Effects Of Food With Different Methyl Content On Anxiety-like And Depressive-like Behavior In Stress-induced Mice And The Role Of M6A Methylation Modification

Objective:The interaction between genetic diathesis and environmental factors can be used to explain the occurrence of stress-related mental diseases,including depression,anxiety,post-traumatic stress disorder and so on.Stress in different life stages,including chronic stress in adulthood and stress in early life,have adverse effects on individuals,and are prone to anxiety and depression.Dietary preference is associated with anxiety and depressive symptoms,and dietary adjustment may improve anxiety and depressive symptoms.In this study,chronic unpredictable mild stress(CUMS)model and maternal separation(MS)model were used in mice during different periods of life,to investigate the effects of different methyl donor diets on anxiety-like behaviors and depression-like behaviors in adult mice induced by environmental stress,and to explore the relationship between m6A-methylation modification and anxiety-like behaviors and depression-like behaviors in stress-induced mice.It provides a new way for further research on diet treatment of emotional disorders and lays a foundation for further research on the relationship between RNA methylation and depression.Methods:1.Establishment of experimental animal model:(1)CUMS model:Male C57BL/6J adult mice aged 7-8 weeks were subjected to unpredictable mild stress for 6 weeks to construct CUMS model,which was randomly divided into 6 groups according to different feeding food:chronic stress+normal food group(CUMS+N),chronic stress+high-methyl food group(CUMS+H),chronic stress+low-methyl food group(CUMS+L),control+normal food group(CON+N),control+high-methyl food group(CON+H),control+low-methyl food group(CON+L).All chronic stress groups were subjected to stress intervention,while mice in the control group were fed mice chow with the corresponding methyl content without interruption every day,and mice in the control group were fed mice chow with the corresponding methyl content without interruption,drank normal water,and cleaned cage regularly.(2)MS model,mice were separated from their mothers for 4 hours a day from day 2 to 14 after birth.After 21 days,they were randomly divided into 4 groups according to feeding differences:maternal separation+normal food group(MS+N),maternal separation+high-methyl food group(MS+H),control+normal food group(N+N),control+high-methyl food group(N+H).Mice in each group were fed with the same living conditions except for different methyl content in food until the end of adult period(70 days).2.Behavioral experiment:the body weight of mice was measured weekly in the experiment.After the experiment,the anxiety and depressive-like behaviors of the mice were evaluated by sucrose preference test(SPT),open field test(OFT),elevated plus maze(EPM)and forced swimming test(FST).3.The expression levels of m6A methylation related enzyme proteins METTL3,FTO and ALKBH5 were detected by Western Blot(WB).Meanwhile,the expression levels of pathway proteins PI3K,AKT and mTOR were detected.4.The mRNA expression levels of PI3K,AKT,mTOR in the pathway were detected by real-time fluorescence quantitative polymerase chain reaction(RT-QPCR).5.The m6A methylation level of PI3K mRNA in hippocampus of CUMS test groups was detected by RNA methylated immuneprecipitation combined with real-time fluorescence quantitative polymerase chain reaction(MeRIP-QPCR).Results:1.In the previous CUMS experiment,it was found that chronic stress in adult mice would lead to obvious anxiety and depressive-like behaviors.Compared with normal control group,the body weight of chronic stress group was lower than that of normal control group(P<0.05),and there was no obvious trend of increase in body weight.However,the weight of the low-methyl food group decreased regardless of whether it was stressed or not,and there was no significant difference between the stressed and non-stressed groups by point comparison(P>0.05).After 6 weeks of CUMS,the sucrose preference index of mice was significantly decreased by stress(P<0.05).The retention time of mice in the central area of the open field was significantly reduced(P<0.05).In the elevated plus maze experiment,the stay time of mice entering the open arm was significantly shortened(P<0.05).The immobility time of forced swimming test was significantly increased(P<0.05).Compared with normal diet,high-methyl diet reversed the changes of behavior in mice under stress,and protected mice from anxiety and depressive-like behavior after chronic stress.At the same time,this study did not find that low-methyl food aggravates the anxiety and depressive-like behaviors induced by stress in mice.It was found that the expression levels of METTL3,FTO and ALKBH5 proteins in the hippocampal tissue of the brain of mice after stress were significantly decreased(P<0.05),and the expression levels of METTL3 protein and FTO and ALKBH5 protein were increased by high-methyl diet compared with normal diet(P<0.05).It causes RNA methylation to change towards methylation.The detection results of m6A methylation level of PI3K gene mRNA showed that stress led to the down-regulation of m6A methylation level of PI3K gene mRNA(P<0.05),and the m6A methylation level of PI3K gene mRNA in mice after stress was increased by high-methyl food compared with normal food(P<0.05).At the same time,chronic stress resulted in decreased mRNA and protein expression levels of PI3K,AKT and mTOR genes in the PI3K-AKT-mTOR pathway(P<0.05).After stress,the mRNA and protein expression levels of PI3K,AKT and mTOR genes were increased in high-methyl diet group compared with normal diet group(P<0.05),which activated this signaling pathway.2.In the mouse MS model,we once again verified the effects of high-methyl food after early life stress on adult anxiety and depressive behaviors of stressed mice and the role of m6A methylation.The results showed that maternal separation stress caused anxiety and depressive-like behaviors in adult mice.The body weight of each group increased gradually with the passage of time,and stabilized after the ninth week.From the 4th week onwards,the weight of mice fed with high-methyl diet was lower than that fed with normal diet at the same time(P<0.05),regardless of whether they had experienced maternal separation or not.After the 9th week,there was no statistically significant difference between the two diets on body weight(P>0.05).Behavioral tests of adult mice showed that there were no significant differences in sucrose preference index and stay time in the central area of the open field(P>0.05).The time to stay in the open arm in the elevated plus maze was significantly shorter in the adult mice that had undergone maternal separation(P<0.05).The immobility time was significantly increased in the forced swimming experiment(P<0.05).In the maternal separation group,the time of entering the open arm in the elevated plus maze was increased by high-methyl diet compared with normal diet(P<0.05).Decreased the immobility time of mice in the forced swimming experiment(P<0.05),indicating that high-methyl diet can reduce the anxiety and depressive-like behaviors in adult mice induced by maternal separation.In addition,we also found that high-methyl diet increased the expression level of METTL3 protein in the hippocampus of adult brain of maternal separation mice(P<0.05),and decreased the expression levels of FTO and ALKBH5 proteins(P<0.05),which may affect the methylation of RNA.Maternal separation stress also led to the decrease of mRNA and protein expression levels of PI3K,AKT,mTOR in the PI3K-AKT-mTOR pathway(P<0.05).After stress,high-methyl diet increased the mRNA and protein expression levels of PI3K,AKT,mTOR in hippocampus of adult mice(P<0.05).Conclusion:Our results suggest that high-methyl diet can reduce anxiety and depressivelike behaviors in mice induced by chronic stress in adulthood or maternal separation stress in early life.High-methyl food may promote the methylation of RNA by regulating the expression level of m6A methylation-related enzymes,and activate the PI3K-AKT-mTOR signaling pathway by increasing the mRNA methylation level of key gene PI3K,which further plays a role in improving stress-induced anxiety and depressive-like behaviors of mice.Therefore,the regulation of m6A methylation may be a potential regulatory mechanism of the anti-anxiety and anti-depression effects of high-methyl foods.