Comprehensive Analysis The Interventional Effect Of Acanthopanax Senticosus On Chronic Unpredictable Mild Stress Mice By Integrating Transcriptomics And Metabolomics

Objective:Chronic unpredictable mild stress(CUMS)model was used to induce depressive mice,and the effects of Acanthopanax senticosus Harms(ASH)on gene expression and metabolites in prefrontal cortex(PFC)of CUMS depressive mice were explored by combining transcriptome with metabolomics.The biological information of co-expressed differential genes and pathway of metabolism in which metabolites were involved in were studied to elucidate the mechanism of ASH in depression treatment.Methods:Firstly,sucrose preference test(SPT)was implemented to screen male ICR mice used for experiment.Subsequencedly,screened mice were divided into normal group(N,n = 10),model group(M,n = 10),ASH intervention groups(HD,400 mg/kg,n = 10;MD,200 mg/kg,n = 11;LD,200 mg/kg,n = 10)and fluxtine positive drug group(PD,10 mg/kg,n = 10).Druing the experiment,bodyweight and food intake of mice were recorded weekly.After 6 weeks of CUMS procedure,the intervention effect of ASH on CUMS mice was evaluated by means of behavioral evaluation,oral glucose tolerance test(OGTT)and detection of serum inflammatory factors and insulin,from the perspective of pharmacodynamics.Secondly,RNA-seq transcriptome sequencing technique was applied to analyze the differential gene expression level in prefrontal cortex(PFC)of N group,M group and optimal dose group of ASH(MD).The differentially expressed genes were further analyzed through GO,KEGG and protein interaction network analysis for bioinformatics interpretation.Lastly,the metabolites in PFC of mice were detected by metabonomics technology based on GC-MS.The differential substances among M and other groups were obtained by multivariate analysis,and finally identified by searching NIST 05,HMDB and KEGG data base.In the meantime,metabolites related metabolic pathway were acquired in combine with Metaboanalyst 4.0 online comprehensive analysis platform.Results:From the fourth week,the bodyweight and food intake of mice in M group were significantly lower than those in N group(P < 0.05).In the sixth week,mice in group M showed reduced sucrose preference(P < 0.05)in sucrose preference test compared to N group.Later,M group mice displayed longer immobility time(P < 0.05,P < 0.001)in forced swimming and tail suspension tests,and lower behavioral score(P < 0.01)in open field test,indicating depression-like behaviors.In elevated plus maze test,the frequency of entering the open arm and the percentage of time spent in open arm in group M mice were decreased(P < 0.05),suggesting appeared anxiety-like symptoms.The results of serum biochemical indexes showed that M group mice had higher fasting insulin level(P < 0.01),tumor necrosis factor-alpha and interleukin-6 levels(P < 0.05)than those in N group.The results of pharmacodynamics study showed that ASH intervention remarkly increased the weight loss and food intake induced by CUMS.It also improved depression and anxiety-like behavior,insulin tolerance and inflammatory state in CUMS mice.It is worth mentioning that the middle dose of ASH has better curative effect and seems more stable.RNA-seq transcriptome sequencing results showed that CUMS model partly changed expression levels of genes,and ASH treatment reversed the expression of 14 genes(P < 0.05,Fold Change value > 1.2 or < 0.83).These 14 genes are involved in biological processes(BP),such as intracellular processes,single-organism and biological regulation;cellular components(CC),such as cell-related components and organelle components;and molecular functions(MF)with binding and catalytic activity categories.KEGG pathway enrichment analysis showed that energy-related metabolic pathways such as glycine,serine and threonine metabolism,valine,leucine and isoleucine degradation,beta-alanine metabolism and pentose phosphate pathway were significantly enriched(P < 0.05).Further studies showed that oligodendrocyte specific protein gene(Opalin)and hemoglobin-related genes(Hba-a1,Hbb-bs and Alas2)play important roles in the development of stressinduced depression,the abnormal expression of Retsat and Plin4 genes are closely related to insulin resistance in M group mice,and the aberrant expression of Hibch and Etnppl genes may lead to metabolic disorders in CUMS mice.Multivariate data analysis methods showed that there were differencial metabolic profilings in PFC between N and M group mice.24 metabolites(VIP > 1,P < 0.05)were screened and identified by OPLS-DA analysis.These 24 differential metabolites are mainly involved in 7 metabolic pathways including:(a)aminoacyl-t RNA biosynthesis,(b)alanine,aspartate and glutamate metabolism,(c)glycine,serine and threonine metabolism,(d)citrate cycle,(e)valine,leucine and isoleucine biosynthesis,(f)pantothenate and Co A biosynthesis,and(g)ubiquinone and other terpenoid-quinones biosynthesis.11,13 and 15 differentially metabolites were reversed in ASH HD,MD and LD groups.Heatmap cluster analysis showed that the improvement effect of MD group is closest to that of fluoxetine.14 metabolites were reversed in fluoxetine-positive group,and alanine,aspartate and glutamate metabolism pathway is specific to fluoxetine.Conclusion:Traditional Chinese medicine ASH has antidepressant effect,and its antidepressant effect is closely related to the improvement of insulin resistance and inflammation.RNA-seq transcriptome analysis showed that ASH could ameliorate gene expression dysfunction in PFC of stress mice induced by CUMS.Among them,oligodendrocytespecific protein genes(Opalin),hemoglobin synthesis(Hba-a1,Hbb-bs and Alas2),insulin resistance(Retsat and Plin4)and PFC metabolic disorders(Hibch and Etnppl)related genes may be involved in the development of depression.Metabolomics analysis showed that metabolic disorders did occur in PFC of mice in group M comparing with N group,and ASH could improve the abnormal metabolism induced by CUMS.