| The clinical manifestations of "Generalized Anxiety Disorder"(GAD)are mainly in the absence of obvious incentives,the patient’s frequent or continuous nervousness and anxiety without a clear object and fixed content,accompanied by autonomic nerve dysfunction symptoms,such as upset,sweating,excessive alertness,sleep problems,and physical discomfort.Generalized anxiety disorder belongs to the category of emotional opathy in traditional Chinese medicine,it is closely related to the "stagnation","palpitations","insomnia" and "dirty irritability" of traditional Chinese medicine.The pathogenesis of generalized anxiety disorder is mostly a mixture of deficiency and excess.Most patients of this type have deficiency of heart and gallbladder and other internal organs,regaining emotional stimulation,dissatisfaction,and long-term anxiety leads to stagnation of liver qi.Stagnation causes anxiety,and Wendan Decoction mainly treats the syndrome of biliary depression and phlegm disturbance.It has the effects of regulating qi and resolving phlegm,as well as promoting choleretic effects on the stomach.Therefore,no matter from the pathogenesis,clinical manifestations,or indications of the disease,it all reflects the therapeutic significance of Wendan Decoction for GAD.Gut microbiota is closely related to anxiety behavior,and some researchers have begun to focus on the treatment of anxiety by changing the gut microbiota.However,there are few experimental studies on the changes of gut microbiota caused by simple generalized anxiety disorder,most of which are combined with other diseases or accompanied by anxiety.Because miRNAs have gene regulation effects,and at the same time linking the core symptoms of generalized anxiety disorder with miRNAs,it can explain the disorder of the anxiety multilevel pathway,and it can also provide new diagnosis and treatment methods for the entire gene network.The pathogenesis of anxiety disorders also plays a very important role,and it can also provide a new target for the development of a new generation of anti-anxiety drugs.Therefore,this experiment uses chronic restraint stress method to prepare GAD model mice,and observes the effects of Wendan Decoction on behavior,gut microbiota and miRNA,so as to explore the effect and mechanism of Wendan Decoction on GAD and provide different entry points for the treatment of generalized anxiety disorder.ObjectiveBy studying the effects of Wendan Decoction on the behavior,gut microbiota and miRNA of mice with generalized anxiety disorder model,the effect and mechanism of Wendan Decoction on the intervention of generalized anxiety disorder were clarified.Methods1.Literature research:Through the retrieval and statistical analysis of the journal literature on TCM or integrative Chinese and clinical medicine differentiation treatment of GAD in databases such as CNKI,Wanfang,etc,to extract the information related to TCM syndrome classification,prescriptions and medicines.Then established database and analyzed the frequency of syndromes and prescriptions,summarized the distribution characteristics of GAD syndromes and the rules of medication,which was provided clinical literature data support for Wendan Decoction in the treatment of GAD.2.Experimental study:The chronic restraint stress method was used to prepare a generalized anxiety disorder model,restrained for 4 hours a day for 14 consecutive days.The control group and the model group were given distilled water for 7 days,and the Wendan Decoction group was given by Wendan Decoction for 7 days.The open field test and elevated plus maze test were performed on each group of mice on the second and third day after the gavage.After the gavage,fresh stool samples of all mice were collected,and 16s rRNA high-throughput sequencing was used to analyze the gut microbiota in the stool samples.At the same time,immediately after the behavioral experiment,the mice were killed and hippocampal tissue was taken,and miRNA and its target genes in the hippocampus were analyzed by miRNA sequencing.Results1.Literature research:A total of 137 literatures were included,involving 27 types of syndromes,with a total frequency of 172 times.The top five types of syndromes that occur frequently are stagnation of liver and qi(37 times,21.51%),deficiency of both heart and spleen(26 times,15.12%),stagnation of qi to transform fire(26 times,15.12%),biliary depression and phlegm disturbance(23 times,13.37%),yin deficiency and fire(14 times,8.14%).A total of 11 syndrome factors were obtained,including 5 disease location syndrome factors and 6 disease syndrome factors,with a cumulative frequency of 445 occurrences.The most common factor of single syndrome is qi stagnation,with a frequency of 56.92%.In the composition of disease location syndrome elements and disease nature syndrome elements,"qi stagnation+liver" is the most common combination form.In the differentiation of the two syndromes,the "qi deficiency+blood deficiency" syndrome element is the most common,with a frequency of 40.63%,followed by "qi stagnation+sputum";the target of "qi deficiency+blood deficiency" is mainly located in the heart and spleen.The target of stagnation+phlegm is mainly located in the gallbladder.The top 3 prescriptions were Wendan Decoction(21 times,14.19%),Guipi Decoction(11 times,7.43%),Chaihu Shugan Decoction(11 times,7.43%).The top 5 traditional Chinese medicines with the highest frequency of use are Poria,Licorice,Suanzaoren,Bupleurum,Polygala.The top three Chinese medicines used most are tonic drugs,tranquilizers,and heat-clearing drugs.2.Experimental research:(1)Behavior test:Compared with the control group,the total distance(P<0.01)and average speed(P<0.05)of the model group in the open field test were significantly higher.However,the time in the center(P<0.05)and the frequecy of crossing(P>0.05)is relatively reduced.Compared with the model group,the total distance and average speed of the Wendan Decoction group and the Paroxetine group were reduced,and the difference was statistically significant(P<0.05),while the Wendan Decoction group and the Paroxetine group stayed in the central time was an increasing trend in the number of times and the frequecy of crossing,but there was no statistically significant difference in the frequecy of crossing(P>0.05).In the central residence time,the paroxetine group increased significantly(P<0.05).Although the Wendan Decoction group increased in the central residence time,the difference was not statistically significant(P>0.05).Compared with the control group,the percentage of open arm entry times in the model group in the elevated plus maze experiment was significantly reduced(P<0.01).Compared with the model group,the paroxetine group and the Wendan Decoction group both increased,while the temperature The increase in Wendan Decoction group was close to that of the normal group and the difference was statistically significant(P<0.01).In the percentage of open arm residence time,the model group was significantly less than the control group,and the difference was statistically significant(P<0.05).At the same time,the paroxetine group and the Wendan decoction group increased compared with the model group,but the difference was not statistically significant(P>0.05).(2)Sequencing of gut microbiota:There were differences in the number of OTUs in each group.The number of unique OTUs in mice increased after the Wendan Decoction was administered,and the diversity of the gut microbiota increased after the treatment of Wendan Decoction.The results of the analysis of the difference of gut microbiota showed that compared with the model group,the Wendan Decoction group had a significant increase in Lactobacillus,Anaerostipes and other beneficial bacteria,and decreased significantly in the harmful bacteria such as Proteobacteria,Ralstonia,Burkholderia,and β-proteobacteria.In addition,the correlation analysis results indicate that the anxiety behavior of GAD mice is mainly positively correlated with harmful bacteria such as Proteobacteria,Ralstonia,Burkholderia,and at the same time with Verrucomicrobia,Actinobacteria,Firmicutes and Akkermania are negatively correlated.(3)miRNA sequencing:A total of 17 overlapping miRNAs were screened out of the differential miRNA between the control group and the model group,the model group and the Wendan Decoction group,including mmu-miR-7a-5p,mmu-miR-148b-3p,mmu-miR-466o-5p,mmu-miR-98-5p,mmu-let-7d-5p,mmu-miR-124-3p,mmu-let-7g-3p,etc.Real-time fluorescent quantitative PCR was used to verify mmu-miR-466o-5p,mmu-miR-7a-5p,and mmu-let-7g-3p,and the results showed that the trend of change was basically the same as that of miRNA sequencing.In addition,the target gene analysis of 17 overlapping miRNAs showed that the differential miRNAs were mostly related to BDNF,Drd3,Cebpb,IL-6,etc.The results of GO enrichment analysis indicate that the target gene is involved in the process of amino acid metabolism in the cell.KEGG pathway analysis results indicate that target genes are mostly involved in the synthesis of steroid hormones,the metabolism of glycine,serine and threonine,and the IL-17 signaling pathway.Conclusion(1)Wendan Decoction has a certain effect on improving the anxiety behavior of mice with generalized anxiety disorder.(2)After the intervention of Wendan Decoction in mice with generalized anxiety disorder,the beneficial bacteria of Lactobacillus and Anaerostipes in the gut microbiota increased and the harmful bacteria of Proteobacteria,Ralstonia,Burkholderia,andβ-proteobacteria decreased.At the same time,the anxiety behavior of the mice was also very related to the gut microbiota.(3)Wendan Decoction has a certain anti-anxiety effect.It may be that mmu-miR-124-3p and mmu-let-7d-5p regulate target genes such as BDNF and Drd3,as well as mmu-miR-124-3 p,mmu-miR-7a-5p,mmu-miR-98-5p,mmu-let-7d-5p,mmu-let-7g-3p regulate the IL-17 signaling pathway,thereby exerting its anti-anxiety effect. |
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