Method for researching antidepressant bacteria intestinal brain axis mechanism through electroacupuncture

Abstract

The invention discloses a method for researching an electroacupuncture anti-depression bacteria intestinal brain axis mechanism, and relates to the technical field of electroacupuncture anti-depression mechanism research. According to the method for researching the antidepressant bacterium intestinal-brain axis mechanism through the electroacupuncture, the situation that acupuncture possibly exerts the antidepressant effect on the basis of a bacterium-intestine-brain axis multi-target mechanism is revealed from the three levels of bacterium / metabolite, intestine and brain in a hierarchical progressive and one-by-one analysis mode, the action level of acupuncture is revealed in multiple aspects, the regulation effect of acupuncture is explored from the macroscopic perspective, and the research on the antidepressant bacterium intestinal-brain axis mechanism is achieved. The research depth of a mechanism for acupuncture therapy of depression is deepened, and an experimental basis is provided for acupuncture therapy of MDD.

Description

Technical Field

[0001] The present invention relates to the technical field of electroacupuncture antidepressant mechanism research, and in particular to a method for studying the electroacupuncture antidepressant bacteria-gut-brain axis mechanism. Background Art

[0002] Depression (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. The definition of depression in traditional Chinese medicine is relatively consistent with the definition of MDD in modern medicine. Traditional Chinese medicine has considerable advantages and potential in the prevention and treatment of MDD. Clinical practice has found that people with subclinical depression symptoms or MDD who are not suitable for Western medicine have a high acceptance of traditional Chinese medicine, acupuncture and other therapies. The most commonly used acupoints for acupuncture treatment of depression are Yintang, Zusanli, Baihui, Sanyinjiao, Taichong, Neiguan, Shenmen, Zhongwan, Qihai, and Shenting. The high-frequency associated acupoint groups are Baihui-Yintang, Baihui-Yintang-Zusanli, Baihui-Neiguan-Taichong, Baihui-Shenting-Taichong, and Baihui-Sanyinjiao. Acupoints on the Du Meridian, Ren Meridian, Stomach Meridian of Foot Yangming, and Kidney Meridian of Foot Shaoyin are commonly used. As an extremely important component of traditional Chinese medicine, acupuncture has made outstanding contributions to the prevention and treatment of depression in clinical practice due to its high safety, significant efficacy, rapid results, and good doctor-patient interaction.

[0003] Due to the complexity of MDD and the characteristics of multi-system involvement, its pathogenesis is still unclear. At present, the main studies on the pathological mechanism of depression include epigenetic theory, neurotransmitter theory, brain-derived neurotrophic factor (BDNF) theory, hypothalamus-pituitary-adrenal (HPA) axis theory, neuroimmunology theory, and bacteria-gut-brain axis theory. Among them, the bacteria-gut-brain axis is a comprehensive physiological concept involving afferent and efferent nerves, endocrine and immune signals between the central nervous system and the gastrointestinal system. The concept of the bacteria-gut-brain axis was originally established mainly through the study of the connection between digestive function and satiety. However, this concept has now developed to describe the gastrointestinal system, especially the interaction between intestinal microorganisms and the central nervous system. The intestine can regulate the emotional activity of the central nervous system, and the central nervous system can also regulate gastrointestinal function in turn. The bacteria-gut-brain axis is completed with the joint participation of microorganisms, the immune system, and the nervous system. It connects the neuroimmunology theory of depression in series, which is a comprehensive and complete chain of evidence with important research value.

[0004] Acupuncture treatment for MDD has been proven to be effective in a large number of studies, but due to the lack of sufficient high-quality evidence, acupuncture has not yet been recommended as a mainstream method for treating depression. CANMAT lists acupuncture as a third-line treatment for mild to moderate MDD, while the first and second-line treatments are still conventional treatments. In 2016, the American College of Physicians (ACP) developed the "Guidelines for the Treatment of Depression with Drug and Non-Drug Therapies", which compared the efficacy and safety of acupuncture therapy and second-generation antidepressants, but due to the low level of evidence, it was ultimately unable to form a recommendation related to acupuncture therapy.

[0005] The clinical efficacy of acupuncture in treating MDD has been widely reported, but the mechanism of action of acupuncture in treating MDD is still under exploration. Current research on the mechanism of acupuncture in treating MDD mainly includes the following aspects: (1) Acupuncture has a regulatory effect on monoamine neurotransmitters and their receptors; (2) Acupuncture can regulate the balance of inhibitory and excitatory amino acids; (3) Acupuncture can increase the expression of neuropeptides; (4) Acupuncture can regulate the function of the hypothalamus-pituitary-adrenocortical and hypothalamus-pituitary-thyrotropin axes through systemic neuroendocrine.

[0006] However, there is currently insufficient research on the comprehensive exploration of the mechanism of acupuncture treatment for depression based on the "bacteria-gut-brain axis" theory. More in-depth research is needed to connect the dots with a more complete chain of evidence, so it has important potential for exploration. Summary of the invention

[0007] One of the purposes of the present invention is to explore whether acupuncture can regulate the conduction of intestinal metabolites and inflammatory factor signals by changing the diversity of the flora and species differences, and reduce the inflammatory response of the central nervous system by inhibiting the TRL4 / NF-κB pathway in the hippocampus, so as to improve depressive-like behavior, based on the theory of "bacteria-gut-brain axis" and taking the intestinal flora as the target.

[0008] The second purpose of the present invention is to explore the effects of electroacupuncture on the chronic mild unpredictable stress (CUMS) rat model based on the "bacteria-gut-brain axis" theory through animal experiments, and to analyze from different levels such as animal behavior, intestinal flora, targeted quantification of metabolites, inflammatory factors, tissue morphology and structure, gene quantification, and protein expression, to explore whether electroacupuncture can treat depression by regulating the host inflammatory cytokine transduction pathway through intestinal microorganisms.

[0009] The third purpose of the present invention is to use the chronic mild unpredictable stimulation rat model as the research object, study the antidepressant effect of electroacupuncture and the intervention of the intestinal flora in depression to regulate the host's humoral pathways, analyze the effects of electroacupuncture on intestinal flora and its metabolites, the effects on inflammatory cytokines and the expression of inflammatory response-related proteins, reveal the mechanism of action of electroacupuncture in improving depression, provide experimental basis for acupuncture treatment of MDD, and enrich the scientific connotation of "acupuncture to regulate the mind".

[0010] To achieve the above-mentioned purpose, the method for studying the mechanism of electroacupuncture on the antidepressant bacteria gut-brain axis of the present invention comprises the following steps: 1) Modeling and intervention SPF healthy male adult Sprague-Dawley rats were divided into a blank group, a model group, an electroacupuncture group, an electroacupuncture combined with antibiotics group, and an electroacupuncture combined with probiotics group. Except for the blank group, rats in the model group, the electroacupuncture group, the electroacupuncture combined with antibiotics group, and the electroacupuncture combined with probiotics group were subjected to chronic mild unpredictable stimulation every day to establish a rat model of depression. After modeling, rats in each group were intervened according to the following methods: rats in the blank group were allowed to eat and drink freely and were gavaged with normal saline; rats in the model group were given chronic mild unpredictable stimulation every day and were gavaged with normal saline; rats in the electroacupuncture group were given chronic mild unpredictable stimulation every day, electroacupuncture stimulation every week and were gavaged with normal saline; rats in the electroacupuncture plus antibiotics group were given chronic mild unpredictable stimulation every day, electroacupuncture stimulation every week and were gavaged with antibiotics; rats in the electroacupuncture plus probiotics group were given chronic mild unpredictable stimulation every day, electroacupuncture stimulation every week and were gavaged with probiotics; Specifically, the electroacupuncture stimulation comprises the following steps: 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; under 2.5% to 3% isoflurane inhalation anesthesia, the Baihui and Yintang points are punctured horizontally, with the Baihui point puncturing toward the back of the head and the Yintang point puncturing toward the nose tip; the needle insertion depth is 2-3mm; the Zusanli point is punctured vertically, with the needle insertion depth of 3-5mm; the acupuncture needles at the Baihui and Yintang points are connected to an electroacupuncture instrument, with a current frequency of 8-12Hz, continuous wave, and a current intensity of 1-3mA, for 15-25min each time; 2) Effects of electroacupuncture on intestinal microorganisms and metabolites in CUMS rats The feces of rats in each group were collected before modeling, after modeling, and after intervention; The effects of electroacupuncture on intestinal microorganisms and metabolites of CUMS rats were explored by performing species community analysis, Alpha diversity analysis, Beta diversity analysis, and species difference analysis on the feces of rats in each group after intervention, and extracting short-chain fatty acids from the feces of rats in each group before, after, and after modeling to conduct targeted SCFAs quantitative analysis. 3) Effects of electroacupuncture on colon morphology and inflammatory cytokines in CUMS rats The colon tissues of rats in each group after intervention were subjected to hematoxylin-eosin staining and enzyme-linked immunosorbent assay to explore the effects of electroacupuncture on colon morphology and inflammatory cytokines in CUMS rats. 4) Effects of electroacupuncture on inflammatory cytokine transduction pathways in the hippocampus of CUMS rats The effects of electroacupuncture on the inflammatory cytokine transduction pathway in the hippocampus of CUMS rats were explored by using hematoxylin-eosin staining, fluorescent quantitative polymerase chain reaction and protein immunoblotting experiments on the brain tissue of rats in each group after intervention.

[0011] Preferably, each group of rats after modeling is intervened according to the following method: Blank group: free food and water intake, 20 min isoflurane anesthesia 5 times a week, and normal saline gavage for 4 weeks; Model group: Chronic mild unpredictable stimulation was performed every day, isoflurane anesthesia was given for 20 min 5 times a week, and normal saline was gavaged for 4 weeks; Electroacupuncture group: daily chronic mild unpredictable stimulation, intragastric administration of normal saline for 4 weeks, electroacupuncture stimulation 5 times a week; The EA plus antibiotic group: daily chronic mild unpredictable stimulation, intragastric administration of antibiotics for 2 weeks, intragastric administration of saline for 2 weeks, and EA stimulation 5 times a week; The electroacupuncture plus probiotics group: chronic mild unpredictable stimulation was performed every day, probiotics were gavaged for 4 weeks, and electroacupuncture stimulation was performed 5 times a week.

[0012] Preferably, the current frequency of the electroacupuncture stimulation is 10 Hz, the current intensity is 2 mA, and each time is 20 minutes.

[0013] Preferably, the stimulation factors of chronic mild unpredictable sexual 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 seven, and the stimulation factors are not repeated in two consecutive days.

[0014] Preferably, in the step 2), after collecting the feces of the rats in each group before modeling, after modeling, and after intervention, the rat feces are placed in sterile enzyme-free EP tubes, first placed in dry ice, and then frozen at -80°C.

[0015] The method for studying the antidepressant bacteria-gut-brain axis mechanism of electroacupuncture, analyzes three levels, bacteria / metabolites, intestines, and brain, in a hierarchical manner, one by one, and reveals that acupuncture may be based on the multi-target mechanism of the "bacteria-gut-brain axis" to exert its antidepressant effect, reveals the action levels of acupuncture from multiple perspectives, explores the regulatory role of acupuncture from a macroscopic perspective, deepens the research on the mechanism of acupuncture treatment for depression, embodies the holistic view of traditional Chinese medicine, and provides an experimental basis for acupuncture treatment of MDD.

[0016] The specific mechanism of action of electroacupuncture in improving depression is as follows: electroacupuncture can regulate the homeostasis of intestinal flora, reduce harmful bacteria, increase beneficial bacteria, repair intestinal mucosal damage by upregulating short-chain fatty acids such as propionate and butyrate, activate the production of anti-inflammatory factor IL-10, inhibit the production of proinflammatory factors IL-6, TNF-α and IL-1β in the intestine, reduce the expression of TLR4, NF-κB and various proinflammatory factors in the hippocampus of the central nervous system, block the occurrence of inflammatory cascade reactions, and improve depressive-like behavioral manifestations. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0018] Figure 1 Acupuncture point diagram of rats stimulated by electroacupuncture according to the present invention; Figure 2 This is a diagram of the open field experiment of rats of the present invention; Figure 3 It is the experimental flow chart of the present invention; Figure 4 This is a comparison of the appearance of CUMS model rats and blank group rats; Figure 5 Comparison of body weight of rats in each group at different time points; Figure 6 This is a comparison of the sugar water preference rate of rats in each group at different time points; Figure 7 This is a comparison chart of the horizontal motor scores of the open field test in each group of rats at different time points; Figure 8 This is a comparison of the vertical movement scores of the open field test in each group of rats at different time points; Fig. 9 The activity trajectory of rats in the open field after modeling (0d) and after intervention (28d); Fig.10 This is the OTU Venn diagram of the intestinal flora of rats in each group; Fig.11 Dilution curve, Shannon curve and Rank-Abundance curve of α-diversity analysis of intestinal flora of rats in each group; Fig.12 This is a pairwise comparison of Chao1 and Simpson index of intestinal flora of rats in each group; Fig.13 The relative abundance diagram of the intestinal flora genus level of rats in each group; Fig.14 This is the species evolution branch diagram of the model group vs. the blank group; Fig.15 Bar chart of LDA values ​​of blank group vs. model group; Fig.16 The species evolution branch diagram of the model group vs. the electroacupuncture group; Fig.17 Bar chart of LDA values ​​of model group vs. electroacupuncture group; Fig.18 The species evolution branch diagram for the model group vs. the electroacupuncture combined with antibiotics group; Fig.19 Bar graph of LDA values ​​of model group vs. electroacupuncture plus antibiotic group; Fig. 20 The species evolution branch diagram for the model group vs. the electroacupuncture plus probiotics group; Fig.21 Bar chart of LDA values ​​of model group vs. electroacupuncture plus probiotics group; Fig. 22 Total ion currents of short-chain fatty acids in mixed standards (A) and samples (B); Fig.23 The graphs show the acetate content in the feces of rats in each group at different time points; Fig.24 Figure 2 shows the fecal propionate content of rats in each group at different time points; Fig.25 Figure 2 is the fecal butyrate content of rats in each group at different time points; Fig.26 This is a comparison of the colon tissue morphology of rats in each group; Fig. 27 This is a comparison of inflammatory factors in colon tissue of rats in each group; Fig.28 This is a comparison of the histomorphology of the hippocampus in each group of rats; Fig.29 This is a comparison of the gene expressions of inflammatory cytokines and related proteins in the hippocampus of rats in each group; Fig.30 The immunoblot images of inflammatory cytokines and related proteins in the hippocampus of rats in each group; Fig.31 The relative expression levels of inflammatory cytokines and related proteins in the hippocampus of rats in each group. DETAILED DESCRIPTION

[0019] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments.

[0020] The English abbreviations involved in the present invention are shown in the following table:

[0021] Section 1 Research Content The content of the present invention on the mechanism of action of electroacupuncture in treating depression is as follows: 1. Effects of electroacupuncture on the behavior of CUMS rats The CUMS animal model, which is widely recognized to be similar to human depression, was used. This experiment used the CUMS method to establish the model for 35 days and then conducted 28 days of electroacupuncture intervention. The success of the CUMS model and the effect of electroacupuncture on the behavior of CUMS model rats were evaluated by observing the changes in rat body weight, sugar water preference rate, and open field test scores. 2. Effects of electroacupuncture on intestinal microorganisms and metabolites in CUMS rat model This experiment used a 35-day CUMS animal model and performed 28 days of electroacupuncture intervention. The fecal bacteria diversity, bacterial genus, and short-chain fatty acid content of rats in each group were compared using 16sDNA sequence detection and gas chromatography-mass spectrometry analysis to explore the effects of electroacupuncture on intestinal microorganisms and their metabolites in CUMS model rats. 3. Effects of electroacupuncture on colon morphology and inflammatory factors in CUMS rats This study used a 35-day CUMS animal model and performed 28 days of electroacupuncture intervention. Hematoxylin and eosin staining and enzyme-linked immunosorbent assay were used to compare the colon morphology and inflammatory cytokine levels of rats in each group to explore the effects of electroacupuncture on colon morphology and inflammatory factors in CUMS model rats. 4. Effects of electroacupuncture on inflammatory cytokine transduction pathways in the hippocampus of CUMS rats This study used a 35-day CUMS animal model and performed 28 days of electroacupuncture intervention. Hematoxylin and eosin staining experiments were used to observe the morphological changes in the hippocampus of CUMS rats treated with electroacupuncture. Fluorescence quantitative polymerase chain reaction experiments and protein immunoblotting experiments were used to compare the phenotypes of inflammatory pathway-related factors in the hippocampus of rats in each group to explore the effect of electroacupuncture on the inflammatory response of the hippocampus of CUMS model rats.

[0022] Section 2 Effects of electroacupuncture on the behavior of CUMS rats 1. Materials 1. Experimental animals and groups 40 SPF-grade healthy male adult Sprague-Dawley (SD) rats, aged 7 weeks 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 access to water (sterile water) and free access to food (growth and reproduction pellet feed). This experiment was reviewed and approved by the Ethics Committee of Zhongke Industry Holdings (Shenzhen) Co., Ltd. (Ethics Approval No.: 2022084). After 7 days of adaptive feeding, the 40 SD rats were randomly divided into a blank group (n=8), a model group (n=8), an electroacupuncture group (n=8), an electroacupuncture combined with antibiotics group (n=8), and an electroacupuncture combined with probiotics group (n=8), as shown in Table 1. Marking with ear tags:

[0023] II) Instruments, Equipment and Reagents The instruments and equipment involved in this section are shown in Table 2:

[0024] The reagents and consumables involved in this section are shown in Table 3:

[0025] 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, EA group, AA group, and AP 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:

[0026] 2. Intervention methods Blank group: The mice were allowed to eat and drink freely, and were anesthetized with isoflurane for 20 min 5 times a week and gavaged with normal saline for 4 weeks.

[0027] Model group: CUMS stimulation was performed every day, see modeling operation for details, isoflurane anesthesia was given for 20 min 5 times a week, and normal saline was gavaged for 4 weeks.

[0028] Electroacupuncture group: CUMS stimulation every day. Normal saline gavage for 4 weeks. Electroacupuncture stimulation 5 times a week. The needles used were 0.2*25mm disposable sterile acupuncture needles from Suzhou Medical Supplies Factory Co., Ltd. The specific locations of acupoints were determined according to relevant literature. Acupoint selection and positioning: Baihui: the middle of the parietal bone; Yintang: 2mm above the midpoint of both eyes; Zusanli: 3mm below the head of the fibula at the posterior and lateral sides of the knee joint. (such as Figure 1 Method: Under 2.5% to 3% isoflurane inhalation anesthesia, insert the needle into Baihui and Yintang acupoints horizontally (put a sterilized cotton ball between the two needle handles to prevent short circuit), the direction of the needle is Baihui toward the back of the head, and the tip of the Yintang needle is toward the tip of the nose; the depth of the needle is 2-3mm. The Zusanli acupoint is inserted vertically, and the depth of the needle is 3-5mm. Connect the needle handles of Baihui and Yintang acupoints to the Han's electroacupuncture instrument, the current frequency is 10Hz, continuous wave, the current intensity is 2mA, and each time is 20min.

[0029] Electroacupuncture plus antibiotic group: CUMS stimulation every day. Antibiotics were gavaged for 2 weeks, freshly prepared daily (mixing method: according to the cocktail mixing method). Normal saline was gavaged for 2 weeks. Electroacupuncture stimulation was performed 5 times a week, the method was the same as above.

[0030] Electroacupuncture plus probiotics group: CUMS stimulation was performed every day. Probiotics were gavaged for 4 weeks. Electroacupuncture stimulation was performed 5 times a week, using the same method as above.

[0031] 3. Behavioral testing 1. 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%; 2. Open field test The open field test was performed before modeling (-35 days), 5 weeks after modeling (0 days), and at the end of intervention (28 days). The rats were placed in an open box with a size of 100 cm*100 cm*50 cm. The box was an opaque black open box with 25 small squares (20*20 cm) of equal size (such as Figure 2The test takes 3 minutes. At the beginning of the test, the rat is placed in the center of the box bottom and the camera is turned on to record the rat's activities. If the rat crosses the grid horizontally within 3 minutes and enters the same grid with at least three paws, it is counted as one horizontal movement and one point; if both front paws are lifted off the ground at the same time (including combing hair), it is counted as one vertical movement and one point. After each rat is tested, 75% alcohol is used to clean the box to avoid interfering with the next rat; 3. Body mass Body weight was measured before modeling (-35 days), 5 weeks after modeling (0 days), and at the end of intervention (28 days). 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.

[0032] (IV) Experimental process The experimental process is as follows Figure 3 shown.

[0033] 5. Statistical 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.

[0034] 3. Results 1. Observation of physical signs of rats after CUMS modeling The appearance, activity and mental state of CUMS model rats were observed. Figure 4As shown, Figure A is a CUMS model rat, and Figure B is a blank group rat. After the CUMS modeling, the blank group rats were well-proportioned and plump, with white and shiny fur, active, docile, less stress response, and oblong stools. The CUMS modeling rats were thin, with yellow and withered fur, slow or overreacted reactions, and occasionally loose stools; 2. Effect of electroacupuncture on body weight of CUMS model rats The body weight of rats in each group at different time points is shown in Table 5. The body weight of rats in each group at different time points is shown in Table 5. Figure 5 (Note: * Comparison between groups P <0.05; **comparison between groups P <0.01). From Table 5, Figure 5 It can be seen that: Before modeling, there was no statistically significant difference between the groups ( P >0.05), indicating that the baseline is balanced and comparable.

[0035] After modeling, the overall differences among the groups were statistically significant. P =0.001; compared with the BC group, the body weight of the MO group, EA group, AA group, and AP group was lower than that of the BC group, and the difference was statistically significant ( P <0.05), and there was no statistically significant difference in the other pairwise comparisons ( P >0.05).

[0036] After intervention, the overall differences among the groups were statistically significant. P＜0.001 Compared with the BC group, the body weight of the MO group, AA group, and AP 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 weight of the AP group and the EA group were higher than that of the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the body weight of the EA group was higher than that of the AA group, and the difference was statistically significant ( P <0.05).

[0037] From the above, we can see that the body weight of CIMS model rats is lower than that of normal rats, and acupuncture treatment can partially reverse the weight loss of depressed rats:

[0038] 3. Effect of electroacupuncture on sugar water preference rate in CUMS model rats The sugar water preference rates of rats in each group at different time points are shown in Table 6. Figure 6 (Note: * Comparison between groups P <0.05; **comparison between groups P<0.01;***comparison between groups P <0.001). From Table 6, Figure 6 It can be seen that: Before modeling, there was no statistically significant difference in the sugar water preference rate among the rats in each group ( P >0.05), indicating that the baseline is balanced and comparable.

[0039] After modeling, the overall differences among the groups were statistically significant. P =0.001; compared with the BC group, the sugar water preference rates of the MO group, EA group, AA group, and AP group were lower than those of the BC group, and the differences were statistically significant ( P <0.05), and there was no statistically significant difference in the other pairwise comparisons ( P >0.05).

[0040] After intervention, the overall difference in sugar water preference rate among the groups was statistically significant. P＜0.001 ; Compared with the BC group, the sugar water preference rate in the MO and AA groups was lower than that in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the AP and EA groups had higher sugar water preference rates than the MO group, and the difference was statistically significant ( P <0.05). Compared with the AA group, the AP group had a higher sugar water preference rate than the AA group ( P <0.05), the difference was statistically significant.

[0041] From the above, we can see that the preference rate of sugar water in CUMS model rats is reduced, and acupuncture treatment can increase the preference rate of sugar water and alleviate the core symptom of "reduced interest" in depressed rats;

[0042] 4. Effect of electroacupuncture on open field scores of CUMS model rats (1) Horizontal movement score in the open field test The horizontal motor scores of the rats in the open field test at different time points in each group are shown in Table 7. The horizontal motor scores of the rats in the open field test at different time points in each group are shown in Table 7. Figure 7 (Note: * Comparison between groups P <0.05; **comparison between groups P <0.01). From Table 7, Figure 7 It can be seen that: Before modeling, there was no statistically significant difference among the rat groups ( P >0.05), indicating that the baseline is balanced and comparable.

[0043] After modeling, the overall differences among rats in each group were statistically significant. P＜0.001; Compared with the BC group, the horizontal motor scores of the MO group, EA group, AA group, and AP group were lower than those of the BC group, and the difference was statistically significant ( P <0.05), and there was no statistically significant difference in the other pairwise comparisons ( P >0.05).

[0044] After intervention, the overall differences among rats in each group were statistically significant. P＜0.001 Compared with the BC group, the horizontal exercise scores of the MO group and the AA group were lower than those of the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the horizontal exercise scores of the AP group and the EA group were higher than those of the MO group, and the difference was statistically significant ( P <0.05). Compared with the AA group, the horizontal movement scores of the AP and EA groups were higher than those of the AA group, and the difference was statistically significant ( P <0.05);

[0045] (2) Vertical movement score of open field test The vertical movement scores of the rats in the open field test at different time points in each group are shown in Table 8. The vertical movement scores of the rats in the open field test at different time points in each group are shown in Table 8. Figure 8 (Note: * Comparison between groups P <0.05; **comparison between groups P <0.01). From Table 8, Figure 8 As shown: Before modeling, there was no statistically significant difference among the rat groups ( P >0.05), indicating that the baseline is balanced and comparable.

[0046] After modeling, the overall difference in vertical movement scores among the groups was statistically significant. P＜0.001 Compared with the BC group, the vertical movement scores of the MO group, EA group, AA group, and AP group were lower than those of the BC group, and the differences were statistically significant ( P <0.05), and the other pairwise comparisons were not statistically significant ( P >0.05).

[0047] After intervention, the overall difference in vertical movement scores among the groups was statistically significant. P＜0.001 ; Compared with the BC group, 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 AP group and the EA group were higher than those of the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the vertical movement scores of the EA and AP groups were higher than those of the AA group, and the difference was statistically significant ( P <0.05);

[0048] The activity trajectories of rats in the open field after modeling (0d) and after intervention (28d) are shown in Figure 2. Fig. 9 As shown, the left picture shows the open field activity trajectory of rats after modeling (0d), and the right picture shows the activity trajectory of rats after intervention (28d) (Note: Picture A is the blank control group, Picture B is the model group, Picture C is the electroacupuncture group, Picture D is the electroacupuncture combined with antibiotics group, and Picture E is the electroacupuncture combined with probiotics group).

[0049] IV. Summary From the above research content, we can know that: 1) Compared with the blank group, the CUMS modeling rats in each group had a lower sugar consumption after 35 days of modeling, and the difference was statistically significant (P < 0.05), body weight decreased, and the difference was statistically significant (P < 0.05), exploratory behavior decreased, and horizontal and vertical movement activities decreased, and the difference was statistically significant (P < 0.05), indicating that the CUMS model group rats had anhedonia and reduced desire for exploration, indicating that the modeling was successful. (2) After intervention, compared with the model group, the EA group and the EA plus probiotics group rats had an increase in sugar consumption and body weight after 28 days of intervention, and the difference was statistically significant (P < 0.05), exploratory behavior recovered, and horizontal and vertical movement activities increased, and the difference was statistically significant (P < 0.05). The above results show that electroacupuncture has a significant reversal effect on the phenomenon of reduced sugar water consumption in rats under CUMS stimulation. The electroacupuncture combined with probiotics group increases the regulatory effect on the target - intestinal microorganisms on the basis of the therapeutic effect of electroacupuncture. The results of this part show that the electroacupuncture combined with probiotics group also has a significant effect on reversing the low consumption of sugar water in CUMS rats; while the electroacupuncture combined with antibiotics group cannot have a significant positive effect on the low consumption of sugar water in CUMS rats. It can be seen that the effectiveness of electroacupuncture cannot resist the disruptive effect of antibiotics on intestinal flora. The results are consistent with the theoretical hypothesis based on the regulation of host behavior by intestinal flora. Secondly, electroacupuncture has a significant improvement effect on the reduction of CUMS rats' exploratory behavior. The electroacupuncture combined with probiotics group also shows significant effectiveness in improving the behavior of CUMS rats. Consistent with the results of the sugar water preference experiment, the electroacupuncture combined with antibiotics group, like the model group, has a significant reduction in exploratory behavior, which is significantly lower than that of the blank group rats. The results of this part of the study show that electroacupuncture can improve the depressive-like behavior of rats. (3) Through the analysis of the body weight of rats in each group, it was found that the body weight of the model group was significantly lower than that of the blank group, the electroacupuncture group, and the electroacupuncture plus probiotics group. Since this study adopted a free-feeding feeding method, it is impossible to accurately understand the intake of each rat. There are two possible reasons for this analysis: (i) Since the body is under continuous stress, it may have a reduced intake. The reduced appetite may be related to the increase of leptin in the body under depression. It is a recognized and key factor regulating eating behavior and energy consumption. It can enter the brain through the blood-brain barrier. The expression of leptin mRNA and protein is positively correlated with the severity of depression. (ii) Changes in the abundance of some intestinal flora species are also related to weight loss. The third section of this study will further detect and analyze the fecal bacteria of each group of rats in order to find the potential reasons behind it.

[0050] Section 3 Effects of electroacupuncture on intestinal microorganisms and metabolites in CUMS rats 1. Materials 1. Experimental animals and groups Same as Section 2.

[0051] 2. Instruments, Equipment and Reagents The instruments and equipment involved in this section are shown in Table 9:

[0052] The reagents and consumables involved in this section are shown in Table 10:

[0053] 2. Methods 1. Feces collection The fecal tissue samples of this experiment were from the rats in Section 2. The feces of the rats were collected before modeling (-35 days), after modeling (0 days), and after intervention (28 days). The rats were placed in a sterilized, clean, and bedding-free cage. After the rats defecate, the feces of the rats were collected with sterilized tweezers. 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 tubes were first placed in dry ice and frozen in a -80°C refrigerator within 2 hours.

[0054] 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.

[0055] 3. PCR amplification Using genomic DNA as template, PCR amplification was performed using specific primers with barcodes and TaKaRa Premix Taq® Version 2.0 (TaKaRa Biotechnology Co., Dalian, China) according to the selection of the sequencing region. The sequencing fragment was: bacterial 16S V4-V5.

[0056] 1. Primer sequences The PCR amplification primer sequences are shown in Table 11:

[0057] 2. Reaction conditions The reaction was performed at 53°C for 30 seconds for 31 cycles. Each sample was repeated three times and the PCR products of the same sample were mixed.

[0058] (IV) 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).

[0059] 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).

[0060] 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.

[0061] 4. Species community analysis (1) Species community structure: R software was used 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.

[0062] 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.

[0063] 6. Beta (β) diversity analysis The components of each sample at different classification levels were calculated based on different distance algorithms (Bray-Curtis and Euclidean, etc.), and the distance difference matrix was calculated. The Anosim analysis method was used to test the significance of the differences in community structure between groups.

[0064] 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.

[0065] 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 perform FDR correction.

[0066] All data analysis in this experiment was completed using the MagiCloud platform (http: / / cloud.magigene.com / ) provided by MagiGene Technology, the testing company.

[0067] 5. Extraction of short-chain fatty acids (SCFAs) from rat feces The experimental samples in this part were fresh feces from rats before modeling (-35d), after modeling (0d) and after intervention (28d). The fecal samples were thawed naturally in an ice bath, and 100 mg of fecal samples were weighed into a 2.0 mL centrifuge tube, 600 μL of deionized water was added, vortexed for 20 s, shaken for 30 min at 4 °C, and centrifuged at 10000 r / min for 10 min. 300 μL of supernatant was transferred to a 2.0 mL centrifuge tube, 20 μL of 5M hydrochloric acid solution was added, and then 300 μL of chromatographic grade ethyl acetate was added, vortexed for 60 s, incubated in an ice bath for 5 min, centrifuged at 10000 r / min for 10 min, and all the ethyl acetate layer solution was aspirated. The lower aqueous solution was extracted once again using the above method. The two ethyl acetate solutions were combined, an appropriate amount of anhydrous sodium sulfate was added, vortexed for 60 s, centrifuged at 10000 r / min for 10 min, and all the ethyl acetate extracts were aspirated for subsequent analysis.

[0068] 6. Targeted SCFAs quantitative analysis based on gas chromatography-mass spectrometry (GC-MS) technology 1. Short-chain fatty acid derivatization reaction Accurately pipette 100 μL of standard curve working solution or fecal ethyl acetate extract into a glass insert tube, add 10 μL of internal standard solution and 5 μL of BSTF derivatization reagent, mix well, and react overnight at room temperature (22 °C) for GC-MS analysis.

[0069] 2. GC-MS analysis conditions The samples were separated using an Agilent HP-5MS capillary column (30 m×0.25 mm×0.25 μm). The carrier gas was nitrogen, the carrier gas flow rate was 1.0 mL / min, the injection port heater temperature was 260 °C, the split ratio was 10:1, and the injection volume was 1 μL. The column temperature program was as follows: the initial temperature was 40 °C for 2 min, then increased to 140 °C at a rate of 15 °C / min and maintained for 1 min, and then increased to 280 °C at a rate of 100 °C / min and maintained for 1 min. The mass spectrometer was an EI ion source, the transfer line temperature was 280 °C, the quadrupole temperature was 150 °C, the full scan and SIM scan modes, the collision electron energy was 70 eV, and the scan range was m / z40~300. The quantitative information is shown in Table 12:

[0070] 3. Data processing The software was used to extract the chromatographic peak area and retention time, calculate the standard curve, and obtain the content of short-chain fatty acids in the sample.

[0071] 7. Statistical methods Same as Section 2.

[0072] 3. 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.

[0073] 1. Effect of electroacupuncture on intestinal flora of CUMS rats 1. Venn diagram of OTUs in each group The number of OTU sequences in each group was analyzed using a Venn diagram, and the similarities and differences in species distribution among different groups were compared from the perspective of the number of OTU sequences. Fig.10 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 electroacupuncture combined with antibiotics group (AA group) and model group (MO group) is second only to the blank group, while the number of specific OTU sequences in the electroacupuncture group (EA group) and electroacupuncture combined with probiotics group (AP group) is relatively small, indicating that different intervention methods have a certain effect on the number of species.

[0074] 2. Analysis of α-diversity of intestinal flora in each group of rats Rarefaction curves, Shannon curves, and Rank-Abundance curves are used to intuitively reflect the test depth of sequencing results, such as Fig.11 As shown in the figure, the rarefaction curve and Shannon curve tend to be flat, indicating that the sequencing depth of each group of samples is sufficient. The Rank-Abundance curve directly reflects the richness of species. The longer the overall length of the curve on the horizontal axis, the richer the species composition; the uniformity of species composition is reflected by the curvature of the curve. The lower the curvature of the curve, the higher the uniformity of species composition. There is no difference in the overall length of the curves of each group on the horizontal axis; the curve of the BC group is relatively flat, indicating that the species uniformity of this group is higher.

[0075] 3. Effect of electroacupuncture intervention on the α-diversity of intestinal flora in CUMS rats 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 Simpson index reflects community diversity and is used to estimate one of the microbial diversity indices in the sample. The larger the Simpson index, the lower the species community diversity. The Chao1 and Simpson indices of each group were compared pairwise. Fig.12 As shown in the results, the Chao1 index was significantly different between the electroacupuncture plus antibiotic group and the electroacupuncture plus probiotic group after intervention. P =0.015), there were no statistically significant differences between the other two groups; there were no statistically significant differences in the Simpson index between the groups ( P >0.05).

[0076] 4. 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. In this experiment, Anosim was used to test the significance of community structure differences between groups. The distance algorithm used was the Canberra algorithm, and the species level was OTU. The significance analysis of community structure differences between β diversity groups is shown in Table 13. From the Anosim analysis results, it can be seen that the species diversity differences between the EA and AP groups are not statistically significant ( P >0.05), the differences in β diversity of the remaining groups of samples were statistically significant ( P <0.05). This indicates that the species structure similarity between the EA group and the AP group is relatively high, and the species structure differences between the other two groups are relatively large;

[0077] 5. 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 Fig.13 As shown by Fig.13It can be seen that at the genus level, electroacupuncture and probiotic intervention can significantly increase the relative abundance of Lactobacillus, with the AP group accounting for the highest proportion (24.44%), followed by the EA group (21.41%), and Clostridium, with the EA group accounting for the highest proportion (8.74%), followed by the AP group (7.37%). The specific species differences between the groups need to be further determined using the LEfSe analysis method.

[0078] 6. 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; (1) Blank group vs. model group Model group vs. blank group species evolutionary branches Fig.14 As shown, the LDA values ​​of the blank group vs. the model group are as follows Fig.15 As shown, compared with the blank group, at the phylum level, the Firmicutes in the model group ( Firmicutes ) abundance decreased; at the class level, the δProteobacteria in the model group ( Deltaprotedbacteria ) abundance increased, Clostridium ( Clostridia ) abundance decreased; at the order level, the Clostridiales ( Clostridiales ) decreased; at the family level, the Lachnospiraceae ( Lachnospiraceae ) decreased in abundance; at the genus level, the model group Lachnospiraceae_NK4A136, Odoribacter、Rumiriclostridium_UCG_005、Eubacterium xylanophilum The abundance of Butyrivibrio spp. Butyrivibrio ), Rikenbacterium RC9 gut ( Rikenellaceae RC9 gut ) increased abundance; (2) Model group vs. electroacupuncture group Evolutionary branches of species in the model group vs. electroacupuncture group Fig.16 As shown, the LDA values ​​of the blank group vs. the model group are as follows Fig.17 As shown, compared with the EA group, at the phylum level, Firmicutes ( Firmicutes ) decreased in abundance, and Verrucomicrobia ( Verrucomicrobia ) abundance increased; at the class level, the model group Verrucomicrobiaceae At the order level, the abundance of Verrucomicrobiales Increase; at the family level, the model group Clostridiaceae_1 The abundance decreases, Akkermansiaceae At the genus level, the abundance of Clostridium_sensu_stricto_1 , Turictbacter , Alloprevotella The abundance decreases, Akkermansia The genus abundance increased; (3) Model group vs. electroacupuncture plus antibiotic group Evolutionary branches of species in the model group vs. electroacupuncture plus antibiotic group Fig.18 As shown, the LDA values ​​of the model group vs. the electroacupuncture combined with antibiotics group are as follows Fig.19 As shown, compared with the electroacupuncture plus antibiotic group, at the phylum level, Epsilonbacteraeeota The abundance of phylum decreased; at the class level, the δProteobacteria in the model group ( Deltaprotedbacteria ) abundance increased; at the order level, the Desulfovibrioles ( Desulfovibrations ) levels increased; at the family level, the model group's Peptostreptococcus family ( Peptostreptococcaceae ) decreased in abundance, and Desulfovibrioideae ( Desulfovibrionaceae ) abundance increased; at the genus level, the model group Romboutsia, ASF356, Anaerotruncus, Butyrivibrio, Lachnoclostridium The genus abundance decreased; (4) Model group vs. electroacupuncture plus probiotics group Species evolution branches of model group vs. electroacupuncture plus probiotics group Fig. 20 As shown, the LDA values ​​of the model group vs. electroacupuncture combined with probiotics group are as follows Fig.21 As shown, compared with the electroacupuncture plus probiotics group, at the phylum level, the model group Tenericutes, Actinobacteria At the class level, the abundance of the model group Bacill The abundance of δProteobacteria was low. Deltaprotedbacteria ) abundance increased; at the order level, the Lactobacillus orders ( Lactobacillales ) decreased in abundance, Mollicutes At the family level, the abundance of Lactobacillus family in the model group ( Lactobacillaceae ) decreased in abundance, and Desulfovibrioideae ( Desulfovibrionaceae ) abundance increased; at the genus level, the model group Lactobacillus, Odoribacter The abundance decreases, Eubacterium coprostanoligenes, Candidatus stoquerfichus Increased abundance.

[0079] 2. Effect of electroacupuncture on the level of short-chain fatty acids (SCFAs) in the colon of CUMS rats 1. Total ion current of short-chain fatty acids GC-MS total ion currents of short-chain fatty acids in mixed standards (A) and samples (B) are shown in Fig. 22 ,It can be seen from the figure that the chromatographic separation of each component to be tested is good, the peak shape is sharp and symmetrical, and each component can be accurately quantified.

[0080] 2. Short-chain fatty acid standard curve The results of the standard curve of short-chain fatty acids are shown in Table 14. The concentration of each component to be tested is used as the horizontal axis (x), and the ratio of the peak area of ​​the component to be tested to the internal standard is used as the vertical axis (y), and the regression equation and correlation coefficient are obtained. The results show that the linear relationship of the components to be tested is good within their respective concentration ranges;

[0081] 3. Quality Control Sample Evaluation Take an appropriate amount of mixed standard solution with a concentration of 1250 μM for each standard, and after processing, obtain a quality control sample (QC). The stability of the detection process is investigated using the QC sample, and a QC sample is injected once every 17 samples. The RSD% results of the QC samples are shown in Table 15. The RSD of each analyte is less than 11.00%, indicating that the experimental data is stable and reliable;

[0082] 4. Quantitative results The short-chain fatty acid targeted quantitative detection method was used to detect the contents of acetate, propionate and butyrate, the main components of the SCFAs group in rat feces, and the quantitative results of each metabolite were analyzed and statistically analyzed.

[0083] (1) Acetate level analysis The fecal acetate content of rats in each group at different time points is shown in Table 16. Fig.23 (Note:*: P <0.05,**: P <0.01,***: P <0.001)as shown: Before modeling, there was no statistically significant difference in acetate levels among the groups ( P >0.05).

[0084] After modeling, the overall differences in acetate levels among the groups were statistically significant P＜0.001 ; Compared with the BC group, the average acetate concentrations in the MO, EA, AA and AP groups were higher than those in the BC group, and the difference was statistically significant ( P <0.05).

[0085] After intervention, the overall difference in acetate levels among the groups was statistically significant. P＜0.001 ; Compared with the BC group, the average acetate content in the EA group was lower than that in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the average acetate level in the EA group was lower than that in the MO group, and the difference was statistically significant (P <0.05); compared with the AA group, the acetate level in the EA group was lower than that in the AA group, and the difference was statistically significant ( P <0.05);

[0086] (2) Propionate content analysis The fecal propionate content of rats in each group at different time points is shown in Table 17. Fig.24 (Note:*: P <0.05,**: P <0.01,***: P <0.001)as shown: Before modeling, there was no statistically significant difference in propionate levels among the groups ( P >0.05).

[0087] After modeling, the propionate levels in each group were lower than those in the BC group in the MO, EA, AA and AP groups, and the difference was statistically significant ( P <0.01).

[0088] After intervention, the propionate levels of each group were lower in MO, EA and AA groups than in BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the propionate levels in the EA group and the AP group were higher than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the propionate level in the AP group was higher than that in the AA group, and the difference was statistically significant ( P <0.05);

[0089] (3) Butyrate content analysis The fecal butyrate content of rats in each group at different time points is shown in Table 18. Fig.25 (Note:*: P <0.05,**: P <0.01,***: P <0.001)as shown: Before modeling, there was no statistically significant difference in butyrate levels among the groups ( P >0.05).

[0090] After modeling, the butyrate levels in each group were lower than those in the BC group in the MO, EA, AA and AP groups, and the difference was statistically significant ( P <0.05).

[0091] After intervention, the overall difference in butyrate levels among the groups was statistically significant. P＜0.001 Compared with the BC group, the average acetate concentration in the MO and AA groups was lower than that in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the average acetate level in the AP group was higher than that in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the butyrate levels in the EA and AP groups were higher than those in the AA group, and the difference was statistically significant ( P <0.05);

[0092] IV. Summary From the results of diversity analysis, we can see that the results of Beta diversity analysis showed that except for the electroacupuncture group and the electroacupuncture plus probiotics group, there was no difference in species structure, and the species differences in the other groups were statistically significant. The results of the α diversity index showed that there was no statistically significant difference in species diversity among the groups, indicating that the number of species was not much different. The diversity results analysis showed that the species structure of the electroacupuncture and electroacupuncture plus probiotics groups showed good consistency. The benign effect of probiotics on gastrointestinal microbial homeostasis has been fully demonstrated. This result of this study shows that electroacupuncture, like probiotics, has a benign improvement effect on intestinal species structure.

[0093] The results of Lefse analysis showed that the model group showed Firmicutes ( Firmicutes ) abundance decreased, indicating that rodents with a low proportion of Firmicutes in the intestine tend to have depressive-like behaviors. Compared with the blank group, electroacupuncture plus probiotics, and electroacupuncture plus antibiotics, the model group showed a decrease in the abundance of δProteobacteria ( Deltaproteobacteria ) abundance increased. Delta Proteobacteria is a pathogenic bacterium whose genome is highly similar to the virulence and host adaptation factors of known human pathogens and is highly correlated with the onset of mucosal inflammation such as periodontal disease. Clostridiales ) decreased. Compared with the blank group, the model group had a decrease in Lachnospiraceae ( Lachnospiraceae ), Lachnospiraceae_NK4A136 The abundance of the genus also decreased. Rumiriclostridium_ UCG_005 Abundance decreased. Eubacterium xylanophilum The abundance of bacteria decreased. Eubacterium xylanophilum It is a strong butyrate-producing bacterium in the intestine. Butyrate has an important stabilizing effect on the integrity of the intestinal mucosa. RC9 gut ( Rikenellaceae RC9 gut ) abundance increased.

[0094] Comparison between the model group and the electroacupuncture group revealed Verrucomicrobiaceae The abundance of the model group and the electroacupuncture group was compared. Clostridiaceae_1The abundance decreased, and Clostridiaceae_1 It is strongly negatively correlated with digestive tract cancer. Alloprevotella belongs to the genus of beneficial bacteria and has advantages in restoring intestinal disorders. This study found that compared with the electroacupuncture group, the model group Alloprevotella In addition, the abundance of Akkermansiaceae , Akkermansia Increased abundance, Akkermansiaceae It is closely related to early cancer cell colonization. Akkermansia It was found to be enriched in the intestines of children with autism and negatively correlated with weight loss. Through the research results in the previous section, we found that the weight of rats in the model group was lighter than that of the other groups, which is consistent with the speculation of this study.

[0095] Compared with the electroacupuncture combined with antibiotics group, the model group had fewer species differences than the other groups, indicating that the species differences between the two groups were small. Although the species differences between the two groups were not large, the harmful species in the model group were still more than those in the electroacupuncture combined with antibiotics group, such as δProteobacteria, Actinobacteria ,in Actinobacteria Most species of the phylum are pathogenic to humans and animals. Desulfovibrio, which is enriched in the model group, can promote bile secretion and the formation of gallstones. In addition, it has been found to increase significantly in the intestines of rats with oxidative stress and myocardial hypertrophy. The model group had fewer beneficial bacterial genera compared with the electroacupuncture combined with antibiotics group, such as Peptostreptococcaceae、ASF356 . Peptostreptococcaceae It was found to reduce the risk of nicotine dependence and was enriched in the intestines of the electroacupuncture combined with antibiotics group in this study. ASF356 It is related to the production of short-chain fatty acids, which was reduced in the intestine of the model group. Romboutsia, Lachnoclostridium In this study, the abundance of CUMS in the intestine of the electroacupuncture combined with antibiotics group increased, and both were found to be enriched in the intestine of patients with depressive disorders. It can be seen that CUMS and antibiotics have a considerable negative impact on the homeostasis of intestinal flora.

[0096] Compared with the electroacupuncture plus probiotics group, the model group had more common pathogenic bacteria in the intestine, such as δProteobacteria, Tenderly , Actinobacteria、Eubacterium coprostanoligenesu、Mollicute Enrichment, Actinobacteria The various pathogenic bacteria contained in the phylum are related to human infection. Eubacterium coprostanoligenesu Positively correlated with the risk of colorectal cancer. Beneficial bacteria in the intestine of the model group Lactobacillus, Odoribacter reduce, Lactobacillus Lactobacillus is a well-known beneficial intestinal bacteria that plays a key role in the stability of intestinal microecology. Clinical studies have found that people with depression Lactobacillus Significantly reduced. Odoric bacteria The effect of inhibiting colitis was achieved by promoting the production of short-chain fatty acids and inducing the increase of IL-10. Candidatus stoquerficusIt was found that it may be related to the repair of intestinal inflammation. The body can also mobilize its repair capacity under continuous stress. However, according to the behavioral results of rats in the model group, it can be seen that the bacteria has limited effect on the improvement of the cognition and behavior of the whole body.

[0097] SCFAs are important substrates for maintaining the integrity of colonic epithelium. By comparing the quantitative results of short-chain fatty acids in each group with the blank group after modeling, it can be seen that the content of acetate in the intestine of rats under stress increased, and the difference was statistically significant ( P <0.05), the contents of propionate and butyrate decreased, and the difference was statistically significant ( P <0.05). After intervention, the acetate content in the electroacupuncture group was lower than that in the blank group, model group and electroacupuncture plus antibiotic group, and the differences were statistically significant ( P <0.05), butyrate was higher than that in the model group and the electroacupuncture combined with antibiotics group, and the difference was statistically significant ( P <0.05); the contents of propionate and butyrate in the electroacupuncture group plus probiotics group were higher than those in the model group and the electroacupuncture combined with antibiotics group, and the difference was statistically significant ( P <0.05). Acetate can be used as one of the precursors of butyrate. According to the isotope tracking experiment of Bergman et al., extracellular acetate can promote the synthesis of butyrate through mutual conversion, and the conversion contribution rate is as high as 60%. It is worth noting that acetate can also affect the process of neurodegeneration by inhibiting the phagocytosis of microglia, affecting the metabolic characteristics of microglia in germ-free mice and enhancing their maturity. Under stable conditions, acetate can inhibit the phagocytosis of microglia, and ultimately lead to an increase in the Aβ load of Alzheimer's mice-inducing the pro-inflammatory phenotype of microglia, increasing the expression of inflammatory factors in cells, and in turn reducing the phagocytosis of microglia. Therefore, except for the blank group, the acetate content of each group increased after modeling, which may lead to the pro-inflammatory phenotype of microglia, thereby having a negative impact on cognition. After intervention, electroacupuncture significantly reduced the acetate content. The high acetate content in the blank group may be due to the need to produce butyrate.

[0098] Propionate can stimulate regulatory T cells to form anti-cytokines, such as interleukin 10 (IL-10). This reaction process is triggered by the short-chain fatty acid receptor GPR43, thereby inhibiting the formation of interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNFα). Propionate and butyrate regulate T cells by inhibiting histone deacetylation (HDAc), thereby producing an anti-inflammatory effect. Therefore, the content of propionate is significantly reduced after modeling, and the body may produce an inflammatory response, which needs to be judged in combination with the level of inflammatory cytokines in the next section. After intervention, electroacupuncture significantly increased the propionate level of CUMS rats, triggering the body's anti-inflammatory mechanism.

[0099] Butyrate plays an important role in regulating oxygen concentration in the host intestine. It can reduce epithelial permeability and enhance epithelial barrier activity through hypoxia-inducible factor (HIF). In addition, butyrate has been found to reduce epithelial permeability and enhance the transepithelial electrical impedance of host tight junctions by regulating the number of claudins occludin, zonulin and interleukin-10 (IL-10) receptors. The increase in butyrate produced by the microbiota can cause the body's anti-inflammatory response, thereby inhibiting pro-inflammatory mechanisms. For example, butyrate can promote the production of downstream pro-inflammatory cytokines, leading to downregulation of nuclear factor κB (NF-κB) expression. In humans, changes in the microbiota of patients with inflammatory bowel disease (IBD) are associated with reduced bacterial diversity and the loss of butyrate-producing bacteria (such as Freundlich). The increase of bacterial lipopolysaccharide (LPS) triggers a pro-inflammatory cascade mediated by toll-like receptor 4 (TLR4) in immune cells (monocytes, macrophages, and Kupffer cells), leading to the activation of downstream signaling pathways such as NF-κB and mitogen-activated protein kinase (MAPK), which can lead to inflammation driven by inflammatory cytokines such as TNF-α and IL-6. Butyrate is currently considered to be the most important regulator of tight junction proteins, and has been shown to enhance intestinal barrier function by increasing the expression of Claudin-1 and Zonula Occludens-1 and the redistribution of occludens. It is a protein that is a key component of tight junction components. In summary, butyrate plays a vital role in the integrity of the intestinal barrier. After modeling, the butyrate levels in each modeling group decreased significantly, which may be accompanied by increased permeability of the intestinal mucosa, forming intestinal leakage, triggering a series of inflammatory responses, and a large amount of lipopolysaccharide entering the periphery. The specific needs to be judged in combination with the intestinal morphological structure in the next section. After intervention, the butyrate content in the electroacupuncture combined with probiotics group was higher than that in the modeling group, and the difference was statistically significant ( P <0.05), indicating that a large number of microorganisms in the intestine produce butyrate, repair the intestinal barrier, and block lipopolysaccharide from passing through the intestinal barrier into the periphery. In addition, after intervention, only the butyrate content of the model group and the electroacupuncture combined with antibiotics group was lower than that of the blank group, and the difference was statistically significant ( P <0.05), there was no significant difference between the electroacupuncture group and the blank group, although the butyrate content in the electroacupuncture group did not show significant difference compared with the model group ( P >0.05), but showed a definite upward trend.

[0100] Through the above analysis of the intestinal flora species in each group, it can be seen that the abundance of Firmicutes in the model group decreased, and many species under the Firmicutes phylum were involved in the production of butyrate, such as Eubacterium xylanophilum, the number of harmful and pathogenic bacteria increased. The levels of propionate and butyrate in the model group and the electroacupuncture combined with antibiotics group were reduced. The abundance of beneficial bacteria related to the production of short-chain fatty acids in the intestines of CUMS rats intervened by electroacupuncture was higher, the number of species was larger, and the abundance of harmful bacteria was reduced. Short-chain fatty acids, metabolites produced by microorganisms, play an important role in maintaining the integrity of the intestinal barrier. Electroacupuncture can significantly increase the propionate content of CUMS rats, and the electroacupuncture combined with probiotics group also showed the same trend. In order to further observe whether microorganisms and their metabolites can induce intestinal inflammatory reactions and structural changes, the next section will conduct in-depth research on this.

[0101] Section 4 Effects of electroacupuncture on colon morphology and inflammatory cytokines in CUMS rats 1. Materials 1. Experimental animals and groups Same as Section 2.

[0102] 2. Instruments, Equipment and Reagents The instruments and equipment involved in this section are shown in Table 19:

[0103] The reagents and consumables involved in this section are shown in Table 20:

[0104] 2. Methods 1. Colon sampling The colon tissue samples of this experiment were from rats in the second section. The samples were collected 28 days after the intervention. 5% sodium pentobarbital solution was used for intraperitoneal anesthesia (0.1-0.2 ml / 100 g). After opening the abdomen, about 5-8 cm of intestinal tissue was cut upward from the anus, and the residual feces in the intestinal cavity were flushed with normal saline. The prepared colon was divided into 3 equal parts, 2 / 3 of the fresh intestinal tissue was stored in liquid nitrogen and promptly moved to a -80℃ refrigerator for enzyme-linked immunosorbent assay; the other 1 / 3 was completely immersed in 4% paraformaldehyde and placed at room temperature for 24 hours, and dehydrated with 15% and 30% sucrose solutions in a gradient manner. When the intestinal tissue completely sank to the bottom of the centrifuge tube, the intestinal tissue was washed three times with an appropriate amount of PBS buffer to prepare for paraffin sections.

[0105] 2. Hematoxylin-eosin staining experiment of paraffin sections of colon tissue (HE) 1. Experimental Procedure (1) Dewaxing of paraffin sections: sequentially place the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 10 min, anhydrous ethanol II for 10 min, 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, 70% alcohol for 5 min, and wash with distilled water; (2) Hematoxylin staining of cell nuclei: Stain sections with Harris hematoxylin for 3-8 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, blue with 0.6% ammonia solution, and rinse with running water; (3) Eosin staining of cytoplasm: Slice into eosin staining solution and stain for 1-3 minutes; (4) Dehydration and sealing: Place the slices in 95% alcohol I for 5 min - 95% alcohol II for 5 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - xylene I for 5 min - xylene II for 5 min to dehydrate and make them transparent. Take the slices out of xylene and let them dry slightly, then seal them with neutral gum. (5) White light slice digital scanning: ①Open the slice scanner and pannoramic scanner scanning software, and place the prepared stained slices, immunohistochemical slices or CISH slices on the scanner slice carrier; ②Operate the pannoramic scanner software to select the bright field scanning mode and load the slices into the scanner; ③ Automatically or manually identify the scanning area, click to start scanning, the scanner will automatically focus and collect the composite image. After the scan is completed, each image will be named according to the slice number.

[0106] 3. Enzyme-linked immunosorbent assay (ELISA) 1. Conditions At room temperature, mix 10 μl of buffer with 100 μl of sample at a ratio of not less than 1:10 and mix well. Prepare the washing solution at a ratio of 1:100 for later use.

[0107] 2. Operation steps The supernatant of PBS homogenate of rat colon tissue was taken to detect the expression of inflammatory cytokines in the supernatant of colon tissue. The specific steps are as shown in Table 21 below:

[0108] 4. Statistical methods Same as Section 2.

[0109] 3. Results 1. Effect of electroacupuncture on colon tissue morphology in CUMS rats HE staining was used to stain the colon tissue sections of rats in each group. Figure 26(Note: 1. (A) is the blank group; (B) is the model group; (C) is the electroacupuncture group; (D) is the electroacupuncture combined with antibiotics group; (E) is the electroacupuncture combined with probiotics group. 2. All are ×200 magnification microscope pictures). In the BC group, the goblet cells were neatly and compactly arranged, with full and clear shapes, and the overall structure of the mucosa was intact; in the MO group, the arrangement order and cell fullness of the epithelial columnar cells deteriorated, the goblet cell gap widened, and the number of lymphocytes increased; in the EA group, the goblet cells were fuller and thicker, arranged closely, and the mucosa had a higher density; in the AA group, the goblet cells were poorly full in shape, and the number of lymphocytes increased; in the AP group, the goblet cells were full, clear, and complete in structure, arranged compactly, and the number of lymphocytes was relatively small.

[0110] 2. Effects of electroacupuncture on colonic inflammatory factors in CUMS rats The levels of inflammatory factors and related proteins in the colon of rats in each group are shown in Table 22. The comparison of inflammatory factors in the colon tissue of rats in each group is shown in Figure 27 (Note:*: P <0.05,**: P <0.01,***: P <0.001); (1) Analysis of NF-κB levels There was no statistically significant difference between the groups P =0.010; compared with the BC group, the NF-κB levels in the MO and AA groups were higher than those in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the NF-κB levels in the AP and EA groups were lower than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the NF-κB levels in the EA and AP groups were lower than those in the AA group, and the difference was statistically significant ( P <0.05); (2) Analysis of TLR4 levels The overall differences among the groups were statistically significant P =0.001; compared with the BC group, the TLR4 levels in the MO and AA groups were higher than those in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the TLR4 levels in the AP and EA groups were lower than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the TLR4 levels in the EA and AP groups were lower than those in the AA group, and the difference was statistically significant ( P <0.05); (3) Analysis of IL-6 levels The overall differences among the groups were statistically significant P =0.001; compared with the BC group, the IL-6 levels in the MO and AA groups were higher than those in the BC group, and the difference was statistically significant (P <0.05); compared with the MO group, the IL-6 levels in the AP, EA and AA groups were lower than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the IL-6 level in the EA group was lower than that in the AA group, and the difference was statistically significant ( P <0.05); (4) Analysis of IL-1β levels The overall differences among the groups were statistically significant P＜0.001 ; Compared with the BC group, the IL-1β levels in the MO and AA groups were higher than those in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the IL-1β levels in the AP and EA groups were lower than those in the MO group, and the difference was statistically significant ( P <0.01); compared with the AA group, the IL-1β levels in the EA and AP groups were lower than those in the AA group, and the difference was statistically significant ( P <0.05); (5) Analysis of TNF-α levels The overall differences among the groups were statistically significant P =0.001; compared with the BC group, the TNF-α levels in the MO and AA groups were higher than those in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the TNF-α levels in the AP and EA groups were lower than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the TNF-α levels in the EA and AP groups were lower than those in the AA group, and the difference was statistically significant ( P <0.05); (6) Analysis of IL-10 levels The overall differences among the groups were statistically significant P＜0.001 ; Compared with the BC group, the IL-10 levels in the EA group and the AP group were higher than those in the BC group ( P <0.05), the difference was statistically significant; compared with the MO group, the IL-10 levels in the EA and AP groups were higher than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the IL-10 levels in the EA and AP groups were higher than those in the AA group, and the difference was statistically significant ( P <0.05);

[0111] IV. Summary The results of the previous study showed that electroacupuncture can increase the content of metabolite propionate by regulating the content of beneficial intestinal bacteria, and the electroacupuncture combined with probiotics group also showed the same trend. This section used HE tissue staining and Elisa experiments to observe the effect of electroacupuncture on the intestinal tissue structure and inflammatory factor levels of CUMS rats.

[0112] The results of HE staining sections showed that the intestinal structure of rats in the model group became less compact, lymphocyte infiltration increased, and the mucosal layer was severely damaged, which was consistent with the results of the levels of inflammatory factors and TLR4 / NF-κB in the colon discussed in this section. The intestinal epithelial tissue of the electroacupuncture combined with antibiotics group also showed obvious inflammatory infiltration and structural destruction, indicating that after CUMS stimulation and antibiotics disturbed the homeostasis of the flora, the benign regulatory effect of electroacupuncture on the host of the intestinal flora was blocked, and therefore the damage to the intestinal mucosal epithelial structure could not be reversed. The intestinal structure of rats in the electroacupuncture group was dense, lymphocyte infiltration decreased, and goblet cells were full and complete. The electroacupuncture combined with probiotics group also showed the same tissue structural characteristics. This result shows that electroacupuncture can improve the intestinal inflammatory infiltration of CUMS rats by regulating the composition of the intestinal flora, the level of short-chain fatty acids and the level of inflammatory factors, and has a certain repair effect on the damaged intestinal barrier.

[0113] Inflammatory factors are important signaling molecules of the body's immune response and are important biomarkers for the occurrence and development of inflammatory reactions. This study found through Elisa quantitative detection results that electroacupuncture can effectively reduce the levels of pro-inflammatory factors IL-6, IL-1β, and TNF-α in the colon, and the difference is statistically significant ( P <0.05), and increased the level of anti-inflammatory factor IL-10, the difference was statistically significant ( P <0.05), reducing the levels of NF-κB and TLR4, proteins closely related to inflammatory response, with statistically significant differences ( P <0.05).

[0114] Animal or clinical studies have shown that pathogens or repeated psychosocial stress can lead to inflammatory responses and induce the enrichment of immune cells and the production of proinflammatory mediators (such as IL-1β, IL-6, and TNF-α). IL-6 participates in the pathophysiology of a part of MDD patients through excessive release of corticotropin-releasing hormone (CRH) and promotion of glucocorticoid receptor resistance, which may eventually impair the negative feedback regulation of the hypothalamus-pituitary-adrenal (HPA) axis. Upregulation of IL-6 levels can inhibit brain-derived neurotrophic factor levels, leading to decreased brain connectivity, which may further lead to depression. Meta-analysis found that compared with healthy controls, the levels of IL-6, IL-1β, and TNF-α were increased in the major depression group. The results of this study indicate that acupuncture can regulate peripheral inflammatory responses by downregulating IL-6 levels in the hippocampus, prefrontal cortex, and serum, activating the cholinergic anti-inflammatory pathway.

[0115] IL-1β also plays an important role in the physiological functions of the central nervous system, and is involved in cognitive, learning and memory processes. IL-1β plays a maintenance function in the steady-state brain. However, the use of IL-1β in the hippocampus can induce memory impairment, and this difference is the result of the complex role of IL-1β in the central nervous system. Cognitive impairment in the brain is closely related to high concentrations of IL-1β. Electroacupuncture stimulation of Zusanli acupoint can significantly downregulate IL-1β levels. This study found that the IL-1β level of the model group rats with depressive-like behavior was higher, and electroacupuncture could significantly reduce the IL-1β level.

[0116] Excessive activation of the proinflammatory cytokine TNF-α in the central nervous system may cause brain damage, such as excitotoxicity and neurotransmission dysregulation. TNF-α levels are elevated in patients with MDD. Under chronic inflammatory conditions, elevated TNF-α levels enhance serotonin transporter-mediated 5-HT uptake and significantly affect available extracellular 5-HT. The results of this study indicate that acupuncture can induce TNF-α downregulation.

[0117] IL-10 is an important immunomodulatory molecule that prevents the development of immunopathological lesions caused by aggravated protective immune responses caused by acute and chronic infections. The secretion of IL-10 by Th1 cells represents a powerful self-regulatory feedback loop that prevents excessive inflammation and potential tissue damage in proinflammatory Th1-driven immune responses to infections. The activation of IL-10 is also closely related to intestinal microorganisms. This study showed that electroacupuncture stimulation of Zusanli acupoint can significantly increase IL-10 levels. It is worth noting that in this study, the IL-10 level of the blank group was significantly lower than that of the model group and the electroacupuncture combined with antibiotics group, which may be due to the fact that under normal feeding conditions without CUMS stimulation, the immune positive feedback loop of the blank group rats was not activated.

[0118] TLRs play an important role in the molecular communication between changes in the intestinal microbiome and the homeostasis of the immune system. Once TLRs are activated, intestinal-associated immune cells produce proinflammatory cytokines such as IL-1β, IL-6, and TNF-α, which can diffuse through the increased permeability of the intestinal mucosa or cross the blood-brain barrier through inflammatory cytokine transporters to reach the brain. Once arriving in the brain, these inflammatory cytokines can act on receptors on microglia and stimulate the further release of inflammatory cytokines and regulate neuronal function. NF-κB is released after being triggered by proinflammatory factors, which can further induce the expression of proinflammatory genes and inflammatory responses, triggering the activation of macrophages and microglia. This process controls the amount of TNF-α or IL-6, which in turn promotes the activation phosphorylation of signal transducers and activators of transcription, promoting the release of NF-κB. Acupuncture can improve ischemia and reduce inflammatory response in neurological deficits by regulating the key signaling molecules in the TLR4 / NF-κB signaling pathway, such as TNF-α, IL-1β, and IL-6 inflammatory cytokine levels; acupuncture significantly inhibits the expression of inflammatory cytokines in the hippocampus and plasma of rats with vascular dementia, significantly downregulates the expression of TLR4, and reverses the overexpression of TLR4 in microglia. The above results show that electroacupuncture can reduce the level of inflammatory factors in the colon. It is worth noting that the levels of TLR4 and NF-κB in the electroacupuncture group and the electroacupuncture combined with probiotics group with good control of proinflammatory factors also showed a consistent downward trend, indicating that electroacupuncture reduces the levels of TLR4 and NF-κB by downregulating proinflammatory factors.

[0119] From the results of HE staining sections, it can be seen that under the stimulation of CUMS, the colon structure of rats was damaged, the inflammatory infiltration increased, and whether the damaged intestinal epithelial cells would release a large number of proinflammatory factors produced in the intestine into the peripheral circulation, pass through the blood-brain barrier, and finally reach the brain, causing inflammation of the central nervous system. Whether electroacupuncture improves the depressive-like behavior of CUMS rats by blocking this inflammatory cascade reaction still needs to be discussed and demonstrated in the next section by analyzing the expression of inflammatory proteins related to the hippocampus of the brain.

[0120] The fifth section: Effects of electroacupuncture on the inflammatory cytokine transduction pathway NF-κB / TLR4 in the hippocampus of CUMS rats 1. Materials 1. Experimental animals and groups Same as Section 2.

[0121] 2. Instruments, Equipment and Reagents The instruments and equipment involved in this section are shown in Table 23:

[0122] The reagents and consumables involved in this section are shown in Table 24:

[0123] 2. Methods 1. Brain tissue collection The brain tissues in this section were from the rats in the second section. They were divided into fresh hippocampus and whole brain tissue after perfusion. The samples were collected 28 days after electroacupuncture intervention.

[0124] 1. Fresh hippocampal tissue 5% sodium pentobarbital solution was used for intraperitoneal anesthesia (0.1-0.2 ml / 100 g). After opening the abdomen and removing the intestines, the head was cut off, the skull was opened, the brain tissue was exposed, and the hippocampus was cut according to the rat brain stereotaxic atlas, placed in a 1.5 ml sterile enzyme-free centrifuge tube, quickly placed in liquid nitrogen, and transferred to a -80°C refrigerator within 2 hours.

[0125] 2. Whole-brain perfusion Fix the anesthetized rat on the dissection table, cut the rat skin and muscle layer with surgical scissors, the length is about 4cm, then open the rat abdominal cavity vertically upward in the center of the incision, cut the left and right diaphragms respectively, then cut the sternum along the diaphragm, and then cut upward in the direction perpendicular to the sternum to expose the heart. Use a small hemostat to peel off the pericardium and expose the aortic arch. Peel off the large and small intestines in the abdomen of the rat, clamp the abdominal aorta with a large hemostat, and place the hemostat on the side away from the stomach. Hold the heart with a small hemostat in the left hand and the needle in the right hand, insert the needle from the right apex of the heart into the aortic arch, clamp the needle in the aortic arch with a small hemostat, and cut the right atrial appendage with small scissors. The needle port was injected with 0℃ iced saline at a constant speed of about 100ml until the blood flowing out of the rat body became transparent, and then about 120ml of room temperature 4% paraformaldehyde fixative was injected. After the rat's limbs twitched, 4% paraformaldehyde was dripped, first fast and then slow. After the liver turned white, the infusion speed was slowed down. After 10 minutes of dripping, each rat was decapitated and the brain was taken out. The whole brain was gently placed on filter paper on ice, rinsed with saline, and the blood vessels on the surface were gently clamped off with curved forceps. The obtained rat brain was soaked in 4% paraformaldehyde fixative (50ml centrifuge tube), and dehydrated with 15% and 30% sucrose solution gradiently. When the rat brain completely sank to the bottom of the centrifuge tube, the rat brain was washed three times with an appropriate amount of PBS buffer, and then the surface water of the rat brain was carefully absorbed with sterile filter paper. The tissue was fixed in the embedding box with OCT embedding agent, and then the embedding box was wrapped with aluminum foil and placed in a -80C refrigerator for storage.

[0126] 2. Brain tissue paraffin sections Colon paraffin sections Hematoxylin-eosin staining experiment (HE) The experimental procedures are the same as the colon paraffin section HE staining experiment in Section 4.

[0127] 3. Fluorescence quantitative polymerase chain reaction (qPCR) 1. Reaction conditions and system The reaction conditions are shown in Table 25, and the reaction system is shown in Table 26:

[0128] 2. Primer sequences The qPCR primer sequences are shown in Table 27:

[0129] 3. Operation steps (1) Sample processing ① Tissue: Grind the tissue in liquid nitrogen. Add 600 μl Buffer RL for every 20-30 mg of tissue (check whether β-mercaptoethanol has been added before use). If the tissue sample is less than 20 mg, add 350 μl Buffer RL. The sample volume should not exceed one tenth of the volume of Buffer RL; ②Monolayer culture cells: directly lyse the cells in the culture flask or process them into a cell suspension, centrifuge to obtain a cell pellet, discard the supernatant, and place the pellets at 6-10 cm 2 Add 600 μl Buffer RL to the culture area, less than 6 cm 2 Add 350 μl BufferRL and pipette up and down several times to fully lyse the cells. ③Cell suspension: Centrifuge at 12,000 rpm (~13,400×g) for 1 minute and discard the supernatant to obtain the cell pellet. 6 -1×10 7 Add 600 μl Buffer RL to cells, less than 5×10 6 Add 350 μl Buffer RL to the cells and pipette them several times to fully lyse them; (2) After the sample is fully lysed, place it at room temperature for 5 minutes to completely separate the protein-nucleic acid complex; (3) Centrifuge at 12,000 rpm for 2-5 min and take the supernatant for the following steps; (4) Add 1 volume (600 μl or 350 μl) of 70% ethanol (prepared with RNase-free water) to the solution obtained in step 3 and mix well. Note: Precipitation may occur after adding ethanol, but it will not affect subsequent experiments; (5) Add all the solution obtained in the previous step to the adsorption column (Spin Columns RM) in the collection tube. If the entire solution cannot be added to the adsorption column at one time, please transfer it twice. Centrifuge at 12,000 rpm for 1 minute and discard the waste liquid. Put the adsorption column back into the collection tube. Note: The maximum loading capacity of the adsorption column is 100 µg. Do not overload, otherwise it will affect the yield and purity of RNA; (6) Add 350 μl of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and return the adsorption column to the collection tube; (7) Prepare DNase I mixed solution: Take 52 μl RNase-Free Water, add 8 μl 10× Reaction Buffer and 20 μl DNase I (1 U / μl), mix well, and prepare a final volume of 80 μl reaction solution; (8) Add 80 µl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 min. (9) Add 200 μl of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and return the adsorption column to the collection tube; (10) Add 500 μl of Buffer RW2 to the adsorption column (check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube; (11) Repeat step 10; (12) Centrifuge at 12,000 rpm for 2 minutes and discard the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to completely dry the anhydrous ethanol in the adsorption column; (13) Transfer the adsorption column to a new centrifuge tube, add 30-50 μl RNase-Free Water to the middle of the adsorption membrane, leave at room temperature for 1 minute, centrifuge at 12,000 rpm for 1 minute, collect the RNA solution, and store the RNA at -70°C to prevent degradation.

[0130] (IV) Western blotting (WB) 1. Sample Preparation Before protein extraction: add protease inhibitors (20uL) and phosphatase inhibitors (20uL) per 1mL RIPA lysis buffer.

[0131] (1) Total protein extraction from tissues ① Cut the tissue into small pieces; ② Add lysis buffer at a ratio of 200 µL per 20 mg of tissue; ③ Homogenize with a glass homogenizer until fully lysed; ④ Lyse on ice for 30 minutes, repeatedly pipetting with a pipette to ensure complete cell lysis; ⑤ Centrifuge at 12000rpm, 4℃ for 10min, collect the supernatant, which is the total protein solution. The storage temperature is -20℃ / -80℃.

[0132] (2) Protein quantification The total protein was quantified using the BCA protein concentration assay kit (enhanced). The steps are as follows: ① Preparation of standard products: Prepare standards as shown in Table 28:

[0133] ② Sample preparation: Dilute 5 times and 10 times for measurement ③ Preparation of working solution: (Required number of wells + 1) / 200 = Volume of A; Volume of A × 49 = Volume of B, mix A + B, add 200uL of working solution to each well, and place at 37℃ for 20-30min; ④ Use an ELISA reader to measure A562 or the absorbance at wavelengths between 540-595nm; ⑤ Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0134] (3) Protein sample denaturation: ① Calculate the required volume of protein solution, water and protein loading buffer (5×) based on the concentration determination; ② Heat in a 100℃ metal bath for 10 minutes to fully denature the protein; ③ After cooling to room temperature, directly load the sample into the SDS-PAGE gel loading well (store at -20℃ / -80℃).

[0135] (4) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ① Preparation of rubber sheet: The glass plate is cleaned with pure water, dried, and the long plate and the short plate are aligned and fixed to the clamp. 12% separation gel, the formula is shown in Table 29. Immediately after mixing, pour the gel, then add 1ml of distilled water to cover, place at room temperature (place in a 37℃ constant temperature incubator in winter) for about 30 minutes. After the gel polymerizes, wash it twice with distilled water, and then dry it with filter paper;

[0136] ② 5% concentrated gel, the formula is shown in Table 30. Immediately after mixing, pour the gel, insert the sample comb to avoid bubbles, wait for the gel to solidify, remove the sample comb, and then rinse the sample wells with distilled water and 1× protein electrophoresis buffer;

[0137] ③ Loading and electrophoresis. Place the gel plate on the electrophoresis device, fill the inner tank with 1× protein electrophoresis buffer, and the 1× protein electrophoresis buffer in the outer tank should exceed the platinum wire. Load the samples in order. Run at 80V constant voltage for about 20 minutes until the sample passes through the concentrated gel, and run at 120V for about 45 minutes until the blue dye reaches the bottom of the gel and then stop the electrophoresis.

[0138] (5) Western blotting ① Take a relative amount of cut PVDF and soak it in methanol for 3-10 minutes; ② Soak the PVDF membrane, filter paper, and sponge pad in transfer buffer in advance; ③ After SDS-PAGE, take out the gel, remove the concentrated gel, and then soak it in transfer buffer for later use. Open the electro-transfer clamp, put a special sponge pad soaked in transfer buffer on each side, and then put three filter papers soaked in transfer liquid on each side. The filter paper and sponge pad are the same size. Put the gel flat on the filter paper on the cathode side, and finally put the PVDF membrane flat on the gel, remove bubbles, and clamp the electro-transfer clamp; ④ Fill the electrophoresis tank with electrotransfer solution, insert the electrotransfer clip (the electrotransfer solution should be pre-cooled in the refrigerator before use), connect the electrodes, turn on the current, and the PVDF membrane of the transfer clip should face the positive electrode of the electrophoresis tank; ⑤ Blocking: Rinse once with 1×TBS. Add 5% BSA blocking buffer and place in a shaking incubator for blocking (room temperature, 60rpm / min, 2-4h); ⑥ Primary antibody hybridization: discard the blocking solution, add the primary antibody hybridization solution diluted with Western primary antibody diluent, place it at 4°C for hybridization overnight, and hybridize it on a shaking incubator the next day (room temperature, 60rpm / min, 1h); ⑦ Recover the primary antibody hybridization solution and wash the membrane 3 times with TBST (place it in a shaking incubator, room temperature, 120 rpm / min, 10 min / each time); ⑧ Discard TBST, add diluted secondary antibody hybridization solution, and place in a shaking shaker for hybridization (room temperature, 60 rpm / min, 1 h); ⑨ Discard the secondary antibody solution and wash the membrane 4 times with TBST (place on a shaker, room temperature, 120 rpm / min, 4 min / each time); ⑩ ECL chemiluminescence and image acquisition and analysis: Perform the experimental operation according to the instructions of the protein blot detection kit. The simple operation process is as follows: Preparation of ECL working solution: Mix equal volumes of reagent 1 and reagent 2 and use them immediately. According to the size of the membrane, add an appropriate amount of ECL working solution to the membrane and leave it for 1 minute. Use a gel imaging system (Tanon 4600) to collect images and perform image analysis.

[0139] (6) Antibody and internal reference dilution ratio The dilution ratio of IL-6, IL-10, TNF-α, P65, P-P65, IL-1β and TLR4 was 1:500; the dilution ratio of GAPDH was 1:5000; the dilution ratio of HRP-labeled goat anti-rabbit and HRP-labeled goat anti-mouse was 1:20000.

[0140] 5. Statistical methods Same as Section 2.

[0141] 3. Results 1. Effects of electroacupuncture on the morphology of the hippocampus in CUMS rats HE staining was used to stain the brain tissue sections of rats in each group, and the hippocampus area was taken as the field of view of interest. Figure 28 (Note: 1. (A) is the blank group; (B) is the model group; (C) is the electroacupuncture group; (D) is the electroacupuncture combined with antibiotics group; (E) is the electroacupuncture combined with probiotics group; 2. All are ×800 magnification microscope pictures). The neurons in the hippocampus of the BC group were complete and full, with clear nuclei visible; the number of necrotic cells in the hippocampus of the MO group increased, probably due to nuclear condensation, the nuclei of the necrotic cells were indistinguishable, and there was infiltration of inflammatory cells; the number of necrotic cells in the hippocampus of the EA group was relatively small, and the neuronal cell structure was relatively intact; the number of necrotic cells in the hippocampus of the AA group was large, and there was also infiltration of inflammatory cells around them; the number of necrotic cells in the hippocampus of the AP group was small, and there were also small inflammatory cells; 2. Effects of electroacupuncture on the expression of genes related to the inflammatory cytokine transduction pathway in the hippocampus of CUMS rats The qPCR method was used to detect the expression levels of genes related to the inflammatory cytokine signaling pathway in the rat hippocampus (TLR4, p-p65, p65, TNF-α, IL-1β, IL-10), and the quantification results were analyzed and statistically analyzed.

[0142] The comparison of inflammatory cytokines and related protein gene expressions in the hippocampus of rats in each group is shown in Table 31. Figure 29 (Note:*: P <0.05,**: P <0.01,***: P <0.001).

[0143] (1) NF-κB The overall differences among the groups were statistically significant P =0.038; compared with the BC group, the NF-κB levels in the MO and AA groups were higher than those in the BC group, and the difference was statistically significant ( P <0.05), the NF-κB level in the AP group was lower than that in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the NF-κB levels in the AP and EA groups were lower than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the NF-κB levels in the EA and AP groups were lower than those in the AA group, and the difference was statistically significant ( P <0.05).

[0144] (2) TLR4 The overall differences among the groups were statistically significant P =0.024; compared with the BC group, the TLR4 levels in the MO group and the AA group were higher than those in the BC group, and the difference was statistically significant ( P <0.05), the level of TLR4 in the AP group was lower than that in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the TLR4 level in the AP group was lower than that in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the TLR4 levels in the EA and AP groups were lower than those in the AA group, and the difference was statistically significant ( P <0.05).

[0145] (3) IL-6 The overall differences among the groups were statistically significant P =0.008; compared with the BC group, the IL-6 levels in the MO and AA groups were higher than those in the BC group, and the difference was statistically significant ( P <0.05), the IL-6 level in the AP group was lower than that in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the IL-6 levels in the AP group and the EA group were lower than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the IL-6 levels in the EA and AP groups were lower than those in the AA group, and the difference was statistically significant ( P <0.05).

[0146] (4) IL-1β The overall differences among the groups were statistically significant P =0.001; compared with the BC group, the IL-1β levels in the MO group and the AA group were higher than those in the BC group, and the difference was statistically significant ( P<0.05), the IL-1β level in the AP group was lower than that in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the IL-1β level in the AP group was lower than that in the MO group, and the difference was statistically significant ( P <0.05)); Compared with the AA group, the IL-1β levels in the EA and AP groups were lower than those in the AA group, and the difference was statistically significant ( P <0.05).

[0147] (5) TNF-α The overall differences among the groups were statistically significant P =0.014; compared with the BC group, the TNF-α levels in the MO and AA groups were higher than those in the BC group, and the difference was statistically significant ( P <0.05), the TNF-α level in the AP group was lower than that in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the TNF-α levels in the AP group and the EA group were lower than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the TNF-α level in the AP group was lower than that in the AA group, and the difference was statistically significant ( P <0.05).

[0148] (6) IL-10 The overall differences among the groups were statistically significant P =0.014; compared with the BC group, the IL-10 levels in the EA group, AA group, and AP group were higher than those in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the IL-10 levels in the AP group and the EA group were higher than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the IL-10 levels in the EA and AP groups were higher than those in the AA group, and the difference was statistically significant ( P <0.05);

[0149] 3. Effect of electroacupuncture on the expression of proteins related to the inflammatory cytokine transduction pathway in the hippocampus of CUMS rats Western blot was used to detect the expression of proteins related to the inflammatory cytokine signaling pathway in the rat hippocampus (TLR4, p-p65, p65, TNF-α, IL-1β, and IL-10), and the quantification results of the grayscale values ​​of each protein were analyzed and statistically analyzed.

[0150] Immunoblotting of inflammatory cytokines and related proteins in the hippocampus of rats in each group Figure 30The relative expression levels of inflammatory cytokines and related proteins in the hippocampus of rats in each group are shown in Figure 31 (Note:*: P <0.05, **: P <0.01,***: P <0.001).

[0151] (1) TLR4 levels Compared with the BC group, the TLR4 level in the MO group was higher than that in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the TLR4 levels in the AP group and the EA group were lower than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the TLR4 levels in the EA and AP groups were lower than those in the AA group, and the difference was statistically significant ( P <0.05).

[0152] (2) p-p65 level Compared with the BC group, the p-p65 levels in the MO and AA groups were higher than those in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, except for the BC group, the other groups had no statistically significant differences ( P >0.05); compared with the AA group, the p-p65 levels in the EA and AP groups were lower than those in the AA group, and the difference was statistically significant ( P <0.05).

[0153] (3) p-65 level Compared with the BC group, the p-65 levels in the MO and AA groups were higher than those in the BC group, and the difference was statistically significant ( P <0.005); compared with the MO group, the p-65 levels in the AP group and the EA group were lower than those in the MO group, and the difference was statistically significant ( P <0.05); Compared with the AA group, the p-65 levels in the EA and AP groups were lower than those in the AA group, and the difference was statistically significant ( P <0.05).

[0154] (4) IL-1β level Compared with the BC group, the IL-1β level in the MO group was higher than that in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the IL-1β levels in the AP group, EA group, and AA group were lower than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the IL-1β levels in the EA and AP groups were lower than those in the AA group, and the difference was statistically significant ( P <0.05).

[0155] (5) TNF-α level Compared with the BC group, the TNF-α levels in the MO and AA groups were higher than those in the BC group, and the differences were statistically significant ( P <0.05); compared with the MO group, the TNF-α levels in the AP group and the EA group were lower than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the TNF-α levels in the EA and AP groups were lower than those in the AA group, and the difference was statistically significant ( P <0.05).

[0156] (6) IL-10 levels Compared with the BC group, the IL-10 levels in the AP and EA groups were higher than those in the BC group, and the difference was statistically significant ( P <0.05); compared with the MO group, the IL-10 levels in the AP group and the EA group were higher than those in the MO group, and the difference was statistically significant ( P <0.05); compared with the AA group, the IL-10 levels in the EA and AP groups were higher than those in the AA group, and the difference was statistically significant ( P <0.05).

[0157] IV. Summary Based on the experimental results in the previous section, it was shown that electroacupuncture has an important regulatory function on the inflammatory factors of intestinal tissue and the protein TLR4 / NF-κB related to the transduction pathway. In order to further illustrate that electroacupuncture affects the behavior of CUMS rats from the three levels of bacteria, intestines, and brain, this section focuses on the hippocampus of the brain. Using HE, q-PCR and WB experimental techniques, we first observe the changes between different groups from the morphological structure, and then demonstrate the argument of "whether electroacupuncture can affect the expression of proteins related to the inflammatory cytokine transduction pathway through the regulatory effect of microorganisms on the host" from the two levels of gene and protein expression.

[0158] The hippocampus is an important part of the brain's limbic system and is considered to be an important brain area for storing memory, controlling cognition and positioning. In the brains of patients with Alzheimer's disease, the hippocampus is the first area to be affected, manifested as memory loss and loss of directional perception. Brain hypoxia and encephalitis can also cause damage to the hippocampus. The hippocampus is the core brain area in charge of cognitive functions and is a key area of ​​focus in the study of mental illnesses.

[0159] This study found that HE staining results showed that the number of necrotic cells in the hippocampus of rats in the CUMS model group increased, accompanied by lymphocyte infiltration, and the hippocampus of the electroacupuncture combined with antibiotics group also showed the same changes; while the hippocampus of the electroacupuncture group had fewer necrotic cells and a more complete neuronal cell structure, and the electroacupuncture combined with probiotics group also showed consistency. This shows that electroacupuncture can reduce neuronal damage, necrosis and inflammatory infiltration in the hippocampus of CUMS.

[0160] At the gene and protein expression levels, the expression of pro-inflammatory factors IL-6, IL-1β, and TNF-α was upregulated in the model group and the electroacupuncture combined with antibiotics group, and the anti-inflammatory factor IL-10 was downregulated, and the difference was statistically significant. The expression of NF-κB and TLR4 increased, and the difference was statistically significant ( P <0.05), indicating that the immune cascade reaction is in an activated state. Electroacupuncture can downregulate the expression of proinflammatory factors IL-6, IL-1β, and TNF-α in the hippocampus of CUMS rats, upregulate the anti-inflammatory factor IL-10, and inhibit the expression of NF-κB and TLR4. The differences are statistically significant ( P <0.05). The expression of related proteins in the hippocampus of rats in the EA plus probiotics group showed the same trend. In addition, in the WB experiment, the semi-quantitative analysis results of p-p65 protein in the EA group and the EA plus probiotics group showed that there was no statistically significant difference between the two groups and the model group ( P >0.05), but the p-p65 gene expression levels in both groups were lower than those in the model group, and the differences were statistically significant ( P <0.05). p-p65 is the phosphorylated state of p-65. When p-p65 is dephosphorylated and enters the nucleus and is activated to p-65, DNA begins to transcribe. By comparing the original bands, it can be observed that the grayscale values ​​of the electroacupuncture group and the electroacupuncture combined with probiotics group are lower than those of the model group and the electroacupuncture combined with antibiotics group, indicating that p-p65 shows a downward trend like p-65. Since the comparison of band grayscale values ​​is a semi-quantitative analysis and is greatly affected by the brightness of the image and the shooting conditions, it is necessary to make a comprehensive judgment based on the results of qPCR.

[0161] TLR4 has a high affinity for bacterial lipopolysaccharide. After binding to LPS, TLR4 promotes signal transduction by activating adaptor proteins and specific intracellular pathways containing the Toll / IL-1R domain adaptor 1. When expressed on the cell membrane, TLR4 exists as a complex with the co-receptor myeloid differentiation protein-2 (MD-2). Binding of the TLR4–MD-2 complex to LPS leads to dimerization, which activates downstream NF-κB and leads to transcriptional activation. The most common NF-κB heterodimer consists of p50 and p65. Under normal circumstances, NF-κB exists in the cytoplasm in an inactive form. When the NF-κB pathway is stimulated by TLR4 polymers, proinflammatory factors (such as TNF-α, IL-6, and IL1β), or bacterial LPS, p65 is transported to the nucleus and activates various transcription factors. After activation, p65 is digested, terminating the activation of transcription factors, further inducing phosphorylation to stabilize p65 and positively regulating the activity of the NF-κB pathway. The continuous activation of the TLR4 / NF-κB pathway may lead to degenerative changes in the central nervous system. Microglia are the first line of immune defense in the central nervous system and play an important role in neuroinflammation. TLR4 and NF-κB are expressed in large quantities in microglia and can induce the activation of microglia.

[0162] Therefore, based on the theory of the humoral pathway of the "bacteria-gut-brain axis", the results of the above four research contents are connected in series. The homeostasis of the intestinal flora of CUMS rats is unbalanced, harmful bacteria increase, beneficial bacteria related to the production of short-chain fatty acids decrease, and the content of propionate and butyrate is downregulated. Short-chain fatty acids, as important metabolites, have an important function in maintaining the integrity of the mucosa. From the HE staining results of the colon, it can be seen that the intestinal blood barrier of the model group is damaged, the structural integrity of the intestinal mucosa is destroyed, and "leaky gut" is formed. The pro-inflammatory factors upregulated in the intestine include IL-6, TNF-α and IL-1β, as well as bacterial lipopolysaccharides, which enter the peripheral blood through the intestinal mucosa with increased permeability, ascend through the blood-brain barrier into the brain, and trigger an inflammatory response in the brain, especially in the hippocampus. TLR4 in the hippocampus of the model group is an important identifier of bacterial lipopolysaccharides. After binding with lipopolysaccharides, TLR4 leads to dimerization, resulting in the upregulation of downstream NF-κB. NF-κB begins to enter the nucleus for transcription, triggering a series of immune cascade reactions. A large number of pro-inflammatory factors are enriched in the hippocampus, including IL-6, TNF-α and IL-1β, and these pro-inflammatory factors will in turn further activate NF-κB nuclear transcription, keeping it in an activated state, microglia are activated in large numbers, and the central nervous system has an inflammatory response, which ultimately has a negative impact on cognition and behavior.

[0163] The electroacupuncture group restored the homeostasis of the intestinal flora by increasing the number of beneficial bacteria and reducing harmful bacteria. With the increase in the content of metabolites such as propionate and butyrate, the integrity of the intestinal mucosa was restored, and bacterial-derived proinflammatory metabolites could not penetrate the intestinal blood barrier and enter the peripheral circulation. At the same time, the proinflammatory factors IL-6, TNF-α and IL-1β in the intestine decreased, the anti-inflammatory factor IL-10 was upregulated, and the content of TLR4 and NF-κB was downregulated. The expression levels of TLR4, NF-κB and various proinflammatory factors in the hippocampus were significantly lower than those in the model group, indicating that the inflammatory cascade reaction in the central nervous system was blocked. The electroacupuncture combined with probiotics group also showed the same trend. It is worth noting that the electroacupuncture combined with antibiotics group did not show the corresponding anti-inflammatory effect of electroacupuncture. Intestinal microorganisms are the target of this study. It is well known that probiotics play an important role in the homeostasis of intestinal microorganisms. In addition to electroacupuncture intervention, electroacupuncture combined with probiotic intervention can also restore the homeostasis of intestinal flora, repair the intestinal mucosa, and inhibit the inflammatory cascade in the brain by upregulating the expression of metabolites (such as butyrate, propionate) and anti-inflammatory factor IL-10. The use of antibiotics has a long-term negative impact on the homeostasis of intestinal microorganisms. Antibiotics disrupt the intestinal microecology, which is equivalent to blocking the target intestinal flora. Therefore, the pathway regulated by electroacupuncture through the "bacteria-gut-brain axis" is inhibited, and ultimately it cannot have an antidepressant effect. This proves that electroacupuncture can treat depression through the humoral pathway of the "bacteria-gut-brain axis".

[0164] The above experimental results of the present invention are as follows: 1. Results of the behavioral effects of electroacupuncture on CUMS rats In this study, the rats in each CUMS modeling group were compared with the blank group. After 35 days of modeling, the sugar water consumption decreased, and the difference was statistically significant ( P <0.05), the body weight was lighter, and the difference was statistically significant ( P <0.05), the exploratory behavior decreased, and the horizontal and vertical movement activities decreased, and the difference was statistically significant ( P <0.05), indicating that the anhedonia and exploration desire of rats in the CUMS model group were significantly reduced, indicating that the model was successfully established; Under 28-day intervention and continuous CUMS stimulation, compared with the model group, the rats in the EA group and EA plus probiotics group had increased sugar water consumption, increased body weight, restored exploratory behavior, and increased horizontal and vertical movement activities after 28 days of intervention, and the differences were statistically significant ( P <0.05), indicating that electroacupuncture has a significant reversal effect on the phenomenon of reduced sugar water consumption in rats under CUMS stimulation, and has a significant improvement effect on restoring the exploratory behavior of rats.

[0165] 2. Results of the effects of electroacupuncture on intestinal microorganisms and metabolites in CUMS rats There was no statistically significant difference in α diversity among the rats in each group after intervention ( P >0.05), except for the electroacupuncture group and the electroacupuncture plus probiotics group, the β diversity of the other groups showed statistically significant differences ( P <0.05). LEfSe was used to compare the species differences between the two groups, and the results were as follows: Compared with the blank group, at the phylum level, the Firmicutes in the model group ( Firmicutes ) abundance decreased; at the class level, the δProteobacteria in the model group ( Deltaproteobacteria ) abundance increased, Clostridium ( Clostridia ) order level abundance decreased; at the order level, Clostridiales ( Clostridiales ) decreased; at the family level, the Lachnospiraceae ( Lachnospiraceae ) decreased in abundance; at the genus level, the model group Lachnospiraceae_NK4A136、 Odoribacter, Rumiriclostridium_UCG_005, Eubacterium xylanophile The abundance of Butyrivibrio spp. Butyrivibrio )、Rikenbacterium RC9 gut( Rikenellaceae RC9 gut ) abundance increased.

[0166] Compared with the EA group, at the phylum level, Firmicutes ( Firmicutes ) decreased in abundance, and Verrucomicrobia ( Verrucomicrobia ) abundance increased; at the class level, the model group Verrucomicrobiasis At the order level, the abundance of Verrucomicrobiales Increase; at the family level, the model group Clostridiaceae_1 The abundance decreases, Akkermansiaceae At the genus level, the abundance of Clostridium_sensu_stricto_1、 Turictbacter、Alloprevotella The abundance decreases, Akkermansia Genera abundance increased.

[0167] Compared with the electroacupuncture plus antibiotic group, at the phylum level, Epsilonbacteriaceae The abundance of phylum decreased; at the class level, the δProteobacteria in the model group ( Deltaproteobacteria ) abundance increased; at the order level, the Desulfovibrioles ( Desulfovibrionales ) levels increased; at the family level, the model group's Peptostreptococcus family ( Peptostreptococcaceae ) decreased in abundance, and Desulfovibrioles ( Desulfovibrionaceae ) abundance increased; at the genus level, the model group Romboutsia、ASF356、Anaerotruncus、Butyrivibrio、Lachnoclostridium The abundance decreased.

[0168] Compared with the electroacupuncture plus probiotics group, at the phylum level, Tenericutes, Actinobacteria At the class level, the abundance of the model group Bacillus The abundance of δProteobacteria was low. Deltaproteobacteria) abundance increased; at the order level, the Lactobacillus orders ( Lactobacilli ) decreased in abundance, Soft-bodied At the genus level, the abundance of Lactobacillus, Odoribacter The abundance decreases, Eubacterium coprostanoligenes Candidatus stoquerficus Increased abundance.

[0169] The quantitative results of short-chain fatty acids in each group after modeling were compared with those in the blank group. It was found that the content of acetate in the intestine of rats under stress increased, and the difference was statistically significant ( P <0.05), the contents of propionate and butyrate decreased, and the difference was statistically significant ( P <0.05). After electroacupuncture intervention, the acetate content in the electroacupuncture group was lower than that in the model group and the electroacupuncture plus antibiotic group, and the difference was statistically significant ( P <0.05), propionate was higher than that in the model group, and the difference was statistically significant ( P <0.05), butyrate content was higher than that in the model group and the electroacupuncture combined with antibiotics group, and the difference was statistically significant ( P <0.05); the propionate content in the electroacupuncture combined with probiotics group was higher than that in the model group, and the difference was statistically significant ( P <0.05), butyrate was higher than that in the model group and the electroacupuncture combined with antibiotics group, and the difference was statistically significant ( P <0.05).

[0170] 3. Results of the effect of electroacupuncture on inflammatory factors and colon morphology in CUMS rats The results of HE staining experiments showed that the intestinal structure of rats in the model group became less compact, lymphocyte infiltration increased, and the mucosal layer was severely damaged. The goblet cells in the electroacupuncture group were fuller and thicker, arranged tightly, and the mucosal density was higher. This shows that electroacupuncture can improve the intestinal inflammatory infiltration of CUMS rats and has a certain repair effect on the damaged intestinal barrier.

[0171] The results of Elisa quantitative detection showed that electroacupuncture can reduce the levels of pro-inflammatory factors IL-6, IL-1β, and TNF-α in the colon, and the difference was statistically significant ( P <0.05), and increased the level of anti-inflammatory factor IL-10, the difference was statistically significant ( P <0.05), reducing the levels of NF-κB and TLR4, proteins closely related to inflammatory response, with statistically significant differences ( P <0.05).

[0172] 4. Effect of electroacupuncture on the transduction pathway of inflammatory cytokines in the hippocampus of CUMS rats This study found that electroacupuncture can reduce neuronal damage, necrosis and inflammatory infiltration in the hippocampus of CUMS rats, and the electroacupuncture combined with probiotics group also showed the same morphological changes.

[0173] At the gene and protein expression levels, the expression of NF-κB and TLR4 increased in the model group and the electroacupuncture combined with antibiotics group, and the difference was statistically significant ( P <0.05), the expression of pro-inflammatory factors IL-6, IL-1β, and TNF-α were upregulated, and the difference was statistically significant ( P <0.05), the anti-inflammatory factor IL-10 was down-regulated, and the difference was statistically significant ( P <0.05). Electroacupuncture can inhibit the expression of NF-κB and TLR4 in the hippocampus of CUMS rats, and the difference is statistically significant ( P <0.05), down-regulated the expression of pro-inflammatory factors IL-6, IL-1β, and TNF-α in the hippocampus, and the difference was statistically significant ( P <0.05), and the anti-inflammatory factor IL-10 was up-regulated, and the difference was statistically significant ( P <0.05). The expression of related proteins in the hippocampus of rats in the electroacupuncture combined with probiotics group showed the same trend.

[0174] In summary, the present study shows that the electroacupuncture group can regulate the homeostasis of intestinal flora, reduce harmful bacteria, increase beneficial bacteria, repair intestinal mucosal damage by upregulating short-chain fatty acids such as propionate and butyrate, activate the production of anti-inflammatory factor IL-10, inhibit the production of proinflammatory factors IL-6, TNF-α and IL-1β in the intestine, reduce the expression of TLR4, NF-κB and various proinflammatory factors in the hippocampus of the central nervous system, block the occurrence of inflammatory cascade reactions, and improve the depressive-like behavior of CUMS rats.

[0175] 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 studying the mechanism of electroacupuncture on the antidepressant gut-brain axis, characterized in that: The following steps are involved: 1) Modeling and intervention SPF healthy male adult Sprague-Dawley rats were divided into a blank group, a model group, an electroacupuncture group, an electroacupuncture combined with antibiotics group, and an electroacupuncture combined with probiotics group. Except for the blank group, rats in the model group, the electroacupuncture group, the electroacupuncture combined with antibiotics group, and the electroacupuncture combined with probiotics group were subjected to chronic mild unpredictable stimulation every day to establish a rat model of depression. After modeling, rats in each group were intervened according to the following methods: rats in the blank group were allowed to eat and drink freely and were gavaged with normal saline; rats in the model group were given chronic mild unpredictable stimulation every day and were gavaged with normal saline; rats in the electroacupuncture group were given chronic mild unpredictable stimulation every day, electroacupuncture stimulation every week and were gavaged with normal saline; rats in the electroacupuncture plus antibiotics group were given chronic mild unpredictable stimulation every day, electroacupuncture stimulation every week and were gavaged with antibiotics; rats in the electroacupuncture plus probiotics group were given chronic mild unpredictable stimulation every day, electroacupuncture stimulation every week and were gavaged with probiotics; Specifically, the electroacupuncture stimulation comprises the following steps: 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; under 2.5% to 3% isoflurane inhalation anesthesia, the Baihui and Yintang points are punctured horizontally, with the Baihui point puncturing toward the back of the head and the Yintang point puncturing toward the nose tip; the needle insertion depth is 2-3mm; the Zusanli point is punctured vertically, with the needle insertion depth of 3-5mm; the acupuncture needles at the Baihui and Yintang points are connected to an electroacupuncture instrument, with a current frequency of 8-12Hz, continuous wave, and a current intensity of 1-3mA, for 15-25min each time; 2) Effects of electroacupuncture on intestinal microorganisms and metabolites in CUMS rats The feces of rats in each group were collected before modeling, after modeling, and after intervention; The effects of electroacupuncture on intestinal microorganisms and metabolites of CUMS rats were explored by performing species community analysis, Alpha diversity analysis, Beta diversity analysis, and species difference analysis on the feces of rats in each group after intervention, and extracting short-chain fatty acids from the feces of rats in each group before, after, and after modeling to conduct targeted SCFAs quantitative analysis. 3) Effects of electroacupuncture on colon morphology and inflammatory cytokines in CUMS rats The colon tissues of rats in each group after intervention were subjected to hematoxylin-eosin staining and enzyme-linked immunosorbent assay to explore the effects of electroacupuncture on colon morphology and inflammatory cytokines in CUMS rats. 4) Effects of electroacupuncture on inflammatory cytokine transduction pathways in the hippocampus of CUMS rats The effects of electroacupuncture on the inflammatory cytokine transduction pathway in the hippocampus of CUMS rats were explored by using hematoxylin-eosin staining, fluorescent quantitative polymerase chain reaction and protein immunoblotting experiments on the brain tissue of rats in each group after intervention.

2. The method for studying the mechanism of electroacupuncture on the antidepressant gut-brain axis according to claim 1, characterized in that: After modeling, rats in each group were intervened according to the following methods: Blank group: free food and water intake, 20 min isoflurane anesthesia 5 times a week, and normal saline gavage for 4 weeks; Model group: Chronic mild unpredictable stimulation was performed every day, isoflurane anesthesia was given for 20 min 5 times a week, and normal saline was gavaged for 4 weeks; Electroacupuncture group: daily chronic mild unpredictable stimulation, intragastric administration of normal saline for 4 weeks, electroacupuncture stimulation 5 times a week; The EA plus antibiotic group: daily chronic mild unpredictable stimulation, intragastric administration of antibiotics for 2 weeks, intragastric administration of saline for 2 weeks, and EA stimulation 5 times a week; The electroacupuncture plus probiotics group: chronic mild unpredictable stimulation was performed every day, probiotics were gavaged for 4 weeks, and electroacupuncture stimulation was performed 5 times a week.

3. The method for studying the mechanism of electroacupuncture on the antidepressant bacteria gut-brain axis according to claim 2, characterized in that: The current frequency of the electroacupuncture stimulation is 10 Hz, the current intensity is 2 mA, and each time is 20 minutes.

4. The method for studying the mechanism of electroacupuncture on the antidepressant bacteria gut-brain axis according to claim 1, characterized in that: The stimulation factors of chronic mild unpredictable sexual 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 seven, and the stimulation factors are not repeated in two consecutive days.

5. The method for studying the mechanism of electroacupuncture on the antidepressant bacteria gut-brain axis according to claim 1, characterized in that: In the step 2), after collecting the feces of the rats in each group before modeling, after modeling, and after intervention, the feces of the rats were placed in sterile enzyme-free EP tubes, first placed in dry ice, and then frozen at -80°C.

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