Method for preparing Ningxiang bacterial liquid for improving chronic colitis and enteritis, bacterial liquid prepared by method and application of bacterial liquid

Abstract

The invention discloses a method for preparing an enteritis Ningxia bacterial liquid for improving chronic colitis, the enteritis Ningxia bacterial liquid prepared by the method and application of the enteritis Ningxia bacterial liquid. According to the present invention, dextran sulfate sodium (DSS) is adopted to intermittently induce mice, a chronic UC mouse disease model is constructed, the treatment effect of the enteritis Ningxia bacterial liquid on chronic colitis is researched, and the research result proves that the enteritis Ningxia bacterial liquid has the treatment and prevention effects on chronic colitis by adjusting intestinal flora. FMT treatment shows a remarkable treatment effect in the aspect of relieving colitis, intervention of FCL remarkably relieves colitis disease symptoms caused by DSS, relieves DSS-induced weight loss, reduces DAI and histological scores, reduces serum inflammatory factors, reduces intestinal permeability, and improves the treatment effect by changing intestinal flora and promoting SCFAs to regulate the steady state of serum cytokines. And the disease symptoms of UC are relieved.

Description

Technical Field

[0001] The invention relates to the field of preparation of Enteritis Ning fecal bacteria liquid, and specifically to a method for preparing Enteritis Ning fecal bacteria liquid for improving chronic colitis, and the prepared Enteritis Ning fecal bacteria liquid and application thereof. Background Art

[0002] Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), are chronic recurrent inflammatory diseases of the gastrointestinal tract, characterized by weight loss, abdominal pain, diarrhea, and blood in the stool. UC is believed to occur in people with a genetic predisposition after environmental exposure, and is closely related to intestinal epithelial barrier defects, intestinal flora, and immune response disorders. In recent years, the incidence of UC, known as a "Western disease," has increased rapidly in my country and has become a common digestive system disease. With the improvement of my country's productivity and per capita disposable income, the dietary structure of residents has become increasingly Westernized, and the proportion of colitis patients has also increased year by year.

[0003] At present, the drugs used clinically for IBD aim to relieve symptoms and improve the quality of life of patients, but these drugs have their own shortcomings. More and more studies have shown that Chinese medicine (compound) has good efficacy in the treatment of UC, whether it is taken orally or by enema, especially in improving the clinical symptoms of patients and improving the quality of life of patients. It has the advantages of low recurrence rate and few side effects. Studies have found that intestinal flora is directly involved in the initiation and development of IBD, and is considered to be an important factor in the pathogenesis of IBD and an important target for the study of IBD therapeutic drugs. It can regulate the life activities of the host, regulate the immune response, and resist intestinal flora imbalance, playing an important role in protecting the health of the host.

[0004] The human intestinal microbiota is a complex ecosystem, and a dynamic balance is maintained between the intestinal flora and the host to maintain the host's physical health. It plays an irreplaceable role in life activities such as metabolism, energy consumption, nutrient absorption, development and regulation of the immune system. Although the diversity and richness of microbial species vary greatly between individuals, the bacterial structure at the phylum level is similar. The human intestinal flora is dominated by Bacteroidetes and Firmicutes, accounting for more than 90% of the entire phylogenetic type. Intestinal microorganisms have multiple metabolic potentials because the number of genes encoded by the genome is rich, 100-150 times that of the human genome. About 1.5 kg of bacteria reside in our intestines, and 50% of our fecal biomass is bacterial cells.

[0005] Intestinal flora is closely related to inflammatory bowel disease. Intestinal flora usually colonizes on the surface of the intestinal mucosa to form a biofilm, which constitutes the biological barrier of the intestine and also affects the intestinal metabolism of nutrients, intestinal permeability and intestinal immune system function. For a healthy body, its innate or adaptive immunity can coexist with normal flora in a friendly manner to prevent harmful flora from causing harm to the body. However, the imbalance of intestinal flora not only causes intestinal inflammatory reactions, but also leads to a rapid increase in harmful bacteria in the intestine. The massive reproduction and adhesion of harmful flora will invade the intestinal mucosa, damage intestinal epithelial cells, and cause damage to the intestinal mucosal barrier. In addition, enterotoxins such as lipopolysaccharides produced by harmful flora will increase the permeability of the intestinal mucosa, and the immunosuppressive proteins released by them will also cause host intestinal immune dysfunction. IBD patients have intestinal flora disorders, such as a decrease in the abundance of beneficial bacteria such as Plasmodium and Roseburia, and an increase in the abundance of potential pathogenic bacteria such as Escherichia coli and Bacteroides fragilis; the metabolites of the intestinal flora also change, such as a decrease in the content of SCFAs, secondary bile acids, tryptophan and its metabolites.

[0006] Changyangning (CYN) tablets and syrup are Chinese herbal medicines with Chinese characteristics, which are included in the Pharmacopoeia of the People's Republic of China (hereinafter referred to as the Chinese Pharmacopoeia). The whole prescription is composed of five Chinese medicinal materials: Herba Dijin, Herba Goldenrod, Elsholtzia, Liquidambar formosana leaves, and Camphor tree roots. It has the effects of clearing heat and dampness, and promoting qi circulation. It is often used for acute and chronic gastroenteritis, diarrhea, bacterial dysentery, and pediatric dyspepsia with the above symptoms. Changyangning granules have the effects of stopping diarrhea, inhibiting gastrointestinal propulsion, and combating acute exudative inflammation; in vitro, it can inhibit the growth of Staphylococcus epidermidis and Escherichia coli. Herba Dijin is commonly used in traditional Chinese medicine for jaundice, dysentery, enteritis, etc., and its ingredients have anti-inflammatory, antioxidant, antifungal, and anti-hepatitis B virus pharmacological activities. Herba Goldenrod has the effects of clearing heat and dampness, detoxifying and reducing swelling. It is often used in traditional Chinese medicine to treat diseases such as cholestasis, hepatitis, and enteritis. Elsholtzia warms the stomach and regulates the middle, promotes water circulation and dispels dampness, and is mainly used to treat abdominal pain, vomiting, diarrhea, etc. Maple leaves dispel wind and dampness, and are mainly used to treat diseases such as dysentery and enteritis. Camphor root is the root of Litsea cubeba, a plant of the Cinnamomum family. It has the effects of dispelling wind and cold, regulating qi and invigorating qi, relieving pain and itching, strengthening the heart and calming spasms, killing insects, resisting inflammation, and inhibiting intestinal peristalsis. It is mainly used to treat rheumatic bone pain, traumatic injuries, colds and headaches.

[0007] In current clinical practice, the traditional Chinese medicine formula CYN is often used alone or in combination with other drugs to treat UC, acute enteritis, acute infectious diarrhea, irritable bowel syndrome, etc. Relevant research and sales have shown that CYN is effective in treating UC.

[0008] For example, Tang Wenling's research shows that CYN has a certain therapeutic effect on IBD, while increasing the diversity of intestinal flora in IBD model animals, regulating the composition and structure of intestinal flora, and restoring flora disorders. (Tang Wenling, Study on the Effect of Changyanning on Intestinal Flora and Therapeutic Mechanism of Inflammatory Bowel Disease, Zhejiang University, 2020)

[0009] Zheng Yanhong's research shows that CYN can improve the symptoms of simple adhesive intestinal obstruction to a certain extent, and has the effect of enhancing intestinal peristalsis and repairing intestinal barrier damage. (Zheng Yanhong, Determination of putrescine in jejunal tissue of rats with intestinal obstruction and study on the effect of Changyanning, Tianjin Medical University, 2015)

[0010] Wang Hairong et al.'s research shows that CYN granules have the effects of stopping diarrhea, inhibiting gastrointestinal propulsion and combating acute exudative inflammation, and can inhibit the growth of Staphylococcus epidermidis and Escherichia coli in vitro. (Wang Hairong et al., Study on the anti-inflammatory and antidiarrheal effects of Changyanning granules on young mice and their in vitro antibacterial effects, Drug Evaluation Research, 2017)

[0011] Liu Yongxiang et al. disclosed the application of the Changyanning preparation in the preparation of drugs for regulating intestinal flora. They found that through the inflammation-improving effect on the inflammatory bowel disease rat model and the bioinformatics study of the rat intestinal flora, the Changyanning preparation prevented and treated intestinal flora imbalance diseases by regulating the abundance and species of intestinal flora, and had a significant improvement effect on the colon crypt damage and deformation, inflammatory cell infiltration, etc. in the Crohn's disease model rats (CN 114601870 A).

[0012] Based on the above literature, it can be shown that CYN has a significant therapeutic effect, but the mechanism by which CYN changes chronic UC is still unclear and has not been reported. In recent years, research on intestinal flora in health and disease has increased significantly. Many studies have shown that human diseases are related to intestinal flora imbalance. Fecal microbiota transplantation experiments using rodent models to study human and animal donors can help us understand the causal relationship between intestinal flora and disease. Summary of the invention

[0013] The invention provides a method for preparing a fecal bacteria liquid for improving chronic colitis, the prepared fecal bacteria liquid and applications thereof.

[0014] The present invention uses 3% dextran sulfate sodium (DSS) to intermittently induce mice to construct a chronic UC mouse disease model, and studies the therapeutic effect of Changyanning fecal bacteria liquid on chronic colitis. The research results prove that Changyanning fecal bacteria liquid has a therapeutic and preventive effect on chronic colitis by regulating intestinal flora.

[0015] The purpose of the present invention is to provide a preparation method and application of a fecal bacteria liquid for treating chronic colitis.

[0016] The technical solution adopted in the present invention is:

[0017] A method for preparing a fecal bacteria liquid for improving chronic colitis comprises the following steps:

[0018] (1) Collection: Feces discharged from chronic colitis mice after 34 to 38 days (more preferably 36 days) of Changyanning treatment were collected using an SPF barrier;

[0019] (2) Stirring: Place mouse feces in a blender, add physiological saline solution, and stir evenly;

[0020] (3) Centrifugation: The obtained fecal solution is centrifuged for 1 to 5 minutes (most preferably 3 minutes), and the supernatant is collected.

[0021] The SPF-level barrier environment in step (1) requires a temperature of 20-24° C., a humidity of 40%-60%, and an alternating light-dark cycle of 12 hours of light and 12 hours of darkness.

[0022] The mouse feces are fresh feces that are immediately excreted from the body of mice under natural conditions.

[0023] The colitis in step (1) refers to a chronic colitis mouse model induced by DSS (dextran sulfate sodium).

[0024] The mouse feces are the feces discharged by chronic colitis mice treated with Changyanning for 36 days, and the colitis symptoms are significantly improved. The feeding dose of Changyanning is 0.25g / kg-1g / kg, further preferably 0.4-0.6g / kg, and preferably 0.5g / kg.

[0025] In the step (2), fresh mouse feces and physiological saline are mixed at a mass:volume ratio of 1:8-11 (g / mL), preferably 1:9 (g / mL).

[0026] The centrifugation conditions in step (3) are 2500-3000 rpm and 1-4°C.

[0027] A fecal bacteria liquid for Changyanning, obtained by the preparation method described in any one of claims 1 to 7;

[0028] The storage temperature of the anti-enteritis Ning bacteria liquid is ≤-80°C, and the storage time is ≤6 months.

[0029] The application is used in the preparation of medicines or preparations for treating inflammatory bowel disease. The dosage of the resistant Enteritis Ning bacteria liquid for intragastric administration is 5-15 mL / kg per mouse, and the intragastric administration time is 36 days.

[0030] The dosage of Enteritis Ning bacterial solution is calculated based on the clinically used human fecal bacterial solution dosage (100-300 mg / kg / d) and the average mouse weight of 22-24 g / mouse.

[0031] The invention provides a method for preparing a Changyanning fecal bacteria liquid and application of the liquid in treating chronic colitis.

[0032] The Changyanning fecal bacteria liquid of the present invention can also be used to prepare medicines for treating symptoms of chronic colitis.

[0033] The present invention verifies through fecal microbiota transplantation experiments that the inhibitory effect of CYN on DSS-induced colon inflammation in mice is achieved by regulating intestinal flora, and that fecal microbiota transplantation treated with Changyanning can treat and prevent chronic colitis.

[0034] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0035] (1) FMT treatment showed significant therapeutic effects in alleviating colitis. Intervention with FCL significantly alleviated the disease symptoms of DSS-induced colitis, alleviated DSS-induced weight loss, reduced DAI and histological scores, downregulated serum inflammatory factors, reduced intestinal permeability, and alleviated the disease symptoms of UC by changing the intestinal flora and promoting the homeostasis of serum cytokines by SCFAs.

[0036] (2) FMT treatment alleviated the symptoms of UC mice by increasing Lachnospiraceae and Oscillospiraceae and beneficial bacteria Dubosiella, Lachnospiraceae_NK4A136_group and uncultured_Bacteroidales_bacterium, Akkermansia_muciniphila; and reducing Muribaculaceae, Erysipelatoclostridiaceae and Lactobacillaceae, Bacteroides and Erysipelatoclostridium, Parasutterella_excrementihominis, Streptococcaceae_bacterium, Ligilactobacillus_murinus, Erysipelatoclostridium_ramosum, Bacteroides_vulgatus, and Bacteroides_uniformis.

[0037] (3) FMT from CL donors significantly alleviated the symptoms of colitis in mice and significantly improved intestinal permeability. However, MC donors also showed the effect of alleviating colitis, but the efficacy was significantly lower than the intervention effect of FCL, indicating that CYN may play a role in alleviating colitis by regulating intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the group modeling scheme.

[0039] Figure 2 This is a flow chart of FMT animal experiments.

[0040] Figure 3 Effects of FMT on body weight (A) and food intake (B) of mice with DSS-induced colitis.

[0041] Figure 4 Effects of FMT on DAI in mice with DSS-induced colitis, including: (A) fecal status; (B) occult blood test; (C) DAI.

[0042] Figure 5 Effects of FMT on colon length and histomorphology of mice with DSS-induced colitis, including: (A) colon length distribution; (B) colon histomorphology; * and # indicate significant differences compared with the MC and FMC groups, respectively (P<0.05).

[0043] Figure 6 Effects of FMT on organ indexes of DSS-induced colitis mice, including: (A) liver index; (B) spleen index; (C) kidney index; * indicates significant difference compared with the MC group (P<0.05).

[0044] Figure 7 Effects of FMT on inflammatory factors in mice with DSS-induced colitis, including the levels of TNF-α (A), IL-1β (B), IL-6 (C), and IL-10 (D) in serum; * and # indicate significant differences compared with the MC and FMC groups, respectively (P<0.05).

[0045] Figure 8 Effects of FMT on intestinal permeability of mice with DSS-induced colitis, including: (A) LPS concentration; (B) DAO concentration; * and # indicate significant differences compared with the MC and FMC groups, respectively (P<0.05).

[0046] Fig. 9 This is the sequencing curve analysis, including: (A) rarefaction curve; (B) Shannon index curve; (C) rank abundance curve; (D) species accumulation curve.

[0047] Fig.10The effect of CYN on the ASV of intestinal flora in mice with fecal microbiota transplantation, including (A) ASV between individuals and (B) ASV between groups; (C) Venn diagram based on ASV; * indicates significant difference compared with the MC group (P<0.05).

[0048] Fig.11 The α diversity of the microbiota of FMT mice, including: (A) Ace index; (B) Chao1 index; (C) Simpson index; (D) Shannon index; (E) PD_whole_tree index; (F) coverage rate; * indicates significant difference compared with the MC group (P<0.05).

[0049] Fig.12 Effect of FMT on the β-diversity of intestinal flora in mice with colitis.

[0050] Fig.13 Figure 3 Effects of FMT on the phylum level of intestinal flora in mice, where (A) phylum-level flora structure; (BF) relative abundances of Bacteroidota, Firmicutes, Firmicutes / Bacteroidota, Proteobacteria, Verrucomicrobiota, and Fusobacteriota, respectively; * and # indicate significant differences compared with the MC and FMC groups, respectively (P<0.05).

[0051] Fig.14 Figure 3 Effects of FMT on the family level of intestinal flora in mice, where (A) is the family level flora structure; (BG) are the relative abundance analysis of significantly different genera; * and # indicate significant differences compared with the MC and FMC groups, respectively (P<0.05).

[0052] Fig.15 Figure 3 Effects of FMT on the genus level of intestinal flora in mice, including (A) family level flora structure; (BG) relative abundance analysis of significantly different genera; * and # indicate significant differences compared with the MC and FMC groups, respectively (P<0.05).

[0053] Fig.16 Figure 3 Effects of FMT on the intestinal flora of mice at the species level, including (A) family level flora structure; (BG) relative abundance analysis of significantly different genera; * and # indicate significant differences compared with the MC and FMC groups, respectively (P<0.05).

[0054] Fig.17 Figure 2: LEfSe analysis of the intestinal flora of colitis mice induced by FMT, including (A) LDA scores of differentially abundant groups; (B) classification branch diagram obtained by LEfSe analysis of 16S sequences.

[0055] Fig.18 Prediction of KEGG functional genes of intestinal flora, including: (A) functional composition bar graph; relative abundance of functional genes of MC and FMC (B), MC and FCL (C), FMC and FCL (D) in the second-level KEGG category.

[0056] Fig.19 Effects of FMT on short-chain fatty acids in mice with DSS-induced colitis, including: (A) acetate; (B) propionate; (C) butyrate; (D) valerate; (E) isobutyrate; (F) isovalerate; (G) total acid; * and # indicate significant differences compared with the MC and FMC groups, respectively (P<0.05).

[0057] Fig. 20 It is the technical roadmap of the present invention. DETAILED DESCRIPTION

[0058] The fresh feces of mice fed with Enteritis Ning are collected through an SPF barrier, mixed with physiological saline in a closed space, homogenized, filtered, and centrifuged. The supernatant is discarded, and the mixture is resuspended in physiological saline. After oscillation and mixing, the centrifugation operation is repeated; the resulting precipitate is resuspended in physiological saline, which is the Enteritis Ning fecal bacterial solution. Fresh feces and physiological saline are mixed according to mass: volume = 1: 8-11 (g / mL). The dosage of Enteritis Ning for feeding is 0.25g / kg-1g / kg, preferably 0.5g / kg. The mouse feces are the feces discharged by mice with chronic colitis after 36 days of Enteritis Ning treatment. The dosage of Enteritis Ning bacterial solution is calculated based on the clinically used human fecal bacterial solution dosage (100-300mg / kg / d) and the average weight of mice 22-24g / mouse.

[0059] Example 1: Construction of mouse model

[0060] (1) Healthy 8-week-old male specific pathogen free (SPF) C57BL / 6 mice (20–22 g) were purchased from Zhejiang Academy of Medical Sciences (Hangzhou, China) with production license number SCXY(Zhejiang)2019-0002 and use license number SYXK(Zhejiang)2022-0007 and were kept at the Experimental Animal Center of Zhejiang University of Technology.

[0061] (2) During animal feeding, the temperature (25±2℃) and humidity (50%±5%) were controlled, and the animals were kept in a 12-h light / dark cycle. The animals were fed with AIN93 standard feed and were allowed to eat freely. The mice were randomly divided into cages with 5 mice / cage. After acclimating to the environment for 7 days, the mice in the normal control group were given normal diet and drinking water without any treatment (NC group); the mice in the chronic colitis model were given a normal diet and 3% DSS solution was given to drink freely every day instead of drinking water for 5 consecutive days, and then sterilized drinking water was used for 7 days. This 12-day cycle was repeated for 3 cycles to induce the chronic UC model in mice ( Figure 1 ). The 3% DSS solution was prepared before use and used immediately after preparation. In order to evenly disperse CYN in the liquid during gavage, 0.5% CMC-Na aqueous solution was used to prepare the CYN gavage solution, which was changed daily. UC model mice were randomly divided into a model control group (MC, n=10, gavage of 0.5% CMC-Na solution), a low-dose group (CL, n=10, gavage of 0.25g / kg CYN), a medium-dose group (CM, n=10, gavage of 0.5g / kg CYN) and a high-dose group (CH, n=10, gavage of 1g / kg CYN). The formal experiment lasted for 36 days. The specific animal groupings are as follows. Figure 1 shown.

[0062] One day before the mice were euthanized, fresh feces of the mice were collected, frozen in liquid nitrogen and stored at -80°C. At the end of the experiment (day 37), the mice were injected with 100 mg / kg of sodium pentobarbital, and blood was taken by cardiac puncture after deep anesthesia, and the mice were euthanized. The serum was obtained by centrifugation at 4°C, 3000g for 20 minutes. At the same time, the liver, spleen, kidney and colon contents of each mouse in each group and the ileum and colon tissues were collected on a sterile operating table, and the organ weight and colon length were measured and recorded. For histological studies, 1 cm long fresh intestinal segments were fixed in 4% (w / v) paraformaldehyde (pH 7.0). The collected serum and other tissue and organ samples were stored at -80°C for further biochemical analysis.

[0063] (3) Design of fecal microbiota transplantation experiment. For fecal microbiota transplantation mice, mixed antibiotics were first added to the drinking water of mice (containing 1g / L neomycin, 0.5g / L vancomycin, 1g / L ampicillin and 1g / L metronidazole) to eliminate the intestinal flora of mice for 1 week (the intestinal flora elimination rate was more than 90% as determined by RT-qPCR). The diet remained unchanged in order to establish a pseudo-sterile mouse model. The mice lost weight and were switched back to drinking water. In the next 36 days, the fecal supernatant of mice in the MC group (FMC group) and the CL group (FCL group) was gavaged (the weight of fresh feces was 1g / kg of mouse body weight), and other treatments and Figure 1 The animals are the same, and the FMT test flow chart is shown in Figure 2 .

[0064] Among them, the fecal bacterial suspension was prepared by adding 0.2 g of freshly collected mouse feces into 2 mL of animal drinking water, mixing thoroughly and centrifuging, and collecting the supernatant as a transplant sample, as shown in Table 1.

[0065] Table 1 Experimental animal groups

[0066]

[0067] Example 2: Effect of CYN on the disease phenotype of chronic UC mice via intestinal flora

[0068] (1) Analysis of body weight and food intake of FMT mice

[0069] The weight of mice was recorded every day, and the weight, food and water intake of mice in each group were recorded. Figure 3 As shown in the figure, the mice with colitis lost weight and their food intake decreased. In the first cycle, the weight of each group decreased significantly, and the weight of the MC group decreased most significantly compared with the initial weight. The weight of the mice in the FMT group was significantly lower than that of the MC group due to antibiotic treatment. The FMT group had a trend of alleviating weight gain compared with the MC group, and the effect of the FCL group was better than that of the FMC group, indicating that gavage of feces from the CL group can alleviate the weight loss of mice.

[0070] (2) Analysis of pathological characteristics of FMT mice

[0071] The weight of mice was recorded every day, and the mental state of mice and whether pathological phenomena occurred were observed. The weight of mice in each group was recorded, and the food and water intake was evaluated. The stool characteristics, occult blood or bloody stool, and death were evaluated. DAI is the combined score of weight loss, stool consistency, and rectal bleeding divided by 3. The DAI index in this experiment was based on the literature.

[60] The scoring strategy in was slightly modified, and the average DAI score of each mouse per day was calculated according to the formula: DAI = (weight loss percentage score + feces viscosity score + feces blood degree score) / 3. For details, please refer to Table 2.

[0072] Table 2 DAI scoring strategy

[0073]

[0074] Effects of FMT on mouse diseases Figure 4 As shown in A and B, the MC group had sparse stools and the darkest color of occult blood test. Compared with the MC and FMC groups, the FCL group had less occult blood, less obvious fecal bleeding, and normal stool in granular form, which indicates that FMT can alleviate some colon inflammation. According to the DAI results ( Figure 4As shown in C), as the experiment progressed, the DAI scores of the three groups of mice were the highest in the MC group, because the MC group had obvious loose stools, and the scores of visible blood in the stool and occult blood were the highest, resulting in the highest DAI. After FMT treatment, the DAI score was significantly reduced, and the score of the FCL group was significantly lower than that of the MC group and the FMC group.

[0075] (3) Analysis of intestinal tissue morphology of FMT mice

[0076] During the experimental period, the weight of mice was recorded every day. After the experiment, the mice were weighed after fasting for 12 hours, and the mice were euthanized. The colon between the ileocecal part and the proximal rectum of each mouse was removed, and its length was immediately measured and recorded.

[0077] Hematoxylin-eosin (H&E) staining was used to observe the tissue damage of the mouse colon. After the mice were euthanized, 0.5-1 cm colon tissue was fixed in 4% paraformaldehyde / PBS (v / v) solution for 24 hours, embedded in paraffin, and H&E staining was performed according to previous literature. Colon histopathological evaluation was performed according to the method of the literature, and a comprehensive evaluation was performed based on the degree of inflammatory cell infiltration and mucosal damage.

[0078] The length of the colon of each group of mice Figure 5 As shown in A, compared with the MC and FMC groups, FCL can alleviate the atrophy and shortening of the mouse colon, and the average length of the colon in the FCL group increased significantly (P<0.05). Figure 5 B, H&E staining results show that compared with the FMC group, the colonic inflammatory cell infiltration and edema were partially alleviated, the glandular morphology of FCL was more regularly arranged and the goblet cells were relatively more than those in the MC group, and only a small amount of inflammatory cell infiltration was observed. Figure 5 As shown in B, the MC group had the highest score. Compared with the MC group, the FMC and FCL pathological scores were significantly reduced (P<0.05); the score of the FMC group was significantly lower than that of the FMC group (P<0.05), which indicates that FCL reduced intestinal mucosal damage and FMT can improve the pathological damage of mouse colon tissue caused by DSS.

[0079] (4) Analysis of organ index of FMT mice

[0080] During the experimental period, the weight of mice was recorded every day. After the experiment, the mice were weighed after fasting for 12 hours and euthanized. After the mice were killed, the organ weight of each mouse was recorded, and the organ index was obtained by dividing the organ mass mg by the mouse weight.

[0081] like Figure 6 As shown, FMT had no significant effect on the liver and kidney (P>0.05), and the spleen index of FMC and FCL was significantly reduced compared with the MC group (P<0.05).

[0082] (5) Analysis of serum inflammatory cytokines in FMT mice

[0083] Frozen serum samples were taken and thawed, and the levels of serum TNF-α, IL-1β, IL-6, and IL-10 were detected using commercial ELISA kits according to the manufacturer's kit instructions.

[0084] like Figure 7 As shown in the results, compared with the MC group, the levels of serum TNF-α, IL-1β, and IL-6 in the FMC and FCL groups were significantly decreased (P<0.05), and the level of IL-10 in the FCL group was significantly increased (P<0.05). Compared with the FMC group, the levels of TNF-α and IL-6 in the FCL group were significantly decreased, and the level of IL-10 was significantly increased (P<0.05); this suggests that FMT has an inhibitory effect on the secretion of pro-inflammatory cytokines and a promoting effect on the release of anti-inflammatory factors.

[0085] (6) Analysis of intestinal permeability in FMT mice

[0086] One day before the end of the experiment, mice were randomly selected from each group and fasted for 4 hours. 4 hours later, 600 mg / kg (body weight) of fluorescein isothiocyanate-labeled dextran (FITC-Dextran) was gavage. 3 hours later, the mice were anesthetized with isoflurane and photographed using the small animal in vivo fluorescence imaging analysis system (excitation wavelength 480nm, emission wavelength: 520nm) to observe the intestinal leakage of the mice; 6 hours later, the tail of the mice was bled, and then centrifuged to obtain serum, and the fluorescence intensity was measured on an ELISA instrument equipped with a fluorescence detector. According to the FITC-Dextran standard curve, the fluorescence intensity of the serum of each group was measured.

[0087] In addition, according to the instructions provided by the manufacturer, ELISA kits were used to measure the levels of LPS and diamine oxidase (DAO) in the serum of animals in each group.

[0088] To explore the effect of FMT on the integrity of the intestinal mucosal barrier induced by DSS in mice, Figure 8 As shown in the figure, compared with the MC group, the serum LPS and DAO levels of the FMT group were significantly reduced (P<0.05); compared with the FMC group, the LPS and DAO levels of the FCL group were significantly reduced (P<0.05). The above data show that FMT can reduce the intestinal permeability of DSS mice.

[0089] Example 3: Effect of CYN on the intestinal flora structure of FMT mice through intestinal flora

[0090] According to the instructions of the commercial kit, total DNA of fecal samples was extracted and amplified after passing the test. The PCR products after amplification were subjected to electrophoresis using 1.8% agarose to detect integrity.

[0091] After the DNA quality test was qualified, the full length of the bacterial 16S r RNA gene was amplified by PCR using universal primers 27F: 5'-AGRGTTTGATYNTGGCTCAG-3' and 1492R5'-TASGGHTACCTTGTTASGACTT-3'. The concentration and band (agarose gel electrophoresis) of the amplified product were tested, and the samples that met the conditions were mixed. For the constructed library, the library construction kit (SMRTbell Template Prep Kit) purchased from PacBio was used to repair damage, repair ends and connect adapters to the mixed product, and the reaction was carried out on the PCR instrument. Finally, it was purified and recovered using AMpure PB magnetic beads to obtain the machine-ready library; the concentration of the final library was detected by Qubit fluorescent dye, and the fragment size was detected by Agilent 2100 to determine whether it met the machine requirements; the machine-ready library was bound to Primer and Polymerase using PacBio Binding kit before being put into the machine; the final reaction product was purified by AMpure PB Beads and entrusted to Biomike Biotechnology Co., Ltd. (Beijing) for sequencing using Sequel II sequencer.

[0092] The results of intestinal flora sequencing were analyzed using the Biocloud platform (http: / / www.biocloud.net / ). The original offline subreads were corrected to obtain CCS (Circular Consensus Sequencing) sequences (SMRTLink, version 8.0), and then the lima (v1.7.0) software was used to identify the CCS sequences of different samples through the arcode sequence, remove chimeras, and obtain high-quality CCS sequences. The DADA2 method in QIIME2 (versoin 2020.6) was used to denoise the data after quality control. By default, 0.005% of all sequence numbers were used as the threshold to filter ASVs (clustering results, equivalent to OTUs with 100% similarity clustering). Species annotation and taxonomic analysis were performed using mainstream databases such as NCBI, Silva, and Greengenes and software such as QIIME2.

[0093] At the ASVs level, the Alpha diversity of intestinal flora was analyzed using the Wilcoxon rank sum test using Mothur software (version 1.31.0); the principal component analysis (PCA) of the samples and the correlation analysis between environmental factors and sample composition (RDA / CCA) were drawn based on the R language platform. Bray-Curtis or unweighted Unifrac distance was used for principal coordinate analysis (PCoA), nonmetric multidimensional scaling (NMDS), and unweighted pair group method with arithmetic mean (UPGMA) analysis using QIIME software (https: / / qiime2.org / ). The biomarker species in each sample were analyzed using the linear discriminant analysis effect size (LEfSe) software (https: / / huttenhower.sph.harvard.edu / galaxy / ). Pearson correlation coefficient analysis was performed using R language (version 4.1.0).

[0094] After sequencing the 18 samples, a total of 142,803 CCS sequences were obtained through Barcode identification. Each sample generated at least 6,821 CCS sequences, with an average of 7,934 CCS sequences. The average Effective-CCS of each sample was 7812, the AvgLen was 1455, and the effectiveness was 98.49%.

[0095] The sequencing curve of the intestinal flora of experimental mice is as follows Fig. 9 As shown. Fig. 9 A dilution curve shows that when the number of sampling sequences is less than 2000, the number of features increases rapidly with the increase of the number of sampling sequences; when the number of sampling sequences is greater than 6000, the number of features increases slowly, indicating that the species in the fecal samples will not increase significantly with the increase of sequencing numbers. The sequencing number is sufficient to reflect the diversity and richness of the species in the samples, and the sample sequences are sufficient to ensure the credibility of the data analysis. Fig. 9 B The Shannon index curve shows that when the number of sampled sequences is greater than 2000, the Shannon index curve has become flat, the data volume is full, the number of sequencing can guarantee the discovery of sufficient species characteristics, and the sequencing enters a plateau period. Fig. 9The C-level abundance curves showed that there were differences in richness and evenness among the groups, indicating that the sequencing depth was sufficient to cover most species. Fig. 9 D The species accumulation curve shows that with the number of sequencing samples in this experiment, species accumulation tends to be flat, indicating that the number of species accumulation in the intestinal flora increases slowly and the sequencing data analysis of this experiment is reliable.

[0096] This study used ASV clustering analysis based on the DADA2 algorithm and clustering with 100% similarity. Effects of CYN on ASV in the intestinal flora of mice Fig.10 As shown, a total of 721 ASVs were identified by classification sequences. Compared with the MC group, the number of ASVs in the FMC and FCL groups increased significantly (P<0.05), and there was no significant difference between the FMC and FCL groups (P>0.05), indicating that FMT treatment caused a significant impact on the intestinal microorganisms of mice. Fig.10 C shows that the number of ASVs shared by the three groups is 141. The number of ASVs unique to the MC group is the least, at 60; the number of ASVs unique to the FMC and FCL groups is 196 and 128, respectively, indicating that FMT has a certain impact on the intestinal microorganisms of mice.

[0097] pass Fig.11 It can be found that compared with the MC group, the ACE index, Chao1 index, Shannon index and PD_whole_tree of the FCL group increased significantly (P<0.05), indicating that oral administration of CL can increase the species richness and diversity of the intestinal flora of mice. Compared with the MC group, the Chao1 index and PD_whole_tree of the FMC group increased significantly (P<0.05), while the ACE index and Shannon index did not differ significantly; compared with the FMC group, the Simpson index and Shannon index of the FCL group did not increase significantly (P>0.05); Fig.11 It can be seen that the ingestion of feces from DSS-treated mice has no effect on the richness and diversity of the mouse microbiota, but the ingestion of feces from CYN-treated mice can increase the species richness and diversity of the DSS mouse microbiota.

[0098] The β diversity results are as follows Fig.12 As shown in the PCA results ( Fig.12 A) It can be seen that the sample community structures of the MC group, FMC group, and FCL group were significantly different, and FMT intervention had a significant effect on β diversity. The results of PCoA and NMDS ( Fig.12 B and Fig.12 C) is similar, further proving that FMT changed the species composition of the intestinal flora of mice, and the intestinal flora of mice fed with FCL was significantly improved. Fig.12D shows that the individual mouse samples in the three treatment groups are close to each other, but there is a clear distinction between the groups. The FMC group and the FCL group are in different branches of the same tree trunk, and the individuals are close to each other, while the MC group is in a different trunk from the above two groups. The microbial structure and composition of the intestinal flora of the FMT group mice are similar, that is, FCL has a significant regulatory effect on the composition of the intestinal flora affected by DSS.

[0099] Effects of FMT on the phylum level of intestinal flora Fig.13 As shown in A, the top four phyla in terms of phylum abundance in each group are Firmicutes, Bacteroidetes, Verrucomicrobia, and Proteobacteria. Fig.13 BF showed that compared with the MC group, FMT treatment significantly reduced the relative abundance of Bacteroidota, Proteobacteria and Fusobacteriota (P<0.05), and significantly increased the relative abundance of F / B and Verrucomicrobiota (P<0.05). Compared with the MC and FMC groups, the abundance of Firmicutes in the FCL group increased significantly (P<0.05), and the abundance of Proteobacteria and Fusobacteriota decreased significantly (P<0.05).

[0100] Depend on Fig.14 It can be seen that among the bacterial communities with an abundance of more than 1% at the family level, the abundance of Muribaculaceae, Erysipelatoclostridiaceae and Lactobacillaceae in the FMC and FCL groups was significantly decreased compared with the MC group (P<0.05), while the abundance of Lachnospiraceae and Oscillospiraceae was significantly increased (P<0.05). Compared with the MC group, the abundance of Erysipelotrichaceae in the FCL group was significantly decreased (P<0.05), and the abundance of unclassified_Clostridia_UCG_014, uncultured_rumen_bacterium and Ruminococcaceae was significantly increased (P<0.05). Compared with the MCL group, the abundance of uncultured_rumen_bacteriu in the FCL group was significantly increased (P<0.05), and the abundance of Erysipelatoclostridiaceae was significantly decreased (P<0.05).

[0101] Based on the genus level, the species composition changes and abundance ratios of each group can be further analyzed. Fig.15It can be seen that compared with the MC group, the abundance of Bacteroides and Erysipelatoclostridium in the FMC and FCL groups was significantly decreased (P<0.05), and the abundance of Dubosiella, Lachnospiraceae_NK4A136_group and uncultured_Bacteroidales_bacterium was significantly increased (P<0.05); compared with the FMC group, the abundance of Bacteroides, Erysipelatoclostridium and [Eubacterium]_fissicatena_group in the FCL group was significantly decreased (P<0.05).

[0102] The most advanced third-generation sequencing technology can reveal better species resolution at the species level. Fig.16 It can be seen that compared with MC, the abundance of Akkermansia_muciniphila in FMC and FCL groups was significantly increased (P<0.05), and the abundance of Parasutterella_excrementihominis, Streptococcaceae_bacterium, Ligilactobacillus_murinus, Erysipelatoclostridium_ramosum, Bacteroides_vulgatus, and Bacteroides_uniformis was significantly decreased (P<0.05); compared with FMC, the abundance of Faecalibaculum_rodentium in FCL group was significantly increased (P<0.05), and the abundance of Parabacteroides_distasonis, Erysipelatoclostridium_ramosum, Bacteroides_vulgatus, Bacteroides_uniformis, and Parabacteroides_distasonis was significantly decreased (P<0.05).

[0103] The present invention sets the LDA threshold to 4, selects the classification level from phylum to species, and uses the Kruskal-Wallis test (P<0.05). Fig.17 As shown. From the LDA value distribution bar graph ( Fig.17A) It can be seen that there are 24, 14 and 19 species with significant differences in MC, FMC and FCL groups, respectively; at the phylum level, Bacteroidota and Proteobacteria in the MC group, Verrucomicrobiota in the FMC group, and Firmicutes in the FCL group can be used as biological biomarker species for these bacteria at the phylum level; at the family level, Muribaculaceae, Sutterellaceae and Streptococcaceae in the MC group, Lachnospiraceae_, Oscillospiraceae in the FMC group, and unclassified_Clostridia_UCG_014, Erysipelotrichaceae, and uncultured_rumen_bacterium in the FCL group can be used as biomarkers for these groups at the family level; at the genus level, Bacteroides, Parasutterella, and Erysipelatoclostridium in the MC group , Streptococcus, Lachnospiraceae_NK4A136_group, Eubacterium__fissicatena_group in the FMC group, uncultured_Bacteroidales_bacterium, Faecalibaculum_rodentium, Dubosiella, and unclassified_Oscillospiraceae in the FCL group can be used as biomarkers of these groups at the genus level; at the species level, Parasutterella_excrementihominis, Streptococcaceae_bacterium, Erysipelatoclostridium_ramosum, Bacteroides_uniformis, Bacteroides_vulgatus in the MC group, and Akkermansia_muciniphila in the FMC group can be used as biomarkers of these species at the species level.

[0104] like Fig.18 A, KEGG pathways were analyzed using PICRUSt2 software to predict potential functional characteristics between different treatment groups. A total of 43 functional genes were predicted, and those with abundance greater than 5% included Amino acid metabolism and Carbohydratemetabolism;

[0105] like Fig.18B, Compared with the MC group, the abundance of Replication and repair, Cancers: Overview, Signaling molecules and interaction, Translation, Transcription, Infectious diseases: Viral, Cardiovascular diseases, Folding, sorting and degradation, Metabolism of cofactors and vitamins, Cell growth and death, Endocrine and metabolic diseases increased significantly, and the abundance of Immune diseases, Carbohydrate metabolism, Metabolism of other amino acids, and Energy metabolism decreased significantly in the FMC group (P<0.01). Fig.18 C, Compared with MC, the proportions of 8 pathways in the FCL group were significantly increased, including Translation, Signaling molecules and interaction, Replication and repair, Cell motility, Transcription, Nucleotide metabolism, Cell growth and death, Metabolism of terpenoids and polyketides, and the proportions of 5 pathways in the FCL group were significantly decreased, including Immune diseases, Carbohydrate metabolism, Energy metabolism, Metabolism of otheramino acids, and Digestive system (P<0.01).

[0106] Depend on Fig.18D showed that there was no significant difference in the predicted functional genes between the FMC group and the FCL group (P>0.05). In summary, CYN regulated the gene function of the intestinal flora of mice with DSS-induced chronic colitis, and FMT significantly (P<0.01) increased Translation, Signaling molecules and interaction, Replication and repair, Transcription, Cell growth and death, and significantly reduced Immune diseases, Carbohydrate metabolism, Energy metabolism, and Metabolism of other amino acids (P<0.01).

[0107] Example 4: Effect of CYN on short-chain fatty acids through intestinal flora

[0108] (1) Sample collection: Accurately weigh 0.2 g of fresh feces from 8 mice in each group, add 5 mL of PBS buffer, and vortex to obtain a 10% (w / v) feces dilution for use.

[0109] (2) Pretreatment: Take 500 μL of fecal dilution into a 1.5 mL centrifuge tube, add 100 μL of crotonic acid-metaphosphoric acid solution, vortex for 2 min, and then place in a -20°C refrigerator to react for 24 h. After the reaction is completed, thaw at 4°C and centrifuge at 10,000 r / min for 5 min at 4°C, and take the supernatant to filter through a 0.45 μm aqueous filter.

[0110] (3) Gas phase detection: Referring to previous studies, a highly polar DB-FFAP column (0.32 mm × 30 m × 0.5 μm, Agilent) was used to detect SCFAs in the intestinal content dilution with crotonic acid as the internal standard.

[59] . Set the injection port temperature to 250℃, carrier gas type N2, injection volume to 1μL, purge flow rate to 3.0ml / L, split ratio to 5:1, pressure to 41.1KPa, linear velocity to 58cm / s, total flow rate to 12.1mL / min, and column flow rate to 2.3mL / min. The column box starts at 75℃, rises to 180℃ at 20℃ / min and holds for 1min, rises to 220℃ at 40℃ / min and holds for 1min, and the program time is 8.05min in total. The parameters of the flame ionization detector are temperature of 250℃, tail gas and flow rate of N2 / H2 / air, 30 / 40 / 400mL / min.

[0111] like Fig.19As shown in the results, compared with the MC group, FMT significantly increased the levels of acetate, propionate, butyrate and total SCFAs in the feces of mice with DSS-induced colitis (P<0.05), and had no significant effect on valerate; compared with the FMC group, the levels of isobutyrate and isovalerate in the FCL group increased significantly (P<0.05). These results indicate that FMT has a regulating effect on fecal SCFAs in mice with DSS-induced colitis.

Claims

1. A method for preparing a fecal bacteria liquid for improving chronic colitis, characterized in that: The following steps are involved: (1) Collection: Feces discharged from chronic colitis mice after 34-38 days of treatment with Changyanning were collected; (2) stirring: placing mouse feces in a blender, adding physiological saline solution, and stirring evenly to obtain a fecal solution; (3) Centrifugation: The obtained fecal solution is centrifuged and the supernatant is collected to obtain the Enteritis Ning fecal bacteria solution.

2. The method according to claim 1, characterized in that In step (1), the feces of the chronic colitis mice after 35 to 37 days of treatment with Changyanning were collected through an SPF barrier.

3. The method according to claim 2, characterized in that In step (1), the environmental requirements of the SPF barrier are: The temperature was 20-24°C, the humidity was 40%-60%, and the light-dark cycle was 12 hours of light and 12 hours of darkness.

4. The method according to claim 1, characterized in that: In step (1), the feces are fresh feces excreted immediately from the mouse under natural conditions.

5. The method according to claim 1, characterized in that In step (1), the chronic colitis mouse refers to a chronic colitis mouse model induced by sodium dextran sulfate.

6. The method according to claim 1, characterized in that In step (1), the feeding dose of the Enteritis Ning is 0.25g / kg-1g / kg.

7. The method according to claim 1, characterized in that In step (2), mouse feces and physiological saline solution are mixed at a mass: volume ratio of 1:8-11 (g / mL).

8. The method according to claim 1, characterized in that In step (3), the centrifugation conditions are 2500-3000 rpm, 1-4°C, and the time is 1 to 5 minutes.

9. A fecal bacteria liquid for Changyanning, characterized in that: Obtained by the preparation method described in any one of claims 1 to 8; The storage temperature of the Enteritis Ning fecal bacteria liquid is ≤-80°C, and the storage time is ≤6 months.

10. Use of the Changyanning fecal bacteria liquid according to claim 9 in the preparation of medicines or preparations for treating inflammatory bowel disease.

Images