Use of butyricum in preparation of a medicine for preventing and treating obesity-related acute pancreatitis

Abstract

This invention discloses the application of *Ruminococcus brucelli* in the preparation of drugs for the prevention and treatment of obesity-related acute pancreatitis, relating to the field of biomedical technology. This invention provides *Ruminococcus brucelli* (… Ruminococcus bromii This invention relates to the use of *Ruminococcus brucelli* in the preparation of medicaments for the prevention and / or treatment and / or adjunctive treatment of obesity-related acute pancreatitis. *Ruminococcus brucelli* is an important class of strictly anaerobic Gram-positive bacteria in the human gut, playing a crucial role in maintaining the intestinal barrier and regulating immune homeostasis. This invention reveals that *Ruminococcus brucelli* can reduce pancreatic tissue edema, pancreatic inflammatory infiltration, pancreatic acinar cell panapoptosis, and systemic inflammatory response in obesity-related acute pancreatitis, and can be used in the preparation of medicaments for the prevention and treatment of obesity-related acute pancreatitis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of Ruminococcus brucelli in the preparation of drugs for the prevention and treatment of obesity-related acute pancreatitis. Background Technology

[0002] Acute pancreatitis (AP) is one of the most common acute abdominal conditions of the digestive system, with an increasing incidence rate each year. It is characterized by rapid onset and progression. Once it progresses to severe acute pancreatitis (SAP), local and systemic complications increase significantly, and the mortality rate can reach 20%-40%. Obesity, as an independent risk factor for SAP, can significantly aggravate pancreatic and peripancreatic fat necrosis, systemic inflammatory response, and multiple organ dysfunction, further increasing the difficulty of clinical treatment and the risk of death in obesity-related SAP. However, current prevention and treatment methods for obesity-related SAP mainly focus on fluid resuscitation and organ function support, lacking specific prevention and treatment strategies that can early halt disease progression and improve prognosis.

[0003] Recent studies have shown that gut microbiota imbalance is closely related to the development and progression of severe acute pancreatitis (SAP). In SAP, intestinal ischemia, mucosal barrier damage, and impaired intestinal motility can lead to gut microbiota dysbiosis, excessive proliferation of opportunistic pathogens, and endotoxin translocation, thereby activating the mononuclear-macrophage system and amplifying the systemic inflammatory storm. To address this pathological mechanism, multiple studies have reported that probiotics such as Bifidobacterium, Lactobacillus, and Akkermansia myxotroph can effectively block secondary pancreatic damage caused by intestinal endotoxins by remodeling intestinal epithelial tight junctions, restoring immune homeostasis, and inhibiting pro-inflammatory signaling pathways, thus reducing pancreatic damage in SAP.

[0004] Ruminococcus bromii (Ruminococcus brucelli, R. bromii Firmicutes (Clostridium perfringens) are an important class of strictly anaerobic Gram-positive bacteria in the human gut, belonging to the class Clostridium of the phylum Firmicutes. As core carbohydrate-metabolizing bacteria in the gut, they excel at degrading resistant starch and efficiently producing short-chain fatty acids such as butyrate, playing a crucial role in maintaining intestinal energy supply, regulating host metabolism, and shaping the immune microenvironment. Current research suggests that... Ruminococcus bromii It exhibits positive biological effects in improving metabolic syndrome, regulating blood glucose homeostasis, and alleviating intestinal inflammation; however, there are no research reports on its application in obesity-related severe acute pancreatitis. Summary of the Invention

[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide the application of Ruminococcus brucelli in the preparation of drugs for the prevention and treatment of obesity-related acute pancreatitis.

[0006] This invention provides R. bromii Application in the preparation of drugs for treating obesity-related acute pancreatitis. This invention is demonstrated experimentally. R. bromii It can prevent and treat obesity-related acute pancreatitis, specifically manifested as: R. bromii This invention can reduce pancreatic tissue edema, decrease pancreatic inflammatory infiltration, and alleviate systemic inflammatory response in obesity-related acute pancreatitis, while also improving energy and amino acid metabolism. It provides a novel strategy for preventing obesity-related acute pancreatitis and has promising clinical application prospects.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides the use of Ruminococcus brucelli in the preparation of a medicament for the prevention and / or treatment and / or adjunctive treatment of obesity-related acute pancreatitis.

[0008] This invention discovers rumenococcus through research ( Ruminococcus bromii Ruminococcus brucelli can significantly improve pancreatic inflammation, necrosis, and systemic inflammatory response caused by acute pancreatitis. It can downregulate serum amylase expression levels, improve the early cytokine storm in acute pancreatitis, reduce pancreatic tissue edema, reduce pancreatic inflammatory cell infiltration, reduce pancreatic acinar cell necrosis, and reduce the levels of inflammatory factors and chemokines in pancreatic tissue. Therefore, using Ruminococcus brucelli to prepare drugs for the prevention and treatment of obesity-related acute pancreatitis provides a new strategy for the prevention and treatment of acute pancreatitis and has good clinical application prospects.

[0009] Preferably, the rumenococcus is... R. bromii ATCC 51896.

[0010] Preferably, the prevention and / or treatment and / or adjunctive treatment includes at least one of the following effects: (a) Improves pathological damage to pancreatic tissue; (b) Reduce serum amylase and / or lipase levels; (c) Inhibit or reduce the early cytokine storm in acute pancreatitis; (d) Reduces apoptosis in pancreatic tissue cells; (e) Reduce the levels of inflammatory factors and / or chemokines in pancreatic tissue.

[0011] Preferably, the improvement of pancreatic tissue pathological damage includes at least one of reducing pancreatic tissue edema, reducing pancreatic inflammatory cell infiltration, and reducing pancreatic acinar cell necrosis.

[0012] Preferably, the inhibition or mitigation of the early inflammatory cytokine storm in acute pancreatitis includes reducing the infiltration or activation of macrophages and / or neutrophils in pancreatic tissue.

[0013] Preferably, the inflammatory factors and / or chemokines are selected from one or more of IL-6, IL-1β, CXCL1, CCL3, IL6, S100a8, and S100a9.

[0014] In a second aspect, the present invention provides a drug for the prevention and / or treatment and / or adjunctive treatment of obesity-related acute pancreatitis, said drug comprising Ruminococcus brucellosis and a pharmaceutically acceptable carrier.

[0015] Preferably, the rumenococcus is... R. bromii ATCC 51896.

[0016] Preferably, the pharmaceutically acceptable carrier includes one or more of the following commonly used medical fillers, binders, humectants, disintegrants, lubricants, and flavoring agents; The dosage forms of the drugs include, but are not limited to, tablets, capsules, solutions, granules, injections, powder for injection, or aerosols.

[0017] Preferably, the pharmaceutically effective dose of the brucellococcus is 10. 9 CFU.

[0018] This invention has at least one of the following beneficial effects: This invention discloses R.bromii Its application in the prevention and treatment of obesity-related severe acute pancreatitis can significantly improve pancreatic inflammation and systemic inflammatory response caused by obesity-related acute pancreatitis. (1) Decreased level of inflammation and degree of damage in pancreatitis: R.bromii After intervention, the levels of serum amylase and lipase, markers of pancreatitis, were significantly reduced, the pathological score of pancreatic tissue was significantly reduced, the infiltration of inflammatory cells in pancreatic tissue was significantly reduced, and the expression of inflammatory factors and chemokines in pancreatic tissue was significantly reduced.

[0019] (2) Decreased systemic inflammatory response level: R.bromii After intervention, the expression levels of pro-inflammatory factors IL-1β and IL-6 in serum were significantly reduced, and differential metabolites were enriched in beneficial functional pathways such as energy metabolism and amino acid synthesis.

[0020] This invention provides a new strategy for the prevention and treatment of obesity-related acute pancreatitis, and has promising clinical application prospects. Attached Figure Description

[0021] Figure 1 This is an electron micrograph of live Ruminococcus brevicornus of the present invention, with a magnification of 20000x and a scale bar of 1µm.

[0022] Figure 2The images show the H&E staining results of mouse pancreatic tissues in each group in Example 1 of this invention, with a magnification of 40x and a scale bar of 50µm.

[0023] Figure 3 This is a statistical chart of the pathological scores of pancreatic tissues in each group of mice in Example 1 of the present invention.

[0024] Figure 4 This is a statistical chart of serum amylase levels in mice in each group in Example 1 of the present invention.

[0025] Figure 5 This is a statistical chart of serum lipase levels in mice in each group in Example 1 of the present invention.

[0026] Figure 6 The images show the MPO staining results of mouse pancreatic tissues in each group in Example 1 of this invention, with a magnification of 100x and a scale bar of 50um.

[0027] Figure 7 This is a statistical graph showing the MPO expression levels in the pancreatic tissues of mice in each group in Example 1 of the present invention.

[0028] Figure 8 The images show the F4 / 80 staining results of mouse pancreatic tissues in each group in Example 1 of this invention, with a magnification of 100x and a scale bar of 50um.

[0029] Figure 9 This is a statistical graph showing the expression levels of F4 / 80 in the pancreatic tissues of mice in each group in Example 1 of the present invention.

[0030] Figure 10 This is a statistical graph showing the expression levels of cytokines CXCL1, CCL3, and IL-6 in the pancreatic tissues of mice in each group of the present invention, as detected by RT-PCR.

[0031] Figure 11 This is a statistical graph showing the expression levels of inflammatory factors S1008 and S100a9 in the pancreatic tissues of mice in each group in Example 1 of the present invention, as detected by RT-PCR.

[0032] Figure 12 This is a statistical graph showing the expression levels of IL-1β and IL-6 in the pancreatic tissues of mice in each group in Example 1 of the present invention, as detected by ELISA.

[0033] Figure 13 The images show the LY6G staining results of mouse pancreatic tissues in each group in Example 1 of this invention, with a magnification of 200x and a scale bar of 100um.

[0034] Figure 14 This is a statistical graph showing the LY6G expression level in the pancreatic tissues of mice in each group in Example 1 of the present invention.

[0035] Figure 15 The images show the TUNEL staining results of mouse pancreatic tissues in each group in Example 1 of this invention, with a magnification of 200x and a scale bar of 100um.

[0036] Figure 16 This is a statistical graph showing the TUNEL expression levels in the pancreatic tissues of mice in Example 1 of this invention.

[0037] Figure 17 This is a KEGG enrichment pathway diagram of serum metabolites in mice in Example 1 of the present invention. The size of the dots represents the number of differentially enriched metabolites in the pathway, and the color represents the p-value. The redder the color, the smaller the p-value and the more significant the enrichment.

[0038] In the statistical chart, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Implementation

[0039] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0040] Ruminococcus bromii (Ruminococcus brucelli, R. bromii Ruminococcus brucelli is an important class of strictly anaerobic Gram-positive bacteria in the human gut, playing a crucial role in maintaining the intestinal barrier and regulating immune homeostasis. The inventors discovered that Ruminococcus brucelli can reduce pancreatic tissue edema, pancreatic inflammatory infiltration, pancreatic acinar cell panapoptosis, and systemic inflammatory response in obesity-related acute pancreatitis. Based on these findings, this invention utilizes Ruminococcus brucelli in the preparation of drugs for the prevention and treatment of obesity-related acute pancreatitis.

[0041] In a first aspect, the present invention provides the use of Ruminococcus brucelli in the preparation of drugs for the prevention and treatment of obesity-related acute pancreatitis.

[0042] Understandably, the drugs mentioned for the prevention, treatment, and / or adjunctive treatment of obesity-related acute pancreatitis refer to drugs for the prevention, treatment, and / or adjunctive treatment of obesity-related acute pancreatitis. Ruminococcus brucelli can downregulate serum amylase expression levels, improve the early cytokine storm in acute pancreatitis, reduce pancreatic tissue edema, reduce pancreatic inflammatory cell infiltration, reduce pancreatic acinar cell necrosis, and decrease the levels of inflammatory factors and chemokines in pancreatic tissue. Serum differential metabolites are mainly enriched in pathways such as energy metabolism and amino acid biosynthesis.

[0043] In some implementations, the acute pancreatitis is severe acute pancreatitis or obesity-related acute pancreatitis.

[0044] In some embodiments, the *Ruminococcus brucellosis* is... R. bromii ATCC 51896.

[0045] In some embodiments, the prevention and / or treatment and / or adjunctive treatment include at least one of the following effects: (a) Improves pathological damage to pancreatic tissue; (b) Reduce serum amylase and / or lipase levels; (c) Inhibit or reduce the early cytokine storm in acute pancreatitis; (d) Reduces apoptosis in pancreatic tissue cells; (e) Reduce the levels of inflammatory factors and / or chemokines in pancreatic tissue.

[0046] In some implementations, the improvement of pancreatic tissue pathological damage includes at least one of reducing pancreatic tissue edema, reducing pancreatic inflammatory cell infiltration, and reducing pancreatic acinar cell necrosis.

[0047] In some implementations, the suppression or mitigation of the early cytokine storm in acute pancreatitis includes reducing the infiltration or activation of macrophages and / or neutrophils in pancreatic tissue.

[0048] In some embodiments, the inflammatory factors and / or chemokines are selected from one or more of IL-6, IL-1β, CXCL1, CCL3, IL6, S100a8, and S100a9.

[0049] Secondly, the present invention provides a drug for the prevention and treatment of obesity-related acute pancreatitis, comprising Ruminococcus brucellosis.

[0050] The drug for preventing and treating obesity-related acute pancreatitis uses Ruminococcus brucelli as the active ingredient and can be formulated into any pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier.

[0051] In some implementations, the drug for preventing and treating obesity-related acute pancreatitis also includes a pharmaceutically acceptable carrier.

[0052] In some embodiments, the pharmaceutically acceptable carrier includes one or more of the following commonly used medical fillers, binders, humectants, disintegrants, lubricants, and flavoring agents.

[0053] In some implementations, the pharmaceutically acceptable carrier is water or physiological saline.

[0054] In some embodiments, the dosage form of the drug for preventing and treating obesity-related acute pancreatitis includes, but is not limited to, tablets, capsules, solutions, granules, injections, powder for injection, or aerosols. Preferably, the dosage form of the drug for preventing and treating obesity-related acute pancreatitis is a dispersible tablet, lozenge, orally disintegrating tablet, sustained-release tablet, soft capsule, or pellet.

[0055] In some embodiments, the *Ruminococcus brucellosis* is... R. bromii (ATCC strain 51896), this bacterium was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. R. bromii The effective concentration is 10 9 CFU.

[0056] In some implementations, when using the aforementioned drug for the prevention and treatment of obesity-related acute pancreatitis, the pharmaceutically effective amount of *Ruminococcus brucellosis* is 10. 9 CFU.

[0057] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following specific embodiments.

[0058] The effectiveness of this invention was verified through the following examples. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials and reagents used are all commercially available. Specifically, SPF-grade 5-6 week old male C57BL / 6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; *Ruminococcus brucellosis* was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.; Caerulein (Cer) was purchased from Tocris and used to induce severe acute pancreatitis in mice; sodium taurocholate (TCA) was purchased from Sigma-Aldrich; and a 60% high-fat diet was purchased from Jiangsu Nantong Trofi Feed Co., Ltd.

[0059] Example 1: Validation experiment on the effect of prophylactic supplementation with Ruminococcus brucellosis in alleviating acute pancreatitis in obese mice. This embodiment provides a validation experiment on the prevention of acute pancreatitis in obese mice by supplementing with Ruminococcus brucellosis, including the following steps: (a) Rumenococcus brucellosis R. bromii Cultivation: The strain used in this example is R. bromii(ATCC51896 strain), purchased from Ningbo Mingzhou Biotechnology Co., Ltd., was prepared using minced meat carbohydrate broth (CMC) + 15% rumen fluid. The components included 30.0g casein peptone, 10.0g beef extract, 5.0g yeast extract, 5.0g dipotassium hydrogen phosphate, 0.5g L-cysteine ​​hydrochloride, 0.0005g resazurin, 4.0g glucose, 1.0g cellobiose, 1.0g maltose, 1.0g starch, and pH 7.0±0.2. Take 57.5g of culture medium and dissolve it in 1000mL of distilled water. Heat to a boil and continue boiling for at least 1 minute. Dispense the mixture into test tubes, add an appropriate amount of beef granules (approximately 1 / 3 of the liquid height) to each tube, autoclave at 121℃ for 30 minutes, cool to below 50℃, and aseptically add 1 vial of heme chloride (0.5mg), 1 vial of vitamin K1 (5mg), and 15% rumen fluid per 100mL. Deaerate and set aside. Incubate the strain at 37℃ anaerobic for 24-48 hours.

[0060] Scanning electron microscopy revealed R. bromii It appears as a coccus, with the bacterial body nearly spherical or slightly oval, with a smooth surface, without protrusions or wrinkles, and is usually arranged in pairs or chains. Figure 1 ).

[0061] (II) SPF-grade 5-6 week old male C57BL / 6 mice were randomly divided into a high-fat diet group (PBS_cer group) and a high-fat diet combined with Ruminococcus brucellis group. R. bromii The _cer group and the _cer group (5 animals per group) were fed a 60% high-fat diet with PBS. R. bromii _cer group mice, and were given 10 daily 9 CFU Steps (1) Gavage administration of cultured Ruminococcus brucellosis. R. bromii Mice in the _cer group were administered an equal volume of physiological saline via gavage daily for 4 weeks. After 4 weeks, mice were induced to develop obesity-related acute pancreatitis by intraperitoneal injection of hygroscopic ...

[0062] (III) Take pancreatic tissue from the model mice and perform H&E staining. The staining method is as follows: After the pancreatic tissue was fixed for 24 hours, it was removed and placed in a fully automatic dehydrator for dehydration. After dehydration, it was transferred to a paraffin embedding machine and the paraffin block was stored at room temperature. When slicing, the paraffin block should be placed in a -20℃ refrigerator about 2 hours in advance, and the slide warmer should be preheated to about 40℃. The thickness of the pathological slide should be set to 4μm. After slicing, the slide should be spread flat on the warm water of the slide warmer and gently picked up with a glass slide for H&E staining. The steps are as follows: (1) Baking: Baking the paraffin pancreatic tissue slide in a 70℃ oven for 1-2 hours; (2) Dewaxing: Placing the tissue slide in xylene I for 10 minutes and xylene II for 10 minutes in sequence; (3) Hydration: Placing the slide in anhydrous ethanol, 95% ethanol and 85% ethanol for 5 minutes in sequence; (4) Washing: Rinsing under running tap water for 15 minutes; (5) Nuclear staining: Staining the nucleus with hematoxylin for 1 minute to 1 minute 30 seconds, and rinsing with tap water 3-5 times; (6) Differentiation: Differentiating with hydrochloric acid alcohol for 3 seconds, and rinsing with tap water 3-5 times; (7) Blue reversal: Blue reversal with lithium carbonate for 3 seconds. (8) Staining: Stain with eosin solution for 1 min 30s-2 min; (9) Dehydration: Place in 85% ethanol, 95% ethanol and anhydrous ethanol for 5 min each; (10) Clearing: Place in xylene I for 10 min and xylene II for 10 min each; (11) Mounting: Dry in a fume hood and then mount with neutral resin; (12) Reading: Observe under an optical microscope and score pathologically.

[0063] The results are as follows Figure 2 and Figure 3 As shown, R.bromii Compared with the PBS-treated cer group, the _cer group mice showed significantly reduced pancreatic damage across multiple dimensions, including pancreatic tissue edema, inflammatory infiltration, and acinar necrosis, with a significantly lower pathological score (total decrease of 19.61%). These results were statistically significant, indicating that... R.bromii It has a protective effect on pancreatic tissue in a high-fat environment (p<0.05).

[0064] (iv) Blood was collected from the model mice to detect serum amylase levels. The detection method is as follows: (1) Add 0.4 mg / mL substrate buffer (C 底物液 Preheat the water bath to 37℃ for 5 minutes, then add 0.5 mL of substrate buffer (V) to both the sample and blank tubes. 底物液 ); (2) Extraction: Remove serum samples from the -80℃ freezer and thaw at 4℃. Then dilute the serum sample to be tested 100 times (N), and add 0.1 mL (V) of the solution to each sample. 样 Pour the mixture into the sample test tube, mix thoroughly, and place in a 37°C water bath for an accurate reaction time of 7.5 min (T). (3) Dilute 0.1 mol / L iodine stock solution 10 times to make iodine application solution. Calculate the required amount according to the sample volume. Add 0.5 mL of iodine application solution and 3 mL of double-distilled water to the sample test tubes respectively, and add 0.5 mL of iodine application solution and 3 mL of double-distilled water to the blank tubes respectively. Mix thoroughly. (4) Set the wavelength of the spectrophotometer to 660 nm and detect the absorbance of each sample and blank tube.

[0065] Calculation formula: AMS Vitality (U / dl) = (A 空白 - A 测定 ) / A 空白 * (C) 底物液 * V 底物液 ) / 10*(30 / T)*(100 / V 样 ) * N.

[0066] The results are as follows Figure 4 As shown, R.bromii The serum amylase level in the _cer group mice was significantly reduced, decreasing by 7.86% compared to the PBS_cer group, suggesting that... R.bromii Intervention can reduce pancreatic tissue damage in mice with obesity-related acute pancreatitis.

[0067] (v) Blood was collected from the model mice, and serum lipase levels were detected using the following method: (1) Take out the serum sample to be tested from the -80℃ freezer and transfer it to the 4℃ freezer to thaw slowly for later use; (2) Take a clean 1.5 mL EP tube and set it as a blank group, a calibration group and a sample group respectively. Add 4 μL of double-distilled water to the blank group, 4 μL of standard to the calibration group and 4 μL of serum to be tested to the sample group. Then add 200 μL of reagent 1 (lipase assay kit) to each group, mix thoroughly and incubate in a constant temperature environment of 37℃ for 5 min. (3) After the reaction was completed, each group was given 50 μL of reagent 2 (lipase assay kit), mixed well and then incubated at 37°C for 5 min. (4) The absorbance of each group of reaction solutions was measured at a wavelength of 580 nm using a spectrophotometer. After the measurement was completed, the solution was incubated at 37℃ for 2 min and the absorbance change was measured again at the same wavelength.

[0068] The calculation formula is as follows: The results are as follows Figure 5 As shown, R.bromiiThe serum amylase level in the _cer group mice was significantly reduced, decreasing by 35.76% compared to the PBS_cer group (p<0.05), suggesting that... R.bromii Intervention can reduce pancreatic tissue damage in mice with obesity-related acute pancreatitis.

[0069] (vi) Take pancreatic tissue from the model mice and perform immunohistochemical detection. The detection method is as follows: (1) The steps of baking, dewaxing, hydration, and washing are the same as those for H&E staining; (2) Antigen retrieval: Boil in citrate buffer on high heat for 15 min, cool to room temperature, and wash the slides 3 times with PBS, 5 min each time; (3) Antigen blocking: Place the slide in 3% H2O2 and let it stand for 8 minutes to block endogenous peroxidase activity. Wash the slide with PBS 3 times, 5 minutes each time. (4) Primary antibody incubation: Use an immunohistochemical pen to draw circles along the edge of the tissue section to cover the tissue, dilute the antibody with 3% BSA to an appropriate concentration and drop it onto the tissue, incubate overnight at 4°C; (5) Secondary antibody incubation: The next day, remove the humidified box and warm it at room temperature for 30 min. Wash the slides with PBS 3 times, 5 min each time. Add the universal immunohistochemical secondary antibody to the slides and react at 37℃ for 30 min. (6) Staining: Wash the slides with PBS 3 times, 5 min each time, add an appropriate amount of DAB staining solution, observe the tissue color change under a microscope, and rinse with tap water for 15 min after staining. (7) Counterstaining: stain with hematoxylin for 10 seconds, then rinse 3 times with running tap water; (8) Differentiation: Differentiate in 1% hydrochloric acid alcohol for 3 seconds, then rinse 3 times with running tap water; (9) Blueing: Lithium carbonate turns blue in 3 seconds, then rinse 3 times with running tap water; (10) Dehydration: Place in 85% ethanol, 95% ethanol and anhydrous ethanol for 5 min each in sequence; (11) Transparency: Place in xylene I for 10 min, then xylene II for 10 min in sequence; (12) Sealing: After drying in a fume hood, seal the film with neutral resin; (13) Review the slides: Observe and photograph the slides under an optical microscope and score them.

[0070] (14) Interpretation of immunohistochemical staining results: Two pathologists performed a double-blind scoring using the German semi-quantitative scoring method. The scoring criteria are shown in the table below: Table 1 Immunohistochemical scoring criteria The percentage of MPO and F4 / 80 positive cells was multiplied by the staining intensity score, and the resulting score was used for semi-quantitative analysis.

[0071] The results are as follows Figures 6-9 As shown, R.bromii Expression of MPO, a marker of neutrophils, in pancreatic tissue of mice in the _cer group ( Figure 6 and Figure 7 ) and macrophage marker F4 / 80 expression ( Figure 8 and Figure 9 The levels were significantly lower in the PBS_cer group, decreasing by 52.00% and 42.86% respectively (p<0.05).

[0072] (vii) Take pancreatic tissue from model mice and perform real-time quantitative PCR detection. The specific method is as follows: 1. cDNA synthesis in mouse pancreatic tissue: (1) Place the experimental consumables in a clean bench and irradiate them with ultraviolet light for 30 minutes in advance. Use a reverse transcription kit to reverse transcribe the extracted RNA into cDNA. (2) Prepare the reaction system for the first step according to the table below; Table 2. Genomic gDNA Reaction System (3) Set the program to 42℃ for 3 minutes, then place it on an ice plate; (4) Prepare the reaction system for the second step according to the table below; Table 3 Reverse Transcription Reaction System (5) After the reaction in (3), add 10 μL of reverse transcription reaction system to the EP tube, shake and centrifuge, set the PCR instrument program to 42℃ for 15 min; 95℃ for 3 min; after the reaction, cDNA can be obtained and stored at -20℃.

[0073] 2. Real-time quantitative PCR (1) The primer sequences used (constructed by Sangon Biotech Co., Ltd.) are as follows: Table 4 (2) Prepare the real-time PCR reaction system according to the table below. Add 2 μL of cDNA and 18 μL of reaction system to the 96-well plate in sequence, and centrifuge at 1000 rpm for 3 min. Table 5. Quantitative Real-Time PCR Reaction System (3) Centrifuge the PCR plate at 1000 rpm for 3 min and then place it in the PCR instrument. Set the program: 95℃, 30 sec; 95℃, 3 sec; 60℃, 20 sec, repeat for 40 cycles; plot the melting curve.

[0074] (4) Based on the CT values ​​of the internal components and the target gene, 2 - Ct The method calculates the relative expression of the sample.

[0075] The results are as follows Figure 10 and Figure 11 As shown, R.bromii The expression levels of inflammatory cytokines CXCL1, CCL3, IL6, S100a8, and S100a9 in the pancreatic tissue of mice in the _cer group were significantly lower than those in the PBS_cer group, decreasing by 58.83%, 44.42%, 47.48%, 78.93%, and 77.69%, respectively (p<0.05). These results indicate... R.bromii Intervention can significantly reduce the level of inflammation in pancreatic tissue.

[0076] (viii) Collect serum from the model mice and perform ELISA experiments. The methods for detecting serum inflammatory factors are as follows: (1) Serum sample preparation: Remove the serum sample from the -80℃ freezer in advance and place it in the 4℃ freezer to thaw. Equilibrate the kit to room temperature and detect the levels of serum inflammatory factors IL-1β and IL-6. Based on previous experience and the detection sensitivity of the kit, dilute the serum 5 times (20μL serum + 80μL sample diluent). (2) Refer to the manufacturer's instructions for preparing the working solution.

[0077] (3) Sample addition: Add 100 μL of different concentrations of standard and serum sample to be tested to the microplate in sequence, cover with membrane and incubate at 37℃ for 90 min; discard the liquid, add 100 μL of biotinylated antibody working solution to each well and incubate at 37℃ for 1 h; discard the liquid, add 350 μL of washing buffer to each well and wash 5 times, then add 100 μL of HRP enzyme conjugate working solution to each well and incubate at 37℃ for 30 min; discard the liquid, wash the plate 5 times, then add 90 μL of substrate solution and incubate at 37℃ in the dark for 15-30 min; add 50 μL of stop solution and measure the OD value at 450 nm wavelength using a microplate reader.

[0078] (4) Calculation: The final concentration is obtained by multiplying the sample concentration calculated from the standard curve by 5.

[0079] The results are as follows Figure 12 As shown, R.bromii The serum levels of the inflammatory cytokines IL-6 and IL-1β in the _cer group mice were significantly lower than those in the PBS_cer group, indicating that... R.bromii Intervention can significantly reduce serum inflammation levels.

[0080] (ix) Take pancreatic tissue from the model mice and perform immunofluorescence detection. The specific method is as follows: (1) The steps of drying, dewaxing, hydrating and washing tissue sections shall be performed in accordance with the aforementioned H&E staining method; (2) Antigen retrieval: Place the tissue sections in citrate buffer, boil at high temperature for 15 min, cool naturally to room temperature, and wash with PBS buffer 3 times for 5 min each time; (3) Endogenous peroxidase blocking: Tissue sections were placed in 3% H2O2 and allowed to stand at room temperature for 8 min, then washed with PBS 3 times for 5 min each time; (4) Non-specific blocking: Mark along the tissue boundary with an immunohistochemistry pen, add 3% BSA solution to the marked area to completely cover the tissue, and incubate at room temperature for 30 min; (5) Tissue permeability: After washing the sections three times with PBS buffer, shake off the excess liquid, add 0.3% Triton-X solution to fully cover the tissue, and incubate at room temperature for 10-15 min; (6) Primary antibody incubation: Wash sections with PBS 3 times for 5 min each time, then prepare a primary antibody solution of the recommended concentration with 3% BSA, add it to the tissue, and incubate overnight at 4°C; (7) Secondary antibody incubation: The next day, the slides were taken out and warmed to room temperature. They were washed with PBS three times for 5 min each time, and then the fluorescently labeled secondary antibody was added in appropriate dilution. The slides were incubated at room temperature in the dark for 60 min. (8) Nuclear staining: Wash 3 times with PBS for 5 min each time, and stain the nuclear cells with Hoechst 33342 staining solution; (9) Mounting: After the tissue sections have dried, seal them around the coverslip with nail polish and store them away from light for later use; (10) Microscopic observation: The slides were observed and photographed under light-protected conditions using a confocal microscope.

[0081] The results are as follows Figures 13-14 As shown, R.bromii Expression of LY6G, a marker of neutrophils, in pancreatic tissue of mice in the _cer group ( Figure 13 The level was significantly lower than that in the PBS_cer group, decreasing by 56.39% (p<0.05).

[0082] (x) Take pancreatic tissue from model mice, perform TUNEL staining, and detect pancreatic tissue apoptosis using TUNEL. The specific method is as follows: (1) The steps of baking, dewaxing, hydrating and washing paraffin sections are the same as those of H&E staining; (2) Draw a circle along the edge of the tissue with an immunohistochemistry pen, add 100 μL of Proteinase K working solution with a concentration of 20 μg / mL to cover the tissue, and react at 37℃ for 20 min; (3) Equilibration: Rinse the slides 3 times with PBS, 5 min each time, add 50 μL of equilibration solution to cover the tissue, and let stand at room temperature for 10 min; (4) Labeling: Rinse the slides 3 times with PBS, 5 min each time. Prepare the incubation buffer according to the instructions, add 56 μL to cover the tissue, and react at 37°C in the dark for 1 h. (5) Nuclear staining: Rinse the slides 3 times with PBS, 5 min each time, and stain the nuclei with Hoechst 33342 staining solution; (6) Covering: Apply nail polish around the coverslip and seal it, then store it away from light; (7) Review the slides: Observe and photograph them under a fluorescence microscope in the dark.

[0083] The results are as follows Figures 15-16 As shown, R.bromii The proportion of pancreatic tissue cells in the _cer group mice was significantly reduced, decreasing by 45.85% compared to the PBS_cer group mice (p<0.05).

[0084] (xi) Serum samples from model mice were collected and analyzed by GC-MS. The specific detection method is as follows: Thaw the samples on clean dry ice. Accurately pipette the samples into 2 mL EP tubes, add 50 μL of 50% H₂SO₄, and then add 200 μL of extraction buffer containing internal standard (methyl tert-butyl ether). Vortex for 30 seconds, shake for 10 minutes, sonicate for 10 minutes, and incubate at -40°C for 60 minutes. Centrifuge the samples at 4°C and 12,000 rpm for 15 minutes. Transfer the supernatant to a sample vial and analyze using an Agilent 7890B-5977B gas chromatography-mass spectrometry (GC-MS). Differential metabolites were identified using Student's t-test with a p-value less than 0.05 and a variable projection importance (VIP) greater than 1. The differential metabolites were functionally annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database. The Rich Factor (the ratio of the number of annotated differential metabolites in a pathway to the total number of metabolites in that pathway) was calculated; a higher Rich Factor indicates greater enrichment.

[0085] The results are as follows Figure 17 As shown, R. bromii The differential metabolites in the serum of _cer group and PBS_cer mice were mainly enriched in the metabolic pathways, biosynthesis of amido acids, and the tricarboxylic acid cycle.

[0086] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Use of Ruminococcus brucelli in the preparation of drugs for the prevention and / or treatment and / or adjunctive treatment of obesity-related acute pancreatitis.

2. The application according to claim 1, characterized in that, The rumenococcus is R. bromii ATCC51896.

3. The application according to claim 1, characterized in that, The preventive and / or therapeutic and / or adjunctive treatments include at least one of the following effects: (a) Improves pathological damage to pancreatic tissue; (b) Reduce serum amylase and / or lipase levels; (c) Inhibit or reduce the early cytokine storm in acute pancreatitis; (d) Reduces apoptosis in pancreatic tissue cells; (e) Reduce the levels of inflammatory factors and / or chemokines in pancreatic tissue.

4. The application according to claim 3, characterized in that, The improvement of pancreatic tissue pathological damage includes at least one of reducing pancreatic tissue edema, reducing pancreatic inflammatory cell infiltration, and reducing pancreatic acinar cell necrosis.

5. The application according to claim 3, characterized in that, The suppression or mitigation of the early cytokine storm in acute pancreatitis includes reducing the infiltration or activation of macrophages and / or neutrophils in pancreatic tissue.

6. The application according to claim 3, characterized in that, The inflammatory factors and / or chemokines are selected from one or more of IL-6, IL-1β, CXCL1, CCL3, IL6, S100a8, and S100a9.

7. A drug for the prevention and / or treatment and / or adjunctive treatment of obesity-related acute pancreatitis, characterized in that, The drug includes Ruminococcus brucelli and a pharmaceutically acceptable carrier.

8. The medicament according to claim 7, characterized in that, The rumenococcus is R. bromii ATCC 51896.

9. The drug according to claim 7, characterized in that, The pharmaceutically acceptable carriers include one or more of the following commonly used medical fillers, binders, humectants, disintegrants, lubricants, and flavoring agents; The dosage forms of the drugs include, but are not limited to, tablets, capsules, solutions, granules, injections, powder for injection, or aerosols.

10. The medicament according to claim 7, characterized in that, The pharmaceutically effective dose of the aforementioned Ruminococcus brucellosis is 10. 9 CFU.

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