CN117965341A - Lactobacillus plantarum FLP-215 and application thereof in ulcerative colitis, diabetes and organ injury - Google Patents

Abstract

The invention discloses a lactobacillus plantarum (Lactobacillus plantarum) FLP-215, wherein the lactobacillus plantarum FLP-215 is stored in the China general microbiological culture collection center (CGMCC) with the preservation number of 28337 in the 9 th month 4 of 2023. The lactobacillus plantarum FLP-215 has the effects of preventing and/or treating ulcerative colitis and diabetes and reducing organ injury, and has a broad market prospect.

Description

Lactobacillus plantarum FLP-215 and application thereof in ulcerative colitis, diabetes and organ injury

Technical Field

The invention belongs to the technical field of microbial degradation. More particularly, it relates to a lactobacillus plantarum FLP-215 and its use in ulcerative colitis, diabetes and organ damage.

Background

Inflammatory Bowel Disease (IBD) is a chronic, non-specific inflammatory disease, mainly comprising both Ulcerative Colitis (UC) and Crohn's Disease (CD), and the specific pathogenesis is currently unknown. The clinical manifestations of patients are diarrhea, hematochezia, weight loss, etc. Ulcerative colitis occupies a large amount of public health resources, and has become a major health problem worldwide due to high occurrence and recurrence, so that prevention and control of ulcerative colitis are of great significance in preventing and treating chronic diseases and guaranteeing public health.

The common medicines for treating ulcerative colitis in clinic are: sulfasalazine salicylic acid preparation, corticosteroid, etc. However, the side effects caused by the use of the medicines are obvious, various related complications can be generated or aggravated, the main symptoms are hypertension, diabetes, osteoporosis, kidney injury and the like, and particularly the complications are diabetes, so that great pain and inconvenience are brought to patients, and the life quality of the patients is reduced. There is an urgent need to find a healthy and effective method of treating ulcerative colitis to improve the current treatment regimen. 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, after ingestion of sufficient amounts, produce beneficial effects on host health. Probiotics have been widely accepted to improve mucosal barrier function, regulate intestinal flora and enhance the immune system of the intestinal tract. Probiotics have many advantages over traditional methods of treatment, safer, low side effects, and the like.

Type 2 diabetes is currently the most common metabolic disease in the world, the cause of the disease is complex, irreversible damage is caused to organisms, various chronic complications are further induced, and the incidence rate in the world is continuously rising. How to effectively prevent and treat diabetes mellitus has become a health problem to be solved in the world. At present, no medicine for radically treating type 2 diabetes is found, a patient can only control blood sugar by means of diet, exercise and the like, and clinical medicines also show various toxic and side effects. Because of life style changes, it is difficult to achieve the expected effect only by changing diet exercise and the like, the aim of treating diabetes is currently achieved mainly by taking injection medicines. These drugs mainly include sulfonylurea and non-sulfonylurea secretagogues for stimulating insulin secretion by islet cells; thiazolidinediones to enhance sensitivity to insulin; biguanides, which reduce hepatic glucose output and improve peripheral insulin resistance; and alpha-glucosidase inhibitors, including acarbose and voglibose, which delay carbohydrate digestion and absorption. Sometimes, in actual treatment, the medicines are combined according to the condition of a patient, so that the purpose of rapidly and effectively reducing blood sugar is achieved. However, the use of the above drugs may cause more or less side effects such as gastrointestinal discomfort, liver diseases, and are limited by renal function and heart diseases. Therefore, people need to find more natural and safe hypoglycemic components and develop safe and effective products or medicaments for assisting in preventing and treating diabetes.

Organ tissue injury refers to pathological injury of the human skin, subcutaneous tissue, synovial capsule, intervertebral disc, peripheral nerves and blood vessels caused by various activities. Organ injury is a special critical state between deterioration and rehabilitation, and is expected to be healed if diagnosis and treatment are timely; conversely, it may deteriorate and even cause organ failure, causing serious damage to the body. Therefore, it is extremely important to diagnose and treat organ and tissue injury, and in recent years, research on liver injury mechanism and prevention and treatment have become one of hot spots of biological and pharmaceutical research at home and abroad.

At present, both the treatment of ulcerative colitis and the treatment of type 2 diabetes or the treatment of organ injury can generate and aggravate complications in the actual treatment process, and when the medicine for treating ulcerative colitis is taken, the diabetes can be aggravated, and the injury of the organ is aggravated; and when the medicine for treating type 2 diabetes is taken, complications such as gastrointestinal discomfort, liver diseases and the like can be caused. In addition, how to treat patients suffering from ulcerative colitis, diabetes and organ damage at the same time in a targeted manner is an important problem to be solved.

Chinese patent (a strain of Lactobacillus plantarum for relieving ulcerative colitis and application thereof) discloses Lactobacillus plantarum which can reduce weight loss during the disease period of ulcerative colitis, improve fecal characteristics and hematochezia, relieve colon length shortening, improve colonic mucosa injury, reduce pro-inflammatory factors in colon and the like. However, the strain only has the effect of relieving ulcerative colitis, but cannot simultaneously treat other complications, such as treatment of diabetes and reduction of organ injury, and has limited functional effects and application scenes.

Disclosure of Invention

Aiming at the prior art problems, the primary aim of the invention is to provide a lactobacillus plantarum FLP-215, wherein the lactobacillus plantarum FLP-215 has the effects of preventing and/or treating ulcerative colitis and diabetes and reducing organ injury, and has wide market prospect.

A second object of the present invention is to provide the use of Lactobacillus plantarum FLP-215 or a bacterial suspension thereof as described above for the preparation of a product for the prevention and/or treatment of ulcerative colitis, diabetes and/or for the reduction of organ damage.

The third object of the present invention is to provide a microbial inoculum comprising the lactobacillus plantarum FLP-215 and/or a bacterial suspension thereof.

A fourth object of the present invention is to provide the use of the above-mentioned microbial inoculum for the preparation of a product for the prevention and/or treatment of ulcerative colitis, diabetes and/or for the reduction of organ damage.

The above object of the present invention is achieved by the following technical scheme:

the invention screens and obtains a lactobacillus plantarum (Lactobacillus plantarum) FLP-215, wherein the lactobacillus plantarum FLP-215 is stored in the China general microbiological culture collection center (CGMCC) with the preservation number of 28337 in the 9 th month 4 of 2023.

The invention discusses the prevention and treatment effect and the action mechanism of lactobacillus plantarum FLP-215 on ulcerative colitis through shotgun metagenome and transcriptome analysis. The study results show that ingestion of lactobacillus plantarum FLP-215 significantly improves dextran sodium sulfate-induced ulcerative colitis in mice, as evidenced by increased body weight, water intake, food intake and colon length in mice, decreased disease activity index, inflammatory factor levels and histopathological scores. In intensive studies, lactobacillus plantarum FLP-215 was found to alleviate inflammation by modulating intestinal microbiota and increasing Short Chain Fatty Acid (SCFAs) production, decreasing the levels of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-a) and interleukin-17A (IL-17A), and increasing the levels of the anti-inflammatory cytokine interleukin-10 (IL-10). In conclusion, lactobacillus plantarum FLP-215 can play a role in preventing and treating the colonitis in a dextran sulfate-induced colonitis mouse model by protecting an intestinal mucosa barrier, weakening inflammatory reaction, regulating intestinal microbiota structure and the like.

The inventor also discovers that lactobacillus plantarum FLP-215 can significantly reduce blood sugar and plays a role in treating diabetes. The inventor finds that lactobacillus plantarum FLP-215 can play a role in treating and preventing type 2 diabetes mellitus by improving a glycolipid metabolism key pathway, reducing fasting blood glucose value, improving glucose intolerance, improving glucagon-like peptide-1 content in serum, reducing insulin resistance index, improving beta cell function and insulin sensitivity detection index and the like. Further, the inventors found that lactobacillus plantarum FLP-215 is capable of playing an excellent role in reducing organ damage, in particular in reducing liver damage, pancreatic damage or colon damage.

The invention provides a lactobacillus plantarum FLP-215 which can effectively treat ulcerative colitis, treat diabetes and reduce organ injury simultaneously, and provides a technical scheme for the combined treatment of ulcerative colitis, diabetes and organ injury.

Furthermore, the invention also claims the application of the lactobacillus plantarum FLP-215 or the bacterial suspension thereof in preparing products for preventing and/or treating ulcerative colitis, diabetes and/or reducing organ damage.

In some embodiments, the product includes, but is not limited to, a drug, a pharmaceutical composition, a functional microbial agent, a food, a health product, a nutritional supplement, a daily chemical skin care product, and the like; in some embodiments, the food product may be a dairy product, a soy product, a fruit and vegetable product; in some embodiments, the dairy product comprises yogurt, cream, cheese, and the like.

In some embodiments, the ulcerative colitis is dextran sodium sulfate induced ulcerative colitis.

In some embodiments, the preventing and/or treating ulcerative colitis comprises at least one of: improving intestinal mucosa barrier, relieving inflammatory reaction or regulating intestinal microbiota structure.

In some embodiments, the reducing the inflammatory response is one or more of increasing IL-10 expression, decreasing IL-1 beta expression, decreasing IL-6 expression, decreasing IFN-gamma expression, decreasing TNF-a expression, or decreasing IL-17A expression.

In some embodiments, the modulating the intestinal microbiota structure is one or more of promoting the growth of probiotics in the intestinal microbiota, increasing the abundance of species capable of producing SCFAs, increasing intestinal microbiota diversity, or inhibiting the growth of pathogenic bacteria in the intestinal microbiota.

In some embodiments, the improving the intestinal mucosal barrier is promoting expression of MUC-2 protein and/or ZO-1 protein.

In some embodiments, more specifically, the efficacy of lactobacillus plantarum FLP-215 in preventing and/or treating ulcerative colitis specifically comprises at least one of the following:

(a) Reducing weight loss due to ulcerative colitis;

(b) Reducing food intake and water intake reduction due to ulcerative colitis;

(c) Significantly increasing colon length;

(d) Significantly reducing disease activity index;

(e) Significantly increasing the anti-inflammatory cytokine levels in serum and decreasing the pro-inflammatory cytokine levels in serum;

(f) Significantly reducing pathology scores of intestinal tract combinations;

(g) Increasing the expression level of zonulin ZO-1 and/or mucin MUC-2 in the intestinal tract;

(h) The content of short-chain fatty acid is obviously improved;

(i) The intestinal flora diversity is remarkably improved;

(j) Promoting the growth of probiotics in the intestinal flora and/or inhibiting the growth of pathogenic bacteria in the intestinal flora.

Because lactobacillus plantarum FLP-215 has any of the above effects, the use of lactobacillus plantarum FLP-215 in the above should also be within the scope of the present application.

In some embodiments, the treating diabetes comprises at least one of: increasing the key pathway of glycolipid metabolism, reducing fasting blood glucose, improving glucose intolerance, reducing insulin content in serum, or increasing glucagon-like peptide-1 content in serum.

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

(a) Reducing blood sugar and blood lipid;

(b) Improving alpha and beta diversity of intestinal flora;

(c) Promoting the growth of probiotics in the intestinal flora and inhibiting the growth of pathogenic bacteria in the intestinal flora;

(d) Improving the key pathway of glycolipid metabolism;

(e) Lowering fasting blood glucose levels;

(f) Improving glucose intolerance;

(g) Reducing the content of insulin in serum;

(h) Increasing the glucagon-like peptide-1 content in serum;

(i) Decreasing insulin resistance index and increasing beta cell function and insulin sensitivity detection index;

(j) Improving lipid metabolism disorder;

(k) Treating and preventing type 2 diabetes;

(l) Reducing the total cholesterol, triglyceride, low density lipoprotein cholesterol and free fatty acid content in serum;

(n) lowering the atherosclerosis index;

(m) increasing the high density lipoprotein cholesterol level in the serum;

(o) reducing interleukin-6 and tumor necrosis factor-alpha content in serum;

(p) decreasing liver index and liver injury;

(q) reducing pancreatic and colon damage;

(r) increasing superoxide dismutase, reducing glutathione, glucose-6-phosphatase content in the liver;

(s) reducing malondialdehyde content in liver.

Because lactobacillus plantarum FLP-215 has any of the above effects, the use of lactobacillus plantarum FLP-215 in the above should also be within the scope of the present application.

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

Furthermore, the invention also claims a microbial inoculum comprising the lactobacillus plantarum FLP-215 and/or a bacterial suspension thereof.

Furthermore, the invention also claims the application of the microbial inoculum in preparing products for preventing and/or treating ulcerative colitis, diabetes and/or reducing organ damage.

The invention has the following beneficial effects: the invention provides a lactobacillus plantarum (Lactobacillus plantarum) FLP-215. The lactobacillus plantarum FLP-215 can play a role in preventing and treating in a dextran sodium sulfate induced colonitis mouse model by protecting intestinal mucosa barrier, weakening inflammatory reaction, regulating intestinal microbiota structure and the like; furthermore, lactobacillus plantarum FLP-215 can play a role in treating diabetes by increasing the key pathway of glycolipid metabolism, reducing fasting blood glucose value, improving glucose intolerance, reducing the content of insulin in serum or increasing the content of glucagon-like peptide-1 in serum. Meanwhile, the lactobacillus plantarum FLP-215 also has excellent treatment effect in reducing organ injury. The invention provides a lactobacillus plantarum FLP-215 which can effectively treat ulcerative colitis, treat diabetes and reduce organ injury simultaneously, and provides a technical scheme for the combined treatment of ulcerative colitis, diabetes and organ injury.

Drawings

FIG. 1 shows the growth pattern of Lactobacillus plantarum FLP-215 on a medium.

FIG. 2 shows the state of Lactobacillus plantarum FLP-215 under a microscope after gram staining.

FIG. 3 shows the 16s rRNA identification result of Lactobacillus plantarum FLP-215.

Fig. 4 to 6 are graphs showing daily body weight, water intake and food intake changes of mice, respectively.

FIG. 7 is a graph showing the daily disease activity index change of mice.

Fig. 8 is a schematic representation of the change in colon length in mice.

FIG. 9 is a graphical representation of the proinflammatory cytokine levels in serum of mice from each group.

FIG. 10 is a schematic representation of H & E stained sections of the colon of each group of mice.

FIG. 11 is a schematic representation of colon histopathological scores for each group of mice.

FIG. 12 is a schematic representation of ZO-1 immunofluorescence sections of the colon of each group of mice.

FIG. 13 is a schematic representation of colon MUC-2 immunofluorescence sections of mice of each group.

FIG. 14 is a graph showing the areal density of the colon ZO-1 and MUC-2 of each group of mice.

FIG. 15 is a graphical representation of Shannon index for each group of mice.

Fig. 16 is a schematic representation of a PCoA assay of the intestinal flora of mice.

FIG. 17 shows various sets of horizontal metagenomic results.

FIG. 18 is a schematic diagram of differential metabolic pathways in Lactobacillus plantarum FLP-215 mice.

Fig. 19 is a schematic diagram of short chain fatty acid levels for each group of mice.

FIG. 20 is a volcanic diagram of gene distribution.

FIG. 21 shows the results of a Gene Ontology (GO) enrichment analysis.

Figure 22 is a graphical representation of body weight of each group of mice over time.

Fig. 23 is a graphical representation of blood glucose levels of each group of mice over time.

FIG. 24 is a schematic of the ability of islet beta cells to regulate blood glucose in mice of each group.

FIGS. 25-28 are insulin, insulin resistance index, beta cell function and insulin sensitivity test index for each group of mice.

Fig. 29 is a schematic representation of HE slice analysis of the pancreas of each group of mice.

FIGS. 30-35 are graphs of serum triglycerides, total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, free fatty acid levels, and atherosclerosis index, respectively, for each group of mice.

FIGS. 36-37 are graphs showing the levels of interleukin-6 and TNF- α in the serum of mice in each group.

FIGS. 38-41 are graphical representations of the levels of superoxide dismutase, reduced glutathione, glucose-6-phosphatase, and malondialdehyde in the liver of mice in each group.

Fig. 42 is a graph showing liver index of each group of mice.

FIG. 43 is HE stained sections of liver from each group of mice.

FIG. 44 is an analysis of the O-stained sections of the oil red of each group of mice.

FIG. 45 shows the Shannon index of the intestinal microorganisms of each group of mice.

FIG. 46 is a schematic representation of species levels of intestinal microorganisms in mice of each group.

FIG. 47 shows the metabolic pathways of mice in each group.

FIG. 48 is a correlation analysis of FLP-215, flora, intestinal metabolites, metabolic pathways and physicochemical indicators.

Detailed Description

The invention is further elucidated in the following in connection with the accompanying drawing and a specific embodiment. The following examples are preferred embodiments of the present invention, but are not intended to limit the scope of the present invention in any way. The invention is mainly described by the strains and based on the application ideas of the strains, the simple parameter substitutions in the embodiments cannot be described in the examples, but are not limited by the examples, and any other changes, modifications, substitutions, combinations and simplifications which do not deviate from the spirit and principle of the invention should be regarded as equivalent substitutions and are included in the scope of the invention.

Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The reagents and materials used in the present invention are commercially available unless otherwise specified.

In the examples below, statistical analysis was performed on the data using GraphPad and R software, all data being expressed as mean ± Standard Deviation (SD). Significance analysis was performed using Wilcoxon, P <0.05 when the value is; * P <0.01; * P <0.001 is considered significant, strongly significant and extremely significant. The ggplot package was used for principal coordinate analysis (PCoA) and the box and bubble diagrams were drawn, respectively. The DESeq2 software package was used for P-value calculation and correction in metabolic pathway bubble diagrams.

EXAMPLE 1 acquisition of Lactobacillus plantarum FLP-215

1. Screening and separating lactobacillus plantarum:

Sampling personnel obtain peasant self-made pickle from Sichuan, ten-time gradient dilution is carried out on the pickle, 100 mu L of samples are respectively taken from the dilutions of-2, -3 and-4 dilution gradients and coated on MRS agar culture medium, the samples are placed in a 37 ℃ incubator for inverted culture for 48 hours, colony separation and streaking with obvious difference in morphology are selected on a plate of-2 dilution gradients to obtain pure single colony, 1 strain with good growth state is obtained through separation, and the strain is named as FLP-215.

2. Identification of lactobacillus plantarum:

the FLP-215 separation and purification plate is observed, colony morphological characteristics are recorded, physiological and biochemical characteristics are studied, and the strain is subjected to taxonomic identification by using a 16s rRNA technology.

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

(1) Morphological features

Lactobacillus plantarum FLP-215 grows well under anaerobic conditions on an MRS agar plate culture medium, obvious colonies can be formed after 48 hours of culture, and the colonies are round and full, milk white, 2-3 mm in diameter, opaque, smooth in edges and surfaces (shown in figure 1). Under the microscope, the strain is purple after gram staining, accords with the staining characteristics of gram-positive bacteria, and has a medium-long rod shape and is arranged independently (as shown in figure 2).

(2) Physiological and biochemical characteristics

Lactobacillus plantarum FLP-215 is free of flagella and spores, is not movable, does not form spores, is facultatively anaerobic, and is negative to catalase. The growth temperature is 30-37 deg.c, and can metabolize galactose, glucose, fructose, mannose and other monosaccharides, maltose, lactose, sucrose and other disaccharides.

(3) Molecular biological characterization results

The 16S rRNA sequence of the lactobacillus plantarum FLP-215 is shown as SEQ ID No. 1. The 16s rRNA molecular biology of FLP-215 identified lactobacillus plantarum (Lactobacillus plantarum) and the identification result is shown in FIG. 3. The strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.28337 in 2023, 9 and 4 days.

EXAMPLE 2 construction of Experimental animal model for ulcerative colitis

50C 57BL/6J mice (black male mice) of 8 weeks old purchased from Hunan Laek Jingda laboratory animals Co., ltd, were randomly divided into 5 groups after 1 week of adaptive feeding: ① Control group (Control group, normal drinking water, 10 mice); ② Model group (Model group, 3% dextran sodium sulfate drinking water, 10 mice); ③ Lactobacillus plantarum FLP-215 preventive group (Pro group, 3% dextran sodium sulfate drinking water+probiotic bacterial suspension, lactobacillus plantarum FLP-215 with probiotic bacterial suspension of 10 ⁹ CFU was resuspended in 300 μl sterile physiological saline, administered by gavage, 10 mice); ④ Lactobacillus plantarum FLP-215 treatment group (Treat group, 3% dextran sodium sulfate drinking water after modeling, given probiotic bacterial suspension, 10 mice); ⑤ Drug group (Drug group, 3% dextran sodium sulfate drinking water after model establishment, sulfasalazine solution, 10 mice). In this procedure, the control group and the model group were given an equivalent amount of physiological saline as a control. And continuously drinking the drinking water containing 3% dextran sodium sulfate for 1 week until the weight is reduced by more than 10% or the mice have the characteristics of loose stool, purulent stool, and the like, so as to judge that the modeling is successful.

The drinking water, the feed and the padding are replaced every day, the environmental temperature of an animal laboratory is controlled at 23+/-1 ℃, the relative humidity is controlled at 52% -60%, and the bright and dark circulation is kept for 12 hours. Mice were fed standard normal commercial mouse feed (consisting essentially of crude protein, crude fiber, crude fat and trace elements). After the end of the experiment, mice were euthanized with 1% sodium pentobarbital solution and various tissue samples were collected, including immune organs, serum, proximal colon, stool, cecal contents, distal colon and other tissues.

EXAMPLE 3 improvement of mouse physiological index and DAI by Lactobacillus plantarum FLP-215

(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; hematocrit levels were assessed according to the mouse fecal occult blood kit instructions and DAI was calculated according to the following table (table 1 is the DAI scoring standard).

TABLE 1

(2) Experimental results

Mice were monitored daily for water intake, food intake, body weight and disease activity index to assess severity of modeling of ulcerative enteritis model and post-supplementation effects of lactobacillus plantarum FLP-215.

Figures 4 to 6 are graphs of daily body weight, water intake and food intake changes of mice, respectively. From the graph, the body weight, food intake and water intake of mice in Model, pro, treat and Drug groups all tended to decrease at 1-7 days, probably because each group was under the same DSS modeling conditions at this stage. The body weight, food intake and water intake of Model group mice still tended to decrease at 8-18 days. In contrast, pro group mice tended to have a plateau in body weight, food intake and water intake, treat group mice had a gradual increase in water intake, food intake and body weight on days 10-18, and showed significant differences (P < 0.05) from the model group.

FIG. 7 is a graph showing the daily disease activity index change of mice. As shown, the disease activity index of each of the other groups was significantly increased, except for the Control group, over 1-10 days, while the disease activity index of mice in the Pro, treat and Drug groups was significantly decreased (P < 0.05) relative to the Model group as the experiment was conducted.

Fig. 8 is a schematic representation of the change in colon length in mice. As shown, the Model group mice had significantly shorter colon lengths than the control group, while the Pro, treat and drug groups had significantly increased colon lengths than the Model group.

EXAMPLE 4 Effect of Lactobacillus plantarum FLP-215 on sodium dextran sulfate-induced ulcerative colitis mouse inflammatory cytokines

(1) Determination of serum cytokines

Blood was collected from the orbital venous plexus with capillaries prior to euthanasia. First, the blood sample was naturally coagulated for 30 minutes. The blood samples were then centrifuged at 3000rpm for 20min at 4℃and serum was collected by separation. Finally, ELISA kits were used to detect levels of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-17A (IL-17A), interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in serum.

(2) Experimental results

The colon injury status was evaluated by quantitatively determining the levels of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-a), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), interleukin-17A (IL-17A) and interferon gamma (IFN-gamma) and the anti-inflammatory cytokine interleukin-10 (IL-10) in the serum of mice.

FIG. 9 is a graphical representation of the proinflammatory cytokine levels in serum of mice from each group. As can be seen, the Pro-inflammatory cytokines TNF-a, IL-1. Beta., IFN-. Gamma., IL-6 and IL-17A were significantly elevated (P < 0.05) in the serum of mice from the Model group compared to the Control group, while the anti-inflammatory cytokine IL-10 was significantly reduced in the serum of mice from the Pro, treat and Drug groups.

EXAMPLE 5 Effect of Lactobacillus plantarum FLP-215 on the index of pathological changes in intestinal tissue of mice

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

The distal colon of the mouse was taken for immunofluorescent protein assay, colon tissue was labeled with mucin MUC-2 and zonulin ZO-1 antibodies, respectively, and immunofluorescent staining was further performed. Fluorescein was combined with antibodies ZO-1 and MUC-2 to form fluorescent antibodies. Characterization and localization of ZO-1 and MUC-2 in intestinal tissues was studied by fluorescence microscopy by specific binding to antigen to form multicomponent complexes. The surface densities of immunofluorescence ZO-1 and MUC-2 were measured and calculated, and the effect of the test strain on improving the barrier to the intestinal membrane was evaluated.

TABLE 2

(2) Experimental results

FIG. 10 is a schematic representation of H & E stained sections of the colon of each group of mice. As shown in the hematoxylin-eosin stained paraffin section results in fig. 10, the intestinal tissue of the Control group mice has clear structures and obvious boundaries; the mucous membrane is complete, the cell morphology is normal, and no obvious inflammatory cell infiltration exists. The Model group intestinal tissue is almost completely ulcerated, no obvious intestinal gland structure is generated, mucous membrane epithelium is detached, the complete necrosis of the intestinal gland is disappeared, the injury invades the serosa, connective tissues are loosely arranged, and more neutrophil granulocytes and lymphocyte infiltrates; the myolayer, serosa, sees a small amount of connective tissue that is infiltrated and proliferated by neutrophils, lymphocytes. In contrast, small-area ulcers can be seen in the intestinal tissue of Pro group mice, the mucous membrane epithelium at the damaged part is complete, a small amount of intestinal gland necrosis disappears, the Pro group mice are replaced by proliferation connective tissue, and a small amount of neutrophil and lymphocyte infiltration is carried out; submucosa is seen with small amounts of neutrophil infiltration and lymphocyte infiltration. Meanwhile, the colon of the Treat group mice can be provided with focal ulcer, mucous membrane epithelium at the damaged part is complete, cytoplasm is loose, a small amount of intestinal gland necrosis disappears, and the mice are replaced by proliferation connective tissue and are infiltrated with neutrophil spots; lesion invasion and submucosa, punctate infiltration of neutrophils was seen. The intestinal tissue of the Drug group mice can be seen as ulcer with larger area, the mucous membrane epithelium at the damaged part is shed, more intestinal gland necrosis disappears, the intestinal gland necrosis is replaced by hyperplasia connective tissue, and more neutrophil granulocyte and lymphocyte infiltrate; lesion invasion and submucosa, small numbers of neutrophil infiltration and lymphocyte infiltration were seen.

Fig. 11 is a schematic representation of colon histopathological scores of mice in each group, which were assessed according to the scoring criteria shown in table 2, and it can be seen from fig. 11 that there was a significant difference between Model group and Control group, whereas Pro group, treat group and Drug group were closer to Control group and there was a significant difference between Model group (P < 0.01).

FIG. 12 is a schematic representation of ZO-1 immunofluorescence sections of the colon of each group of mice. As can be seen from FIG. 12, the immunofluorescence analysis results show that the MUC-2 protein and ZO-1 protein are rich in the Control group, while the intestinal mucosa is destroyed in the Model group, so that the tight junction proteins remain almost unchanged, and the ZO-1 distribution can be clearly seen in the sections of mice in the Pro group and the Treat group, the expression level is obviously higher than that in other groups, and the Drug group is also partially expressed.

FIG. 13 is a schematic representation of immunofluorescence sections of the colon MUC-2 of mice. Similarly, the tight junction protein MUC-2, the Model group and the Control group are greatly different, and meanwhile, the MUC-2 in the Pro group and the Treat group is distributed in a large quantity, so that the expression quantity of the Model group is obviously more than that of the Model group, and more expression exists in the Drug group. The expression of the zonulin ZO-1 and MUC-2 was further quantitatively analyzed and a scatter plot of the areal density was plotted. FIG. 14 is a graph of the areal densities of the mouse colon ZO-1 and MUC-2, and it can be seen that the trend of the areal densities of ZO-1 and MUC-2 are similar, the Pro and Treat groups are closer to the Control group than the other groups, and there is a significant difference (P < 0.05) from the Model group.

Example 6 Effect of Lactobacillus plantarum FLP-215 on the modulation of the intestinal flora and SCFAs in mice

(1) Short chain fatty acid assay: after freeze-drying the mouse faeces, short Chain Fatty Acids (SCFAs) were extracted and the levels of short chain fatty acids in each group of samples were quantitated. The method comprises the following steps: at the end of the experiment, the colon contents of 6 golden yellow mice were randomly selected for SCFA assay per group. First, the freeze-dried colon contents were weighed at 40mg, and 600mL of physiological saline (85%) was added. The samples were then placed on a shaker for 5min, thoroughly mixed and centrifuged (8000 rpm,5 min). 200mL of the supernatant was then acidified by adding 100mL of sulfuric acid (50%). Short chain fatty acids were extracted by adding 400 μl of n-hexane. The solution was finally transferred to a vial through an organic film. The concentration of SCFAs, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, was analyzed by gas chromatography-mass spectrometry (GCMS; agilent-7890, santa clara, california, usa). The concentration of SCFAs was calculated by external standard methods.

Fecal DNA extraction, metagenomic sequencing and data quality control: mouse faeces are collected at the end of the test, total DNA of faecal samples is extracted according to the instruction of a faecal DNA extraction kit, and shotgun metagenome sequencing is performed after detecting the purity and the integrity of the DNA by using 0.8% agarose gel electrophoresis, and a sequencing library is prepared. And obtaining abundance data and metabolic pathway data of the intestinal microorganisms of each group of mice by utilizing humann database, qualitatively and quantitatively analyzing alpha diversity and beta diversity of the intestinal microorganisms of each group of mice by using R software, and simultaneously calculating differential flora and differential metabolic pathways among the groups of mice.

FIG. 15 is a graphical representation of Shannon index for each group of mice. The diversity analysis results showed that the Shannon index was significantly reduced for each group of mice under the same modeling conditions as compared to the Control group.

Fig. 16 is a schematic representation of a PCoA assay of the intestinal flora of mice. Meanwhile, the beta diversity results show that the groups are obviously separated from the control group, the Pro group, the Treat group and the Drug group are obviously separated from the model group, and obvious strain abundance differences exist.

The species level metagenome results are shown in fig. 17, with the Model group potential colonitis pathogens such as ESCHERICHIA COLI, aeromonas caviae, clostridium perfringens and Enterococcus faecalis significantly increased, while decreasing in the Pro and Treat groups; potentially beneficial bacteria, such as Bifidobacterium pseudolongum, AKKERMANSIA MUCINIPHILA, anaerotruncus sp G3 2012, were significantly reduced in the Model group and significantly increased in the Pro and Treat groups.

(2) Effect of Lactobacillus plantarum FLP-215 on the modulation of SCFAs

FIG. 18 is a schematic diagram of differential metabolic pathways in mice that ingest Lactobacillus plantarum FLP-215. Analysis of mouse intestinal flora diversity has found an increased abundance of species capable of producing Short Chain Fatty Acids (SCFAs) in the Pro and Treat groups. Metabolic pathway analysis results showed that the abundance of 7 microbial metabolic pathways associated with SCFAs production was significantly increased in the Pro and Treat groups compared to the Model group, including the superpathway III of acetyl-coa biosynthesis, the fermentation of pyruvate to acetate and lactate ii, the fermentation of pyruvate to acetate and S-lactate i, glycolysis III (from glucose), fatty acid biosynthesis, and acetyl-coa biosynthesis i and acetyl-coa biosynthesis ii.

Fig. 19 is a schematic diagram of short chain fatty acid levels for each group of mice. To confirm the above results, we further examined the content of each group of SCFAs using Gas Chromatography (GC). The results show that the SCFAs (including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid) content of the Model group was significantly reduced compared to the Control group. In contrast, the Pro and Treat group acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid content was significantly increased (P < 0.05) after ingestion of Lactobacillus plantarum FLP-215, as compared to the Drug group. From the above results, we speculate that Lactobacillus plantarum FLP-215 increases the content of SCFAs by affecting the abundance of SCFAs-producing microorganisms and the metabolic pathways of SCFAs-producing microorganisms.

Example 7 comparison of mice intestinal epithelial cell transcriptome study

(1) Transcriptome sequencing: after euthanasia, the distal colon of the mice was taken for transcriptome measurements. The total RNA of the sample is extracted according to Trizol extraction instruction, the extraction effect is detected by using 0.8% agarose gel electrophoresis, mRNA fragmentation treatment is purified, and a sequencing library is prepared. The denaturation treatment produces single-stranded DNA fragments, which are amplified by PCR to produce DNA clusters, and linearization of the DNA amplicons to single strands. Adding modified DNA polymerase and dNTPs with 4 fluorescent labels, reading the types of nucleotides polymerized by the first round of reaction of each template sequence, counting the collected fluorescent signal results of each round, obtaining the sequence of the template DNA fragment, and detecting the change of the expression quantity. Library construction and quality control were then performed and the RNA-seq raw data was filtered. After constructing the RNA library, sequencing was performed using Illumina Novaseq 6000,6000 and the gene expression level was estimated using featucounts.

(2) Experimental results

FIG. 20 is a volcanic diagram of gene distribution. As shown, the distribution of genes was analyzed using colon transcriptome data. The volcanic plot shows that ingestion of lactobacillus plantarum FLP-215 significantly affects gene expression profiles. To further explore the effects of these Differentially Expressed Genes (DEGs), we analyzed the pathways associated with DEGs.

FIG. 21 shows the results of a Gene Ontology (GO) enrichment analysis. The results can be divided into three major categories, namely biological processes, cellular components and molecular functions. The GO analysis results show that DEGs of the Model and Control groups are mainly involved in biological processes such as cellular components like cytoplasm, extracellular space, extracellular area, cell cycle, and molecular functions like metal ion binding. Whereas DEGs of the Treat group and Model group are mainly involved in biological processes such as immune response, immunoglobulin production and positive regulation of transcription of the RNA polymerase II promoter, cellular components such as extracellular space, and molecular functions such as DNA binding and RNA polymerase II core promoter near sequence specific DNA binding.

Example 8 construction of Experimental animal model for diabetes

(1) Experimental animal

24 Healthy male C57BL/6 mice with the age of 6 weeks, the body mass of 18-20g, are purchased from Beijing Veitz Lihua laboratory animal technology Co., ltd (Beijing, china) and the animal license number is SCXK (Beijing) 2021-0006. The animals were given adequate diet, drinking water and free play during the experiment. The animal laboratory feeding environment meets SPF-grade hygienic standards (temperature 25+ -2deg.C, humidity 70% -80%), and the experimental period meets 12 hours of light-dark cycle. Both normal feeds and high-sugar high-fat feeds were purchased from Jiangsu province collaborative biotechnology limited (Jiangsu, china). The formula of the high-sugar and high-fat feed is as follows: 38% of common feed, 28% of lard, 5.6% of sucrose and whole milk powder: 10.8%, 11.5% casein, microcrystalline cellulose: 1.9 percent of premix for experimental animals: 2%, calcium bicarbonate 1.8% and stone powder 0.4%.

(2) Chemical and reagent

STZ was purchased from sigma chemistry, glucometer and blood glucose test paper were purchased from roche germany, glucose, human insulin, and citric acid buffer were purchased from beijing solebao technologies, inc, and various kits were purchased from shanghai yu biotechnology, inc.

(3) Test strain

Lactobacillus plantarum (Lactobacillus plantarum) FLP-215 obtained by the screening in example 1 was used.

(4) Establishment and experimental design of diabetic mouse model

The experiment uses high-fat diet combined with STZ small-dose multiple injection to establish a model of a type II diabetes mouse. The 18 mice were randomly assigned to normal (Control, n=8), model (Model, n=8), FLP-215 prophylaxis (FLP-215, n=8) after one week of adaptive feeding. The normal group was fed basal feed, the diabetes prevention group was fed high-sugar high-fat feed and the stomach was irrigated daily (1×10 ⁹ CFU, dissolved using 300 μl of physiological saline) based on probiotics for 6 weeks. The concentration of the citrate buffer was 0.1mol/L and ph=4.5, and 230mgSTZ was dissolved per 100ml of the citrate buffer when used. After 6 weeks, the rest groups except the normal group were respectively subjected to STZ intraperitoneal injection at a dose of 45mg/kg, once a day for 3 days; the normal group was subjected to control treatment by injecting the same volume of citrate buffer according to body weight. The symptoms associated with polyphagia, polydipsia, polyuria and significant weight loss associated with 2 to 3 consecutive measures of glucose levels greater than 11.1mmol/L in fasted mice in the model group, 1 week after STZ injection, for more than 6 hours, were considered successful in modeling diabetes. Food intake, water intake, body weight and fasting blood glucose were monitored weekly during the experiment.

The lactobacillus plantarum FLP-215 intervenes until the day before the test is finished, and the feces of each mouse are collected and put into a 2ml sterile centrifuge tube, and are immediately put into a refrigerator at the temperature of minus 80 ℃ for freezing and preservation so as to facilitate the subsequent analysis of the metagenome sequencing and metabolic pathways of the feces; all mice were prohibited from eating feed but were free to drink sterile distilled water. After euthanizing the mice, all mice were subjected to orbital bleeding, left standing at room temperature for 1h for blood clotting, centrifuged at 4 ℃,3500rmp for 45min, and the mouse serum was carefully collected using a pipette and sub-packaged for storage at-80 ℃ for later 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). Placing the cut part of liver into 4% paraformaldehyde solution for tissue fixation, and placing the rest part of liver into liquid nitrogen for quick freezing and storing in a refrigerator at-80 ℃; placing colon content into liquid nitrogen for quick freezing, storing in a refrigerator at-80deg.C, placing part of colon into 4% paraformaldehyde solution for tissue fixation, and placing the rest part into liquid nitrogen for quick freezing, and storing in a refrigerator at-80deg.C; pancreas was filled with 4% paraformaldehyde solution for tissue fixation.

Example 9 Lactobacillus plantarum FLP-215 on weight change, fasting blood glucose levels, oral glucose tolerance experiments in diabetic mice

(1) The body weight of each group of mice was measured once a week during the experiment, and the physiological activity of the mice was observed and the change in body weight was recorded. When the fasting blood glucose level of the mice is measured, the mice are fasted for 12 hours before night, the tail ends of the mice are disinfected and blood is taken, and the tail parts of the mice are disinfected again after the value is measured by a Rogowski blood glucose meter.

On the first day after the seventh week, all mice were fasted without water withdrawal for 12 hours, then glucose solutions (2 g/kg. Bw) were orally taken, then blood glucose levels at this time were measured by tail vein bleeding after ingestion of glucose 0, 15, 30, 60, 120min, respectively, and a graph of blood glucose fluctuation was drawn. Area Under Curve (AUC) values for glucose were calculated using the trapezoidal method, with the following formula: G-AUC (h mmol/L) = (g0+g120)/2+g15+g30+g60.

(2) Experimental results

Sustained weight loss is one of the typical conditions of type 2 diabetes. Figure 22 is a graphical representation of body weight of each group of mice over time. As shown in the figure, from week 0 to week 6, the Control group was fed with normal diet, the Model group and the FLP-215 group were fed with high-fat diet, the mice in the Control group were found to have the slowest weight increase, the Model group had the greatest weight increase, and the FLP-215 group had a weight increase of less than that of the Model group and greater than that of the normal group, which may be related to the ingested Lactobacillus plantarum, indicating that the ingestion of Lactobacillus plantarum may slow the weight increase of the mice due to the ingestion of high-fat diet; after three consecutive injections of STZ at week six, the mice in each group had a decrease in body weight, except the Control group; by comparing the body weight values of the Model group at weeks 6 and 7, a very significant difference (P < 0.0001) occurred, indicating that the diabetes modeling was successful from a body weight perspective. Compared with the Model group, the weight reduction degree of the FLP-215 group is smaller than that of the Model group, and experimental results show that the lactobacillus plantarum FLP-215 can effectively prevent the weight reduction of mice suffering from type 2 diabetes and prevent diabetes symptoms.

Higher fasting blood glucose levels are the most representative feature of type 2 diabetic mice, and the level of fasting blood glucose is also the gold standard for judging the success of modeling of a mouse diabetes model. To investigate the effect of lactobacillus plantarum FLP-215 on blood glucose levels, fasting blood glucose was monitored weekly from the beginning of the experiment. Fig. 23 is a graphical representation of blood glucose levels of each group of mice over time. As shown in the figure, after STZ injection after week 6, the blood glucose level of the blood glucose level Control group at week 7 is significantly higher than that of the Model group (p < 0.0001), the blood glucose level of the FLP-215 group is significantly lower than that of the Model group (p < 0.0001), and the experimental result shows that the Lactobacillus plantarum FLP-215 can significantly reduce the level of diabetic mice.

The function and glucose regulation ability of the human pancreatic islet beta cells were evaluated by OGTT, and FIG. 24 is a schematic diagram showing the glucose regulation ability of the pancreatic islet beta cells in mice of each group. As shown in the figure, after the mice in each group are infused with the gastric glucose solution, the fasting blood glucose value of each group of mice has a severe fluctuation, a blood glucose peak value appears at 30min, and the blood glucose of the mice in the FLP-215 group is reduced at the highest speed from 30min to 120 min; at the end of the OGTT experiment, all mice in FLP-215 group had significantly lower blood glucose than the Model group (p < 0.001). At the same time, the area under the OGTT curve (AUC) was significantly reduced in the FLP-215 group compared to the Model group (p < 0.05, p < 0.01, p < 0.05). Experimental results show that lactobacillus plantarum FLP-215 can significantly improve the glucose tolerance of diabetic mice.

EXAMPLE 10 prevention of Lactobacillus plantarum FLP-215 against insulin resistance in type 2 diabetic mice and pancreatic HE section analysis

(1) After serum was obtained as described above, insulin levels of each group of mice were determined according to the kit instructions, and insulin resistance index (HOMA-IR), insulin sensitivity test index (QUICKI) and beta cell function (HOMA-beta) were calculated according to the formulas as follows: HOMA-IR = FBG x pins/22.51; quacki=1/(lgFBG + lgFINS); HOMA- β=20×pins/(FBG-3.5).

(2) Experimental results

Insulin is the only hormone known to the body to lower blood glucose and insulin resistance is a prominent feature of type 2 diabetes. To further evaluate insulin resistance, figures 25-28 are insulin, insulin resistance index, beta cell function, and insulin sensitivity detection index for each group of mice. As shown, the insulin level was significantly higher in the Model group than in the Control group (p < 0.0001) and FLP-215 group (p < 0.0001). After 6 weeks of Lactobacillus plantarum prophylaxis, the Model group had significantly higher insulin resistance index than the Control group (p < 0.0001); at the beta 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 insulin sensitivity detection index, the Model group was significantly lower than the Control group (p < 0.0001) and the FLP-215 group (p < 0.001). These results indicate that lactobacillus plantarum FLP-215 plays an important role in slowing the rise of insulin in diabetic mice, slowing the rise of insulin resistance, preventing the decline of beta cell function, and preventing the decline of insulin sensitivity detection index.

To further explore changes in islet tissue in diabetic mice, we performed HE slice analysis of the pancreas of the mice. Fig. 29 is a schematic diagram of HE section analysis of pancreas of each group of mice, islet cells of Control group mice are in a cluster rope shape, the islet and peripheral tissue border is clear, cells in the islets are dense, and the arrangement is ordered. The islet structure of Model mice was significantly impaired, the number of cells within the islets was significantly reduced and the arrangement was irregular, the islet tissue was atrophic, and there were vacuoles and inflammatory cell infiltrates inside. After 6 weeks of probiotic intervention, the FLP-215 group effectively delays the damage of islet structures of the diabetic mice and slows down the atrophy of islet tissues and the cavitation of islet cells.

EXAMPLE 11 determination of Lactobacillus plantarum FLP-215 Biochemical index on type 2 diabetic mice

(1) Serum glucagon-like peptide (GLP-1), triglyceride (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), interleukin 6 (IL-6) were measured using a kit provided by Shanghai Yu Biotechnology Co., ltd. And liver tissue Malondialdehyde (MDA), superoxide dismutase (SOD), reduced Glutathione (GSH) were measured using a kit provided by Shanghai Yu Biotechnology Co., ltd.

(2) Experimental results

Previous studies have shown that type 2 diabetics have a relatively disturbed lipid metabolism. Thus, FIGS. 30-35 are schematic diagrams of serum triglycerides, total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, free fatty acid levels, and atherosclerosis index, respectively, for each group of mice. The significant increase in total cholesterol, triglycerides, low density lipoprotein cholesterol and free fatty acids in the Model group serum compared to Control group, while the significant decrease in high density lipoprotein cholesterol (p < 0.0001) indicates a significant decrease in lipid metabolism in Model group mice after a 6 month high fat diet. Compared to the Model group, the FLP-215 group showed a significant decrease in total cholesterol, triglycerides, low density lipoprotein cholesterol and free fatty acids, while the high density lipoprotein cholesterol was significantly increased (p < 0.01). Studies have shown that Lactobacillus plantarum FLP-215 is effective in preventing the lipid production of diabetic mice.

To further explore the protective effect of probiotics on inflammation in diabetic mice, we examined interleukin-6 and tumor necrosis factor-alpha in the serum of mice. Fig. 36-37 are graphs showing the levels of interleukin-6 and tumor necrosis factor- α in the serum of mice from each group, and it can be seen that the levels of interleukin-6 and tumor necrosis factor- α in the serum of mice from the Model group were significantly increased (P < 0.0001) and that FLP-215 was significantly decreased (P < 0.01) compared to the Model group.

EXAMPLE 12 analysis and evaluation of Lactobacillus plantarum FLP-215 on organ sections of type 2 diabetic mice

(1) Fixing liver tissue, pancreas and colon specimens in 4% neutral paraformaldehyde solution, embedding the samples after dehydration, slicing the samples to be 5um thick, and finally staining the samples with H & E; liver tissue was additionally stained with oil red O and observed for changes in slice morphology.

(2) Experimental results

Long-term high-fat diet induces the occurrence of fatty liver in mice, resulting in the occurrence of liver injury, leading to oxidative stress in the organism. The content of superoxide dismutase, reduced glutathione, glucose-6-phosphatase and malondialdehyde in the liver of the mouse is determined by referring to the specification of ELISA kit after grinding the liver of the mouse.

Superoxide dismutase is an antioxidant enzyme taking free radicals as specific substrates, and can remove organism toxins through combination reaction with the free radicals, so that the risk of damage of the liver by the free radicals is reduced. The activity of superoxide dismutase in the liver is closely related to the health of the body.

FIGS. 38-41 are graphical representations of the levels of superoxide dismutase, reduced glutathione, glucose-6-phosphatase, and malondialdehyde in the liver of mice in each group. As shown in the figure, the superoxide dismutase enzyme activity of the Model group mice showed a significant decrease (P < 0.0001) compared with the Control group mice, and the decrease trend of the superoxide dismutase enzyme activity of the FLP-215 group was effectively controlled and the activity was close to the normal level (P < 0.001) after 6 months of probiotic prevention.

Glutathione helps to maintain normal immune system function, and has antioxidant and integral detoxification effects. Compared to Control mice, model mice showed a significant decrease in glutathione activity (P < 0.0001), and after 6 months of probiotic prophylaxis, no significant difference was observed in FLP-215 compared to Model mice (P > 0.05).

Glucose-6-phosphatase releases glucose into the blood by hydrolysis of glucose-6-phosphate release in liver tissue, so that liver glycogen can supplement blood glucose upon starvation, maintaining blood glucose balance. It follows that this enzyme is a key enzyme for gluconeogenesis. Compared with Control mice, the G6P enzyme activity of Model mice shows a significant reduction (P < 0.0001), and the reduction trend of the G6P enzyme activity of FLP-215 mice is effectively controlled and the activity is close to the normal level (P < 0.001) after 6 months of probiotic prevention.

The liver malondialdehyde content was significantly increased in Model mice (P < 0.0001) compared to Control mice, and MDA content was significantly reduced in FLP-215 mice (P < 0.000) compared to Model mice. In conclusion, lactobacillus plantarum FLP-215 can reduce oxidative stress injury of T2DM mice.

Meanwhile, we calculated liver index from liver weight and body weight of each group of mice, which is obtained by using the mass (mg)/body weight (g) ×100% of the liver of the mice. Normal liver index is between 0 and 40, with high liver index generally indicating liver damage. FIG. 42 is a graph showing the liver index of each group of mice, wherein the liver index of Model group mice is significantly increased and significantly different (P < 0.01) from that of Control group mice; the liver index was significantly reduced (P < 0.05) in the FLP-215 group compared to the Model group.

The liver plays an important role in fat metabolism. To further explore the fat accumulation and liver damage of diabetic mice, we performed HE-stained and oil red O-stained sections of the mouse liver. FIG. 43 is a HE stained slice of the liver of each group of mice, and FIG. 44 is an O stained slice analysis of the liver of each group of mice, showing that the liver cells of the Model group of mice showed edema necrosis, severe fatty vacuoles and inflammatory cell infiltration, and liver cell arrangement disorder, as compared to the Control group of mice; liver oil red O staining sections further show that a large amount of fat is accumulated in the liver of the model mouse, and the area of red fat drops is obviously increased; liver fat accumulation and red lipid drop area were significantly reduced in the FLP-215 group compared to the Model group.

EXAMPLE 13 type 2 diabetes mouse fecal microorganism metagenome Shutgun sequencing

(1) Metagenome Shutgun was sequenced from each group of 6 mouse fecal samples, and the post-genomic DNA was extracted from the fecal samples using QIAGENDNAMINI-Kit (QIAGEN, hilden, germany) and homogenized by bead beating. The concentration and integrity of the DNA was then checked by 0.8% agarose gel electrophoresis. Sequencing work is carried out by Beijing vitamin Tonglihua gene technology Co., ltd, raw data is processed by a sliding window method, low-quality sequences in the raw sequences are removed, high-quality clean readings are obtained, and finally SFIFLE software is used for cutting and modifying the readings.

(2) Experimental results

Previous studies have shown that biochemical indicators in type 2 diabetic mice are closely related to intestinal microflora. Thus, we examined and analyzed the effects of four probiotics on species composition and metabolic pathways of type 2 diabetic mouse intestinal microorganisms by metagenomic sequencing. Through careful screening and alignment we identified 6 phylum, 14 classes, 15 orders, 28 families, 43 genera, 80 species and 366 absolute metabolic pathways altogether. We expressed the alpha diversity of the intestinal microorganisms of each group of mice by Shannon index. FIG. 45 is a graph showing that the Shannon index of the enteric microorganisms of each group of mice is significantly reduced (P < 0.05) compared to the Control group; whereas the FLP-215 group showed statistical differences compared to the Model group and was close to the Control group (P < 0.05). Meanwhile, the analysis of beta diversity at the seed level showed that 3 groups separated on the PCoA map, with Control group and FLP-215 group being far from the other groups, respectively. At the species level, we use a heat map to reveal the species level of intestinal microorganisms in each group of mice. FIG. 46 is a graph showing that the levels of species of intestinal microorganisms in mice of each group, and that the ingestion of FLP-215 significantly increases the abundance of Lactobacillus pentosus, lactobacillus-del brueckii, leuconostoc mesenteroides, and Lactobacillus lactis in the intestinal tract of mice of FLP-215, and decreases the abundance of Bifido bacterium _ pseudolongum, prevotella _sp_MGM1, lactobacillus reuter, lachnospiraceae _bacteria_3_2, and Lachnospiraceae _bacteria_COE1 (P < 0.05) compared to Model groups.

In terms of metabolic pathways, we screened 22 metabolic pathways with the greatest differences (fig. 47). The Model groups were each up-regulated polyisoprenoid biosynthesis(E.coli)、superpathwayofL-methioninebiosynthesis(bysulfhydrylation)、glycogendegradationII、starchbiosynthesis、sucrosebiosynthesisII、homolacticfermentation、superpathwayofL-cysteinebiosynthesis(mammalian)、purinenucleotidesdegradationII(aerobic)、L-argininebiosynthesisI(viaL-ornithine)、L-argininebiosynthesisII(acetylcycle)、L-ornithinebiosynthesisI、L-histidinebiosynthesis、purineribonucleosidesdegradation、superpathwayofL-aspartateandL-asparaginebiosynthesis、L-lysinebiosynthesisI、methylerythritolphosphatepathwayI、D-galactosedegradationI(Leloirpathway)、guanosinenucleotidesdegradationII、guanosinenucleotidesdegradationI、superpathwayofpurinedeoxyribonucleosidesdegradation, down-regulated tRNAprocessing compared to the Control groups. Notably, the intake of FLP-215 was down-regulated L-argininebiosynthesisI(viaL-ornithine)、L-argininebiosynthesisII(acetylcycle)、L-ornithinebiosynthesisI、L-histidinebiosynthesis、phospholipases、superpathwayofL-methioninebiosynthesis(bysulfhydrylation)、sucrosebiosynthesisII、purineribonucleosidesdegradation, and up-regulated tRNAprocessing. Overall, FLP-215 intake significantly altered specific metabolic pathways of gut microorganisms in diabetic mice.

To reveal the possible mechanism of probiotics for alleviating type 2 diabetes, we performed correlation analysis of FLP-215, intestinal bacteria, metabolic pathways of intestinal microorganisms and various biochemical indexes, we analyzed the experimental data by using Pearson correlation coefficients, and constructed a correlation network graph of the same. Based on PearsonCC value comparisons, correlation analysis was performed on FLP-215, flora, intestinal metabolites, metabolic pathways and physicochemical indexes. FIG. 48 is a correlation analysis of FLP-215, flora, intestinal metabolites, metabolic pathways and physicochemical indicators, as shown in the figure, FLP-215 uptake can up-regulate Lactobacillusdelbrueckii abundance, down-regulate Bifidobacteriumpseudolongum and PrevotellaspMGM1 abundance, while Lactobacillusdelbrueckii promotes fatty acid recovery, mixed acid fermentation, mixed lactic acid fermentation, sucrose degradation (sucrose invertase), sucrose anaerobic degradation superpathway, glucose and pentose degradation superpathway, glycolysis superpathway up-regulation. These pathways all promote the secretion of acetic acid, and acetic acid is inversely related to these physiological indexes FBG, FINS, TG, TC, LDL-C, FFA, TNF-alpha, IL-6 and MDA, and positively related to GLP-1 and HDL-C, SOD, GSH. It was demonstrated that FLP-215 group can modulate these metabolic pathways to produce acetic acid by altering the intestinal flora, thereby improving the glycolipid metabolic capacity of mice.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. The lactobacillus plantarum (Lactobacillus plantarum) FLP-215 is characterized in that the lactobacillus plantarum FLP-215 is stored in the China general microbiological culture Collection center (CGMCC) with the preservation number of 28337 at the 9 th month 4 of 2023.

2. Use of lactobacillus plantarum FLP-215, or a bacterial suspension thereof, according to claim 1, for the preparation of a product for the prevention and/or treatment of ulcerative colitis, diabetes and/or for the reduction of organ damage.

3. The use according to claim 2, wherein the ulcerative colitis is dextran sodium sulfate induced ulcerative colitis.

4. The use according to claim 2, wherein said preventing and/or treating ulcerative colitis comprises at least one of: improving intestinal mucosa barrier, relieving inflammatory reaction or regulating intestinal microbiota structure.

5. The use of claim 4, wherein the reducing the inflammatory response is one or more of increasing expression of IL-10, decreasing expression of IL-1 β, decreasing expression of IL-6, decreasing expression of IFN- γ, decreasing expression of TNF-a, or decreasing expression of IL-17A.

6. The use according to claim 4, wherein the modulation of gut microbiota structure is one or more of promoting the growth of probiotics in the gut microbiota, increasing the abundance of SCFAs-producing species, increasing gut microbiota diversity or inhibiting the growth of pathogenic bacteria in the gut microbiota; the improvement of intestinal mucosa barrier is to promote the expression of MUC-2 protein and/or ZO-1 protein.

7. The use according to claim 2, wherein the prevention and/or treatment of diabetes comprises at least one of: increasing the key pathway of glycolipid metabolism, reducing fasting blood glucose, improving glucose intolerance, reducing insulin content in serum, or increasing glucagon-like peptide-1 content in serum.

8. The use of claim 2, wherein the organ injury is one or more of liver injury, pancreatic injury, or colon injury.

9. A microbial agent comprising lactobacillus plantarum FLP-215 and/or a bacterial suspension thereof according to claim 1.

10. Use of the microbial agent of claim 9 for the preparation of a product for preventing and/or treating ulcerative colitis, diabetes and/or reducing organ damage.