospinal fluid of female and male rats in normal low leucine diet group,MS stress normal diet group and MS stress low leucine diet group was lower than that in normal growth diet group,while that in MS stress high leucine diet group was significantly higher than that in MS stress low leucine diet group(P<0.05,P<0.01).The open field score and object recognition index of female and male rats in control low leucine diet group,MS stress normal diet group,MS stress low leucine diet group and MS stress high leucine diet group were lower than those in control normal diet group.The open field score and object recognition index in male MS stress high leucine diet group were significantly higher than those in MS stress low leucine diet group.The open field score and object recognition index of female MS high leucine diet group were higher than those of MS low leucine diet group,and the difference was not statistically significant.In Morris water maze test,the times of crossing the platform of female and male rats in MS normal diet group and MS low leucine diet group were significantly lower than those in normal diet group(P<0.01).The frequency of crossing platform in MS high leucine diet group was higher than that in MS low leucine diet group,and the difference was not statistically significant.The latent time of female and male rats in low-leucine diet group,MS normal diet group,MS low-leucine diet group,MS high-leucine diet group and MS low-leucine diet group was significantly higher than that of control normal diet group,while the latency of female and male rats in MS high-leucine diet group was significantly lower than that in MS low-leucine diet group.Under electron microscope,autophagosomes were found in the hippocampus of male control low-leucine diet group and MS low-leucine diet group,no autophagosomes were found in normal growth diet group and MS high-leucine diet group,and no autophagosomes were found in female rats with different doses of leucine diet.Tunel fluorescence detection showed that there was no apoptosis in the hippocampus of rats in each group.The expression of autophagy-related protein Beclin-1 in hippocampus of male rats was increased in MS normal diet group and MS low-leucine diet group,and decreased in MS high-leucine diet group compared with MS low-leucine diet group.The difference was statistically significant(P<0.05,P<0.01).There was no significant change in Beclin-1 expression in female rats.The expression of apoptosis-related proteins BCL-2 and Bax in hippocampus of rats was detected.The ratio of BCL-2/Bax in normal low-leucine diet group,MS normal diet group and MS low-leucine diet group decreased significantly(P<0.05).Ⅲ.Regulatory mechanism of cognitive impairment mediated by leucine deficiency.To observe the effect of leucine deficiency on neuronal autophagy,and to verify that the damage caused by excessive autophagy mediated by leucine deficiency may be an important reason for the decrease in the number of hippocampal neurons induced by MS stress.Establish cell model: control group neuron standard medium culture(CON+N),control group neuron leucine deficiency medium culture(CON+NOLEU),GC intervention neuron standard medium culture(GC+N),GC intervention neuron leucine deficiency medium culture(GC+NOLEU),autophagy inhibitor(Baflomycin A1)induced neuron leucine deficiency medium culture(NOLEU+B-A1).Autophagy inhibitors induced neuronal culture in standard medium(CON+B-A1).Autophagy flow at the level of Beclin-1 and LC3II/I was detected by automatic Western Blot quantitative Analysis system(WES).The expression of LC3 in neurons infected with m RFP-GFP-LC3 lentivirus was detected by immunofluorescence(IF).Cytotoxicity LDH Assay Kit-WST detection reagent was used to detect the release of LDH.The apoptosis rate of neurons was detected by flow cytometry(Annexin-V and PI double staining kit).The results showed that the protein expression levels of Beclin-1,LC3 II and LC3 I and the ratio of LC3II/I were higher than that of CON group,GC+NOLEU was higher than that of GC+N group,leucine of GC+ was lower than that of GC+NOLEU,and the fluorescence intensity of Red+Green increased in CON+NOLEU group and decreased in other groups after autophagy double labelled lentivirus m RFP-GFP-LC3 infection.When autophagy double-labeled lentivirus m RFP-GFP-LC3 infected neurons of each group,the fluorescence intensity of GC+NOLEU group increased,while that of other groups decreased with the increase of concentration.When neurons were cultured in leucine deficiency medium,LC3II/I in CON+NOLEU group was higher than that in CON+N group,and the difference was statistically significant.LC3II/I in different CON+ gradients of leucine group was significantly lower than that in CON+NOLEU group(P<0.05,P<0.01).The release of LDH in NOLEU and NOLEU+B-A1 groups was higher than that in CON group,while that in NOLEU+B-A1 group and CON+B-A1 group was lower than that in NOLEU group(P<0.05).The apoptosis rate in NOLEU group was higher than that in CON group,while that in NOLEU+B-A1 and CON+B-A1 groups was lower than that in NOLEU group(P<0.01).To sum up: MS stress induced cognitive impairment in rats,and the degree of damage in females was slighter than that in males,which may be related to the degree of pathological damage of neurons in the hippocampus.MS stress causes central and peripheral amino acid metabolism disorder,and the difference of cognitive function between male and female caused by stress may be related to urine and threonine metabolism in dogs.Abnormal metabolism of lysine,leucine,5-hydroxylysine and aspartic acid may be important mediators or molecular basis of cognitive impairment induced by MS stress,in which leucine is the key functional amino acid.Low-leucine diet can aggravate cognitive impairment caused by MS stress,and leucine supplementation can improve the degree of stress-induced cognitive impairment.MS stress and low leucine diet can induce autophagy activation and apoptosis in hippocampus of male rats.Leucine deficiency induces excessive activation of autophagy,apoptosis and neuronal injury.Leucine supplementation can alleviate the injury effect.Abnormal autophagy may be an important pathological mechanism of cognitive impairment induced by leucine-mediated MS stress.