Effect And Mechanism Of MiRNA-mediated Motor Preconditioning On Depressive Behavior In SATD Mice

Background: Depression is a chronic and recurrent mental disease featured by low feeling,social isolation and anhedonia.According to the WHO,depression poses a significant challenge to the quality of life of individuals and to social and economic problems.Therefore,its pathogenesis and treatment measures need to be further studied.miRNA is a kind of non-coding small RNA,which mainly works in the regulation of post-transcriptional gene expression.Previous studies have found that miRNA dysregulation exists in both rodent depressive models and depressed patients.Through miRNA chip analysis,it was found that dysregulation of miRNA was involved in neurotrophic factor signaling pathway,AMPK signaling pathway and m TOR signaling pathway,reducing hippocampal plasticity and activating central inflammation.A variety of exercise methods based on skeletal muscle contractile activity have significant effect on the prevention and treatment of depression.Peroxisome proliferator-activated receptor-γ coactivator-1α（PGC-1α）mediated "muscle-brain Crosstalk" plays an important role in exercise antidepressant.miR-696 is an motion-sensitive miRNA involved in PGC-1α translation regulation and skeletal muscle metabolism in mice.Skeletal muscle PGC-1α can induce the production of anti-inflammatory factors in skeletal muscle to exert anti-inflammatory effects directly or through the regulation of IL-10 and nuclear transcription factor κB（NF-κB）signaling,which may have a series of positive effects on depression.However,the "Crosstalk" regulation mechanism between exercise-induced skeletal muscle adaptation and central inflammation needs to be further studied.Objective: In this study,based on the simplified acute tryptophan depletion depression（SATD）animal model,we investigated the effects of treadmill exercise preconditioning on depressive behavior and miRNA expression profile in hippocampus of mice,and search for key miRNA that mediate the regulation of depressive behavior.To further explore the inflammatory response mechanism of miRNA-696 /PGC-1α/ NF-κB pathway in skeletal muscle mediating exercise anti-depression,and provide theoretical basis for the possible way of preventing and improving depression through exercise.Methods: A total of 4-week-old C57 male mice were divided into 8 groupse randomly.According to the research purpose,it is divided into three parts.The first part : NC group（control group,gavage equal amount of normal saline）and SATD group（model group,gavage neutral amino acid mixture without tryptophan）,to explore the effect of SATD on depressive behavior and its mechanism.Part two : SS group（SATD model + saline injection）and and SSE group（SATD model + saline injection after 6-week treadmill exercise）,to investigate the effect of exercise preconditioning on SATD mice depressive behavior and its mechanism.Part III : SAC group（SATD model + miR-696 control agent injected into skeletal muscle）;SCE group（ATD model + miR-696 control agent injected into skeletal muscle after6 weeks of treadmill exercise）;SA group（SATD model + miR-696 stimulator injected into skeletal muscle）;SAE group（6 weeks treadmill exercise,SATD modeling + skeletal muscle injection with miR-696 agonist）to investigate whether skeletal muscle miR-696 is the dependent of exercise preconditioning.The exercise program of SSE,SAE and SCE groups was as follows: slope 0°,speed 10 m/min,60min/ day,6 days/week,consecutive exercise for6 weeks.At the sixth week of treadmill exercise,miR-696 control agent and agonist were injected into gastrocnemius muscle with a single injection of 50 ul,5nmol/ mouse.The control group was injected with equal saline.SATD modeling was performed at the seventh week: the experimental animals were fasted 15 hours before modeling and fed with neutral amino acid mixture without tryptophan twice（90min apart）.Behavior tests included tail suspension test（TST）and forced swimming test（FST）were carried out 5 hours after the modeling,and death was performed.Blood and tissue samples were taken.The contents of 5-HT and inflammatory factors in serum were detected by ELISA.The m RNA and protein expressions of miRNA-696/PGC-1α/NF-κB related pathways were detected by quantitative real-time PCR and WES techniques.Finally,miRNA sequencing technology was used to detect miRNA expression in the hippocampus of NC,SATD and SSE groups,and bioinformatics methods were used to screen the differential miRNA and analyze the enrichment pathway.Results:1.Behavioral results revealed that contrasted with NC group,the immobility time of SATD mice was significantly increased in the TST（P < 0.05）.Compared with SS group,the immobility time of SSE mice was significantly decreased（P < 0.05）,and the struggle time was significantly increased（P < 0.05）in the FST.In the TST,the immobility time of SSE mice was significantly decreased（P < 0.05）,and the struggle time was significantly increased（P < 0.05）.In the FST,miR-696 agomir significantly increased the immobility time（P < 0.05）and significantly decreased the struggle time（P < 0.01）.In the TST,miR-696 Agomir significantly increased the immobility time of mice（P < 0.01）,and significantly decreased the struggle time（P < 0.05）.ELISA results showed that compared with NC,5-HT content in hippocampus of STAD mice was significantly decreased（P < 0.01）.Compared with SS group,the content of 5-HT in hippocampus of SSE mice increased significantly（P < 0.01）.miR-696 agonist significantly reduced the content of 5-HT in hippocampus of mice（P < 0.05）.2.m RNA and protein results related to miRNA-696 /PGC-1α/NF-κB inflammatory pathway: The m RNA level of PGC-1α in gastrocnemius of SATD mice was significantly decreased（P < 0.05）,the contents of CRP,IL-1β and NF-κB in serum were significantly increased（P < 0.01）,and the m RNA level of NF-κ B in hippocampus was significantly increased（P < 0.05）.The miR-696 m RNA level was significantly decreased（P < 0.05）,the PGC-1α m RNA level was significantly increased（P < 0.05）,and the contents of CRP,IL-1βand NF-κB in serum were significantly increased（P < 0.01）.The m RNA levels of miR-124,miR-696,PGC-1α and NF-κB in hippocampus were significantly decreased（P < 0.05）.The level of p105/ P50 protein in hippocampus of SATD mice was significantly decreased by exercise preconditioning（P < 0.05）.miR-696 agonist significantly increased the contents of CRP and IL-1β in serum（P < 0.01）,and did not affect the m RNA and protein levels of PGC-1α and NF-κB in gastrocnemius and hippocampus.3.miRNA sequencing results revealed that contrasted with NC group,18 unlikely expressed miRNAs were screened in SATD group,and 13 miRNAs were up and 5 miRNAs were down.Compared with SATD group,8 unlikely expressed miRNAs were screened in SSE group,and 4 miRNAs were up and 4 miRNAs were down.The screening criteria for differential miRNAs were P < 0.05.Compared with NC,the differentially expressed miRNAs target genes in SATD group were mainly involved in cell metabolism,localization,signal regulation,decomposition and protein assembly.Compared with SATD group,target genes differentially expressed miRNAs in SSE group were mainly involved in metabolism,macromolecular modification,phosphorus metabolism,phosphate complex related process and assembly process.Compared with NC group,target genes differentially expressed miRNAs in SATD group were involved in synaptic vesicle cycle,neurotrophic factor signaling pathway,m RNA monitoring pathway,insulin signaling pathway,glycosyl phosphatidylinositol biosynthesis,axon transduction,ABC transporter,etc.Compared with the SATD group,the difference of SSE group of miRNAs expression of target genes involved in signaling pathways mainly include the regulation of actin cytoskeleton,phenylalanine,tyrosine and tryptophan biosynthesis,the reciprocal transformation of pentose and uronic acid,oxytocin signaling pathways,m TOR signaling pathways,insulin signaling pathway,adrenergic myocardial cell signaling,etc.Conclusion:1.SATD induced depression behavior in mice;Long-term exercise preconditioning can prevent depression behavior induced by SATD,suggesting the preventive effect of exercise on depression.2.Exercise preconditioning can activate PGC-1α in skeletal muscle and inhibit NF-κB inflammatory signaling pathway.However,miR-696 interference in skeletal muscle cannot affect central inflammatory response through PGC-1α/NF-κB signaling pathway,and may be involved in the anti-depressive mechanism of exercise through other pathways.3.SATD and exercise preconditioning can change miRNA expression profile in mice hippocampus.This may be related to synaptic vesicle circulation,neurotrophic factor,actin cytoskeleton regulation,phenylalanine,tyrosine and tryptophan biosynthesis and other signaling pathways.