A strain of Lactobacillus plantarum FLP-215 and its application in ulcerative colitis, diabetes and organ damage

Abstract

The present invention discloses a strain of Lactobacillus plantarum FLP-215, which was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on September 4, 2023, with a deposit number of CGMCC No. 28337. The Lactobacillus plantarum FLP-215 has the effects of preventing and / or treating ulcerative colitis, diabetes and reducing organ damage, and has broad market prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial degradation, and more specifically, relates to a strain of Lactobacillus plantarum FLP-215 and its application in ulcerative colitis, diabetes and organ damage. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic nonspecific inflammatory disease, mainly including ulcerative colitis (UC) and Crohn's disease (CD). There is no consensus on the specific pathogenesis. The clinical manifestations of patients include diarrhea, blood in the stool, weight loss, etc. Ulcerative colitis occupies a large amount of public health resources. Due to its high incidence and recurrence, it has become a major health problem worldwide. The prevention and control of ulcerative colitis is of great significance to the prevention and treatment of chronic diseases and the protection of public health.

[0003] Common drugs for the treatment of ulcerative colitis in clinical practice include: sulfasalazine salicylic acid preparations, corticosteroids, etc. However, the side effects of the above drugs are also obvious, which can lead to the occurrence or aggravation of various related complications, mainly manifested in hypertension, diabetes, osteoporosis and kidney damage, especially the occurrence of complications of diabetes, which brings great pain and inconvenience to patients and reduces the quality of life of patients. Therefore, it is urgent to find a healthy and effective method for the treatment of ulcerative colitis to improve the current treatment methods. Studies have shown that differences in intestinal flora are one of the key factors in ulcerative colitis. Probiotics are a class of living microorganisms that can have beneficial effects on host health after taking in sufficient amounts. Probiotics have always been widely recognized to improve mucosal barrier function, regulate intestinal flora, and enhance the immunity of the intestinal system. Probiotics have many advantages over traditional treatments, such as being safer and having fewer side effects.

[0004] Type 2 diabetes is the most common metabolic disease in the world. Its causes are complex and cause irreversible damage to the body, which in turn induces various chronic complications. The incidence rate continues to rise worldwide. How to effectively prevent and treat diabetes has become a global health problem that needs to be solved urgently. At present, there is still no drug that can effectively cure type 2 diabetes. Patients can only control blood sugar through diet, exercise and other means, and clinical drugs also show various toxic side effects. Due to changes in lifestyle, it is difficult to achieve the expected effect by changes such as diet and exercise alone. At present, the purpose of treating diabetes is mainly achieved by taking injectable drugs. These drugs mainly include sulfonylurea and non-sulfonylurea secretagogues to stimulate pancreatic cells to secrete insulin; thiazolidinediones to enhance insulin sensitivity; biguanides to reduce liver glucose output and improve peripheral insulin resistance; and α-glucosidase inhibitors including acarbose and voglibose that can delay carbohydrate digestion and absorption. Sometimes in actual treatment, the above drugs are used in combination according to the patient's condition to achieve the purpose of rapid and effective hypoglycemia. However, the use of the above drugs will more or less produce some side effects, such as gastrointestinal discomfort, liver disease, and is limited by kidney function and heart disease. Therefore, people need to find more natural and safe blood sugar-lowering ingredients and develop safe and effective products or drugs to assist in the prevention and treatment of diabetes.

[0005] Organ tissue damage refers to pathological damage to human skin, subcutaneous tissue, synovial bursa, intervertebral disc, peripheral nerves and blood vessels caused by various activities. Organ damage is a special critical state between deterioration and recovery. If diagnosed and treated in time, it is expected to be cured; otherwise, it may deteriorate or even cause organ failure, causing serious damage to the body. Therefore, the diagnosis and treatment of organ tissue damage is extremely important. In recent years, the study of the mechanism of liver damage, as well as its prevention and treatment, has become one of the hot topics in biological and medical research at home and abroad.

[0006] At present, whether it is the treatment of ulcerative colitis, the treatment of type 2 diabetes, or the treatment of organ damage, complications will occur and worsen during the actual treatment process. When taking drugs to treat ulcerative colitis, it may lead to the aggravation of diabetes and organ damage; when taking drugs to treat type 2 diabetes, it may cause complications such as gastrointestinal discomfort and liver disease. In addition, how to provide targeted treatment for patients who suffer from ulcerative colitis, diabetes, and organ damage at the same time is also an important issue that needs to be solved urgently.

[0007] The Chinese patent "A strain of Lactobacillus plantarum for relieving ulcerative colitis and its application" discloses a strain of Lactobacillus plantarum that can reduce weight loss during ulcerative colitis, improve stool characteristics and blood in stool, relieve shortening of colon length, improve colon mucosal damage, reduce pro-inflammatory factors in the colon, etc. However, this strain only has the effect of relieving ulcerative colitis, and cannot simultaneously treat other complications, such as the treatment of diabetes and the reduction of organ damage. Its functional effects and application scenarios are limited. Summary of the invention

[0008] In view of the above-mentioned existing technical problems, the primary purpose of the present invention is to provide a strain of Lactobacillus plantarum FLP-215, which has the effects of preventing and / or treating ulcerative colitis, diabetes and reducing organ damage, and has broad market prospects.

[0009] The second object of the present invention is to provide the use of the above-mentioned Lactobacillus plantarum FLP-215 or its bacterial suspension in the preparation of products for preventing and / or treating ulcerative colitis, diabetes and / or reducing organ damage.

[0010] The third object of the present invention is to provide a bacterial agent comprising the above-mentioned Lactobacillus plantarum FLP-215 and / or its bacterial suspension.

[0011] The fourth object of the present invention is to provide the use of the above-mentioned bacterial agent in the preparation of products for preventing and / or treating ulcerative colitis, diabetes and / or reducing organ damage.

[0012] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0013] The present invention screened and obtained a strain of Lactobacillus plantarum (Lactobacillus plantarum) FLP-215, and the Lactobacillus plantarum FLP-215 was preserved in the General Microbiology Center of China Microorganism Culture Collection Administration on September 4, 2023, with the preservation number of CGMCC No.28337.

[0014] The present invention explores the preventive and therapeutic effects of Lactobacillus plantarum FLP-215 on ulcerative colitis and its mechanism of action through shotgun metagenomic and transcriptomic analysis. The results show that the intake of Lactobacillus plantarum FLP-215 significantly improves ulcerative colitis in mice induced by sodium dextran sulfate, as shown by increased weight, water intake, food intake and colon length of mice, and decreased disease activity index, inflammatory factor levels and histopathological scores. In-depth research has found that Lactobacillus plantarum FLP-215 relieves inflammation by regulating intestinal microbiota and increasing the production of short-chain fatty acids (SCFAs), reducing the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon γ (IFN-γ), tumor necrosis factor-α (TNF-a) and interleukin-17A (IL-17A), and increasing the content of anti-inflammatory cytokine interleukin-10 (IL-10). In summary, Lactobacillus plantarum FLP-215 can play a preventive and therapeutic role in the dextran sulfate sodium-induced colitis mouse model by protecting the intestinal mucosal barrier, attenuating the inflammatory response, and regulating the structure of the intestinal microbiota.

[0015] The inventors also found that Lactobacillus plantarum FLP-215 can significantly reduce blood sugar and play a role in treating diabetes. The inventors found that Lactobacillus plantarum FLP-215 can treat and prevent type 2 diabetes by improving key pathways of sugar and lipid metabolism, reducing fasting blood sugar levels, improving glucose intolerance, increasing serum glucagon-like peptide-1 levels, reducing insulin resistance index, and improving beta cell function and insulin sensitivity detection index. Further, the inventors found that Lactobacillus plantarum FLP-215 can play an excellent role in reducing organ damage, especially reducing liver damage, pancreatic damage or colon damage.

[0016] The present invention provides a strain of Lactobacillus plantarum FLP-215, which can effectively treat ulcerative colitis, treat diabetes and reduce organ damage at the same time, and provides a technical solution for the combined treatment of ulcerative colitis, diabetes and organ damage.

[0017] Furthermore, the present invention also seeks to protect the use of the above-mentioned Lactobacillus plantarum FLP-215 or its bacterial suspension in the preparation of products for preventing and / or treating ulcerative colitis, diabetes and / or reducing organ damage.

[0018] In some embodiments, the ulcerative colitis is dextran sulfate sodium-induced ulcerative colitis.

[0019] In some embodiments, the prevention and / or treatment of ulcerative colitis comprises at least one of the following: improving the intestinal mucosal barrier, alleviating inflammatory response, or regulating the intestinal microbiota structure.

[0020] In some embodiments, the relieving inflammatory response is one or more of increasing the expression of IL-10, decreasing the expression of IL-1β, decreasing the expression of IL-6, decreasing the expression of IFN-γ, decreasing the expression of TNF-a, or decreasing the expression of IL-17A.

[0021] In some embodiments, the regulating the structure of the intestinal microbiota is one or more of promoting the growth of probiotics in the intestinal flora, increasing the abundance of bacteria that can produce SCFAs, increasing the diversity of the intestinal flora, or inhibiting the growth of pathogenic bacteria in the intestinal flora.

[0022] In some embodiments, the improvement of the intestinal mucosal barrier is to promote the expression of MUC-2 protein and / or ZO-1 protein.

[0023] In some embodiments, more specifically, the efficacy of Lactobacillus plantarum FLP-215 in preventing and / or treating ulcerative colitis specifically includes at least one of the following:

[0024] (a) reduce weight loss due to ulcerative colitis;

[0025] (b) reduce the reduced food and water intake caused by ulcerative colitis;

[0026] (c) Significant increase in colon length;

[0027] (d) Significantly reduce the disease activity index;

[0028] (e) Significantly increased the levels of anti-inflammatory cytokines in serum and reduced the levels of pro-inflammatory cytokines in serum;

[0029] (f) Significantly reduced intestinal pathology scores;

[0030] (g) increase the expression of tight junction protein ZO-1 and / or mucin MUC-2 in the intestine;

[0031] (h) significantly increase the content of short-chain fatty acids;

[0032] (i) Significantly improve the diversity of intestinal flora;

[0033] (j) promoting the growth of probiotics in the intestinal flora and / or inhibiting the growth of pathogenic bacteria in the intestinal flora.

[0034] Since Lactobacillus plantarum FLP-215 has any of the above effects, the application of Lactobacillus plantarum FLP-215 in the above should also be within the protection scope of the present application.

[0035] In some embodiments, the treatment of diabetes includes at least one of the following: improving key pathways of glucose and lipid metabolism, reducing fasting blood glucose levels, improving glucose intolerance, reducing serum insulin levels, or increasing serum glucagon-like peptide-1 levels.

[0036] In some embodiments, more specifically, the efficacy of Lactobacillus plantarum FLP-215 in treating diabetes specifically includes at least one of the following:

[0037] (a) Lowering blood sugar and blood lipids;

[0038] (b) improve the α and β diversity of the intestinal flora;

[0039] (c) promoting the growth of probiotics in the intestinal flora and inhibiting the growth of pathogenic bacteria in the intestinal flora;

[0040] (d) improving key pathways of glucose and lipid metabolism;

[0041] (e) reduce fasting blood sugar levels;

[0042] (f) Improve glucose intolerance;

[0043] (g) reduce serum insulin levels;

[0044] (h) increasing the level of glucagon-like peptide-1 in serum;

[0045] (i) Reduce insulin resistance index and improve β-cell function and insulin sensitivity test index;

[0046] (j) Improve lipid metabolism disorders;

[0047] (k) treatment and prevention of type 2 diabetes;

[0048] (l) Reduce serum total cholesterol, triglycerides, low-density lipoprotein cholesterol and free fatty acid levels;

[0049] (n) Reduce atherosclerosis index;

[0050] (m) Increase the level of high-density lipoprotein cholesterol in serum;

[0051] (o) Reduce serum interleukin-6 and tumor necrosis factor-α levels;

[0052] (p) Reduce liver index and liver damage;

[0053] (q) Reduce pancreatic and colon damage;

[0054] (r) Increase the levels of superoxide dismutase, reduced glutathione, and glucose-6-phosphatase in the liver;

[0055] (s) Reduce the content of malondialdehyde in the liver.

[0056] Since Lactobacillus plantarum FLP-215 has any of the above effects, the application of Lactobacillus plantarum FLP-215 in the above should also be within the protection scope of the present application.

[0057] In some embodiments, the organ injury is one or more of liver injury, pancreatic injury, or colon injury.

[0058] Furthermore, the present invention also seeks to protect a bacterial agent, which comprises the above-mentioned Lactobacillus plantarum FLP-215 and / or its bacterial suspension.

[0059] Furthermore, the present invention also seeks to protect the use of the above-mentioned bacterial agent in the preparation of products for preventing and / or treating ulcerative colitis, diabetes and / or reducing organ damage.

[0060] The present invention has the following beneficial effects: The present invention provides a strain of Lactobacillus plantarum FLP-215. The Lactobacillus plantarum FLP-215 can play a preventive and therapeutic role in the colitis mouse model induced by sodium dextran sulfate by protecting the intestinal mucosal barrier, reducing the inflammatory response, regulating the structure of the intestinal microbiome, etc.; further, Lactobacillus plantarum FLP-215 can play a role in treating diabetes by improving the key pathways of sugar and lipid metabolism, reducing fasting blood sugar values, improving glucose intolerance, reducing the content of insulin in serum, or increasing the content of glucagon-like peptide-1 in serum. At the same time, the Lactobacillus plantarum FLP-215 also has an excellent therapeutic effect in reducing organ damage. The present invention provides a strain of Lactobacillus plantarum FLP-215, which can effectively treat ulcerative colitis, treat diabetes and reduce organ damage at the same time, and provides a technical solution for the combined treatment of ulcerative colitis, diabetes and organ damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is the growth morphology of Lactobacillus plantarum FLP-215 on the culture medium.

[0062] Figure 2 This is the state of Lactobacillus plantarum FLP-215 under a microscope after Gram staining.

[0063] Figure 3 This is the 16s rRNA identification result of Lactobacillus plantarum FLP-215.

[0064] Figures 4 to 6 The following are the daily changes in body weight, water intake and food intake of mice.

[0065] Figure 7This is a graph showing the daily changes in the disease activity index of mice.

[0066] Figure 8 Schematic diagram of the changes in mouse colon length.

[0067] Fig. 9 Schematic diagram of the pro-inflammatory cytokine levels in the serum of each group of mice.

[0068] Fig.10 Schematic diagram of H&E staining sections of the colon of mice in each group.

[0069] Fig.11 Schematic diagram of colon histopathological scoring of mice in each group.

[0070] Fig.12 Schematic diagram of ZO-1 immunofluorescence sections of the colon of mice in each group.

[0071] Fig.13 Schematic diagram of MUC-2 immunofluorescence sections in the colon of each group of mice.

[0072] Fig.14 Schematic diagram of the surface density of ZO-1 and MUC-2 in the colon of each group of mice.

[0073] Fig.15 Schematic diagram of the Shannon index of intestinal flora in each group of mice.

[0074] Fig.16 Schematic diagram of PCoA analysis of mouse intestinal flora.

[0075] Fig.17 These are the species-level metagenomic results for each group.

[0076] Fig.18 Schematic diagram of the differential metabolic pathways in mice in the Lactobacillus plantarum FLP-215 group.

[0077] Fig.19 Schematic diagram of the short-chain fatty acid levels of each group of mice.

[0078] Fig. 20 Volcano plot of gene distribution.

[0079] Fig.21 Results of Gene Ontology (GO) enrichment analysis are shown.

[0080] Fig. 22 Schematic diagram of the changes in body weight of mice in each group over time.

[0081] Fig.23 Schematic diagram of the changes in blood glucose levels of mice in each group over time.

[0082] Fig.24Schematic diagram of the blood glucose regulation ability of pancreatic β cells in each group of mice.

[0083] Figure 25-28 These are the insulin, insulin resistance index, β-cell function and insulin sensitivity detection index of each group of mice.

[0084] Fig.29 Schematic diagram of HE section analysis of pancreas of mice in each group.

[0085] Figure 30-35 Figure 2 shows the levels of serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, free fatty acids, and a schematic diagram of the atherosclerosis index in each group of mice.

[0086] Figure 36-37 Schematic diagram of interleukin-6 and tumor necrosis factor-α levels in the serum of mice in each group.

[0087] Figure 38-41 Schematic diagram of the contents of superoxide dismutase, reduced glutathione, glucose-6-phosphatase and malondialdehyde in the liver of each group of mice.

[0088] Fig.42 is the liver index of mice in each group.

[0089] Fig.43 HE-stained sections of the liver of mice in each group.

[0090] Fig.44 Oil Red O stained sections were analyzed in each group of mice.

[0091] Fig.45 is the Shannon index of intestinal microorganisms in each group of mice.

[0092] Fig.46 Schematic diagram of the species level of intestinal microorganisms in each group of mice.

[0093] Fig.47 Metabolic pathways of mice in each group.

[0094] Fig.48 To analyze the correlation between FLP-215, flora, intestinal metabolites, metabolic pathways and physicochemical indicators. DETAILED DESCRIPTION

[0095] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The following examples are preferred embodiments of the present invention, but do not limit the scope of protection of the present invention in any form. The present invention mainly describes the strain and the application ideas based on the strain. The replacement of simple parameters in the implementation mode cannot be repeated one by one in the examples, but the present invention is not limited thereto. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention should be regarded as equivalent replacement methods and should be included in the scope of the present invention.

[0096] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0097] In the following examples, GraphPad and R software were used to perform statistical analysis on the data, and all data were expressed as mean ± standard deviation (SD). Wilcoxon was used for significance analysis, and values ​​of *, P < 0.05; **, P < 0.01; ***, P < 0.001 were considered significant, strongly significant, and extremely significant. The ggplot2 package was used for principal coordinate analysis (PCoA) and to draw box plots and bubble plots. The DESeq2 software package was used for P value calculation and correction in the metabolic pathway bubble plot.

[0098] Example 1 Acquisition of Lactobacillus plantarum FLP-215

[0099] 1. Screening and separation of Lactobacillus plantarum:

[0100] Sampling personnel obtained homemade kimchi from farmers in Sichuan, diluted the kimchi tenfold, and spread 100 μL of samples from each of the three dilution gradients -2, -3, and -4 on MRS agar medium. The samples were placed in an incubator at 37°C and incubated upside down for 48 hours. Colonies with obvious morphological differences were selected on the -2 dilution gradient plate and streaked to obtain pure single colonies. A strain with good growth status was isolated and named FLP-215.

[0101] 2. Identification of Lactobacillus plantarum:

[0102] The FLP-215 isolation and purification plates were observed, the colony morphology was recorded, its physiological and biochemical characteristics were studied, and the 16srRNA technology was used to identify the strain taxonomy.

[0103] Lactobacillus plantarum FLP-215 has the following morphological, physiological and biochemical characteristics and molecular biological identification results:

[0104] (1) Morphological characteristics

[0105] Lactobacillus plantarum FLP-215 grows well under anaerobic conditions on MRS agar plate medium. After 48 hours of culture, it can form obvious colonies. The colonies are round and full, milky white, 2 to 3 mm in diameter, opaque, with smooth edges and surfaces and moist (such as Figure 1 Under the microscope, the Gram staining is purple, which is consistent with the staining characteristics of Gram-positive bacteria. The bacteria are medium-long rods and arranged individually (such as Figure 2 ).

[0106] (2) Physiological and biochemical characteristics

[0107] Lactobacillus plantarum FLP-215 has no flagella and spores, is immobile, does not form spores, is facultatively anaerobic, and is catalase negative. The suitable growth temperature is 30℃~37℃, and it can metabolize monosaccharides such as galactose, glucose, fructose, and mannose, as well as disaccharides such as maltose, lactose, and sucrose.

[0108] (3) Molecular biological identification results

[0109] The 16S rRNA sequence of the plant lactobacillus FLP-215 is shown in SEQ ID: No. 1. The 16S rRNA molecular biological identification result of FLP-215 is Lactobacillus plantarum. Figure 3 The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 4, 2023, with the deposit number CGMCC No.28337.

[0110] Example 2 Construction of an ulcerative colitis experimental animal model

[0111] Fifty 8-week-old C57BL / 6J mice (black male mice) were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. After one week of adaptive feeding, they were randomly divided into 5 groups: ① Control group (normal drinking water, 10 mice); ② Model group (3% dextran sulfate sodium drinking water, 10 mice); ③ Lactobacillus plantarum FLP-215 prevention group (Pro group, 3% dextran sulfate sodium drinking water + probiotic suspension, probiotic suspension is 10 9CFU of Lactobacillus plantarum FLP-215 was resuspended in 300 μL sterile saline and administered by gavage, 10 mice); ④ Lactobacillus plantarum FLP-215 treatment group (Treat group, 3% sodium dextran sulfate drinking water was used to establish the model and then the probiotic suspension was given, 10 mice); ⑤ Drug group (Drug group, 3% sodium dextran sulfate drinking water was used to establish the model and then sulfasalazine solution was given, 10 mice). During this process, the control group and the model group were given an equal amount of saline as a control. After drinking drinking water containing 3% sodium dextran sulfate for 1 week, the model was judged to be successful until the body weight decreased by more than 10% or the mice showed loose stools, pus and blood in the stool, etc.

[0112] Drinking water, feed, and bedding were changed every day. The temperature of the animal laboratory was controlled at 23±1°C, the relative humidity was controlled at 52%-60%, and a 12-hour light-dark cycle was maintained. All mice were fed with standard normal commercial mouse feed (mainly composed of crude protein, crude fiber, crude fat, and trace elements). After the experiment, the mice were euthanized by intraperitoneal injection of 1% sodium pentobarbital solution, and various tissue samples were collected, including immune organs, serum, proximal colon, feces, cecal contents, distal colon, and other tissues.

[0113] Example 3 Improvement of Physiological Indexes and DAI of Mice by Lactobacillus plantarum FLP-215

[0114] (1) The body weight, disease activity index, stool characteristics, colon length, food intake and water intake of each group of mice in Example 2 were recorded; the fecal blood level was evaluated according to the instructions of the mouse fecal occult blood kit, and the DAI was calculated according to the following table (Table 1 is the DAI scoring standard).

[0115] Table 1

[0116]

[0117] (2) Experimental results

[0118] The water intake, food intake, body weight and disease activity index of the mice were monitored daily to evaluate the severity of the ulcerative colitis model and the effect of Lactobacillus plantarum FLP-215 supplementation.

[0119] Figures 4 to 6The following are the daily changes in body weight, water intake and food intake of mice. As can be seen from the figure, from 1 to 7 days, the body weight, food intake and water intake of mice in the Model group, Pro group, Treat group and Drug group all showed a downward trend, and the degree was similar. This may be because the groups were under the same DSS modeling conditions at this stage. From 8 to 18 days, the body weight, food intake and water intake of mice in the Model group still showed a downward trend. On the contrary, the body weight, food intake and water intake of mice in the Pro group tended to be stable, and the water intake, food intake and body weight of mice in the Treat group gradually increased from 10 to 18 days, and there was a significant difference with the model group (P<0.05).

[0120] Figure 7 The figure shows the daily changes in the disease activity index of mice. As shown in the figure, from 1 to 10 days, except for the Control group, the disease activity index of the other groups increased significantly, and as the experiment progressed, the disease activity index of mice in the Pro group, Treat group and Drug group decreased significantly compared with the Model group (P<0.05).

[0121] Figure 8 The figure is a schematic diagram of the changes in the length of the mouse colon. As shown in the figure, compared with the control group, the length of the colon of the mice in the Model group was significantly shortened, while the length of the colon of the mice in the Pro group, Treat group and Drug group was significantly increased compared with the Model group.

[0122] Example 4 Effect of Lactobacillus plantarum FLP-215 on inflammatory cytokines in mice with ulcerative colitis induced by sodium dextran sulfate

[0123] (1) Determination of serum cytokines

[0124] Blood was collected from the orbital venous plexus using a capillary tube before euthanasia. First, the blood samples were naturally coagulated for 30 minutes. Then, the blood samples were centrifuged at 3000 rpm for 20 minutes at 4°C to separate and collect the serum. Finally, ELISA kits were used to detect the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-17A (IL-17A), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in the serum.

[0125] (2) Experimental results

[0126] Colonic injury was evaluated by quantitatively measuring the levels of proinflammatory cytokines tumor necrosis factor-α (TNF-a), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-17A (IL-17A), and interferon gamma (IFN-γ) and the anti-inflammatory cytokine interleukin-10 (IL-10) in mouse serum.

[0127] Fig. 9 The figure shows the content of pro-inflammatory cytokines in the serum of mice in each group. As shown in the figure, compared with the Control group, the pro-inflammatory cytokines TNF-a, IL-1β, IFN-γ, IL-6 and IL-17A in the serum of mice in the Model group were significantly increased (P<0.05), and the anti-inflammatory cytokine IL-10 was significantly reduced, while the opposite was true in the serum of mice in the Pro group, Treat group and Drug group.

[0128] Example 5 Effects of Lactobacillus plantarum FLP-215 on intestinal pathological parameters of mice

[0129] (1) After euthanasia, the distal colon of the mice was taken for H&E staining, sectioning and histopathological scoring. The specific steps of staining were as follows: the colon samples were washed with PBS, fixed in 4% (w / v) paraformaldehyde for 24 h, then dehydrated, embedded in paraffin, and sectioned (3 μm). Next, the sections were stained with hematoxylin-eosin and observed and evaluated under an optical microscope according to the histopathological scoring table (the colon histopathological scoring criteria are shown in Table 2 below).

[0130] The distal colon of mice was taken for immunofluorescence protein determination, and the colon tissue was labeled with mucin MUC-2 and tight junction protein ZO-1 antibodies, respectively, and further immunofluorescence staining was performed. Fluorescein binds to antibodies ZO-1 and MUC-2 to form fluorescent antibodies. Through specific binding to antigens to form multi-component complexes, the characterization and localization of ZO-1 and MUC-2 in intestinal tissues were studied using fluorescence microscopy. The surface density of immunofluorescent ZO-1 and MUC-2 was measured and calculated to evaluate the improvement effect of the test strains on the colon intestinal membrane barrier.

[0131] Table 2

[0132]

[0133] (2) Experimental results

[0134] Fig.10 Schematic diagram of H&E staining sections of the colon of each group of mice. Fig.10The results of paraffin sections stained with hematoxylin-eosin showed that the intestinal tissue layers of mice in the Control group had clear structures and distinct boundaries; the mucosa was intact, the cell morphology was normal, and there was no obvious inflammatory cell infiltration. In the Model group, the intestinal tissue was almost completely ulcerated, there was no obvious intestinal gland structure, the mucosal epithelium was sloughed, the intestinal glands were completely necrotic and disappeared, the damage invaded the serosa, the connective tissue was loosely arranged, accompanied by a large number of neutrophils and lymphocytes infiltration; a small amount of neutrophils, lymphocytes infiltration and hyperplastic connective tissue were seen in the muscular layer and serosa. In contrast, small areas of ulcers were observed in the intestinal tissue of mice in the Pro group, the mucosal epithelium at the lesion was intact, a small amount of intestinal glands were necrotic and disappeared, and were replaced by hyperplastic connective tissue, accompanied by a small amount of neutrophils and lymphocytes infiltration; a small amount of neutrophils and lymphocytes infiltration were seen in the submucosal layer. At the same time, focal ulcers were observed in the colon of mice in the Treat group, the mucosal epithelium at the lesion was intact, the cytoplasm was loose, a small amount of intestinal glands disappeared due to necrosis and were replaced by proliferative connective tissue, accompanied by neutrophil punctate infiltration; the lesions invaded the submucosal layer, and neutrophil punctate infiltration was observed. In contrast, larger areas of ulcers were observed in the intestinal tissue of mice in the Drug group, the mucosal epithelium at the lesion was sloughed, and many intestinal glands disappeared due to necrosis and were replaced by proliferative connective tissue, accompanied by more neutrophil and lymphocyte infiltration; the lesions invaded the submucosal layer, and a small amount of neutrophil and lymphocyte infiltration was observed.

[0135] Fig.11 The colon histopathological scores of mice in each group were evaluated according to the scoring criteria shown in Table 2. Fig.11 It can be seen that there are significant differences between the Model group and the Control group, while the scores of the Pro group, Treat group and Drug group are closer to the Control group and are significantly different from the Model group (P<0.01).

[0136] Fig.12 Schematic diagram of ZO-1 immunofluorescence sections of the colon of mice in each group. Fig.12 It can be seen that the results of immunofluorescence analysis showed that the content of MUC-2 protein and ZO-1 protein was abundant in the Control group, while in the Model group, due to the destruction of the intestinal mucosa, there were few tight junction proteins left. In the sections of mice in the Pro and Treat groups, the distribution of ZO-1 could be clearly seen, and the expression level was significantly higher than that in other groups. There was also some expression in the Drug group.

[0137] Fig.13Schematic diagram of the immunofluorescence section of mouse colon MUC-2. Similarly, for the tight junction protein MUC-2, there is a huge difference between the Model group and the Control group. At the same time, MUC-2 is distributed in large quantities in the Pro group and the Treat group, which is significantly more than the expression level in the Model group. There is also a large amount of expression in the Drug group. The expression of tight junction proteins ZO-1 and MUC-2 was further quantitatively analyzed, and a scatter plot of their surface density was drawn. Fig.14 The figure is a schematic diagram of the surface density of ZO-1 and MUC-2 in mouse colon. It can be seen that the surface density trends of ZO-1 and MUC-2 are similar. Compared with other groups, the Pro group and Treat group are closer to the Control group, and there are significant differences with the Model group (P<0.05).

[0138] Example 6 Effects of Lactobacillus plantarum FLP-215 on the intestinal flora and SCFAs regulation in mice

[0139] (1) Determination of short-chain fatty acids: After freeze-drying the mouse feces, short-chain fatty acids (SCFAs) were extracted, and the levels of short-chain fatty acids in each group of samples were quantitatively analyzed. The method is as follows: At the end of the experiment, the colon contents of 6 golden hamsters were randomly selected from each group for SCFA determination. First, 40 mg of freeze-dried colon contents were weighed and added to 600 mL of normal saline (85%). The sample was then placed on a shaker for 5 minutes, thoroughly mixed and centrifuged (8000 rpm, 5 minutes). Then 200 mL of the supernatant was taken and 100 mL of sulfuric acid (50%) was added for acidification. 400 microliters of n-hexane were added to extract short-chain fatty acids. The solution was finally passed through an organic membrane and transferred to a vial. The concentration of SCFAs, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and hexanoic acid, was analyzed by gas chromatography-mass spectrometry (GCMS; Agilent-7890, Santa Clara, California, USA). The concentration of SCFAs was calculated by the external standard method.

[0140] Fecal DNA extraction, metagenomic sequencing and data quality control: At the end of the experiment, mouse feces were collected, and total DNA from fecal samples was extracted according to the instructions of the fecal DNA extraction kit. After detecting DNA purity and integrity by 0.8% agarose gel electrophoresis, shotgun metagenomic sequencing was performed and sequencing libraries were prepared. The abundance data and metabolic pathway data of intestinal microorganisms in each group of mice were obtained using the humann2 database, and R software was used to qualitatively and quantitatively analyze the α diversity and β diversity of intestinal microorganisms in each group of mice, and the differential flora and differential metabolic pathways between the groups of mice were calculated.

[0141] Fig.15The figure is a schematic diagram of the Shannon index of the intestinal flora of each group of mice. The results of diversity analysis showed that compared with the Control group, under the same modeling conditions, the Shannon index of each group of mice decreased significantly.

[0142] Fig.16 The figure is a schematic diagram of PCoA analysis of mouse intestinal flora. At the same time, the β diversity results showed that each group was significantly separated from the control group, and the Pro group, Treat group and Drug group were significantly separated from the model group, with significant differences in species abundance.

[0143] Species-level metagenomic results are as follows Fig.17 As shown, potential colitis pathogens such as Escherichiacoli, Aeromonas caviae, Clostridium perfringens and Enterococcus faecalis increased significantly in the Model group, while decreased in the Pro and Treat groups; potential beneficial bacteria such as Bifidobacterium pseudolongum, Akkermansiamuciniphila, Anaerotruncus sp G3 2012 decreased significantly in the Model group, while increased significantly in the Pro and Treat groups.

[0144] (2) Effect of Lactobacillus plantarum FLP-215 on the regulation of SCFAs

[0145] Fig.18 Schematic diagram of differential metabolic pathways in mice fed Lactobacillus plantarum FLP-215. Analysis of the diversity of intestinal flora in mice found that the abundance of bacteria that can produce short-chain fatty acids (SCFAs) increased in the Pro and Treat groups. Metabolic pathway analysis results showed that compared with the Model group, the abundance of seven microbial metabolic pathways related to the production of SCFAs increased significantly in the Pro and Treat groups, including superpathway III for acetyl-CoA biosynthesis, pyruvate fermentation to produce acetate and lactate II, pyruvate fermentation to produce acetate and S-lactic acid I, glycolysis III (from glucose), fatty acid biosynthesis, and acetyl-CoA biosynthesis I and acetyl-CoA biosynthesis II.

[0146] Fig.19Schematic diagram of the short-chain fatty acid levels of mice in each group. To confirm the above results, we further used gas chromatography (GC) to detect the content of SCFAs in each group. The results showed that compared with the Control group, the content of SCFAs (including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid) in the Model group was significantly reduced. Compared with the Model group, after taking Lactobacillus plantarum FLP-215, the content of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid in the Pro and Treat groups was significantly increased (P<0.05), which was close to the level of the Drug group. Based on the above results, we speculate that Lactobacillus plantarum FLP-215 increases the content of SCFAs by affecting the abundance of microorganisms that can produce SCFAs and the metabolic pathways of microorganisms that can produce SCFAs.

[0147] Example 7 Comparative study of mouse intestinal epithelial cell transcriptome

[0148] (1) Transcriptome sequencing: After euthanasia, the distal colon of mice was taken for transcriptome determination. Total RNA was extracted from the samples according to the Trizol extraction instructions, and the extraction effect was detected by 0.8% agarose gel electrophoresis. The purified mRNA was fragmented and the sequencing library was prepared. Single-stranded DNA fragments were generated by denaturation treatment, PCR amplification was performed to generate DNA clusters, and the DNA amplicons were linearized into single strands. Modified DNA polymerase and dNTPs with four fluorescent labels were added to read the types of nucleotides polymerized in the first round of reaction for each template sequence, and the fluorescence signal results collected in each round were counted to obtain the sequence of the template DNA fragment and detect the change in expression. Then, library construction and quality control were performed, and the RNA-seq raw data was filtered. After the RNA library was constructed, Illumina Novaseq 6000 was used for sequencing, and featucounts was used to estimate gene expression.

[0149] (2) Experimental results

[0150] Fig. 20 The volcano plot of gene distribution. As shown in the figure, the gene distribution was analyzed using colon transcriptome data. The results of the volcano plot showed that the intake of Lactobacillus plantarum FLP-215 significantly affected the gene expression distribution. To further explore the effects of these differentially expressed genes (DEGs), we analyzed the pathways associated with the DEGs.

[0151] Fig.21The results of gene ontology (GO) enrichment analysis are shown. The results can be divided into three categories, namely biological processes, cellular components, and molecular functions. The results of GO analysis showed that the DEGs in the Model and Control groups were mainly involved in biological processes such as cellular components such as cytoplasm, extracellular space, extracellular region, cell cycle, and molecular functions such as metal ion binding. While the DEGs in the Treat and Model groups were mainly involved in biological processes such as immune response, immunoglobulin production, and positive regulation of RNA polymerase II promoter transcription, cellular components such as extracellular space, and molecular functions such as DNA binding and sequence-specific DNA binding in the proximal region of the core promoter of RNA polymerase II.

[0152] Example 8 Construction of diabetic experimental animal model

[0153] (1) Experimental animals

[0154] Twenty-four 6-week-old healthy male C57BL / 6 mice, weighing 18-20 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, China) with an animal license number of SCXK (Beijing) 2021-0006. During the experiment, the animals were given adequate food, drinking water and free movement. The animal laboratory breeding environment met the SPF grade hygiene standards (temperature 25±2℃, humidity 70%-80%), and a 12-h light-dark cycle was met during the experiment. Both ordinary feed and high-sugar and high-fat feed were purchased from Jiangsu Collaborative Biotechnology Co., Ltd. (Jiangsu, China). The formula of high-sugar and high-fat feed is as follows: ordinary feed 38%, lard 28%, sucrose 5.6%, whole milk powder: 10.8%, casein 11.5%, microcrystalline cellulose: 1.9%, experimental animal premix: 2%, calcium bicarbonate 1.8%, stone powder 0.4%.

[0155] (2) Chemicals and reagents

[0156] STZ was purchased from Sigma Chemical Company, blood glucose meters and blood glucose test strips were purchased from Roche, Germany, glucose, human insulin, and citrate buffer were purchased from Beijing Solebow Technology Co., Ltd., and various test kits were purchased from Shanghai Xinyu Biotechnology Co., Ltd.

[0157] (3)Tested strain

[0158] The Lactobacillus plantarum FLP-215 obtained by screening in Example 1 was used.

[0159] (4) Establishment of diabetic mouse model and experimental design

[0160] In this experiment, a high-fat diet combined with multiple injections of low-dose STZ was used to establish a type 2 diabetic mouse model. After one week of adaptive feeding, 18 mice were randomly divided into a normal group (Control, n = 8), a model group (Model, n = 8), and a FLP-215 prevention group (FLP-215, n = 8). The normal group was fed with a basic diet, and the diabetes prevention group was fed with a high-sugar and high-fat diet and gavage with probiotics (1×10 9 CFU, dissolved in 300 μL of normal saline) and continued for 6 weeks. The concentration of citric acid buffer was 0.1 mol / L and pH = 4.5. 230 mg STZ was dissolved in 100 ml of citric acid buffer. After 6 weeks, except for the normal group, the other groups were injected with STZ intraperitoneally at a dose of 45 mg / kg, once a day, for 3 consecutive days; the normal group was injected with the same volume of citric acid buffer according to body weight for control treatment. One week after STZ injection, fasting for more than 6 hours, 2 to 3 consecutive measurements of fasting blood glucose in the model group mice were greater than 11.1 mmol / L sugar level accompanied by polyphagia, polydipsia, polyuria and significant weight loss. Symptoms were considered to be successful diabetes modeling. Food intake, water intake, body weight and fasting blood glucose were monitored weekly during the experiment.

[0161] Lactobacillus plantarum FLP-215 intervention was continued until the day before the end of the experiment. The feces of each mouse were collected and placed in a 2 ml sterile centrifuge tube and immediately placed in a -80 ° C refrigerator for subsequent fecal metagenomic sequencing and metabolic pathway analysis; all mice were prohibited from eating feed but could freely drink sterile distilled water. After euthanasia, all mice were blooded from the eye sockets, left at room temperature for 1 hour to allow blood coagulation, centrifuged at 4 ° C, 3500 rpm for 45 minutes, and the mouse serum was carefully collected with a pipette and stored in aliquots at -80 ° C for subsequent index analysis. After dissection, the liver, kidney, intestine, and pancreas of the mice were collected and weighed, and the organ index was expressed as organ weight / body weight (mg / g). Part of the liver was placed in a 4% paraformaldehyde solution for tissue fixation, and the remaining part was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator; the contents of the colon were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator, and part of the colon was placed in a 4% paraformaldehyde solution for tissue fixation, and the remaining part was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator; the pancreas was placed in a 4% paraformaldehyde solution for tissue fixation.

[0162] Example 9 Effects of Lactobacillus plantarum FLP-215 on body weight changes, fasting blood glucose levels, and oral glucose tolerance test in diabetic mice

[0163] (1) During the experiment, the weight of each group of mice was measured once a week, and the physiological activities of the mice were observed and their weight changes were recorded. When measuring the fasting blood glucose level of mice, the mice were fasted but not watered for 12 hours the night before. The tail of the mice was disinfected and blood was collected. The value was measured with a Roche blood glucose meter and the tail of the mice was disinfected again.

[0164] On the first day after the seventh week, all mice were fasted for 12 hours but not water, and then orally administered with glucose solution (2 g / kg.bw). Then, blood was collected from the tail vein at 0, 15, 30, 60, and 120 minutes after glucose intake to measure blood glucose levels at that time, and a blood glucose fluctuation curve was drawn. The trapezoidal method was used to calculate the area under the curve (AUC) value of glucose, and the formula was as follows: G-AUC (h*mmol / L) = (G0+G120) / 2+G15+G30+G60.

[0165] (2) Experimental results

[0166] Sustained weight loss is one of the hallmark symptoms of type 2 diabetes. Fig. 22 The figure is a schematic diagram of the weight changes of mice in each group over time. As shown in the figure, from week 0 to week 6, the Control group was fed with ordinary feed, and the Model group and FLP-215 group were fed with high-fat feed. It can be found that the weight of mice in the Control group increased the slowest, the weight of the Model group increased the most, and the weight increase of the FLP-215 group was less than that of the Model group but greater than that of the normal group, which may be related to the intake of plant lactobacillus, indicating that the intake of plant lactobacillus may slow down the increase in the weight of mice caused by eating high-fat feed; after three consecutive injections of STZ in the sixth week, the weight of mice in each group decreased except for the Control group; by comparing the weight values ​​of the Model group in the 6th and 7th weeks, there was a very significant difference (P < 0.0001), indicating that the diabetes modeling was successful in terms of weight. Compared with the Model group, the weight loss of the FLP-215 group was less than that of the Model group. The experimental results show that plant lactobacillus FLP-215 can effectively prevent the weight loss of mice with type 2 diabetes and prevent diabetic symptoms.

[0167] A high fasting blood sugar level is the most representative feature of type 2 diabetic mice, and the level of fasting blood sugar is also the gold standard for judging the success of the mouse diabetes model. In order to explore the effect of Lactobacillus plantarum FLP-215 on blood sugar levels, fasting blood sugar was monitored once a week from the beginning of the experiment. Fig.23The figure is a schematic diagram of the changes in blood glucose levels of mice in each group over time. As shown in the figure, after STZ injection in the 6th week, the blood glucose level of the Control group was significantly higher than that of the Model group in the 7th week (p < 0.0001), and the blood glucose level of the FLP-215 group was significantly lower than that of the Model group (p < 0.0001). The experimental results show that Lactobacillus plantarum FLP-215 can significantly reduce the level of diabetic mice.

[0168] OGTT is used to evaluate the function of pancreatic β cells and their ability to regulate blood sugar. Fig.24 It is a schematic diagram of the ability of pancreatic beta cells in each group of mice to regulate blood sugar. As shown in the figure, after each group of mice was gavaged with glucose solution, the fasting blood sugar values ​​of each group of mice fluctuated dramatically, and a blood sugar peak appeared at 30 minutes. From 30 minutes to 120 minutes, the blood sugar of the FLP-215 group of mice dropped the fastest; at the end of the OGTT experiment, the blood sugar of the FLP-215 group of mice was significantly lower than that of the Model group (p<0.001). At the same time, compared with the Model group, the area under the OGTT curve (AUC) of the FLP-215 group was significantly reduced (p<0.05, p<0.01, p<0.05). The experimental results show that Lactobacillus plantarum FLP-215 can significantly improve the glucose tolerance of diabetic mice. Example 10 Preventive effect of Lactobacillus plantarum FLP-215 on insulin resistance in type 2 diabetic mice and pancreatic HE section analysis

[0169] (1) After obtaining serum according to the above method, the insulin level of each group of mice was measured according to the instructions of the kit, and the insulin resistance index (HOMA-IR), insulin sensitivity detection index (QUICKI) and β-cell function (HOMA-β) were calculated according to the following formulas: HOMA-IR = FBG × FINS / 22.51; QUICKI = 1 / (lgFBG + lgFINS); HOMA-β = 20 × FINS / (FBG-3.5).

[0170] (2) Experimental results

[0171] Insulin is the only hormone known to the body that can lower blood sugar. Insulin resistance is a prominent feature of type 2 diabetes. To further evaluate insulin resistance, Figure 25-28The insulin, insulin resistance index, β-cell function and insulin sensitivity test index of each group of mice. As shown in the figure, the insulin level of the Model group was significantly higher than that of the Control group (p < 0.0001) and the FLP-215 group (p < 0.0001). After 6 weeks of Lactobacillus plantarum prevention, the insulin resistance index of the Model group was significantly higher than that of the Control group (p < 0.0001); in terms of β-cell function level, the Model group was significantly lower than the Control group (p < 0.001) and the FLP-215 group (p < 0.01); from the analysis of the insulin sensitivity test index, the Model group was significantly lower than the Control group (p < 0.0001) and the FLP-215 group (p < 0.001). These results show that Lactobacillus plantarum FLP-215 plays an important role in slowing down the increase of insulin in diabetic mice, slowing down the increase of insulin resistance, preventing the decline of β-cell function, and preventing the decline of insulin sensitivity test index.

[0172] In order to further explore the changes in pancreatic islet tissue in diabetic mice, we performed HE section analysis on the pancreas of mice. Fig.29 The following is a schematic diagram of the HE section analysis of the pancreas of each group of mice. The pancreatic islet cells of the mice in the Control group were in the form of clusters and cords, with clear edges between the islets and the surrounding tissues, dense cells in the islets, and regular and orderly arrangement. The pancreatic islet structure of the mice in the Model group was significantly damaged, the number of cells in the islets was significantly reduced and irregularly arranged, the islet tissue was atrophied, and there were vacuoles and inflammatory cell infiltration inside. After 6 weeks of probiotic intervention, the FLP-215 group effectively delayed the damage to the pancreatic islet structure, slowed down the atrophy of the islet tissue, and slowed down the phenomenon of islet cell vacuoles in diabetic mice.

[0173] Example 11 Determination of biochemical indices of type 2 diabetic mice by Lactobacillus plantarum FLP-215

[0174] (1) Serum glucagon-like peptide (GLP-1), triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), free fatty acids (FFA), tumor necrosis factor (TNF-α), and interleukin 6 (IL-6) were measured using the kits provided by Shanghai Xinyu Biotechnology Co., Ltd., and liver tissue malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) were measured using the kits provided by Shanghai Xinyu Biotechnology Co., Ltd.

[0175] (2) Experimental results

[0176] Previous studies have shown that the lipid metabolism function of patients with type 2 diabetes is relatively disordered. Figure 30-35The following are the levels of serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and free fatty acids in each group of mice, as well as a schematic diagram of the atherosclerosis index. Compared with the Control group, the total cholesterol, triglycerides, low-density lipoprotein cholesterol, and free fatty acids in the serum of the Model group were significantly increased, while the high-density lipoprotein cholesterol was significantly decreased (p < 0.0001), indicating that after 6 months of high-fat diet, the lipid metabolism ability of the Model group mice was significantly decreased. Compared with the Model group, the total cholesterol, triglycerides, low-density lipoprotein cholesterol, and free fatty acids in the FLP-215 group were significantly decreased, while the high-density lipoprotein cholesterol was significantly increased (p < 0.01). Studies have shown that Lactobacillus plantarum FLP-215 can effectively prevent lipid metabolism in diabetic mice.

[0177] To further explore the preventive effect of probiotics on inflammation in diabetic mice, we measured interleukin-6 and tumor necrosis factor-α in the serum of mice. Figure 36-37 The figure is a schematic diagram of the interleukin-6 and tumor necrosis factor-α levels in the serum of mice in each group. It can be seen from the figure that compared with the Control group, the interleukin-6 and tumor necrosis factor-α in the serum of mice in the Model group were significantly increased (****P<0.0001), while those in the FLP-215 group were significantly lower than those in the Model group (**P<0.01).

[0178] Example 12 Analysis and evaluation of organ slices of Lactobacillus plantarum FLP-215 on type 2 diabetic mice

[0179] (1) The liver tissue, pancreas and colon specimens were fixed in 4% neutral paraformaldehyde solution, dehydrated and embedded in paraffin, sliced ​​into 5 μm thick sections, and finally stained with H&E. The liver tissue was also stained with Oil Red O to observe the morphological changes of the sections.

[0180] (2) Experimental results

[0181] Long-term high-fat diet can induce fatty liver in mice, leading to liver damage and oxidative stress in the body. The mouse liver was ground and the contents of superoxide dismutase, reduced glutathione, glucose-6-phosphatase and malondialdehyde in the mouse liver were determined according to the instructions of the ELISA kit.

[0182] Superoxide dismutase is an antioxidant enzyme that uses free radicals as specific substrates. It removes toxins from the body by combining with free radicals and reduces the risk of free radical damage to the liver. The activity of superoxide dismutase in the liver is closely related to the health of the body.

[0183] Figure 38-41The figure is a schematic diagram of the contents of superoxide dismutase, reduced glutathione, glucose-6-phosphatase and malondialdehyde in the liver of each group of mice. As shown in the figure, compared with the mice in the Control group, the superoxide dismutase activity of the mice in the Model group showed a significant decrease (P < 0.0001). After 6 months of probiotic prevention, the decreasing trend of superoxide dismutase activity in the FLP-215 group was effectively controlled, and the activity was close to the normal level (p < 0.001).

[0184] Glutathione can help maintain normal immune system function and has antioxidant and integrated detoxification effects. Compared with the mice in the Control group, the glutathione activity of the mice in the Model group showed a significant decrease (P < 0.0001). After 6 months of probiotic prevention, there was no significant difference between the FLP-215 group and the Model group (p > 0.05).

[0185] Glucose-6-phosphatase releases glucose into the blood by hydrolyzing glucose-6-phosphate in liver tissue. Therefore, when hungry, liver glycogen can replenish blood sugar and maintain blood sugar balance. This shows that this enzyme is a key enzyme for gluconeogenesis. Compared with the mice in the Control group, the G 6P enzyme activity of the mice in the Model group showed a significant decrease (P < 0.0001). After 6 months of probiotic prevention, the decreasing trend of G 6P enzyme activity in the FLP-215 group was effectively controlled, and the activity was close to the normal level (P < 0.001).

[0186] Compared with the Control group, the MDA content in the liver of the Model group was significantly increased (P < 0.0001), and the MDA content in the FLP-215 group was significantly decreased (P < 0.000) compared with the Model group. In conclusion, Lactobacillus plantarum FLP-215 can reduce oxidative stress damage in T2DM mice.

[0187] At the same time, we calculated the liver index based on the liver weight and body weight of each group of mice, which is the mass of the mouse liver (mg) / body weight (g) × 100%. The normal liver index is between 0 and 40, and a high liver index usually indicates liver damage. Fig.42 is the liver index of mice in each group. As can be seen from the figure, compared with the Control group, the liver index of the Model group was significantly increased and there was a significant difference (P < 0.01); compared with the Model group, the liver index of the FLP-215 group was significantly decreased (P < 0.05).

[0188] The liver plays an important role in fat metabolism. To further explore the fat accumulation and liver damage in diabetic mice, we analyzed the liver sections stained with HE and Oil Red O. Fig.43HE staining sections of the liver of mice in each group. Fig.44 This is the Oil Red O staining section analysis of each group of mice. As can be seen from the figure, compared with the Control group, the liver cells of the Model group mice showed edema and necrosis, severe fat vacuoles and inflammatory cell infiltration, and disordered arrangement of liver cells; the Oil Red O staining of the liver sections further showed that there was a large amount of fat accumulation in the liver of the model mice, and the area of ​​red fat droplets was significantly increased; compared with the Model group, the liver fat accumulation and red fat droplet area of ​​the FLP-215 group were significantly reduced.

[0189] Example 13 Shutgun sequencing of fecal microbial metagenomics of type 2 diabetic mice

[0190] (1) Six mouse fecal samples were collected from each group for metagenomic Shutgun sequencing. Genomic DNA was extracted from fecal samples using QIAGEN DNA Mini-Kit (QIAGEN, Hilden, Germany) and homogenized using a bead beating method. The concentration and integrity of the DNA were then tested using 0.8% agarose gel electrophoresis. The sequencing work was performed by Beijing Weitong Lihua Gene Technology Co., Ltd. The raw data was processed using a sliding window method to remove low-quality sequences in the raw sequence and obtain high-quality clean reads. Finally, the reads were trimmed and modified using SFIFLE software.

[0191] (2) Experimental results

[0192] Previous studies have shown that biochemical indicators in type 2 diabetic mice are closely related to the intestinal microbial flora. Therefore, we tested and analyzed the effects of four probiotics on the species composition and metabolic pathways of intestinal microorganisms in type 2 diabetic mice through metagenomic sequencing. After careful screening and comparison, we identified 6 phyla, 14 classes, 15 orders, 28 families, 43 genera, 80 species and 366 absolute metabolic pathways. We used the Shannon index to represent the alpha diversity of intestinal microorganisms in each group of mice. Fig.45 is the Shannon index of intestinal microorganisms of mice in each group. As shown in the figure, compared with the Control group, the Shannon index of the Model group mice was significantly reduced (P < 0.05); while the FLP-215 group showed a statistical difference compared with the Model group and was close to the Control group (P < 0.05). At the same time, the results of the β diversity analysis at the species level showed that the three groups were separated on the PCoA graph, among which the Control group and the FLP-215 group were far away from the other groups. At the species level, we used a heat map to display the species level of intestinal microorganisms of each group of mice. Fig.46It is a schematic diagram of the species level of intestinal microorganisms of each group of mice. As shown in the figure, compared with the Model group, the intake of FLP-215 significantly increased the abundance of Lactobacillus_pentosus, Lactobacillus_del brueckii, Leuconostoc_mesenteroides and Lactococcus_lactis in the intestines of the FLP-215 group mice, and reduced the abundance of Bifido bacterium_pseudolongum, Prevotella_sp_MGM1, Lactobacillus_reuter, Lachnospi raceae_bacterium_3_2 and Lachnospiraceae_bacterium_COE1 (P < 0.05).

[0193] In terms of metabolic pathways, we screened out 22 metabolic pathways with the greatest differences ( Fig.47)。Compared with the Control group, the Model group up-regulated polyisoprenoid biosynthesis (E. coli), superpathway of L-methionine biosynthesis (by sulfhydrylation), glycogen degradation II, starch biosynthesis, sucrose biosynthesis II, homolactic fermentation, superpathway of L-cysteine biosynthesis (mammalian), purine nucleotides degradation II (aerobic), L-arginine biosynthesis I (via L-ornithine), L-arginine biosynthesis II (acetyl cycle), L-ornithine biosynthesis I, L-histidine biosynthesis, purine ribonucleosides degradation, superpathway of L-aspartate and L-asparagine biosynthesis, L-lysine biosynthesis I, methylerythritol phosphate pathway I, D-galactose degradation I (Leloir pathway), guanosine nucleotides degradation II, guanosine nucleotides degradation I, superpathway of purine deoxyribonucleosides degradation, and down-regulated tRNA processing.Notably, FLP-215 intake downregulated L-arginine biosynthesis I (via L-ornithine), L-arginine biosynthesis II (acetyl cycle), L-ornithine biosynthesis I, L-histidine biosynthesis, phospholipases, superpathway of L-methionine biosynthesis (by sulfhydrylation), sucrose biosynthesis II, purineribonucleoside degradation, and upregulated tRNA processing. Overall, FLP-215 intake significantly altered specific metabolic pathways of the intestinal microbiota of diabetic mice.

[0194] In order to reveal the possible mechanism of probiotics in alleviating type 2 diabetes, we conducted a correlation analysis between FLP-215, intestinal bacteria, intestinal microbial metabolic pathways and various biochemical indicators. We used the Pearson correlation coefficient to analyze the data obtained from the experiment and constructed their correlation network diagram. According to the comparison of PearsonCC values, the correlation analysis of FLP-215, flora, intestinal metabolites, metabolic pathways and physicochemical indicators was carried out. Fig.48 For the correlation analysis of FLP-215, flora, intestinal metabolites, metabolic pathways and physicochemical indicators, as shown in the figure, the intake of FLP-215 can upregulate the abundance of Lactobacillusdelbrueckii and downregulate the abundance of Bifidobacteriumpseudolongum and PrevotellaspMGM1, while Lactobacillusdelbrueckii promotes the upregulation of fatty acid recovery, mixed acid fermentation, mixed lactic acid fermentation, sucrose degradation (sucrose invertase), sucrose anaerobic degradation superpathway, glucose and pentose degradation superpathway, and glycolysis superpathway. These pathways can promote the secretion of acetate, and acetate is negatively correlated with physiological indicators such as FBG, FINS, TG, TC, LDL-C, FFA, TNF-α, IL-6, and MDA, and positively correlated with GLP-1, HDL-C, SOD, and GSH. This shows that the FLP-215 group can regulate these metabolic pathways to produce acetate by changing the intestinal flora, thereby improving the glucose and lipid metabolism ability of mice.

[0195] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum FLP-215, characterized in that: The plant lactobacillus FLP-215 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on September 4, 2023, with the deposit number CGMCC No.28337.

2. The use of Lactobacillus plantarum FLP-215 or its bacterial suspension according to claim 1 in preparing a product for preventing and / or treating ulcerative colitis, diabetes and / or reducing organ damage, characterized in that: The organ damage is one or more of liver damage, pancreatic islet structure damage or colon damage.

3. The application according to claim 2, characterized in that: The ulcerative colitis is ulcerative colitis induced by dextran sulfate sodium.

4. The application according to claim 2, characterized in that: The prevention and / or treatment of ulcerative colitis is at least one of the following: improving the intestinal mucosal barrier, alleviating inflammatory response or regulating the intestinal microbial community structure.

5. The application according to claim 4, characterized in that: The relieving of inflammatory response is one or more of increasing the expression of IL-10, reducing the expression of IL-1β, reducing the expression of IL-6, reducing the expression of IFN-γ, reducing the expression of TNF-a or reducing the expression of IL-17A.

6. The use according to claim 4, characterized in that: The regulating the intestinal microbial community structure is one or more of promoting the growth of probiotics in the intestinal flora, increasing the abundance of bacteria that can produce SCFAs, increasing the diversity of the intestinal flora, or inhibiting the growth of pathogenic bacteria in the intestinal flora; the improving the intestinal mucosal barrier is promoting the expression of MUC-2 protein and / or ZO-1 protein.

7. The use according to claim 2, characterized in that: The prevention and / or treatment of diabetes mellitus is at least one of the following: improving key pathways of glucose and lipid metabolism, reducing fasting blood glucose levels, improving glucose intolerance, reducing serum insulin levels, or increasing serum glucagon-like peptide-1 levels.

8. A bacterial agent, characterized in that Contains the Lactobacillus plantarum FLP-215 and / or its bacterial suspension according to claim 1.

9. Use of the bacterial agent according to claim 8 in the preparation of a product for preventing and / or treating ulcerative colitis, diabetes and / or reducing organ damage, characterized in that: The organ damage is one or more of liver damage, pancreatic islet structure damage or colon damage.

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