| Objective:1 To investigate the theoretical connotation of "Yi" and "Si" under the category of"Spleen storing Yi and dominates Si",the relationship between Spleen,Yi and Si,and the significance of this theory for the clinical application of generalized anxiety disorder.2 To investigate the regulatory effects of Wendan Decoction on intestinal flora and protein expression of glutamatergic synapse pathway in a fecal transplantation model of mice with generalized anxiety disorder.3 To investigate the biological mechanism of the pathway of Wendan Decoction-key flora-gut-brain axis-glutamatergic synapse pathway,and further supplement the modern scientific interpretation of "the Spleen storing the Yi".Methods:1 Literature research:To further clarify the connotation of Yi and Si under the category of "the Spleen storing Yi and dominates Si",to investigate the relationship and functional basis of the spleen,Yi and Si,and to explore the implications of this theory for the treatment of generalized anxiety disorder.The theoretical basis of treating anxiety disorders from the relationship between the gallbladder and the spleen,stomach are also discussed with the example of Wendan Decoction.Based on the theory of "the Spleen storing the Yi dominates Si" and previous studies,we propose the scientific hypothesis that "Wendan Decoction is involved in regulating anxiety disorders through the key flora-gut-brain axis-glutamatergic synapse pathway",aiming to further supplement the modern scientific interpretation of "the Spleen storing the Yi".2 Experimental Research:5 normal subjects and 5 subjects with generalized anxiety disorder who met the inclusion criteria in the clinic were collected,and the fecal samples of the subjects were collected and made into fecal bacteria suspension for distribution,and the normal human and anxiety model mice were prepared by the fecal bacteria transplantation method.The randomized group method was used to divide the control group,control model group,anxiety model group and Wendan Decoction group according to body weight,10 animals in each group,and the formal experiment lasted for 4 weeks.The experimental group was treated with antibiotic elution for 14 days and five fecal transplants for 14 days,and the drug was administered on the last 7 days.After the experiment,anxiety-like changes were detected by open field test and elevated plus maze test;The 16S rRNA high-throughput sequencing method was used to analyze the composition of intestinal flora in fecal bacteria suspensions of normal and anxiety patients and animal experimental groups,and to identify the key bacterial genus groups involved in the formation of anxiety disorders and the regulation of Wendan Decoction.Spearman correlation analysis was used to identify key genus groups that are closely related to behavior.To further investigate their effects on the central nervous system,Western blot experiment was conducted to verify the protein expression of eight target genes,EAAT2,GNAI2,NMDAR2B,PSD-95 and so on;To further clarify the effect of key flora on significantly differentially expressed proteins,Spearman correlation analysis was applied to correlate nine key flora,including Verrucomicrobia,Akkermansia,and Rothia,with EAAT2 and GNAI2 differential proteins.Results:1 Literature research:"Yi" includes three aspects:memory,thinking and attention;"thinking" includes two parts:thinking and consideration;Yi is hidden inside and dominates the activity of thinking,and thinking can hurt the spleen and affect Yi.The spleen and stomach regulate the qi of the five internal organs and transport the essence of water and grain as the basis of their functions,and dysfunction can lead to the development of generalized anxiety disorder.The theoretical connotation of the regulating effect of Wendan Decoction on the middle jiao is " Wen Tong",which can be summarized as opening up the Yu,unimpede and smooth the qi,so as to harmonize.In summary,it can be divided into two aspects:the influence of the gallbladder on the function of the spleen and stomach,and the influence of the gallbladder and spleen and stomach as a hub on the peripheral qi.Therefore,Wendan Decoction is more suitable as a therapeutic medicine.2 Experimental Research:In the open field test,the total distance(P<0.05),time of central zone(P<0.01),and number of zone(P<0.05)were significantly lower in the anxiety model group compared to the control model group.Except for the total distance,the rest were significantly higher after treatment with WenDan Decoction(P<0.05).In the elevated plus maze test,the percentage of entries to the open arms(P<0.05)and the percentage of time spent in the open arms(P<0.01)were significantly lower in the anxiety model compared to the control model group,and both were significantly higher after treatment with WenDan Decoction(P<0.01).The results of intestinal flora analysis of human-derived samples showed that Actinobacteria,Gemmatimonadetes,Deferribacteres and Proteobacteria were significantly up-regulated and Verrucomicrobia were significantly down-regulated in anxious patients at the gate level compared to normal human intestinal flora;at the genus level,anxious patients compared to normal human intestinal Sutterella,Megamonas,Mucispirillum and others were significantly up-regulated in abundance;Butyricimonas,Akkermansia and others were significantly down-regulated.The total intestinal flora of human samples and animal experimental samples at the gateway level was 100%,and the regulatory direction was consistent about 56%,of which the two experimental anxiety models were more consistent than the normal model regulatory direction about 50%,combined with behavioral analysis indicates that this experimental transplantation was successful.The phylum-level regulatory direction consistent flora included Verrucomicrobia,Tenericutes,Deferribacteres,Cyanobacteria and Bacteroidetes,and the genus-level regulatory direction consistent flora included Ruminococcus,Mucispirillum,Bacteroides,Akkermansia and other 15 genera.At the phylum level,Deferribacteres was significantly upregulated in the anxiety model group compared to the control model group,and the anxiety model group did not contain Verrucomicrobia The relative abundance of Butyricimonas was significantly lower,Ruminococcus,Mucispirillum and Allobaculum were significantly higher in the anxiety model group compared to the control model group,and Akkermansia was absent in the anxiety model group.After treatment with Wendan Decoction,Verrucomicrobia was significantly up-regulated at the Phylum level;at the Genus level,Allobaculum and Rothia were significantly down-regulated and Odoribacter and Akkermansia were significantly up-regulated.To further clarify the effect of key flora on behavior,correlation analysis was conducted between them,Verrucomicrobia,Akkermansia and Butyricimonas were significantly positively correlated with behavior,and Allobaculum and Rothia were significantly negatively correlated with behavioral indicators;The results of WB experiments showed that EAAT2 and GNAI2 protein expression were significantly down-regulated in the anxiety model group compared with the control model group,and significantly up-regulated after treatment with Wendan Decoction.EAAT2 was significantly positively correlated with Verrucomicrobia and Akkermansia,and GNAI2 was significantly negatively correlated with Rothia.Notably,Allobaculum,although not directly correlated with EAAT2 and GNAI2,was significantly negatively correlated with Verrucomicrobia and Akkermansia.Conclusion:1 Literature Research:"the Spleen storing Yi and dominates Si",mainly includes three aspects:memory,thinking and attention;"Si" includes two parts:thinking and contemplation,as an extension of the connotation of Yi,which is the activity of thinking about things,and contemplation is the emotional change after thinking;the relationship between the spleen,Yi and Si is that Yi is hidden inside and dominates the activity of Si,and Si can hurt the spleen and thus affect Yi.The functions of the Yi and Si are closely related to those of the spleen,and the functional characteristics of the spleen are also of the nature of "Tu De",so they are attributed to the spleen."The function of the spleen and stomach is to control the qi of the five internal organs,and when the qi is in harmony with the Yi,the spleen and stomach transport the water and grain essence,and store the Ying to store the Yi.The malfunction of"the Spleen storing Yi and dominates Si" can lead to the occurrence of generalized anxiety disorder,such as the malfunction of attention,which is manifested as "excessive and persistent worrying that is difficult to control",and negative thinking tendency is an important cause of generalized anxiety disorder.The treatment is to regulate the "Yi" to cure the Yu disorder and to restore the elevation function of the spleen and stomach to cure the Yu disorder.The two are not isolated but complementary to each other.Generalized anxiety disorder is a type of Yu disorder in Chinese medicine,and the treatment of Yu disorder should pay special attention to the regulation of the spleen and stomach,and the first step is to regulate the middle and smooth the qi.In this experiment,the medicine used is Wendan Decoction.From the point of view of unblocking the qi flow in the middle jiao,the combination of all the medicines aims to open the stagnation in the middle Jiao,while taking into account the elements of phlegm,heat,stagnation and deficiency,etc.If the function of the spleen and stomach can be restored,the spirit will be peaceful and the Yi will be stored,and the stagnation can be removed.Different dynasties have identified different characteristics of the symptoms,and the intent of the formula has changed from "warming and tonifying" to "warming and harmonizing","warming and smoothing",and the pathogenesis has changed from gallbladder cold to heart and gallbladder deficiency and timidity to qi stagnation and salivation,changing the formula of Wendan Decoction from being applied alone to a certain formula to harmonize the qi mechanisms of heart,gallbladder and spleen and stomach.The idea of the formula for Wendan Decoction implies the close and complex relationship between the gallbladder,the spleen and stomach,which is manifested in the following four aspects:the gallbladder draws a straight furrow,helping the spleen to make decisions;the gallbladder holds the essence juice,helping the spleen and stomach to transportation and transformation;gallbladder holds the fire,helping the spleen and stomach to decompose water and grain;the gallbladder is the beginning of growth,helping the spleen and stomach to turn the pivot.At the same time,the gallbladder is the pivot of the six meridians and works with the Spleen and Stomach,which are the pivots of the five internal organs,to regulate the body’s qi,so that the body functions normally and all diseases are energized.Combining the physiological functions of the spleen,stomach and gallbladder and the close connection between the two,Wendan Decoction aims to open up and smooth the qi,and to harmonize with the deficiency,the core pathogenesis of the disease is Yu,the deficiency is deficiency cold or deficiency heat,the positivism is qi Yu,phlegm Yu,and can also be seen due to the deficiency of Yu,so its treatment should be warming and clearing heat,warming to nourish the deficiency and opening up the Yu,aiming to harmonize the gallbladder and spleen and stomach,and restore the qi elevation of the gallbladder,spleen and stomach.2 Experimental Research:Generalized anxiety disorder is associated with upregulation of Deferribacteres and downregulation of Verrucomicrobia,and with decreased abundance of Akkermansia and Butyricimonas and upregulation of Ruminococcus,Mucispirillum and Allobaculum.The use of Wendan Decoction was able to restore part of the microecological environment,mainly in two ways:on the one hand,it led to a significant increase in the abundance of probiotic bacteria such as Akkermansia.On the other hand,the abundance of antagonistic bacteria(e.g.Allobaculum)of Akkermansia or conditionally pathogenic bacteria(e.g.Rothia)and other probiotics was significantly down-regulated,which affected the protein expression of EAAT2 and GNAI2 of glutamatergic synapse pathways in neuronal cells of cortical brain area,leading to dysregulation of glutamatergic neurotransmission and impairment of neuronal cell and synaptic functions and pathogenesis.This affects the dysregulation of glutamatergic neurotransmission and impairs neuronal cells and synaptic function.3 Combining theoretical and experimental research to explain the modern scientific connotation of "the Spleen storing the Yi dominates Si".3.1 Intestinal flora is involved in the pathological state of"Si hurts the Spleen" and "Si leads to qi stagnation"The dysregulated intestinal flora in subjects with generalized anxiety disorder is involved in the pathological state of"Si hurts the Spleen" and "Si leads to qi stagnation",which is associated with the down-regulation of the abundance of the probiotics Akkermansia and Butyricimonas and the up-regulation of the conditionally pathogenic bacteria Ruminococcus,Mucispirillum and Allobaculum.3.2 Intestinal flora is involved in the process of "Spleen storing Ying"The intestinal flora and its metabolites can produce or participate in the synthesis of neurotransmitters,and their metabolites play an important physiological function,which can be regarded as a kind of "Ying".Long-term chronic stress leads to dysbiosis of the intestinal flora,derangement of transport function,and disturbance of neurotransmitter or its precursor production,a process similar to the derangement of water and grain metabolism.3.3 Intestinal flora is involved in the process of rising and falling of spleen and stomach qiIntestinal flora can restore the permeability of the intestinal mucosa and blood-brain barrier,which increases due to long-term chronic stress,and harmful molecules enter the circulatory system in the lumen.When the intestinal mucosa is restored,nutrients and water are properly absorbed and harmful molecules in the intestinal cavity are reduced or excreted in a timely manner,similar to the rise and fall of the spleen and stomach.3.4 Effective treatment of anxiety disorders from the middle-jiao viewpointIn combination with previous studies,it has been found that the treatment of anxiety disorders from the middle-jiao is effective from the viewpoint of behavior,pathological section and molecular biology experiments.Therefore,the treatment of depression should pay particular attention to the regulation of the spleen and stomach,and the addition and reduction of medicine to the internal organs of the disease on the basis of the regulation of the spleen and stomach. 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