Intestinal bacterial liquid, preparation method and application of intestinal bacterial liquid in depression relieving medicine

Abstract

The invention discloses intestinal bacterial liquid, a preparation method and application of the intestinal bacterial liquid in depression relieving drugs, and relates to the technical field of development of anti-depression drugs. The method comprises the following steps: 1) performing CUMS stimulation on a CUMS rat every day, performing electroacupuncture stimulation for five times every week, and then collecting rat feces; and 2) mixing the rat feces with the PBS solution, and carrying out oscillation, precipitation, centrifugation and filtration to obtain the intestinal bacterial liquid. The intestinal flora of CUMS rats is intervened by a specific electroacupuncture stimulation method, the intervened intestinal flora is extracted from the rat feces to obtain the intestinal bacterial liquid, and the intestinal bacterial liquid has the effect of relieving depressive symptoms through verification.

Description

Technical Field

[0001] The present invention relates to the technical field of antidepressant drug development, and in particular to an intestinal bacterial liquid, a preparation method and application thereof in a drug for alleviating depression. Background Art

[0002] Major depressive disorder (MDD) is one of the most common mental and behavioral disorders in the world, mainly caused by a variety of factors, such as family history, chronic health problems and major life changes. MDD patients show symptoms such as sadness, difficulty concentrating, loss of appetite, loss of interest, sleep disorders, etc. If the condition worsens, they may also engage in self-harming behaviors.

[0003] Currently, antidepressants are the first choice for the treatment of MDD, including serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants amitriptyline, and monoamine oxidase inhibitors. However, the currently widely used antidepressant treatment regimens have various disadvantages, including drug tolerance, long onset time, unsatisfactory clinical response rate, various side effects, and long treatment courses.

[0004] In addition to medication, conventional first-line treatments for MDD also include psychotherapy and physical therapy for patients to choose from, but conventional treatments also have shortcomings. In order to achieve the expected therapeutic effect of psychotherapy, in addition to sufficient treatment frequency and course of treatment, the counselor must also have rich treatment experience and establish a good doctor-patient trust relationship, so it requires the joint efforts of multiple factors to achieve. Neurostimulation physical therapy, such as electroconvulsive therapy and transcranial magnetic stimulation therapy, although the effect is significant and rapid, but due to the large side effects of the therapy, the treatment effect requires a long course of treatment and a considerable frequency to maintain, and the symptom recurrence rate is high.

[0005] Therefore, it is urgent to find drugs with fewer side effects and lower prices.

[0006] Gut microbes play an extremely important role in depression. Previous studies have shown that the intestinal flora of patients with depression will change, and adjusting the intestinal flora of MDD patients may help improve depressive symptoms. However, there are many types of intestinal microbial flora and their proportions are also different. How to obtain intestinal microbes that are beneficial to improving depressive symptoms and inhibiting unfavorable flora is a difficult problem in the current development of intestinal microbial antidepressant drugs. Summary of the invention

[0007] In order to solve the above technical problems, the purpose of the present invention is achieved through the following technical solutions.

[0008] Provided is a method for preparing intestinal bacterial fluid in rats subjected to CUMS (Chronic Mild Unpredictable Stress) by electroacupuncture intervention, comprising the following steps: 1) Perform CUMS stimulation on CUMS rats every day and electroacupuncture stimulation at least 5 times a week, and then collect rat feces; preferably, electroacupuncture stimulation is performed for at least four weeks; The electroacupuncture stimulation is specifically as follows: using acupuncture needles to puncture the Baihui point in the middle of the parietal bone, the Yintang point 2mm above the midpoint of both eyes, and the Zusanli point 3mm below the fibular head and posterior and lateral to the knee joint; the puncture direction of the Baihui point is toward the back of the head, the Yintang point is toward the tip of the nose, and the needle insertion depth is 2-3mm; the Zusanli point is punctured directly, and the needle insertion depth is 3-5mm; the acupuncture needles at the Baihui point and the Yintang point are connected to the electroacupuncture instrument, the current frequency is 8-12Hz, continuous wave, the current intensity is 1-3mA, and each time is 15-25min; 2) The rat feces was mixed with PBS solution, and after shaking, precipitation, centrifugation and filtration, the intestinal bacterial solution was obtained.

[0009] Preferably, the current frequency is 10 Hz, the current intensity is 2 mA, and each time is 20 minutes.

[0010] In some specific embodiments, the stimulation factors of the CUMS stimulation include swimming in 4°C ice water for 5 minutes, reversing day and night, fasting for 24 hours, depriving water for 24 hours, restraining for 2 hours, wet bedding for 24 hours, and clamping the tail for 3 minutes, a total of 7 factors, and the stimulation factors are not repeated in two consecutive days.

[0011] Preferably, the ratio of rat feces to PBS solution is 0.1 g: 1-2 ml.

[0012] In some specific embodiments, the step 1) further includes placing the rat feces in a sterile enzyme-free EP tube, first placing it in dry ice, and then freezing it at -80°C; the step 2) further includes thawing the frozen rat feces at room temperature and then mixing it with the PBS solution.

[0013] The present invention also provides an intestinal bacterial liquid prepared by the above method.

[0014] In the above-mentioned intestinal bacterial fluid, at the genus level, the relative abundance of Lactobacillus was 26.0-27.0%; the relative abundance of Akkermansia was 19.0-20.0%; the relative abundance of Clostridium_sensu_stricto_1 was 12.0-13.0%; the relative abundance of Ruminococcaceae_UCG-01 was 10.0-11.0%; the relative abundance of Romboutsia was 8.0-9.0%; the relative abundance of Allobaculum was 7.0-8.0%; the relative abundance of uncultured was 6.0-7.0%; the relative abundance of Turicibacter was 3.0-4.0%; the relative abundance of Lachnospiraceae_NK4A136_group was 1.0-2.0%; and the relative abundance of Alistipes was 1.0-2.0%.

[0015] In a specific intestinal bacterial fluid, at the genus level, the relative abundance of Lactobacillus was 26.8%; the relative abundance of Akkermansia was 19.0%; the relative abundance of Clostridium_sensu_stricto_1 was 12.2%; the relative abundance of Ruminococcaceae_UCG-01 was 10.8%; the relative abundance of Romboutsia was 8.4%; the relative abundance of Allobaculum was 7.0%; the relative abundance of uncultured was 6.2%; the relative abundance of Turicibacter was 3.4%; the relative abundance of Lachnospiraceae_NK4A136_group was 1.9%; and the relative abundance of Alistipes was 1.2%.

[0016] The intestinal bacterial liquid of the present invention can be used in drugs for relieving depression.

[0017] The present invention also provides a drug for relieving depression, comprising the above-mentioned intestinal bacterial liquid. In some specific embodiments, the above-mentioned intestinal bacterial liquid can also be made into freeze-dried powder.

[0018] The present invention intervenes in the intestinal flora of CUMS rats through a specific electroacupuncture stimulation method, and then extracts the intervened intestinal flora from the rat feces to obtain intestinal flora liquid. It has been verified that the intestinal flora liquid has the effect of alleviating depressive symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0020] Figure 1Acupuncture point diagram of rats in the electroacupuncture group of the present invention; Figure 2 This is the OTU Venn diagram of the intestinal flora of rats in each group; Figure 3 This is a comparison chart of Chao1 index of each group; Figure 4 This is a comparison of the relative abundance of the intestinal flora genus level in each group of rats; Figure 5 This is the species evolution branch diagram of the model group vs. the blank group; Figure 6 Bar chart of LDA values ​​of blank group vs. model group; Figure 7 The species evolution branch diagram of the model group vs. the electroacupuncture group; Figure 8 Bar chart of LDA values ​​of model group vs. electroacupuncture group; Fig. 9 The species evolution branch diagram of the blank group vs. the electroacupuncture group; Fig.10 It is a bar chart of LDA values ​​of the blank group vs. the electroacupuncture group. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0023] Example 1 Electroacupuncture stimulation of CUMS rats

[0024] 1. Materials 1. Experimental animals and groups Twenty-four SPF healthy male adult Sprague-Dawley (SD) rats, 7 weeks old and weighing 200-250 g, were purchased from Zhuhai Baishitong Biotechnology Co., Ltd. [License No.: SCXK (Guangdong) 2020-0051]. The feeding conditions were a barrier animal room with a room temperature of 25 °C, a humidity of 40%, a light period of 8:00-20:00, and a dark environment of 20:00-8:00. During the adaptive feeding period and the non-fasting and water-deprivation period, the rats were given free water (sterile water) and free food (growth and reproduction pellet feed). After 7 days of adaptive feeding, the 24 SD rats were randomly divided into a blank group (n=8), a model group (n=8), and an electroacupuncture group (n=8). Ear tags were used for marking.

[0025] Table 1 Animal groups

[0026] 2. Instruments, Equipment and Reagents Table 2 Instruments and equipment Table 3 Reagents and consumables

[0027] 2. Methods 1. Modeling This experiment used 35 days of chronic mild unpredictable stimulation (Chronic Unpredictable MildStress, CUMS) to establish a rat model of depression. Except for the BC group, the MO group and the EA group were all caged and subjected to CUMS stimulation. The 7 stimulation factors in this study included: ice water swimming (4℃, 5 minutes), day and night reversal (12 / 12 hours), fasting (24 hours), water deprivation (24 hours), restraint (2 hours), wet bedding (24 hours), and tail clamping (3 minutes). There was no regular pattern throughout the experiment (except the day of behavioral testing), and the stimulation factors were not repeated on two consecutive days. The total stimulation was 63 days, as shown in Table 4: Table 4 CUMS modeling schedule

[0028] 2. Behavioral testing 1. Body mass Body weight was measured before modeling (-35 days), 5 weeks after modeling (0 days), and at the end of intervention (28 days). The rats were placed on an electronic balance and the values ​​were recorded when they were in a relatively resting state and the values ​​on the electronic balance screen were relatively stable; 2. Sugar water preference experiment Sugar water preference experiments were conducted before modeling (-35 days), 5 weeks after modeling (0 days), and at the end of intervention (28 days). The sugar water preference experiment took a total of 3 days: on the first day, the rats were adapted to sugar water. Two drinking fountains with the same appearance were placed in each cage, one bottle was for rats to drink sterile water, and the other bottle was for sucrose water (sucrose: water = 1:100), which were collected after 24 hours; on the second day, the rats were deprived of water for 24 hours and were allowed to eat freely; on the third day, two drinking fountains with the same appearance were placed in each cage, one bottle was for rats to drink sterile water, and the other bottle was for sucrose water (sucrose: water = 1:100), and they were weighed separately and collected and weighed after 24 hours. The formula for calculating sugar water consumption in rats is: Sugar water consumption (%) = sugar water consumption (g) / total liquid consumption (g) × 100%; 3. Open field experiment The open field test was conducted before modeling (-35d), 5 weeks after modeling (0d), and at the end of intervention (28d). The rats were placed in an open box of 100cm*100cm*50cm without a lid. The box was an opaque black open box with 25 small squares of equal size (20*20cm) on the bottom. The test took 3 minutes. At the beginning of the test, the rat was placed in the center of the bottom of the box and the camera was turned on to record the rat's activities. If the rat crossed the grid horizontally within 3 minutes and at least three paws entered the same square, it was counted as one horizontal movement, which was counted as 1 point; if both front paws were lifted off the ground at the same time (including combing), it was counted as one vertical movement, which was counted as 1 point. After each rat was tested, 75% alcohol was used to clean the box to avoid interfering with the next rat.

[0029] 3. Intervention methods Blank group: free access to food and water, 20 min isoflurane anesthesia 5 times a week; Model group: CUMS stimulation was performed every day, see the modeling procedure for details, and isoflurane anesthesia was given for 20 minutes 5 times a week; Electroacupuncture group: CUMS stimulation was performed every day. Electroacupuncture stimulation was performed 5 times a week starting from 5 weeks of modeling (0d), and continued for 4 weeks (28d). The needles used were 0.2*25mm disposable sterile acupuncture needles from Huatuo Suzhou Medical Supplies Factory Co., Ltd. Acupoint selection and positioning: Baihui: the center of the parietal bone; Yintang: 2mm above the midpoint of both eyes; Zusanli: the posterolateral side of the knee joint, 3mm below the head of the fibula. Methods: Under 2.5-3% isoflurane inhalation anesthesia, the Baihui and Yintang acupoints were pierced horizontally (sterilized cotton balls were placed between the two needle handles to prevent short circuits), the direction of acupuncture was Baihui toward the back of the head, and the Yintang needle tip was toward the tip of the nose; the depth of the needle was 2-3mm. Zusanli was pierced vertically, with a depth of 3-5mm. Figure 1 Connect the needle handles of Baihui and Yintang acupoints to Han's electroacupuncture instrument, with a current frequency of 10 Hz, continuous wave, and a current intensity of 2 mA, for 20 minutes each time.

[0030] 3. Data Analysis Methods IBM SPSS Statistics 26.0 software was used for data collation and statistical analysis. Shapiro-Wilk To test for normal distribution, use Levene's Statistic Test for homogeneity of variance. For data that conform to normal distribution, the mean plus or minus the standard deviation ( ) showed normally distributed data, and one-way analysis of variance was used for comparison among multiple groups with equal variance. LSD The data that did not conform to the normal distribution or had uneven variance were presented as median and interquartile range (M(IQR)) and analyzed by rank sum test. Kruskal Wallis If there are many types of normally distributed data in the table, for the sake of uniformity, If there are many non-normal distribution data types in the table, they are displayed as (M(IQR)) for uniformity. P The values ​​are all Bonferroni Correction method, the output is corrected P The above tests are all two-tailed tests. P A value of ≤0.05 was considered statistically significant.

[0031] IV. Experimental Results 1. Body mass analysis From the results in Table 5, it can be seen that there was no statistically significant difference in body mass among the groups before modeling (P>0.05), indicating baseline balance; after modeling, compared with the BC group, the body mass of the MO group and the EA group was lower than that of the BC group, and the difference was statistically significant (P<0.05); after intervention, compared with the BC group, the body mass of the MO group was lower than that of the BC group, and the difference was statistically significant (P<0.05); compared with the MO group, the body mass of the EA group was higher than that of the MO group, and the difference was statistically significant (P<0.05).

[0032] From the above, we can see that acupuncture can improve the body weight of depression model rats compared with the model group rats;

[0033] 2. Analysis of sugar water preference rate From the results in Table 6, it can be seen that there was no statistically significant difference in the sugar water preference rate among the groups before modeling ( P >0.05), indicating baseline balance. After modeling, compared with the BC group, the sugar water preference rate in the MO group and the EA group was lower than that in the BC group, and the differences were statistically significant ( P <0.05); after intervention, the sugar water preference rate in the EA group was higher than that in the BC group and the MO group, and the difference was statistically significant (P <0.05).

[0034] From the above, we can see that acupuncture can increase the sugar water preference rate of depression model rats compared with the model group rats;

[0035] 3. Open field experiment data analysis 1. Horizontal movement From the results in Table 7, it can be seen that there was no statistically significant difference in the horizontal exercise scores between the groups before modeling ( P >0.05); after modeling, the horizontal movement scores of the MO and EA groups were lower than those of the BC group, and the difference was statistically significant ( P <0.05), there was no significant difference between the MO group and the EA group ( P >0.05); after intervention, the horizontal motor score of the MO group was lower than that of the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the horizontal exercise scores of the EA group were higher than those of the MO group, and the difference was statistically significant ( P <0.05).

[0036] From the above, we can see that acupuncture can improve the horizontal movement of depression model rats compared with the model group rats;

[0037] 2. Vertical Movement From the results in Table 8, it can be seen that there was no statistically significant difference in the vertical movement scores of each group before modeling ( P >0.05); the overall difference in vertical movement scores after modeling was statistically significant ， Compared with the BC group, the vertical movement scores of the MO group and the EA group were lower than those of the BC group, and the differences were statistically significant ( P <0.05), there was no statistically significant difference between the MO group and the EA group ( P >0.05); after intervention, the vertical movement score of the MO group was lower than that of the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the vertical movement scores of the EA group were higher than those of the MO group, and the difference was statistically significant ( P <0.05).

[0038] From the above, we can see that acupuncture can improve the vertical movement ability of depression model rats compared with the model group rats;

[0039] Therefore, based on the above research content, it can be seen that acupuncture can improve the depressive-like behavior of depression model rats.

[0040] Example 2 Effect of electroacupuncture on intestinal microorganisms in CUMS rats 1. Experimental Animals and Grouping The experimental animals and groups were the same as those in Example 1.

[0041] 2. Instruments, Equipment and Reagents Table 9 Instruments and Equipment Table 10 Reagents and consumables

[0042] III. Methods 1. Feces collection The fecal tissue samples of this experiment were derived from the rats in Example 1. After the intervention (28 days), the rat feces were collected and placed in a sterilized, clean, bedding-free cage. After the rat defecated, the rat feces were collected with sterilized tweezers, and a 1.5 ml sterile enzyme-free EP tube was used to hold one feces. The cage and tweezers were disinfected with 75% alcohol for the next animal. The collected EP tube was first placed in dry ice and frozen to -80°C within 2 hours; 2. DNA extraction from rat feces The samples in this part of the experiment were from fresh feces of rats after intervention (28 days). After genomic DNA was extracted using the DC306-09 magnetic bead soil DNA extraction kit, the purity and concentration of DNA were tested using Thermo Nano Drop One; (III) Analysis process 1. Library construction and sequencing The library was constructed according to the standard procedure of NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NewEngland Biolabs, USA). The constructed amplicon library was sequenced by PE250 using the Illumina Nova 6000 platform; (1) Paired-end Raw Reads sequencing data filtering: Use fastp to perform sliding window quality trimming (-W 4 -M 20) on the paired-end Raw Reads data. At the same time, according to the primer information at both ends of the sequence, use cutadapt software to remove the primers to obtain the paired-end Clean Reads after quality control. (2) Paired-end Clean Reads splicing: For paired-end sequencing data, it is necessary to use usearch -fastq_mergepairs to filter out non-compliant tags based on the overlap relationship between PE reads to obtain the original spliced ​​sequence (Raw Tags); (3) Raw Tags sequence quality filtering: Use fastp to perform sliding window quality trimming on the Raw Tags data (-W 4-M 20) to obtain valid splicing fragments (Clean Tags).

[0043] 2. OTU clustering and species annotation (1) OTU clustering: The clustering method is UPARSE: UPARSE (RC Edgar. Highly accurate OTU sequences from microbial amplicon reads. Nature methods, 2019, 10 (10): 996); (2) Species annotation of representative sequences: Use usearch -sintax to compare the representative sequence of each OTU with the SILVA (16S), RDP (16S), Greengenes (16S), SILVA (18S), and Unite (ITS) databases to obtain species annotation information (the confidence threshold is set to 0.8 by default, and the default databases are SILVA (16S), SILVA (18S), and Unite (ITS)), so as to understand the species origin of all sequences. The taxonomy results of species annotation are divided into 7 levels, namely kingdom (L1), phylum (L2), class (L3), order (L4), family (L5), genus (L6), and species (L7).

[0044] 3. OTU statistics (1) OTU_Table: Based on the OTU_table after removing singleton OTUs, chimeras, and contaminated OTUs, count the number of reads and OTUs contained in the sample or group; (2) Pan_Core species analysis: R software (V5.1.3) was used to count the union (Pan) and intersection (Core) of the target taxonomic level in different sample numbers to assess whether the sample size was sufficient.

[0045] 4. Species community analysis (1) Species community structure: Use R software to perform common and unique species statistics, community composition analysis, and species abundance cluster analysis; (2) System evolution analysis: ① Single sample-system evolution analysis at each classification level; ② Systematic evolutionary analysis of all samples at each classification level (default parameters): Select the representative OTU sequences ranked in the top 20 in terms of overall relative abundance, use FastTree software to infinitely compare and build a tree, and combine the relative abundance of each OTU and the species annotation confidence information of its representative sequence, and use the ggtree software package for visualization; (3) Analysis of significance of community structure differences among groups: The anosim function, mrpp function, adonis function and Amova of the vegan and pegas packages of the R software were used to perform Anosim, MRPP, Adonis and Amova significance analyses.

[0046] 5. Alpha (α) diversity analysis (1) Alpha diversity index statistics: Based on the OTU abundance table, use search -alpha_div to calculate the diversity index (chao1, Simpson); (2) Dilution curve: Based on the OTU abundance table, use usearch -alpha_div_rare to calculate the dilution curve of the above diversity index; (3) Rank Abundance curve: Based on the OTU abundance table, Rank Abundance calculation was performed using a self-built python script (python v2.7); (4) Index difference test between groups: Use R software to analyze the difference between the alpha diversity index groups. The difference between the alpha diversity index groups will be analyzed by parametric and non-parametric tests. If there are only two groups, select Student's t-test or Wilcox Rank sum test, if there are more than two groups, choose Kruskal-Wallis Kruskal-Wallis test or one-way ANOVA.

[0047] 6. Beta (β) diversity analysis The component composition of each sample at different taxonomic levels was calculated based on different distance algorithms (Bray-Curtis and Euclidean, etc.), and the Anosim analysis method was used to test the significance of community structure differences among groups.

[0048] 7. Species Difference Analysis LEfSe analysis: Based on the homogenized abundance table OTU_table of each species level, LEfse software was used to perform significant difference analysis of species between groups. First, the non-parametric factorial Kruskal Wallis (KW) sum-rank test was used to detect species with significant abundance differences between different groups. Then, the grouped Wilcoxon rank sum test was used to judge the differences between the two groups. Finally, linear discriminant analysis (LDA) was used to achieve dimensionality reduction and evaluate the effect size of species with significant differences (i.e., LDA Score). The default screening value of LDA Score was set to 2, and the results were the biomarkers in each group.

[0049] 8. Statistical methods (1) Comparison among multiple groups: Based on the OTU abundance table, R software was used to Kruskal_Wallis Rank sum test or One-way ANOVA with FDR correction; (2) Comparison between the two groups: Based on the OTU abundance table, R software was used to Wilcox Rank sum test, Wilcoxon Signed rank test, Student's Inspection or Welch's Test and FDR correction were performed.

[0050] All data analysis in this experiment was completed using the MagiCloud platform (http: / / cloud.magigene.com / ) provided by MagiGene Technology, the testing company.

[0051] IV. Results The 16sDNA amplicon experimental analysis method was used to analyze the species abundance, species diversity and species composition of intestinal microorganisms in each group of rats after intervention, and the results are as follows.

[0052] 1. Venn diagram of OTUs in each group The number of OTU sequences in each group was analyzed using the Venn diagram, and the similarities and differences in species distribution between different groups were compared from the perspective of the number of OTU sequences. The results are as follows Figure 2 As shown. Figure 2 It can be seen that there are 766 OTU sequences in each group, accounting for more than 71.39% of the total number of sequences in each group, indicating that the number of species units of the overall rat intestinal flora tends to be stable under different intervention conditions. The blank group (BC group) has the most specific OTU sequences, indicating that the species structure of the rat intestinal flora has shown a large change after CUMS stimulation. The number of specific OTU sequences in the model group (MO group) is second only to the blank group, while the number of specific OTU sequences in the electroacupuncture group (EA group) is relatively small, indicating that acupuncture has a certain effect on the number of species.

[0053] 2. Effect of electroacupuncture intervention on α-diversity of intestinal flora in CUMS rats The comparison of Chao1 index of each group is shown in the figure Figure 3 As shown. Figure 3 It can be seen that the Chao1 index reflects the abundance of species communities. Its algorithm estimates the total number of species in the sample based on the OTU index contained in the sample. The larger the Chao1 value, the more species there are. The results showed that after the intervention, there was no significant statistical difference in the Chao1 index between the groups. P =0.659).

[0054] 3. Effect of electroacupuncture intervention on the β-diversity of intestinal flora in CUMS rats β diversity reflects the differences in species structure between different samples. As shown in Table 11, this experiment used Anosim to test the significance of community structure differences between groups, the distance algorithm used the Canberra algorithm, and the species level was OTU. The Anosim analysis results show that the differences in β diversity of the remaining groups of samples are statistically significant ( P <0.05).

[0055] Table 11 Analysis of the significance of community structure differences among β diversity groups

[0056] 4. Effect of electroacupuncture intervention on the species classification of intestinal flora in CUMS rats The relative abundance of intestinal flora in each group of rats Figure 4 As shown. Figure 4 It can be seen that at the genus level, electroacupuncture can significantly increase the content of Lactobacillus. The EA group had the highest content of Lactobacillus (26.82%), followed by the MO group (19.94%). The relative abundance of Akkermansia was the highest in the MO group (19.05%) and the EA group (19.04%).

[0057] Table 12 Statistical table of relative abundance distribution of species in each group (first 15 items)

[0058] 5. Effect of electroacupuncture intervention on differences in intestinal flora of CUMS rats LEfSe (Linear discriminant analysis Effect Size) was used to compare the species differences between the two groups to find species (i.e., biomakers) with significant differences in abundance between the groups. This section uses LDA value bar graphs and species evolution branch diagrams to present the results of this part, where the length of the bar in the LDA value bar graph represents the weight of the species; the size of the circle corresponding to the group color in the species evolution branch diagram at the same classification level is positively correlated with the relative abundance of the species in the group.

[0059] 1. Blank group vs. Model group The evolutionary branch diagram of species in the model group vs. blank group is shown in the figure below. Figure 5 As shown, the LDA value bar chart of the blank group vs. model group is as follows Figure 6 Compared with the blank group, the abundance of Firmicutes, Clostridia, Clostridiales, Lachnospiraceae, Lachnospiraceae_NK4A136, Odoribacter, Rumiriclostridium_UCG_005, and Eubacterium xylanophilum in the model group decreased; the abundance of Deltaprotedbacteria, Butyrivibrio, and Rikenellaceae RC9 gut in the model group increased.

[0060] 2. Model group vs. electroacupuncture group The evolutionary branches of species in the model group vs. electroacupuncture group are shown in the figure below. Figure 7 As shown in the bar chart of LDA values ​​of the model group vs. electroacupuncture group Figure 8 Compared with the EA group, the abundance of Firmicutes, Clostridiaceae_1, Clostridium_sensu_stricto_1, Turictbacter, and Alloprevotella in the model group decreased; the abundance of Verrucomicrobia, Verrucomicrobiaceae, Verrucomicrobiales, and Akkermansiaceae in the model group increased.

[0061] 3. Blank group vs. electroacupuncture group The evolutionary branches of species in the blank group vs. electroacupuncture group are shown in the figure below. Fig. 9 As shown, the LDA value bar chart of the blank group vs. electroacupuncture group is as follows Fig.10Compared with the EA group, the abundance of Bacilli and Lactobacillaceae in the blank group decreased, while the abundance of Lactobacillales, Lachnospiraceae, Lactobacillus, and Clostridium_sensu_stricto_1 in the EA group increased.

[0062] Example 3 Study on the treatment of CUMS rats with intestinal bacterial fluid 1. Materials 1. Experimental animals and groups Twenty-four SPF healthy male adult Sprague-Dawley (SD) rats, 7 weeks old and weighing 200-250 g, were purchased from Zhuhai Baishitong Biotechnology Co., Ltd. [License No.: SCXK (Guangdong) 2020-0051]. The breeding conditions were a barrier animal room with a room temperature of 25 °C, a humidity of 40%, a light period of 8:00-20:00, and a dark environment of 20:00-8:00. During the adaptive feeding period and the non-fasting and water-deprivation period, the rats were given free water (sterile water) and free food (growth and reproduction pellet feed). After 7 days of adaptive feeding, the 24 SD rats were randomly divided into a blank bacterial solution treatment group (n=8), a model bacterial solution treatment group (n=8), and an electroacupuncture bacterial solution treatment group (n=8). Ear tags were used for marking.

[0063] 2. Instruments, Equipment and Reagents Table 13 Experimental reagents Table 14 Test equipment

[0064] 2. Methods 1. Establishment of sterile rat model with antibiotics 1. Antibiotic preparation method In order to eliminate the intestinal microbiota and allow for better colonization of fecal bacteria, the "cocktail antibiotic" method was used for 7 consecutive days of gavage. Specifically, ampicillin (200 mg / kg), vancomycin (100 mg / kg), metronidazole (200 mg / kg) and neomycin (200 mg / kg) were administered. The gavage volume for each was 0.2 ml, and physiological saline was drawn with a sterile syringe.

[0065] (Reference: CAESAR R, TREMAROLI V, KOVATCHEVA-DATCHARY P, et al. Crosstalk between gut microbiota and dietary lipids aggravates WATinflammation through TLR signaling [J]. Cell metabolism, 2015, 22(4): 658-68) 2. Preparation method of intestinal bacterial solution The fecal samples after intervention in the model group, the electroacupuncture group, and the blank control group in the experiment of Example 1 were used as donor fecal samples. The specific configuration operation was as follows: 1. Take 2 fecal samples from each rat in each group as donor fecal samples. After completely thawing at room temperature, mix them according to the ratio of fecal sample: PBS solution = 0.1g: 1ml; 2. Oscillate the mixed fecal bacteria solution on an oscillator for 5 minutes and then let it settle for 5 minutes; 3. Centrifuge the fecal bacteria liquid, adjust the centrifuge parameters to 4°C, centrifuge speed to 3000 rpm, half a minute, and take the fecal solid centrifugation as the benchmark; 4. Take the upper filtered slurry and mix them, then store them in a refrigerator at -80℃. All the above operations are performed aseptically.

[0066] Based on the above steps, three groups of fecal bacterial fluid were obtained, namely, the blank group fecal bacterial fluid, the model group fecal bacterial fluid, and the electroacupuncture group fecal bacterial fluid.

[0067] 3. Experiment on the treatment of depression model rats with intestinal bacterial fluid 1. CUMS modeling in germ-free mice The method was the same as that of the modeling part in Example 1. That is, the three groups of animals were all subjected to CUMS modeling for a total of 5 weeks; 2. Behavioral Observation Indicators The observation indexes were the same as those in the "behavioral observation indexes" in the method section of Example 1. That is, body weight analysis, sugar water preference rate, and open field test were tested before and after intragastric administration; 3. Intervention measures (1) Intragastric administration method: Fix the rat's head and body with your left hand, with the body in a straight line, and insert the needle from one corner of the mouth, along the upper palate, the posterior wall of the pharynx, and into the stomach along the swallowing action. If there is no obvious struggle or choking, it indicates that the gavage is successful. Observe for 30 minutes after gavage; (2) Intragastric administration volume The fecal bacterial suspensions of the three groups, namely the blank group fecal bacterial liquid (BC group), the model group fecal bacterial liquid (MO group), and the electroacupuncture group fecal bacterial liquid (EA group), were melted at room temperature. The same gavage needle was used for each group to avoid cross-use, and 0.2 ml was gavaged each time. The gavage was continued for 14 days, and the gavage method was the same as before.

[0068] 3. Experimental Results 1. Analysis of body weight of rats in different groups From the results in Table 15, it can be seen that there is no significant difference in the body weight of each group in the baseline period (before treatment) ( P >0.05); there was no significant difference in body weight among the groups after oral administration of antibiotics ( P >0.05); after CUMS treatment and intragastric administration of fecal bacteria solution, the body weight of the MO group was significantly lower than that of the BC group ( P <0.05); compared with the MO group, the body weight of the EA group was significantly higher than that of the MO group ( P <0.05).

[0069] It can be seen that compared with the depression model rats gavaged with fecal microbial fluid in the model group (MO group), the fecal microbial fluid in the electroacupuncture group (EA group) can improve the body weight of the depression model rats;

[0070] 2. Analysis of sugar water preference rate As shown in Table 16, there was no significant difference in the sugar water preference rate among the groups during the baseline period (before treatment). P >0.05); there was no significant difference in the sugar preference rate among the groups after oral administration of antibiotics ( P >0.05); after CUMS treatment and intragastric administration of fecal bacteria solution, the sugar water preference rate in the MO group was significantly lower than that in the BC group ( P <0.05); compared with the MO group, the sugar water preference rate in the EA group was significantly higher than that in the MO group ( P <0.05).

[0071] It can be seen that compared with the depression model rats gavaged with fecal microbial fluid in the model group (MO group), the fecal microbial fluid in the electroacupuncture group (EA group) can increase the sugar water preference rate of the depression model rats;

[0072] 3. Open field experiment (1) Horizontal sports score As shown in Table 17, there were no significant differences in the open field test scores between the groups during the baseline period (before treatment). P >0.05); there was no significant difference in the overall horizontal movement score after intragastric administration of antibiotics (P =0.395>0.05); after CUMS treatment and intragastric administration of fecal bacteria solution, the horizontal motor score of the MO group was significantly lower than that of the BC group ( P <0.05); compared with the MO group, the horizontal motor score of the EA group was higher than that of the MO group ( P <0.05).

[0073] It can be seen that compared with the depression model rats gavaged with fecal microbial fluid in the model group (MO group), the fecal microbial fluid in the electroacupuncture group (EA group) can improve the horizontal movement of the depression model rats;

[0074] (2) Vertical Movement Score As shown in Table 18, there were no significant differences in the vertical movement scores of the open field test among the groups during the baseline period (before treatment). P >0.05); there was no significant difference in the overall vertical movement score after intragastric administration of antibiotics ( P =0.199>0.05); after CUMS treatment and intragastric administration of fecal bacteria solution, the vertical movement score of the MO group was significantly lower than that of the BC group ( P <0.05); compared with the MO group, the vertical movement score of the EA group was higher than that of the MO group ( P <0.05).

[0075] It can be seen that compared with the depression model rats gavaged with fecal microbial fluid in the model group (MO group), the fecal microbial fluid in the electroacupuncture group (EA group) can improve the vertical movement ability of the depression model rats;

[0076] Therefore, based on the above research content, it can be seen that fecal bacteria fluid in the electroacupuncture group can improve the depressive-like behavior of depression model rats.

[0077] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for preparing intestinal bacterial liquid by electroacupuncture intervention in CUMS rats, characterized in that: The following steps are involved: 1) CUMS rats were stimulated with CUMS every day and electroacupunctured at least 5 times a week, and then the feces of the rats were collected; The electroacupuncture stimulation is specifically as follows: using acupuncture needles to puncture the Baihui point in the middle of the parietal bone, the Yintang point 2mm above the midpoint of both eyes, and the Zusanli point 3mm below the fibular head at the posterior and lateral sides of the knee joint; the puncture direction of the Baihui point is toward the back of the head, the Yintang needle tip is toward the nose tip, and the needle insertion depth is 2-3mm; the Zusanli point is punctured directly, and the needle insertion depth is 3-5mm; the acupuncture needles at the Baihui point and the Yintang point are connected to the electroacupuncture instrument, the current frequency is 8-12Hz, continuous wave, the current intensity is 1-3mA, and each time is 15-25min; 2) The rat feces was mixed with PBS solution, and after shaking, precipitation, centrifugation and filtration, the intestinal bacterial solution was obtained.

2. The method according to claim 1, characterized in that: The current frequency is 10 Hz, the current intensity is 2 mA, and each time is 20 minutes.

3. The method according to claim 1, characterized in that The stimulation factors of the CUMS stimulation include swimming in 4°C ice water for 5 minutes, day and night reversal, fasting for 24 hours, water deprivation for 24 hours, restraint for 2 hours, wet bedding for 24 hours, and tail clamping for 3 minutes, a total of 7 factors, and the stimulation factors are not repeated in two consecutive days.

4. The method according to claim 1, characterized in that The ratio of rat feces to PBS solution is 0.1g:1-2ml.

5. The method according to claim 1, characterized in that The step 1) further comprises placing the rat feces in a sterile enzyme-free EP tube, first placing it in dry ice, and then freezing it at -80°C; the step 2) further comprises thawing the frozen rat feces at room temperature and then mixing it with the PBS solution.

6. An intestinal bacterial liquid, characterized in that: The method is prepared according to any one of claims 1 to 5.

7. The intestinal bacterial liquid according to claim 6, characterized in that At the genus level, the relative abundance of Lactobacillus was 26.0-27.0%; the relative abundance of Akkermansia was 19.0-20.0%; the relative abundance of Clostridium_sensu_stricto_1 was 12.0-13.0%; the relative abundance of Ruminococcaceae_UCG-01 was 10.0-11.0%; the relative abundance of Romboutsia was 8.0-9.0%; the relative abundance of Allobaculum was 7.0-8.0%; the relative abundance of uncultured was 6.0-7.0%; the relative abundance of Turicibacter was 3.0-4.0%; the relative abundance of Lachnospiraceae_NK4A136_group was 1.0-2.0%; and the relative abundance of Alistipes was 1.0-2.0%.

8. Use of the intestinal bacterial liquid as claimed in claim 6 or 7 in a drug for relieving depression.

9. A drug for relieving depression, characterized in that: Contains the intestinal bacterial liquid as described in claim 6 or 7.

10. A drug for relieving depression, characterized in that: A freeze-dried powder comprising the intestinal bacterial liquid as claimed in claim 6 or 7.

Images