Lactobacillus johnsonii for modulating the endocannabinoid system in the colon and uses thereof

Abstract

This invention discloses a strain of *Lactobacillus johnsonii* that regulates the colonic endocannabinoid system and its applications, belonging to the field of microbial technology. This invention is the first to discover and verify that *Lactobacillus johnsonii* strain LJEH8 can significantly promote the expression of DGLβ in colonic epithelial cells and colonic tissue, thereby effectively regulating the colonic endocannabinoid system. This strain provides a new potential target and microbial resource for the prevention and intervention of intestinal diseases, and has good application prospects. Animal experiments show that in a model of intestinal flora disorder induced by combined antibiotic treatment, colonization of *Lactobacillus johnsonii* LJEH8 can significantly restore colonic DGLβ protein levels, confirming its stable and effective regulatory function in vivo.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Lactobacillus johnsonii that regulates the colonic endocannabinoid system and its applications. Background Technology

[0002] Intestinal diseases have an extremely high incidence rate worldwide. Among them, inflammatory bowel disease (IBD), a chronic and relapsing inflammatory bowel disease, is characterized by its prolonged and difficult-to-cure course, severely impacting patients' quality of life and significantly increasing the risk of colorectal cancer. Developing safe and effective new intervention strategies has become an urgent need in the field of intestinal disease prevention and treatment.

[0003] Endocannabinoids are widely present in the digestive system and play an important role in maintaining gastrointestinal homeostasis and regulating the progression of related diseases. Their involvement in the occurrence and development of diseases such as inflammatory bowel disease is often accompanied by the remodeling of the intestinal biological barrier, suggesting that the development and utilization of gut microbiota targeting the endocannabinoid system may be a potential therapeutic strategy for alleviating inflammatory bowel disease.

[0004] In recent years, the interaction between the gut microbiota and the endocannabinoid system has gradually become a research hotspot. Gut microorganisms can regulate the activity of the endocannabinoid system through metabolites or by directly acting on intestinal epithelial cells, thereby affecting gut homeostasis. However, probiotic strains that can specifically target and regulate the colonic endocannabinoid system are still very scarce, and the relevant mechanisms of action are not yet fully understood. Therefore, screening and developing probiotic strains with clear regulatory effects has important theoretical significance and practical value for the prevention and treatment of intestinal diseases. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, this invention provides a strain of *Lactobacillus johnsonii* that regulates the colonic endocannabinoid system and its applications. This strain is safe and non-toxic, effectively maintaining intestinal homeostasis and providing new targets and methods for the prevention and treatment of intestinal diseases such as inflammatory bowel disease.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: A *Lactobacillus johnsonii* strain that regulates the colonic endocannabinoid system is provided. This strain, named LJEH8, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026895 and a deposit date of May 8, 2026. The regulation of the colonic endocannabinoid system refers to promoting the expression of diacylglycerol lipase β in the colon.

[0007] The present invention also provides the use of the above-mentioned Lactobacillus johnsonii strain in the preparation of products for regulating the colonic endocannabinoid system.

[0008] Furthermore, the product is a drug or a functional food.

[0009] The present invention also provides the use of the above-mentioned Lactobacillus johnsonii strain in the preparation of a medicament for the prevention and / or treatment of intestinal diseases.

[0010] Furthermore, the intestinal disease mentioned is inflammatory bowel disease.

[0011] The present invention also provides a microbial agent for regulating the colonic endocannabinoid system, comprising the above-mentioned Lactobacillus johnsonii strain LJEH8 as an active ingredient.

[0012] Furthermore, the viable count of Lactobacillus johnsonii strain LJEH8 in the microbial inoculant was 2 × 10⁸. 8 CFU / g.

[0013] Furthermore, microbial agents may also contain pharmaceutically acceptable carriers or food-acceptable excipients.

[0014] Furthermore, the dosage form of the microbial agent is an oral preparation.

[0015] The beneficial effects of this invention are as follows: This invention is the first to discover and verify that *Lactobacillus johnsonii* strain LJEH8 can significantly promote the expression of DGLβ in colonic epithelial cells and colonic tissue, thereby effectively regulating the colonic endocannabinoid system. This strain provides a new potential target and microbial resource for the prevention and intervention of intestinal diseases, and has good application prospects. Animal experiments show that in a gut microbiota dysbiosis model induced by combined antibiotic treatment, colonization of *Lactobacillus johnsonii* LJEH8 can significantly restore colonic DGLβ protein levels, confirming its stable and effective regulatory function in vivo. Attached Figure Description

[0016] Figure 1 This is a comparison diagram of the effects of different antibiotic treatments on the expression of endocannabinoid system-related metabolic enzyme genes in mouse duodenal and colonic tissues in Example 1. Figure 2 The effect of ampicillin treatment on DGLβ protein expression in mice, as shown in Example 1; Figure 3 This refers to the effect of ampicillin treatment on the levels of DGLβ upstream and downstream metabolites in mouse serum and colon, as shown in Example 1. Figure 4 The effect of ampicillin treatment and fecal microbiota transplantation complementation on DGLβ expression in mouse colon in Example 1; Figure 5 LEfSe analysis of the differences in gut microbiota in mice treated with ampicillin in Example 1; Figure 6 This is a comparison of the regulation of DGLβ protein expression in intestinal epithelial cells by different Lactobacillus strains screened in Example 2. Figure 7To investigate the regulatory effect of Lactobacillus johnsonii strain LJEH8 on the expression of DGLβ protein in MC-38 colon cancer cells; Figure 8 To investigate the regulatory effect of Lactobacillus johnsonii strain LJEH8 on the expression of DGLβ protein in mouse colorectal cancer cells CMT-93; Figure 9 This is a comparison diagram of the effect of Lactobacillus johnsonii LJEH8 colonization on the expression of endocannabinoid system metabolic enzyme genes in the colon of mice treated with compound antibiotics in Example 4. Figure 10 The effect of Lactobacillus johnsonii LJEH8 colonization on DGLβ protein expression in the colon of mice treated with compound antibiotics in Example 4. Detailed Implementation

[0017] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0018] Example 1: Screening and identification of target functional strains By combining antibiotic intervention with gut microbiota sequencing, the key gut microbiota species regulating colonic DGLβ expression were identified, providing a basis for the screening of target strains.

[0019] Specifically, 48 six-week-old SPF-grade female ICR mice were randomly divided into 6 groups (n=8) and treated for 2 weeks with 1 g / L ampicillin (AMP), 1 g / L neomycin (NEO), 0.5 g / L vancomycin (VAN), 1 g / L metronidazole (MET), a mixed antibiotic (ABX), or normal drinking water (CON). The expression of endocannabinoid system-related metabolic enzymes was detected by qRT-PCR and Western blot.

[0020] The qRT-PCR detection process involved collecting duodenal and colonic tissues, extracting RNA from the colonic tissues according to the EZBioscience Total RNA Extraction Kit instructions, and synthesizing cDNA according to the ABclonal Reverse Transcription Kit instructions.

[0021] The reverse transcription reaction system used is shown in Table 1 below; Table 1

[0022] Add the reaction mixture to an RNase-free PCR tube on ice, mix well and centrifuge briefly. Use the following reaction program: 37℃, 15 min; 95℃, 5 s; store at 4℃.

[0023] Using β-actin as an internal reference gene, the relative expression levels of diacylglycerol lipase β (Dglb), diacylglycerol lipase α (Dgla), fatty acid amide hydrolase (Faah), monoacylglycerol lipase (Magl), and N-acylphosphatidylethanolamine-specific phospholipase D (Napepld) were calculated using the 2-(ΔΔCt) method. Metabolic enzymes related to various endocannabinoid systems and... β -The primer sequences for actin are shown in Table 2 below; Table 2

[0024] qRT-PCR test results are as follows Figure 1 As shown, where, Figure 1 The values ​​a through e in the figures represent the expression levels of diacylglycerol lipase β (Dglb), diacylglycerol lipase α (Dgla), fatty acid amide hydrolase (Faah), monoacylglycerol lipase (Magl), and N-acylphosphatidylethanolamine-specific phospholipase D (Napepld) in mouse duodenal (Duodenum) and colon (Colon) tissues, respectively. Figure 1 It was found that the expression of endocannabinoid system-related enzymes differed in different intestinal segments. After ampicillin treatment, the expression of DGLβ decreased in different intestinal segments, with the most significant difference observed in the colon tissue. Therefore, Western blot analysis was performed on mice treated with ampicillin and mice that drank normal water.

[0025] Western blot analysis was performed as follows: Cell and tissue samples were lysed using RIPA lysis buffer (P0013B, Beyotime). Protein concentration was determined using a BCA protein assay kit (P0012, Beyotime). Equal volumes of protein were loaded for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred from the gel to a PVDF membrane and blocked with 5% skim milk powder (P0216, Beyotime) for 2 hours. The membrane was then co-incubated with primary and secondary antibodies and developed using a chemiluminescent reagent. The optical density of the signal on the film was quantitatively analyzed using ImageJ software. Unless otherwise specified, β-actin was used as an internal control protein. Primary antibody information is shown in Table 3 below. Table 3

[0026] Information on secondary antibody is shown in Table 4 below; Table 4

[0027] Western blot results are as follows Figure 2 As shown, by Figure 2The results of Western blot analysis were consistent with those of qRT-PCR analysis, indicating that DGLβ is a core target of gut microbiota in regulating the endocannabinoid system, and its expression is mainly regulated by ampicillin-sensitive bacteria.

[0028] To further verify the effect of ampicillin-associated microbes on DGLβ expression, the contents of arachidonic acid (AA) and 2-arachidonic glycerol (DAG) upstream and downstream of DGLβ were measured. Serum and colon tissue samples from mice in the ampicillin group and the normal drinking water group were used for metabolomics analysis. The results are as follows: Figure 3 As shown, where, Figure 3 The left side of 'a' in the figure shows a comparison of the 2-arachidonic glycerol content in the colon tissue of two groups of mice. Figure 3 The right side of 'a' in the figure shows a comparison of the arachidonic acid content in the colon tissue of two groups of mice. Figure 3 The left side of b in the figure shows the DAG (16∶1 / 18∶2) content in the serum of two groups of mice, which is a comparison of the content of 2-arachidonic glycerol linked to one molecule of palmitoleic acid (16 carbons, 1 double bond) and one molecule of linoleic acid (18 carbons, 2 double bonds). Figure 3 The middle 'b' represents a comparison of the DAG (16∶2 / 18∶2) content in the serum of two groups of mice, i.e., the content of 2-arachidonic glycerol linked to two molecules of linoleic acid (18 carbons, 2 double bonds). Figure 3 The right side of b in the figure shows a comparison of the arachidonic acid content in the serum of the two groups of mice.

[0029] Depend on Figure 3 It is evident that ampicillin, by eliminating specific gut microbiota, not only alters the gene and protein expression of DGLβ, but also substantially affects the metabolic flux of endocannabinoids, demonstrating that the regulation of this system by gut microbiota is functional, rather than simply gene expression regulation.

[0030] To further confirm the regulatory role of ampicillin-associated flora on DGLβ expression, mice were treated with 1 g / L ampicillin in their drinking water for 2 weeks (AMP), and a fecal microbiota transplantation (FMET) complement group (AMP+CC) was established. Colon samples were collected at 9 weeks of age and analyzed by quick-return sampling (qRT). PCR and Western blot were used to detect the expression level of DGLβ.

[0031] See results Figure 4 ,in, Figure 4 'a' in qRT PCR test results Figure 4 In this context, 'b' represents the Western blot detection result, derived from... Figure 4It was found that, compared with the control group (CON), ampicillin treatment significantly reduced the transcription and protein levels of DGLβ in the mouse colon; while after transplantation of colon contents from normal mice, the expression level of DGLβ significantly increased, suggesting that ampicillin-associated gut microbiota are involved in regulating DGLβ expression.

[0032] As shown above, ampicillin-associated microbes are related to changes in DGLβ in endogenous cannabinoids. Further research was conducted to investigate the impact of ampicillin-associated microbes on DGLβ expression. 16S rRNA amplicon sequencing was performed on colon contents samples from both the control and ampicillin groups to analyze differences in gut microbiota structure and its correlation with DGLβ expression levels.

[0033] The results are as follows Figure 5 As shown, by Figure 5 It was found that, compared with the control group (CONCC), the abundance of Lactobacillus johnsonii and Lactobacillus reuteri in the intestine of mice treated with ampicillin (AMPCC) was significantly reduced; correlation analysis showed that the abundance of Lactobacillus johnsonii was significantly positively correlated with the expression level of DGLβ.

[0034] Example 2: Methods for the isolation and identification of Lactobacillus johnsonii strains Feces were collected from ordinary ICR mice, and 1 mL of PBS was added to every 100 mg of feces to prepare a fecal microbiota suspension; the fecal microbiota suspension was diluted to 1×10⁻⁶. -5 1×10 -6 and 1×10 -7 Three gradients were applied and inoculated onto MRS solid agar plates, and cultured at 37°C for 48 h. Single colonies were picked and repeatedly streaked onto MRS plates for purification, resulting in multiple purified strains. The 16S rRNA gene of the selected strains was amplified and sequenced, and BLAST comparison with the NCBI database confirmed that all purified strains were *Lactobacillus johnsonii*, numbered LJEH3, LJEH5, LJEH6, LJEH7, LJEH8, LJEH10, LJEH13, and LJEH16.

[0035] Example 3: Verification of in vitro upregulation of DGLβ expression by Lactobacillus johnsonii LJEH8 Mouse colon cancer cells MC-38 were seeded into 12-well plates. When the cell confluence reached 80%, a bacterial suspension (MOI=100∶1) prepared from Lactobacillus johnsonii selected in Example 2 was added. An equal volume of sterile PBS was added to the control group. The plates were co-cultured at 37°C and 5% CO2 for 2 hours.

[0036] Cells were collected, and the transcription level of the Dglb gene was detected by qRT-PCR. The results are as follows: Figure 6 As shown, by Figure 6 It was found that, compared with the control group (CON), the DGLβ protein level in cells of each Lactobacillus johnsonii treatment group was significantly increased (P<0.01), with the LJEH8 group showing the largest increase, confirming that Lactobacillus johnsonii LJEH8 can effectively upregulate the expression of DGLβ in intestinal epithelial cells in vitro.

[0037] To further validate this, Western blot was used to detect the expression level of DGLβ protein in the LJEH8 group (with β-actin as an internal control). The results are as follows: Figure 7 As shown, co-culture with mouse colorectal cancer cells CMT-93 was also used for verification, and the results are as follows. Figure 8 As shown, by Figure 7 and Figure 8 It can be seen that, compared with the control group, the L. johnsonii strain LJEH8 treatment group showed the most significant upregulation of DGLβ protein level, indicating that L. johnsonii strain LJEH8 is a positive regulator of the endocannabinoid system balance.

[0038] Example 4: Validation of the in vivo regulation of the colonic endocannabinoid system by Lactobacillus johnsonii LJEH8 Sixteen 6-week-old SPF-grade female ICR mice were randomly divided into two groups (n=8). The first group was treated with a mixed antibiotic regimen containing 1 g / L ampicillin, 1 g / L metronidazole, 1 g / L neomycin, and 0.5 g / L vancomycin in drinking water for two weeks to clear the pre-existing intestinal flora. Subsequently, the LJEH8 strain obtained in Example 2 was administered via gavage at a dose of 2 × 10⁻⁶ g / day. 8 200 μL of CFU / mL bacterial suspension was administered to mice, while the control group was administered an equal volume of sterile PBS by gavage. After two weeks of continuous intervention, colon tissue was collected from mice and analyzed using qRT. PCR and Western blot were used to detect the expression level of DGLβ protein.

[0039] qRT PCR test results as follows Figure 9 As shown, where, Figure 9 The values ​​a through e in the figures represent the expression levels of diacylglycerol lipase β (Dglb), diacylglycerol lipase α (Dgla), fatty acid amide hydrolase (Faah), monoacylglycerol lipase (Magl), and N-acylphosphatidylethanolamine-specific phospholipase D (Napepld) in mouse colon tissue, respectively. Western blot results are shown below. Figure 10 As shown, Depend on Figure 9 and Figure 10 It was found that, compared with the control group, the DGLβ protein level in the colon tissue of mice in the LJEH8 group was significantly increased (P<0.01), confirming that this strain can effectively upregulate colonic DGLβ expression and regulate the balance of the endocannabinoid system in vivo.

Claims

1. A *Lactobacillus johnsonii* strain that regulates the colonic endocannabinoid system, characterized in that... The Lactobacillus johnsonii, named LJEH8, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026895 and deposit date of May 8, 2026. The regulation of the colonic endocannabinoid system refers to promoting the expression of diacylglycerol lipase β in the colon.

2. The use of the Lactobacillus johnsonii strain of claim 1 in the preparation of a product for regulating the colonic endocannabinoid system.

3. The application according to claim 2, characterized in that, The product is a medicine or a functional food.

4. The use of the Lactobacillus johnsonii strain of claim 1 in the preparation of a medicament for the prevention and / or treatment of intestinal diseases.

5. The application according to claim 4, characterized in that, The intestinal disease mentioned is inflammatory bowel disease.

6. A microbial agent for regulating the colonic endocannabinoid system, characterized in that, It contains the Lactobacillus johnsonii strain LJEH8 as described in claim 1 as an active ingredient.

7. The microbial agent according to claim 6, characterized in that, The viable count of Lactobacillus johnsonii strain LJEH8 in the microbial agent was 2 × 10⁸. 8 CFU / g.

8. The microbial agent according to claim 6, characterized in that, The microbial agent also contains a pharmaceutically acceptable carrier or a food-acceptable excipient.

9. The microbial inoculant according to any one of claims 6 to 8, characterized in that, The microbial agent is in the form of an oral preparation.

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