Impact Of Traditional Chinese Medicine Treatment On Chronic Unpredictable Mild Stress-induced Depression-like Behaviors And Fecal Microbiota

Depression is a widespread and debilitating mental disorder.So far,as the etiology of depression is still unclear,there is a lack of effective approaches to cure this disease.Therefore,looking for an effective anti-depressant strategy has important scientific significance.Xiaoyaosan formula,as a traditional Chinese medicine（TCM）for treating depression in China,has been used in the management of neuropsychiatric diseases for more than 1000 years.However,as the active ingredients of this formula is very complicated,the mechanism of this formula is unclear.Recently,an increasing number of researches revealed that gut microbiota plays an important role in the development and treatment of depression,which have become a new target for prevention and treatment of depression.In addition,more and more evidence indicate that a variety of traditional Chinese medicine formula might perform their health benefits via influencing and reshaping the gut microbiota.Accordingly,we proposed that TCM formula may regulate the alpha diversity,structure,abundance and function of the gut microbiota,thereby present an anti-depressant effect.Therefore,a randomized,controlled trial was conducted to explore the potential mechanism of TCM on the improvement of depression symptoms based on the 16 S rRNA V4-V5 amplicon sequencing,LC-MS targeting quantitative technology and metagenome sequencing technology using Chronic Unpredictable Mild Stress（CUMS）rats,a well-established rodent model for depression.The experiments and results are as follows:a)An array of behavior tests（sucrose preference test（SPT）,open filed test（OPT）and light/dark test（LDT））were used to explore the behavior changes of depressed rats before and after intervention with TCM.Results revealed that SPT and depression-like behavior tests（e.g.,sucrose preference ratio,open field rearing and crossing numbers,and total time spent in the dark zone）of the CUMS-induced rats were significantly different compared with those in the HC group after 8 weeks of model development.After 6 weeks of TCM treatment,TCM reversed the behaviors of depressive and anxiety,including recovering sucrose preference ratio,improving the activity rates,and significantly decreasing the time spent in the dark zone of the CUMS-induced rats.b)The profiles of the fecal microbiota were assessed by Illumina HiSeq high-throughput sequencing technology.16 S rRNA V4-V5 sequencing results revealed that chronic stress significantly decreased the α diversity of the rat gut microbiota and changed the structure of the rat gut microbiota.Moreover,TCM significantly reversed the gut intestinal structure and the taxa levels of the CUMS rats,especially reversed the changes of Ruminococcus₂ and Roseburia reportedly being associated with relieving psychiatric disorders.c)Based on the liquid chromatography-mass spectrometry（LC-MS）technology to quantify the levels of brain neurotransmitters after intervention with TCM.LC-MS targeted metabolomics revealed that the levels of brain neurotransmitters（5-HT,5-HIAA,GABA,and Ach）perturbed by CUMS were reversed by TCM treatment,and Spearman’s correlation analysis illustrated strong correlation between brain neurotransmitters and perturbed fecal microbiota genera.For example,5-HIAA showed a strong positive correlation with the genera Lachnospiraceae_UCG-006,norank_oMollicutes_RF9,Ruminococcaceae_UCG-004,while the genera Lactobacillus,Blautia,and Ruminococcus₂ were negatively correlated with 5-HIAA;Lachnospiraceae_UCG-006 and [Eubacterium]_coprostanoligenes_group were positively correlated with GABA;Blautia was negatively correlated with acetylcholine,while Lachnospiraceae_UCG-006 was positively correlated with acetylcholine.d)The intestinal microbial function was assessed by shotgun sequencing based metagenomic analysis.Metagenome analysis results showed that chronic stress not only significantly changed the levels of function gene（COG0582,COG1674,COG4646,ENOG411221 R,ENOG410Y01Z,ENOG4112DF3,ENOG410XQ4 N,K03205,K03497,K06941,K04763,K03581,K03310,M00202,GH13₃,CBM60,PL17₁,ZP₀4049838,ZP₀2429913,YP₀01896735 and P78218 were significantly decreased in CUMS group relative to HC group,COG5433,M00535,M00502 and AA2 were significantly increased）,but also perturbed the amino acid metabolism and notably evaluated the abundance of bile acid biosynthesis gene.TCM-treated rats showed strikingly alleviate the CUMS-induced changing of the functional genes（COG0582,ENOG411221 R,ENOG410Y01Z,ENOG4112DF3,ENOG410XQ4 N,COG5433,K03205,K033110 and M00202 were significantly decreased in TCM group relative to CUMS group,M00535 and M00502 were significantly increased）,and elevate the metabolism of cysteine and methionine potentially correlated to the relief of oxidative stress.Overall,depressive and anxiety-like behaviors were observed in CUMS rats,then significantly reversed in CUMS rats received TCM and it is maybe correlated to reversing CUMS-induced intestinal microbiota dysbiosis and changes in brain neurotransmitters.These findings set up the foundation to further reveal the exact causal relationship among the TCM formula,host responses,gut microbiota dysbiosis and the levels of brain metabolites,and enabled scientific interpretation of the therapeutic function of classic the TCM formula.