| Depression is a syndrome of emotional disorders primarily characterized by low mood.According to data from the World Health Organization(WHO)and the Global Burden of Disease(GBD),the number of people suffering from depression has reached 322 million,with an increase of over 50%between 1990 and 2017,the global coronavirus outbreak has led to more people suffering from depression,with a dramatic 25%increase in anxiety and depression worldwide in the first year of the pandemic.Furthermore,depression is the leading cause of disability worldwide and a major factor contributing to the overall burden of disease.In extreme cases,depression could lead to suicide,with severe depression patients having a suicide rate of 10-15%,and it is expected that by 2030,depression will become the leading cause of death worldwide.Therefore,providing safe and effective treatment options for patients with depression is urgent.Zhi-Zi-Chi decoction(ZZCD),derived from the book "Treatise on Febrile Diseases",is a representative Chinese herbal formula for treating depression,consisting of two traditional Chinese medicines,Fructus gardeniae and Semen sojae praeparatum.It has the function of clearing heat,reducing irritability,and relieving depression,but the mechanism of its antidepressant effect is not yet clear.Our previous study found that feeding ZZCD to depressed rats significantly increased the content of intestinal butyric acid,and butyric acid has been proved to have an antidepressant effect,suggesting that ZZCD may play an antidepressant role by regulating the production of butyric acid by gut microbiota,but the specific types of gut microbiota regulated by ZZCD remain to be studied.Some researchers have found that the gut microbiota structure of patients with depression undergoes significant changes compared to healthy individuals,with an increase in opportunistic pathogens and a decrease in butyrate-producing bacteria(BPB).BPB,as an important functional group in the gut microbiota,can produce butyrate through the metabolism of nutrients such as dietary fiber that the human body cannot break down.Butyrate could reduce the permeability of the blood-brain barrier,reduce the activation of microglia and promote the expression of brain-derived neurotrophic factor,and regulate host brain function and behavior through the microbiota-gut-brain axis,thus achieving the goal of treating depression and other psychiatric disorders.In summary,based on the preliminary studies of ZZCD treating depression by changing the abundance and diversity of the gut microbiota,we focus on BPB from a variety of gut microbiota significantly altered by depression intervention,and conduct isolation and identification of BPB and evaluate its antidepressant function,and further study the material basis of ZZCD promoting the growth of BPB.The details are as follows:1.The effect of ZZCD on expression of butyric acid producing gene in gut microbiota of depressed rats.The chronic unpredictable mild stress(CUMS)depression rat model was established,and ZZCD was administered to the depressed rats,and sugar water preference,tail suspension test and forced swimming test were used as evaluation indicators.The results showed that ZZCD significantly improved the depressive behavior,which proved that ZZCD had antidepressant effect.Then,the cecal contents of the rats were taken for gene sequencing of the gut microbiota,and the V3-V4 hypervariable region of the bacterial 16S rRNA was sequenced using the IlluminaMiseq high-throughput sequencing method,based on the 16S rRNA gene sequencing data of the cecum contents,the functional abundance was predicted using PICRUSt2,the results showed that ZZCD significantly promoted the expression of hbd gene,which encodes 3-hydroxybutyryl-CoA dehydrogenase,a key enzyme in the metabolic pathway of butyric acid production by gut microbiota.This part of the work proves the antidepressant effect of ZZCD,and uses genomics technology to confirm the antidepressant effect of ZZCD by enhancing the butyric acid production function of intestinal BPB.It also provides a theoretical foundation for the study of antidepressant effect of BPB.2.Isolation,screening and identification of BPB from rat feces.According to the method of anaerobic culture,88 strains of bacteria were isolated from rat feces.The metabolites of all bacteria were detected by GC-MS,and 3 strains producing butyric acid were screened successfully.The 3 strains of BPB obtained were further screened by in vitro bacterial growth experiment:inoculated into the liquid medium containing the extract of ZZCD,by detecting the OD600 value of the bacterial liquid at the end of the logarithmic phase,the strain with significantly higher OD600 value was screened.Finally,one BPB strain was selected as strain B33.The strain was sequenced according to the 16S rRNA gene sequence and compared with the gene sequence of the strain on the NCBI website,the result of preliminary identification was Lacticaseibacillus rhamnosus(100.00%similarity).Strain B33 was then subjected to a comprehensive strain identification and the results were consistent with those reported for Lacticaseibacillus rhamnosus.Therefore,strain B33 was identified as Lactobacillus rhamnosus.3.The antidepressant function of Lacticaseibacillus rhamnosus B33 was evaluated by animal model.Experimenting with the CUMS depression rat model,sugar water preference,tail suspension test and forced swimming test were used as evaluation indicators.Lacticaseibacillus rhamnosus B33 was given intragastric administration to depressed rats.The results showed that Lacticaseibacillus rhamnosus B33 significantly improved the behavioral indexes of sugar water preference,tail suspension test and forced swimming test.Through the Nissl staining of the hippocampus of each group,the results showed that Lacticaseibacillus rhamnosus B33 could greatly improve the vertebral cells in the CA1 region of the hippocampus of depressed rats,the vertebral cells were arranged more neatly,and the Nissl bodies in the cell bodies could be seen clearly.The detection of butyric acid in rat cecal contents by GC-MS showed that Lacticaseibacillus rhamnosus B33 could reverse the decrease of intestinal butyric acid content in rats caused by depression.All the results showed that Lacticaseibacillus rhamnosus B33 played an antidepressant role.4.Using gene sequencing to study the effect of Lacticaseibacillus rhamnosus B33 on the gut microbial community structure of rats.Determination of gut microbial community structure and microbial species diversity in cecal contents using Illumina Miseq highthroughput sequencing.From the aspects of Alpha diversity,Beta diversity and species composition,it was found that the diversity of intestinal microbial species in CUMS rats decreased,and the gut microbial community structure became imbalanced,while the Lacticaseibacillus rhamnosus B33 can increase species diversity to a certain extent,improve the imbalance of gut microbial community structure,increase the relative abundance of Lactobacillus、Ruminococcus and Eubacterium in the intestinal tract,reducing the relative abundance of Alloprevotella,remodeling the homeostasis of gut microbiota.5.In vitro bacterial growth experiment was used to screen out the effective parts of ZZCD that can promote the proliferation of Lacticaseibacillus rhamnosus B33.Adding daidzin and other 13 kinds of active components of ZZCD into the blank liquid medium to obtain medium with concentrations of 2.5 μg/mL,5 μg/mL and 10 μg/mL respectively,and use the blank liquid medium as the blank control group,the Lacticaseibacillus rhamnosus B33 was inoculated in parallel in each medium for anaerobic culture,the OD600 value at the end of the logarithmic phase was determined,and the active component that could significantly increase the OD600 value were screened out.The results showed that compared with the blank control group,glycitin could significantly increase the OD600 value of the bacterial fluid at a concentration of 2.5μg/mL,and genistin,shanzhiin methyl ester,daidzin and crocin Ⅲ could significantly increase the OD600 value of the bacterial fluid at a concentration of 5 μg/mL.In conclusion,this study used CUMS-induced rats as model of depression,systematically evaluated the antidepressant effect of ZZCD from the perspective of gut microbiota and intestinal metabolites,revealed the antidepressant mechanism of ZZCD based on improving the abundance of intestinal BPB,and identified the material basis of the antidepressant effect of ZZCD.By establishing a set of culture-detection system for microbial isolation and identification and screening of fermentation products,Lactobacillus rhamnosus regulated by ZZCD was successfully screened and identified.CUMS-induced rats were used as the animal model of depression,and the antidepressant efficacy of Lacticaseibacillus rhamnosus B33 was evaluated.Finally,by studying the effects of extracts and active components of ZZCD on the growth curve of the strains cultured in vitro,the active components and optimal concentration that can promote the growth of Lacticaseibacillus rhamnosus B33 were selected successfully.The theoretical basis and scientific connotation of the antidepressant function of ZZCD were revealed from the perspective of gut microbiota,and found the pharmacodynamic material basis of ZZCD regulating BPB.By targeting intestinal BPB,a new breakthrough was found for the application of traditional Chinese medicine in antidepressant treatment. |
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