Lactobacillus reuteri HNU872 and application thereof in preparation of intestinal inflammation related products

Abstract

The invention provides lactobacillus reuteri HNU872 and application of the lactobacillus reuteri HNU872 in preparation of intestinal inflammation related products. The invention discloses how the Lactobacillus reuteri HNU872 effectively alleviates the phenotype of DSS-induced mouse enteritis. The invention also discloses a preparation method of the Lactobacillus reuteri HNU872. Research results show that the Lactobacillus reuteri HNU872 can significantly relieve weight loss caused by enteritis, improve colon length, significantly improve the level of anti-inflammatory cytokines in serum and reduce the concentration of pro-inflammatory cytokines. In addition, the expression of the intestinal tight junction protein ZO-1 is obviously promoted. The lactobacillus reuteri HNU872 can regulate diversity and structure of intestinal microbiota of mice, inhibit enrichment of pathogenic bacteria and relieve inflammatory response. According to the invention, the action mechanism of the lactobacillus reuteri HNU872 in improving enteritis is clarified, and a solid scientific basis is provided for developing a microbial product based on probiotics as an alternative treatment strategy for ulcerative colitis.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to Lactobacillus reuteri HNU872 and application thereof in preparing intestinal inflammation related products. Background Art

[0002] Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, has become one of the chronic diseases that seriously threaten human health with its increasing incidence worldwide. Currently, millions of people worldwide are affected by IBD, and its incidence is still rising year by year. Studies have shown that IBD patients have a 1.4 to 1.7 times increased risk of colorectal cancer compared with the general population, further increasing the burden of the disease. Clinical symptoms of IBD usually include recurrent diarrhea, abdominal pain, intestinal bleeding / bloody stools, fever, weight loss, and fatigue, which seriously affect the patient's quality of life and social function. However, despite a large number of studies devoted to revealing the etiology and pathogenesis of IBD, its exact pathogenic mechanism has not yet been fully clarified. Current studies indicate that the occurrence of IBD is closely related to multiple factors, including genetic susceptibility, immune system abnormalities, external environmental stress, and dysbiosis of the intestinal microbiota. Significant imbalances in the intestinal microbiome have been widely reported in IBD patients. Healthy individuals usually have a rich, diverse, and functionally coordinated intestinal microbiota, but in IBD patients, this balance is broken. The number of beneficial microbial populations, such as Bifidobacterium, Lactobacillus, Roseburia, and Faecalibacterium prausnitzii, was significantly reduced, while the abundance of some harmful microorganisms, such as Ruminococcus palatineus, Fusobacterium nucleatum, pathogenic Escherichia coli, and enterotoxigenic Bacteroides fragilis, was significantly increased. This imbalance of the microbiome not only exacerbates the intestinal inflammatory response, but may also promote further progression of the disease. Therefore, intervention strategies targeting the intestinal microbiome have become an important direction for the treatment of IBD.

[0003] In recent years, the potential of probiotics in regulating the intestinal microbiome has received increasing attention. Probiotics can not only regulate the balance of the intestinal microbiome by inhibiting the growth of harmful microorganisms and enhancing the number of beneficial microorganisms, but also reduce intestinal inflammatory responses by maintaining the integrity of the intestinal barrier, blocking the production of proinflammatory cytokines, and promoting the production of beneficial metabolites. In addition, as a natural intervention, probiotics have the significant advantages of low cost and no side effects, which makes it a promising option for the treatment of IBD. Summary of the invention

[0004] Based on the above, the present invention provides Lactobacillus reuteri HNU872 and its use in the preparation of intestinal inflammation related products. This application takes Lactobacillus reuteri HNU872 as the research object, and systematically explores its preventive and alleviating effects on DSS-induced enteritis in a mouse model, hoping to provide new insights into the probiotic intervention strategy for IBD and lay the foundation for the development of more effective IBD treatment plans in the future.

[0005] In order to achieve the technical purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides a strain of Lactobacillus reuteri HNU872, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on August 30, 2024, with a deposit number of GDMCC NO: 65076, and a deposit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, China, and a taxonomic name: Limosilactobacillus reuteri.

[0007] The present invention also provides the use of the Lactobacillus reuteri HNU872 in the preparation of intestinal inflammation related products.

[0008] Preferably, the Lactobacillus reuteri HNU872 can significantly reduce weight loss and colon shortening caused by colitis.

[0009] Preferably, the Lactobacillus reuteri HNU872 is capable of improving colon lesions and repairing the intestinal barrier.

[0010] Preferably, the Lactobacillus reuteri HNU872 can significantly inhibit the level of pro-inflammatory cytokines and increase the level of anti-inflammatory cytokines; the pro-inflammatory cytokines include TNF-α, IL-1β and IFN-γ, and the anti-inflammatory cytokines include IL-10.

[0011] Preferably, the Lactobacillus reuteri HNU872 can regulate the intestinal flora disorder caused by colitis and regulate the diversity and structure of the intestinal microbial flora.

[0012] Preferably, Lactobacillus reuteri HNU872 is capable of inhibiting the enrichment of pathogenic bacteria and the decrease in the relative abundance of beneficial bacteria caused by colitis.

[0013] Preferably, Lactobacillus reuteri HNU872 is capable of significantly downregulating pro-inflammatory metabolic pathways in colon tissue.

[0014] More preferably, the product comprises a probiotic agent and a medicament.

[0015] More preferably, the intestinal inflammation comprises inflammatory bowel disease IBD.

[0016] The beneficial effects of the present invention are:

[0017] The present invention reveals how Lactobacillus reuteri HNU872 effectively alleviates the enteritis phenotype of mice induced by dextran sulfate sodium (DSS) through shotgun metagenomic sequencing and RNA transcriptome sequencing. The results show that Lactobacillus reuteri HNU872 can significantly alleviate the weight loss caused by enteritis, improve colon length, significantly increase the level of anti-inflammatory cytokines in serum, and reduce the concentration of pro-inflammatory cytokines. In addition, Lactobacillus reuteri HNU872 also significantly promotes the expression of intestinal tight junction protein ZO-1. Metagenomic and transcriptome sequencing results further show that Lactobacillus reuteri HNU872 can regulate the diversity and structure of mouse intestinal microbiota, inhibit the enrichment of pathogenic bacteria Mucispirillumschaedler and Escherichia coli, and alleviate inflammatory response. The invention clarifies the mechanism of action of Lactobacillus reuteri HNU872 in improving enteritis, and provides a solid scientific basis for the development of probiotic-based microbial products as an alternative treatment strategy for ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shown are the changes in body weight (A) and colon length (B) of mice in different groups.

[0019] Figure 2 Shown are the results of hematoxylin-eosin-stained paraffin sections (A), ZO-1 antibody staining (B), and integrated fluorescence signals (C) of mice in different groups.

[0020] Figure 3 Shows the measurement results of immune factor indicators in the serum of mice in different groups.

[0021] Figure 4 The Simpson index and Shannon index (A), principal coordinate analysis results of the Bray-Curtis distance (B), and Bray-Curtis distance (C) of the intestinal microbiota of mice in different groups are shown.

[0022] Figure 5 The species annotation results of mice in different groups are shown.

[0023] Figure 6 Shows the colon transcriptome results of mice in different groups (A) and enrichment analysis results (B). DETAILED DESCRIPTION

[0024] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0025] Example

[0026] 1. Materials and Methods

[0027] 1.1 Bacterial culture

[0028] A strain of Lactobacillus reuteri HNU872, which was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on August 30, 2024, with the deposit number GDMCC NO: 65076, and the deposit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, China, taxonomic name: Limosilactobacillus reuteri.

[0029] Limosilactobacillus reuteri HNU872 was cultured on MRS agar medium at 37°C for 24 hours. The colonies were milky white, 0.1-0.2 cm in diameter, with a smooth, moist, raised surface and neat edges. They were rod-shaped when observed under an optical microscope.

[0030] 1.2 Experimental animals and in vivo experimental design

[0031] C57BL / 6J male mice (9 weeks old) were obtained from Sibeifu (Beijing) Biotechnology Co., Ltd. and were kept under specific pathogen-free conditions with a 12-h daylight cycle, a temperature of 22±2°C, and a relative humidity of 55%±10%. During the feeding phase, mice were provided with ad libitum diet and water. The mice were randomly divided into 3 groups, 6 in each group, namely: ① control group (normal drinking water, daily gavage with sterile PBS); ② model group (drinking water containing 2% DSS, daily gavage with sterile PBS); ③ probiotic group (drinking water containing 2% DSS, daily gavage with an equal amount of Lactobacillus reuteri HNU872). The gavage dose of Lactobacillus reuteri HNU872 was 5Log10 CFU (dissolved in sterile PBS). During the entire experiment, mice in each group were fed the same feed, and their body weight was measured every 3 days. The experiment lasted for a total of 14 days.

[0032] 1.3 Sample collection and measurement

[0033] At the end of the experiment, mouse feces, colon tissue and serum samples were collected and stored at -80°C until use. Feces were used for metagenomic determination (Beijing Novogene Technology Co., Ltd.), and serum samples were used for determination of immune factor indicators (ELISA kit, Shanghai Xinyu Biotechnology Co., Ltd.). After measuring the length of the colon tissue, it was divided into two parts: one part was rinsed with 0.85% saline and fixed in a paraformaldehyde solution, and then used to determine the colon tissue section (Wuhan Sevier Biotechnology Co., Ltd.). The other part was quickly frozen with liquid nitrogen and stored at -80°C for determination of colon tissue immunofluorescence (Wuhan Sevier Biotechnology Co., Ltd.). The fixed colon tissue was embedded in paraffin and then stained with hematoxylin-eosin (H&E) or ZO-1 antibody for analysis (anti-ZO1 tight junction protein rabbit pAb, Wuhan Sevier Biotechnology Co., Ltd.). After incubation with the primary antibody, the corresponding secondary antibody was added, and after incubation, DAPI (DAPI staining reagent, Wuhan Sevier Biotechnology Co., Ltd.) was used to counterstain the cell nucleus. Finally, images were collected after sealing, and the integrated fluorescence signal of each image was calculated using ImageJ software (v1.54g) (the threshold and image size settings were kept consistent).

[0034] 1.4 Fecal DNA extraction and high-throughput metagenomic sequencing

[0035] Fecal metagenomic DNA was extracted using the QIAamp DNA stool mini kit (Qiagen) following the manufacturer's protocol. The quality of genomic DNA was determined using agarose gel electrophoresis. Shotgun metagenomic sequencing was performed using the Illumina novaseq 6000 platform. Paired-end reads of 150 bp were generated. The raw sequencing reads were quality filtered and trimmed to remove the host genome. For metagenomic species annotation, qualitative and quantitative analysis of bacterial species was performed using metaphlan software to obtain relative abundance at the species level.

[0036] 1.5 Colon tissue transcriptome analysis

[0037] The quality control of the sequence data was performed using Fastp (v0.23.1) software. The sequencing data were aligned to the mouse genome using STAR software (v2.7.8a), and the gene expression levels were quantified using Features software (v2.0.6).

[0038] 1.6 Statistical analysis

[0039] Data were statistically analyzed using R software. Box plots were generated using the “ggplot2” package. The alpha diversity of the microbiome and the similarity between samples were calculated using the “vegan” package. Principal coordinate analysis was performed based on the Bray-Curtis distance. Histograms were prepared using GraphPad 8.0 statistical software, and values ​​are expressed as mean ± SEM. The Wilcoxon rank sum test was used to calculate the significance of the differences, *P < 0.05, **P < 0.01, ***P < 0.001.

[0040] 2. Results

[0041] 2.1 Supplementation with Lactobacillus reuteri HNU872 significantly alleviated weight loss and colon shortening in mice with DSS-induced colitis

[0042] DSS-induced colitis mice were used to evaluate the effect of Lactobacillus reuteri HNU872 supplementation by recording the weight and colon length of the mice. During the experiment, the weight of the mice in the model group gradually decreased and was significantly lower than that of the control group ( Figure 1 A). In contrast, supplementation with Lactobacillus reuteri HNU872 significantly prevented DSS-induced weight loss and was significantly higher than the model group. Moreover, on day 14, it was found that DSS treatment significantly shortened the colon length of mice, while supplementation with Lactobacillus reuteri HNU872 significantly increased the colon length of mice ( Figure 1 B).

[0043] 2.2 Supplementation of Lactobacillus reuteri HNU872 improves colonic lesions and repairs the intestinal barrier in mice with DSS-induced colitis

[0044] The results of paraffin sections stained with hematoxylin and eosin showed that the intestinal tissue of the mice in the control group had normal morphology, clear structure, intact mucosa, and no obvious inflammatory cell infiltration. However, the intestinal tissue of the mice in the model group was almost completely ulcerated, with no obvious intestinal gland structure ( Figure 2 A). However, supplementation with Lactobacillus reuteri HNU872 significantly improved the pathological changes in the intestinal tissue of mice. Although small ulcers were observed, the intestinal gland structure was still maintained and the mucosal epithelium was intact. In addition, the expression of ZO-1 protein in the control group was significantly higher than that in the model group, while supplementation with Lactobacillus reuteri HNU872 significantly increased the expression of ZO-1 ( Figure 2 B and C). These results further indicate that supplementation with Lactobacillus reuteri HNU872 can improve DSS-induced intestinal tissue structural damage and repair the mechanical barrier of intestinal epithelial cells.

[0045] 2.3 Supplementation of Lactobacillus reuteri HNU872 can improve inflammation in mice with DSS-induced colitis

[0046] The levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon-γ (IFN-γ) and anti-inflammatory cytokine interleukin-10 (IL-10) in mouse serum showed that the levels of pro-inflammatory cytokines in the model group mice were significantly increased and the levels of anti-inflammatory cytokines were significantly decreased after DSS treatment ( Figure 3 ). However, supplementation with Lactobacillus reuteri HNU872 significantly suppressed the levels of pro-inflammatory cytokines and increased the levels of anti-inflammatory cytokines. These results suggest that supplementation with Lactobacillus reuteri HNU872 can regulate host immunity and inhibit the level of inflammation caused by DSS treatment.

[0047] 2.4 Supplementation of Lactobacillus reuteri HNU872 can regulate intestinal flora disturbance in DSS-induced colitis mice

[0048] From the perspective of microbiome richness, DSS treatment led to a significant decrease in the Simpson index and Shannon index of the intestinal microbiome of mice, while supplementation with Lactobacillus reuteri HNU872 significantly increased the richness of the intestinal microbiome of mice ( Figure 4 A). The results of principal coordinate analysis based on Bray-Curtis distance showed that DSS treatment caused the structure of the intestinal microbiome of mice to move away from the control group, while supplementation with Lactobacillus reuteri HNU872 could inhibit the structural deviation of the microbiome ( Figure 4 B). Further quantification of the Bray-Curtis distance showed that the probiotic group had a more similar structure to the control group ( Figure 4 C). Therefore, supplementation with Lactobacillus reuteri HNU872 could regulate the disordered intestinal microbiome structure induced by DSS treatment.

[0049] 2.5 Supplementation of Lactobacillus reuteri HNU872 can inhibit the enrichment of pathogenic bacteria caused by DSS treatment

[0050] Based on the species annotation results, DSS treatment significantly enriched pathogenic bacteria such as Mucispimllumschaedler and Escherichia coli in the mouse intestine, while supplementation with Lactobacillus reuteri HNU872 could significantly downregulate their relative abundance. In addition, the relative abundance of some beneficial species, such as Limosilactobacillus reuteri and Faecalibaculum rodentium, decreased significantly after DSS treatment, while in the probiotic group, the relative abundance of these beneficial species was significantly upregulated ( Figure 5 ).

[0051] 2.6 Supplementation of Lactobacillus reuteri HNU872 can significantly downregulate the pro-inflammatory metabolic pathways in the colon tissue of mice

[0052] The reasons why probiotics resist IBD were explored. The results of mouse colon transcriptome showed that among the 35 genes with significant differences between the probiotic group and the model group, 27 genes were significantly downregulated in the probiotic group, and 8 genes were significantly upregulated ( Figure 6 A). Enrichment analysis of 27 genes significantly downregulated in the probiotic group showed that these genes were involved in pathways related to inflammatory response, neutrophil chemotaxis, innate immune response, antigen processing and presentation, and negative regulation of immune system processes ( Figure 6 B), which indicated that the inflammatory environment in the model group was more severe and that the inflammation was significantly alleviated by probiotic supplementation.

[0053] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus reuteri HNU872 was deposited in the Guangdong Provincial Microbiological Culture Collection Center GDMCC on August 30, 2024, with the deposit number GDMCC NO: 65076. The deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, China. The taxonomic name is Limosilactobacillus reuteri.

2. Use of Lactobacillus reuteri HNU872 according to claim 1 in preparing products related to intestinal inflammation.

3. The use according to claim 2, characterized in that: The Lactobacillus reuteri HNU872 can significantly reduce the weight loss and colon shortening caused by colitis.

4. The use according to claim 2, characterized in that: The Lactobacillus reuteri HNU872 can improve colon lesions and repair the intestinal barrier.

5. The use according to claim 2, characterized in that: The Lactobacillus reuteri HNU872 can significantly inhibit the level of pro-inflammatory cytokines and increase the level of anti-inflammatory cytokines; the pro-inflammatory cytokines include TNF-α, IL-1β and IFN-γ, and the anti-inflammatory cytokines include IL-10.

6. The use according to claim 2, characterized in that: The Lactobacillus reuteri HNU872 can regulate the intestinal flora disorder caused by colitis and regulate the diversity and structure of the intestinal microbial flora.

7. The use according to claim 2, characterized in that: The Lactobacillus reuteri HNU872 can inhibit the enrichment of pathogenic bacteria and the decrease in the relative abundance of beneficial bacteria caused by colitis.

8. The use according to claim 2, characterized in that: The Lactobacillus reuteri HNU872 can significantly downregulate the pro-inflammatory metabolic pathways in colon tissue.

9. The use according to any one of claims 2 to 8, characterized in that: The products include probiotics and medicines.

10. The use according to any one of claims 2 to 8, characterized in that: The intestinal inflammation includes inflammatory bowel disease IBD.

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