Application of Roseburia inulinivorans in preparation of medicine for treating diabetes

Abstract

The invention belongs to the technical field of biomedical treatment, and particularly relates to an application of Roseburia inulinivorans. The invention provides an application of Roseburia inulinivorans in the preparation of a medicine for treating and / or preventing diabetes mellitus, and a preparation method of the Roseburia inulinivorans. The intestinal bacteria Roseburia inulinivorans can relieve type 2 diabetes mellitus by adjusting intestinal microorganisms and metabolites thereof, protecting the integrity of intestinal barriers and improving the inflammatory state and insulin resistance, particularly, the curative effect of the intestinal bacteria Roseburia inulinivorans is close to the therapeutic effect of a clinical conventional medicine metformin, and the intestinal bacteria Roseburia inulinivorans are small in side effect, more stable in therapeutic effect, small in economic burden and more suitable for application and popularization.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of Roseburia inulinivorans in the preparation of medicaments for the treatment and / or prevention of diabetes. Background Technology

[0002] Diabetes mellitus is a syndrome caused by a relative or absolute deficiency of insulin, leading to disorders in the metabolism of carbohydrates, fats, and proteins. Statistics show that its global incidence has risen significantly, becoming the third leading cause of chronic non-communicable diseases. Furthermore, as the disease progresses, it can induce various serious complications such as diabetic nephropathy, diabetic foot lesions, and diabetic retinopathy, posing a serious threat to human health. Therefore, timely and effective treatment of diabetes is crucial. Clinically, treatment generally involves choosing hypoglycemic agents such as metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, SGLT-2 inhibitors, and GLP-1 receptor agonists, either alone or in combination, depending on the patient's specific condition. Insulin injections may be necessary in some cases. However, these drug treatments have various side effects, thus we urgently need to find a safe and effective hypoglycemic therapy.

[0003] The gut bacteria *Roseburia inulinivorans*, belonging to the Firmicutes phylum, are beneficial bacteria primarily found in the colons of humans and animals. Gut microbiota is closely related to the development and progression of diabetes. Studies have shown that probiotics not only improve gut microbiota, enhance immunity, and reduce inflammation, but also lower blood sugar and maintain glycemic homeostasis. Human and animal experiments have confirmed that probiotics can reduce the risk of type 2 diabetes. Targeted regulation of the gut microbiota by probiotics may be an effective approach to treat and prevent the large number of patients with type 2 diabetes. Although there are probiotic preparations for the prevention and treatment of type 2 diabetes in relevant national patents, their therapeutic effects have not been compared with those of the clinically common drug metformin, and current probiotic preparations only have a certain control effect on blood sugar levels, possibly not achieving a significant hypoglycemic effect. This situation completely limits the future clinical translation of probiotic preparations. Furthermore, current probiotics have not been evaluated for their positive effects on blood glucose and lipids, inflammation, oxidative stress, apoptosis, and insulin resistance.

[0004] It has been reported that the abundance of *R. inulinivorans* in the gut is significantly reduced in patients with type 2 diabetes. Currently, no studies have reported that *R. inulinivorans* can regulate gut microbiota and its metabolites, lower blood glucose levels, or treat type 2 diabetes. Summary of the Invention

[0005] This invention is the first to discover that *Roseburia inulinivorans* can lower blood glucose levels by regulating gut microbiota and its metabolites, thereby alleviating the pathological symptoms of type 2 diabetes. In particular, its efficacy is close to that of metformin, a commonly used clinical drug. Simultaneously, *R. inulinivorans* also possesses advantages such as improving systemic inflammation levels, anti-oxidative stress, anti-apoptosis, and reducing insulin resistance.

[0006] R. inulinivorans, commonly known as the inulin-eating Rossi bacterium, belongs to the genus R. inulinivorans of the family Cladosporidae in the class Clostridium of the phylum Firmicutes in the domain Bacteria. It is a Gram-positive bacterium (G7). + This strain is strictly anaerobic, capable of forming spores, possesses flagella, and is motile. As one of the core gut microbiota, this strain can efficiently degrade and utilize inulin, converting it into beneficial metabolites (especially butyric acid).

[0007] This invention provides the use of Roseburia inulinivorans in the preparation of medicaments for the treatment and / or prevention of diabetes.

[0008] Preferably, the diabetes is type 2 diabetes. Roseburia inulinivorans can significantly reduce blood glucose levels, increase glucose tolerance, promote insulin secretion, reduce insulin resistance, and significantly reduce serum TC, TG, and LDL concentrations. It can also increase insulin and its receptor expression and promote PI3K / AKT pathway activation.

[0009] Preferably, the drug further includes pharmaceutically acceptable excipients or carriers. The carrier can be one that reduces drug degradation and loss, and minimizes side effects, such as micelles, microemulsions, or gels.

[0010] Excipients can be materials added to make drugs into suitable dosage forms, such as buffers, lyophilization excipients, etc. Liquid formulations are generally buffer solutions, isotonic solutions, and aqueous solutions.

[0011] The pharmaceutically acceptable excipients include any one or more combinations of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surfactants, or stabilizers.

[0012] The excipients are selected from at least one of the following: fillers, diluents, disintegrants, binders, lubricants, flow aids, surfactants, solvents, flavoring agents, stabilizers, colorants, or preservatives.

[0013] The fillers or diluents include sugars such as lactose, sucrose, glucose, mannitol, sorbitol, and dextrin; starches such as starch, pregelatinized starch, and dextrin; celluloses such as microcrystalline cellulose, gum arabic, fenugreek gum, and dextran; and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, pharmaceutical-grade calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminosilicate.

[0014] The lubricant, flow aid, or anti-sticking agent includes stearic acid; metal stearate salts such as calcium stearate or magnesium stearate; talc; colloidal silica; micronized silica gel; hydrogenated vegetable oil; polyethylene glycol; lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; silicates such as silicic anhydride or silicate hydrates, etc.

[0015] The adhesives include distilled water, ethanol of different concentrations, starch paste, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, methylcellulose, ethylcellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, polyethylene glycol, and compounds similar to the excipients described above.

[0016] The disintegrants include cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, or croscarmellose sodium; croscarmellose; and chemically modified starch / cellulose, such as carboxymethyl starch or sodium carboxymethyl starch.

[0017] The antioxidants mentioned include sodium bisulfite, sodium metabisulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine, thiourea, phosphoric acid, citric acid, etc.

[0018] The preservatives or antibacterial agents mentioned include benzoic acid and sodium benzoate, sorbic acid, ethanol, parabens, benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenylethanol, sodium propionate, sorbic acid, eucalyptus oil, cinnamon oil, and peppermint oil, etc.

[0019] Examples of suitable dosage forms include tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, patches, aerosols, nasal sprays, and sterile solutions that can be used for injection. The drugs of this invention can be formulated as solutions or lyophilized powders for parenteral administration. The powder can be reconstituted by adding appropriate solvents or other carriers before use. Liquid formulations are generally PBS buffer, isotonic saline solution, and aqueous solutions.

[0020] This invention provides a medicament for treating diabetes, the medicament comprising Roseburia inulinivorans. Preferably, the medicament further comprises pharmaceutically acceptable excipients.

[0021] The dosage of Roseburia inulinivorans in pharmaceutical formulations can vary within a wide range, and those skilled in the art can easily determine it based on objective factors such as the type of disease, the severity of the condition, the patient's weight, the dosage form, and the route of administration.

[0022] The beneficial effects of this invention are: This invention comprehensively evaluated the efficacy of Roseburia inulinivorans in type 2 diabetes, finding that it possesses functions such as weight reduction, blood glucose and lipid reduction, anti-inflammation, anti-oxidative stress, anti-apoptosis, improvement of insulin resistance, regulation of short-chain fatty acid levels, and restoration of intestinal ecology imbalance. In particular, its therapeutic effect is close to that of the clinically common drug metformin. Furthermore, Roseburia inulinivorans has no side effects, while metformin commonly has side effects. Therefore, Roseburia inulinivorans is very likely to become a good candidate drug for improving type 2 diabetes. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Images of body weight changes, food intake, water intake, epididymal white fat, and liver, along with their corresponding weights, are shown for each group of mice. (*) p < 0.05;** p < 0.01; *** p <0.001).

[0025] Figure 2 The levels of FBG, OGTT, AUC, HOMA-IR, and insulin in the blood of mice in each group, as well as the serum levels of C-peptide, GLP-1, TC, TG, LDL, and HDL (*) are displayed. p < 0.05;** p < 0.01; *** p <0.001).

[0026] Figure 3 The levels of IL-1β, TNF-α, IL-6, and LPS in the serum of mice in each group were shown (*). p < 0.05;** p < 0.01; *** p<0.001).

[0027] Figure 4 The image shows HE staining and NAFLD scores of the liver in each group of mice, Oil Red O staining and lipid region quantification of the liver, and HE staining of the pancreas (*). p < 0.05;** p < 0.01; *** p <0.001).

[0028] Figure 5 The text displays the protein expression of TLR4 and Myd88, the gene expression of TNF-α, IL-1β, and IL-6, and the relative levels of SOD and MDA in the livers of mice in each group (*). p < 0.05;** p < 0.01; *** p <0.001).

[0029] Figure 6 The protein expression of ZO-1, Occludin, and TLR4 in the colon of mice in each group is shown (*). p < 0.05;** p <0.01; *** p <0.001).

[0030] Figure 7 The gene expression of MCP-1, IFN-γ, and IL-1β, and the protein expression of Bcl-2 and Cacepase-3 in the pancreas of mice in each group are shown (*). p < 0.05;** p < 0.01; *** p <0.001).

[0031] Figure 8 The protein expression of insulin, INS-R, PI3K, and AKT in the pancreas of mice in each group is shown (*). p < 0.05;** p < 0.01; *** p <0.001).

[0032] Figure 9 The gene expression of GPR41, PC1 / 3, and GLP-1 in the colon of mice in each group is shown, as well as the relative concentrations of total short-chain fatty acids, butyric, acetic, and propionic fatty acids (*). p < 0.05;** p < 0.01; *** p <0.001).

[0033] Figure 10 Displaying the gut microbiota α diversity index of mice in each group, Venn plot, principal component analysis (PCA) and principal coordinate analysis (PCOA) plots (*). p < 0.05;** p < 0.01; *** p <0.001).

[0034] Figure 11 Principal component analysis (PCA) score plots and volcano plots for each group of fecal samples under positive and negative ion modes. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0036] In this embodiment of the invention, 3×10 are used daily. 9 db / db mice were treated orally with CFUs of Roseburia inulinivorans or metformin at a dose of 0.012 g / kg for 6 weeks.

[0037] Six weeks later, a comprehensive evaluation of the efficacy and mechanism of Roseburia inulinivorans in the treatment of type 2 diabetes was conducted: (1) Evaluation of treatment effect: body weight, food intake, water intake, weight of white fat in liver and epididymis of mice, serum blood glucose related indicators (FBG, OGTT, AUC, HOMA-IR, insulin, C-peptide, GLP-1), blood lipid related indicators (TC, TG, LDL, HDL) and inflammation related indicators (IL-1β, TNF-α, IL-6, LPS); (2) Evaluation of anti-inflammatory and antioxidant effects in the liver: enzyme-linked immunosorbent assay (ELISA) was used to analyze the relative content of SOD and MDA in liver homogenate, immunohistochemistry was used to analyze the protein expression of TLR4 and Myd88 in the liver, and PCR was used to analyze the gene expression of TNF-α, IL-1β and IL-6 in the liver. (3) Evaluation of intestinal barrier function and anti-inflammatory effect in colon: Immunofluorescence analysis of the expression of colonic tight junction proteins ZO-1 and Occludin, and immunohistochemical analysis of the expression of TLR4 in colon; (4) Evaluation of anti-inflammatory effects in the pancreas: PCR analysis of gene expression of IL-1β, MCP-1 and IFN-γ in pancreatic tissue; (5) Evaluation of anti-apoptosis and PI3K / AKT pathway in pancreas: Immunohistochemical analysis of protein expression of apoptosis-related pathways (Bcl-2, Case-3) and PI3K / AKT pathways (Insulin, INS-R, TLR4, NF-κB, PI3K, AKT) in pancreatic tissue; (6) Assessment of the regulation of GLP-1 expression level in colon: PCR analysis of gene expression levels of GPR41, PC1 / 3 and GLP-1 in colon tissue; (7) Assessment of the regulation of short-chain fatty acid levels in feces: The relative levels of butyric acid, acetic acid, propionic acid, isobutyric acid, isovaleric acid, valeric acid and hexanoic acid in feces were detected and analyzed by gas chromatography-mass spectrometry (GC-MS) or high performance liquid chromatography (HPLC). (8) Assessment of regulation of intestinal microecological balance: Mouse feces were collected for 16S rRNA sequencing of intestinal flora, and then the bio-cloud platform of Meiji Biotechnology Co., Ltd. was used to analyze the diversity and richness of intestinal flora, composition of intestinal flora, function and phenotype of intestinal flora. (9) Assessment of the regulation of intestinal metabolite levels: Collect mouse feces for metabolite analysis.

[0038] Finally, the efficacy of Roseburia inulinivorans in alleviating db / db mice was comprehensively evaluated and compared with the therapeutic effect of the clinical drug metformin.

[0039] The *Roseburia inulinivorans* used in this embodiment of the invention were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, DSMZ catalog number: 16841 (type strain), strain identifier: A2-194, other accession numbers or WDCM numbers: NCIMB 14030, CIP 109405, JCM17584; risk level: 1 (classified according to the German TRBA standard). It was isolated in 1997 from human fecal samples in Aberdeen, Scotland, United Kingdom of Great Britain and Northern Ireland. GenBank accession number: 16S rRNA gene: AJ270473, whole genome shotgun sequencing sequence: ACFY00000000, culture conditions: medium 412, anaerobic, 37°C.

[0040] M2GSC culture medium formulation composition table of the present invention

[0041] Example 1 Preparation of Roseburia inulinivorans (1) Prepare M2GSC medium: Weigh 1g of tryptone, 0.25g of yeast extract, 0.4g of NaHCO3, 0.045g of K2HPO4, 0.045g of KH2PO4, 0.09g of (NH4)2SO4, 0.09g of NaCl, 0.009g of MgSO4·7H2O, 0.009g of CaCl2, and 0.1mg of azurite and dissolve them in 50ml of triple-distilled water. Stir thoroughly to mix well. Autoclave at 120℃ for 20 minutes, then cool in a laminar flow hood for later use. Separately, weigh 0.2g of glucose, 0.2g of cellobiose, and 0.2g of soluble starch, dissolve them in 15ml of triple-distilled water, stir thoroughly, autoclave at 115℃ for 15 minutes, and cool in a laminar flow hood for later use. Weigh 0.1g of cysteine, dissolve it in 5ml of triple-distilled water, and filter sterilize in a laminar flow hood. Take 30ml of clear rumen fluid and filter sterilize. Transfer the above 15ml glucose solution, 5ml cysteine ​​solution, and 30ml clear rumen fluid into 50ml of culture medium, mix thoroughly, and obtain 100ml of M2GSC medium.

[0042] (2) Inoculation with bacterial culture: Take out 1 ml of Roseburia inulinivorans bacterial culture stored in the laboratory at -80℃ and slowly thaw it at room temperature. In a clean bench, slowly transfer the dissolved bacterial culture into the prepared M2GSC medium, and perform aseptic operation throughout the process.

[0043] (3) Remove oxygen: Place the inoculated culture medium into the corresponding anaerobic bottle, connect the ANOXOMAT MARK II anaerobic microaerophilic culture system to remove oxygen until the oxygen is completely removed.

[0044] (4) Culture: Place the deoxygenated culture medium in a 37℃ constant temperature incubator and culture in the dark for 2-3 days. Observe the bottom of the culture medium. If there is fine sand-like sediment, it is Roseburia inulinivorans.

[0045] (5) Preparation of freeze-dried bacterial powder: In a clean bench, transfer the cultured medium into a sterile 50ml centrifuge tube, centrifuge at 3000xg for 15min at 4℃, discard the supernatant, wash 3 times with sterile PBS, and place the resulting precipitate in a Heto PowerDry freeze dryer. Freeze-dry at -80℃ for 24h until the precipitate is honeycomb-like, then twist it into powder to prepare 63×10 10 Freeze-dried powder with CFUs / g.

[0046] Example 2: Animal Experiments and Design Thirty 6-week-old male db / db mice and ten 6-week-old male C57BL / 6J mice were housed in a pathogen-free environment at 21–25°C and 55 ± 10% humidity, with a 12-hour light / dark cycle, and were provided with distilled water and food. After one week of acclimatization, the C57BL / 6J mice were divided into a normal group (N group), while the db / db mice were randomly divided into a model group (D group), a metformin group (DM group), and a probiotic group (D-RI group), with 10 mice in each group.

[0047] Within a fixed time period, mice in the normal and model groups were administered sterile PBS by gavage daily, mice in the metformin group were administered metformin (200 μl, 0.012 g / kg / d) by gavage daily, and mice in the probiotic group were administered Roseburia inulinivorans (200 μl, 3×10⁻⁶ g / kg / d) by gavage daily. 9 CFU / mouse / day) for 6 weeks, during which the body weight and fasting blood glucose levels of the four groups of mice were recorded once a week.

[0048] In week six, an oral glucose tolerance test (OGTT) was performed. After fasting for 12 hours, mice in four groups were injected with the corresponding amount of glucose solution at 2 g / kg body weight. Blood glucose levels were then measured at 0 min, 30 min, 60 min, and 120 min. After collecting feces from each group of mice, they were euthanized by anesthesia with pentobarbital. The epididymal white fat and liver were removed for photography, and blood, pancreas, liver, colon, and other tissues were collected for analysis.

[0049] like Figure 1 As shown, compared to the metformin group, the probiotic group significantly reduced the body weight, food intake, and water consumption of db / db mice, and decreased the weight of epididymal fat, but had no significant effect on liver weight. Therefore, Roseburia inulinivorans can improve related indicators such as body weight, food intake, water consumption, and epididymal fat weight in mice.

[0050] Example 3 Biological Experimental Methods 3.1 Oral Glucose Tolerance Test (OGTT) After fasting for 12 hours, the mice in each group were accurately weighed. A glucose solution of appropriate concentration (generally 20%-30%) was prepared using sterile saline. The dosage for each mouse was calculated based on a glucose concentration of 2 g / kg body weight. The glucose solution was administered to the mice via gavage. Blood samples were collected from the tail tip at 0, 15, 30, and 45 minutes after glucose administration. Blood glucose levels were measured and recorded using blood glucose test strips at each time point. Blood glucose curves were plotted for each group of mice, and the area under the curve (AUC) and other indicators were calculated to assess the mice's glucose tolerance.

[0051] Table 1. Results of blood glucose and blood lipid tests in mice of each group

[0052] like Figure 2 As shown in Table 1, compared with the metformin group, the probiotic group significantly reduced blood glucose levels and increased glucose tolerance in db / db mice, while promoting insulin secretion and reducing insulin resistance. It also significantly reduced serum TC, TG, and LDL concentrations, thereby alleviating diabetes symptoms. Therefore, Roseburia inulinivorans can reduce blood glucose and lipid levels in mice.

[0053] Table 2 Quantitative analysis of serum inflammatory factors in each group of mice

[0054] like Figure 3 As shown in Table 2, the expression levels of inflammatory factors IL-1β, TNF-α, IL-6, and LPS in the serum of db / db mice were significantly increased, and immune homeostasis was severely disrupted. RI supplementation could significantly reduce the concentration of TNF-α and LPS and restore the body's immune homeostasis.

[0055] 3.2 Immunohistochemistry and immunofluorescence staining Tissues were fixed for 48 h, embedded in paraffin, and cut into 4 µm sections. Colon, liver, and pancreas paraffin sections were dewaxed and hydrated. They were first incubated in hydrogen peroxide solution to eliminate endogenous peroxidase activity, then heated in a microwave oven in 0.1 mol / L sodium citrate buffer for 10 min, blocked with 5% BSA at room temperature for 1 h, stained overnight with diluted primary antibody at 4°C, washed with PBS, and incubated with HRP-conjugated secondary antibody at room temperature for 30 min. Color development was achieved by incubating sections with the HRP substrate 3,5-diaminobenzothiazide. Positive staining was indicated by a brownish-yellow or brownish-red signal observed under a microscope, and quantified using ImageJ software.

[0056] Colonic paraffin sections were dewaxed and hydrated. The sections were first heated in a microwave oven in 0.1 mol / L sodium citrate buffer for 10 min, then blocked with 5% BSA at room temperature for 1 h. Diluted primary antibody was used for staining overnight at 4°C. After rinsing with PBS, the sections were incubated with Alexa Fluor 555-conjugated goat anti-rabbit secondary antibody at room temperature in the dark for 40 min. Finally, the nuclei were stained with DAPI at room temperature in the dark for 10 min, followed by rinsing with PBS. The sections were observed under a fluorescence microscope, and positive results were quantified using ImageJ software.

[0057] like Figure 4As shown, both the metformin group and the probiotic group significantly improved the pathological damage of liver tissue, reduced lipid accumulation in hepatocytes, and greatly restored the histological structure of pancreatic islets, thereby alleviating the pathological symptoms in db / db mice. This demonstrates that Roseburia inulinivorans can alleviate the pathological damage of liver and pancreatic tissues in mice.

[0058] 3.3 Enzyme-linked immunosorbent assay (ELISA) Blood samples from each group of mice were allowed to coagulate naturally for 2 hours, then centrifuged at 3000 rpm for 15 minutes at 4°C, and serum was collected. Liver and fecal samples from each group of mice were weighed at 0.1 g and ground to prepare homogenates. Blank wells, standard wells, and sample wells were prepared. 50 μL of standard solution and sample wells were added to each well and mixed thoroughly. The plate was sealed with a sealing film and incubated at 37°C for 1 hour. The liquid in the wells was discarded, and the plate was patted dry on absorbent paper. 200 μL of washing buffer was added, and the plate was allowed to stand for 1 minute. The washing buffer was discarded and the plate was patted dry. This process was repeated 5 times. 50 μL of solution A and 50 μL of solution B were added to each well. The plate was sealed with a sealing film and incubated at 37°C for 15 minutes. 50 μL of stop solution was added to terminate the reaction, and the absorbance of each well was measured at 450 nm.

[0059] Table 3 Results of liver inflammation and oxidative stress markers in mice of each group

[0060] like Figure 5 As shown in Table 3, the probiotic group significantly reduced the protein expression of TLR4 and Myd88 in the liver tissue of db / db mice, reduced the gene expression of TNF-α, IL-1β and IL-6, and effectively improved the level of inflammation in the liver. At the same time, the probiotic treatment can also reduce the level of oxidative stress in the liver, thereby maintaining the immune homeostasis of the liver tissue.

[0061] 3.4 Quantitative Real-Time PCR Total RNA was extracted and purified from colon, liver, and pancreas tissues using the RNAeasy™ Animal RNA Isolation Kit, following the manufacturer's instructions.

[0062] RNA concentration was determined using a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA). cDNA was then synthesized and amplified using the Revertaid First Strand cDNA Synthesis Kit, the iTaq™ Universal SYBR@Green Supermix Kit, and specific primers.

[0063] RT-PCR was performed using a BIO-RAD MyIQ 2 real-time PCR system (Hercules, CA, USA) under the following amplification conditions: initial denaturation at 94°C for 1 minute, followed by 40 cycles of 95°C for 10 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 6 seconds. (The last sentence appears to be incomplete and possibly refers to a specific method or process.) -ΔΔCt The relative mRNA expression of Occludin, ZO-1, ZO-2, FXR, and CYP7A1 was calculated. β-actin was used as a normalized internal control.

[0064] Table 4. Assessment of colonic barrier function and inflammation in mice of each group

[0065] Figure 6 Table 4 shows the protein expression of ZO-1, Occludin, and TLR4 in the colon of mice in each group. The results show that the expression levels of ZO-1 and Occludin in the colon of db / db mice were significantly reduced, while the expression level of TLR4 was significantly increased. After treatment with probiotics, the above indicators were close to normal levels, indicating that probiotics RI can alleviate the pathological symptoms of db / db mice by restoring the integrity of the intestinal barrier and reducing colonic inflammation.

[0066] Table 5. Detection results of pancreatic inflammation and apoptosis markers in each group of mice.

[0067] Figure 7 Table 5 shows the gene expression of MCP-1, IFN-γ, and IL-1β, and the protein expression of Bcl-2 and Caspase-3 in the pancreas of each group of mice. The results show that the gene expression levels of MCP-1, IFN-γ, and IL-1β and the protein expression level of Caspase-3 were significantly increased in db / db mice, while the protein expression level of Bcl-2 was significantly decreased. However, the probiotic intervention reversed the expression trends of the above indicators to varying degrees. This indicates that the probiotic RI effectively alleviates the pathological damage in db / db mice by inhibiting inflammation and apoptosis activation.

[0068] Table 6. Detection of insulin and PI3K / AKT pathway protein expression levels in the pancreas of mice in each group.

[0069] Figure 8Table 6 shows the protein expression of insulin, INS-R, PI3K, and AKT in the pancreas of mice in each group. The results indicate that probiotic intervention can significantly increase the protein expression levels of insulin and INS-R in the pancreas, while also increasing the protein expression levels of PI3K and AKT. This suggests that RI can improve the symptoms of db / db mice by increasing the expression of insulin and its receptor and promoting the activation of the PI3K / AKT pathway.

[0070] Figure 9 The study showed the gene expression of GPR41, PC1 / 3, and GLP-1 in the colon of mice in each group, as well as the relative concentrations of total short-chain fatty acids, Butyric, Acetic, and Propionic. The results indicated that after probiotic treatment, the gene expression of GPR41, PC1 / 3, and GLP-1 in the colon tissue of db / db mice was significantly increased, and the level of total short-chain fatty acids in the intestine was significantly increased, especially the concentrations of Butyric, Acetic, and Propionic, which promoted insulin secretion and the maintenance of intestinal barrier function.

[0071] 3.5 Gut microbiota analysis Gut microbiota assessment was performed using QIIME and R software package 3.5.1. Alpha diversity indices and bacterial abundance data across different groups were compared using the Kruskal-Wallis test followed by paired Mann-Whitney U comparisons. The resulting p-values ​​were then corrected using the Bonferroni method. Furthermore, after complex data ordering, Venn diagrams were used to visualize the core gut microbiota species shared by 78% of mice across all groups. Simultaneously, β-diversity analysis was performed using the UniFrac distance metric to investigate structural differences in the microbial community among samples, and the results were visualized using principal coordinate analysis (PCoA). Additionally, differences in UniFrac distance between groups were determined using similarity analysis (ANOSIM). To compare the relative levels of abundant taxa at the phylum, family, and genus levels among groups, one-way ANOVA and post-hoc minimum significance tests were performed using IBM SPSS Statistics 19.0 software. In addition, LEfSe (Linear Discriminant Analysis (LDA) effect size) was performed to identify biomarkers of abundant taxa and functional pathways by calculating LDA scores (greater than 4) for each group, and heatmaps were generated using the R package 3.5.1. Furthermore, a community phylogenetic survey (PICRUSt) based on unobserved state OTU reconstruction was used to predict the abundance of functional categories using orthologs (KOs) from the Kyoto Genome and Encyclopedia of Genomes (KEGG), while bacterial composition was predicted using BugBase based on sequencing results. However, the relative abundance of KOs meta-related to SCFA was further analyzed using one-way ANOVA. Additionally, Pearson correlation analysis was performed to assess the correlation between taxa and factors such as colitis index, hematologic parameters, bacterial translocation, and protein and gene expression; data are expressed as mean ± standard error of mean (SEM), and p < 0.05 was set as the significance threshold.

[0072] Figure 10 The results showed that the probiotic intervention significantly restored the reduced α diversity and decreased OTU abundance of the gut microbiota in db / db mice. Meanwhile, β diversity analysis showed that the composition of gut microbiota differed among different groups, while the probiotic intervention significantly increased the richness and diversity of the gut microbiota.

[0073] 3.6 Intestinal Metabolite Analysis Fresh fecal samples were collected and completely frozen in liquid nitrogen. An appropriate amount of sample was added to a pre-cooled methanol / acetonitrile / water solution (2:2:1, volume ratio). The sample was vortexed, sonicated at low temperature for 30 minutes, allowed to stand at -20°C for 10 minutes, and then centrifuged at 14000g for 20 minutes at 4°C. The supernatant was collected and dried under vacuum. For mass spectrometry analysis, 100 μL of acetonitrile (acetonitrile:water = 1:1, volume ratio) was added, vortexed, and centrifuged at 14000g for 15 minutes at 4°C. The supernatant was used as the analytical sample. The samples were separated on an Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) HILIC column, and primary and secondary spectra were collected using an AB Triple TOF 6600 mass spectrometer. Principal component analysis (PCA) was performed on the peaks extracted from all experimental and quality control samples.

[0074] 3.7 Short-chain fatty acid (SCFA) analysis The content of SCFAs in feces was determined by gas chromatography. Samples were centrifuged in 2 mL centrifuge tubes with 50 μL of 15% phosphoric acid and 125 μg / mL internal standard (isocaproic acid). 100 μL of the internal standard (isocaproic acid) solution was then homogenized with 400 μL of diethyl ether for 1 min, followed by centrifugation at 12000 rpm for 10 min at 4 °C. The supernatant was then used for analysis. Samples were injected separately onto an Agilent HP-INNOWAX capillary column (30 m * 0.25 mm ID * 0.25 μm). Six samples were repeatedly processed according to their metabolic status.

[0075] Figure 11 Principal component analysis (PCA) score plots and volcano plots are presented under four positive and negative ion modes. In the PCA score plots of the positive and negative ion modes, metabolites were well separated among the N group, D group, DM group, and D-RI group.

[0076] Except for the detailed description of 16S rRNA data, all data obtained in the embodiments of this invention are expressed as mean ± SEM. Furthermore, all statistical analyses were performed using IBM SPSS Statistics software 19.0 for Windows, and differences between two groups were assessed using unpaired two-tailed t-tests. Additionally, one-way ANOVA was used, followed by Newman-Keuls post-hoc tests to evaluate more than two groups. It should be noted that a p-value greater than 0.05 was considered statistically significant.

[0077] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Use of Roseburia inulinivorans in the preparation of medicines for the treatment and / or prevention of diabetes.

2. The application according to claim 1, characterized in that, The diabetes mentioned is type 2 diabetes.

3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, The pharmaceutically acceptable excipients include any one or more combinations of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surfactants, and stabilizers.

5. A drug for treating diabetes, characterized in that, The drug includes Roseburia inulinivorans.

6. The medicament for treating diabetes according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that, The pharmaceutically acceptable excipients include any one or more combinations of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surfactants, and stabilizers.

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