Use of em in preparation of drugs for preventing and treating enteritis-related neural inflammation and improving anxiety-like behavior

Abstract

The application discloses application of EM bacteria in preparation of medicines for preventing and treating intestinal inflammation related neural inflammation and improving anxiety-like behavior, and relates to the technical field of microbial pharmaceuticals.The application provides application of EM bacteria in preparation of medicines for preventing and treating intestinal inflammation related neural inflammation and improving anxiety-like behavior, wherein the EM bacteria are a composite microbial viable bacteria preparation, which is composed of fungi and bacteria, and main components include Lactobacillus paracasei, Hellemannia sp., Sediminibacter sp., Lactobacillus sp., Paraburkholderia sp., Lactobacillus mui, Methylobacterium sp., Mesorhizobium sp., Agrobacterium sp., and Saccharomyces uvarum.The application verifies, for the first time, the prevention and treatment effect of the EM bacteria on intestinal inflammation related neural inflammation, and proves that the EM bacteria can not only significantly relieve intestinal inflammation, but also effectively inhibit central nervous inflammation in a DSS-induced ulcerative colitis mouse model, thereby widening the application range of the EM bacteria and filling the technical blank of the EM bacteria in the field of neural immune regulation.

Description

Technical Field

[0001] This invention belongs to the field of microbial pharmaceutical technology, specifically the application of EM bacteria in the preparation of drugs for the prevention and treatment of enteritis-related neuroinflammation and the improvement of anxiety-like behaviors. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of diseases characterized by chronic, relapsing intestinal inflammation, primarily including ulcerative colitis (UC) and Crohn's disease (CD). In recent years, with in-depth research into the mechanisms of the gut-brain axis, IBD patients often experience central nervous system symptoms such as neuroinflammation, anxiety, and depression, suggesting that peripheral inflammation can affect central nervous system function through immune, neural, and endocrine pathways. Specific mechanisms include: impaired intestinal barrier function leading to the entry of gut microbiota products into the circulatory system, activating peripheral immune cells, which in turn induce microglial activation and inflammasome pathway activation in the brain through disruption of the blood-brain barrier or the vagus nerve pathway, ultimately resulting in neuroinflammation and anxiety-like behaviors.

[0003] Currently, the main treatments for IBD include aminosalicylic acid preparations, glucocorticoids, immunosuppressants, and biologics. However, these drugs have issues such as inconsistent efficacy, significant side effects, and a high relapse rate. In particular, in clinical treatment guidelines for UC, probiotic supplementation is only listed as an adjunctive therapy and is not recommended as first-line treatment. The main reason is that most existing probiotics are single-species strains or simple combinations of lactic acid bacteria, lacking the ability to systemically regulate multiple targets along the gut-brain axis.

[0004] EM (Effective Microbiota) is a complex live bacterial preparation composed of various bacteria and fungi, traditionally used primarily in agriculture, animal husbandry, and environmental remediation. Although studies have shown that EM has a certain regulatory effect on the host's gut microbiota, whether it can alleviate neuroinflammation and anxiety-like behaviors induced by enteritis by repairing the intestinal barrier, inhibiting peripheral and central inflammation, and improving the integrity of the blood-brain barrier has not been publicly reported. Therefore, exploring the application of EM in the prevention and treatment of enteritis-related neuroinflammation and anxiety-like behaviors has significant scientific value and clinical translational prospects.

[0005] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide the application of EM bacteria in the preparation of drugs for the prevention and treatment of enteritis-related neuroinflammation and the improvement of anxiety-like behaviors, in order to solve the problems in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: the application of EM bacteria in the preparation of drugs for preventing and treating enteritis-related neuroinflammation and improving anxiety-like behaviors. The EM bacteria are a compound live microbial preparation composed of fungi and bacteria, and the main components include Lactobacillus lentigines, Hellmann's bacterium, Bacillus subtilis, Lactobacillus, Burkholderia paraknifediformis, Lactobacillus sphaeroides, Methylbazella, Rhizobium mesenteriae, Agrobacterium tumefaciens, and Saccharomyces cerevisiae.

[0008] Preferably, the concentration of the EM bacteria is 10. 9 CFU / mL, diluted with physiological saline, and used immediately.

[0009] Preferably, the enteritis-related neuroinflammation is a central nervous system inflammation secondary to ulcerative colitis, Crohn's disease, or other inflammatory bowel diseases.

[0010] Preferably, the improvement of anxiety-like behavior includes increasing the number of open arm entries and the total distance traveled in the elevated cross maze experiment, and increasing the central area exploration time and the total distance traveled in the open field experiment.

[0011] Preferably, the EM bacteria alleviate intestinal inflammation and repair the intestinal barrier by improving intestinal barrier function, reducing the expression levels of IL-1β, TNF-α, and Caspase-1 in colonic tissue, and upregulating the expression of intestinal barrier proteins ZO-1 and Claudin5.

[0012] Preferably, the EM bacteria inhibit neuroinflammation and repair the blood-brain barrier by reducing the expression of IL-1β, TNF-α, NLRP3, Caspase-1 and Iba1 proteins in brain tissue and upregulating the expression of brain barrier-related proteins.

[0013] Preferably, the EM bacteria are administered in the form of an oral live bacterial preparation, including but not limited to gavage solution, lyophilized powder, capsules or emulsion.

[0014] Preferably, the EM bacteria simultaneously exert both peripheral and central anti-inflammatory effects.

[0015] The present invention also provides a pharmaceutical composition for preventing and treating enteritis-related neuroinflammation and improving anxiety-like behavior, comprising an effective amount of EM bacteria and a pharmaceutically acceptable carrier or excipient.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. This invention is the first to verify the preventive and therapeutic effects of EM bacteria on enteritis-related neuroinflammation. It confirms that in a DSS-induced ulcerative colitis mouse model, EM bacteria can not only significantly alleviate intestinal inflammation, but also effectively inhibit central nervous system inflammation, thus broadening the application scope of EM bacteria and filling the technological gap in the field of neuroimmune regulation.

[0018] 2. This invention systematically reveals that EM bacteria achieve dual anti-inflammatory effects in both the peripheral and central nervous systems by repairing the intestinal barrier, inhibiting the expression of IL-1β, TNF-α, and Caspase-1 in colonic tissue, reducing inflammatory markers such as NLRP3, Caspase-1, and Iba1 in brain tissue, and repairing blood-brain barrier-related proteins. It clarifies the dual regulatory mechanism of the gut-brain axis and demonstrates its unique advantages in multi-target intervention of the gut-brain axis.

[0019] 3. Through classic behavioral tests such as the elevated cross maze and open field test, this invention demonstrates for the first time that EM bacteria can significantly increase the number of open arm entries and total movement distance, and increase the exploration time and movement distance in the central area, indicating that it has a clear ameliorative effect on enteritis-induced anxiety-like behavior, providing a potential new treatment strategy for patients with IBD and mood disorders.

[0020] 4. This invention breaks through the traditional research and development approach of probiotics based on single strains or lactic acid bacteria, and proposes and verifies the feasibility of compound microbial preparations with coexisting bacteria and fungi in the prevention and treatment of gut-brain axis-related diseases, providing a new direction for the future development of microbial preparations. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0022] Figure 1 A schematic diagram of the microbial community composition of EM live bacteria preparation;

[0023] Figure 2 A schematic diagram illustrating how live EM bacteria preparations improve DSS-induced intestinal pathological changes in mice.

[0024] Figure 3 A schematic diagram illustrating how live EM bacteria preparations improve DSS-induced brain and intestinal barrier damage in mice.

[0025] Figure 4 A schematic diagram illustrating how live EM bacteria preparations improve DSS-induced anxiety-like behavior in mice;

[0026] Figure 5 A schematic diagram illustrating how live EM bacteria preparations improve DSS-induced intestinal and brain inflammation in mice. Detailed Implementation

[0027] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Interventional effect of EM bacteria on DSS-induced ulcerative colitis and neuroinflammation in mice

[0029] 1. Experimental Materials and Preparation

[0030] EM (Effective Microorganisms) preparations: EM is a compound live microbial preparation, with main components such as... Figure 1 As shown. The viable cell concentration was determined to be 1 × 10⁻⁶ using the plate count method. 9 CFU / mL, diluted to working concentration with sterile physiological saline, and used immediately after preparation.

[0031] Dosage: 10 10 Mice were administered CFU / kg body weight (i.e., 10 mL / kg) via gavage once daily until the end of the model cycle.

[0032] Animal model: SPF-grade male C57BL / 6J mice, 6-8 weeks old and weighing 18-22 g, were used. After one week of acclimatization, an acute ulcerative colitis model was induced by two cycles of free access to 3% (w / v) sodium dextran sulfate (DSS) solution. Each cycle lasted 7 days, with mice given normal drinking water for 2 days in each cycle, followed by 5 days of 3% DSS solution.

[0033] Grouping and intervention steps:

[0034] Control group (CON): Received an equal volume of physiological saline by gavage daily and drank normal water.

[0035] Model group (UC): DSS modeling, daily gavage administration of an equal volume of physiological saline.

[0036] EM treatment group (EM): DSS modeling, daily gavage administration of EM bacterial solution, 10 10 CFU / kg.

[0037] The experiment lasted for 14 days, with daily records of body weight, fecal characteristics, and blood in the stool. Mice were euthanized at the end of the experiment, and cecal contents and feces were collected for omics analysis. Colon and brain tissue samples were also collected for morphological and biochemical analysis.

[0038] 2. Detection Indicators and Methods

[0039] (1) Intestinal inflammation and intestinal barrier related indicators:

[0040] Colon length measurement;

[0041] HE staining was used to observe the pathological morphology of colon tissue and to score mucosal damage.

[0042] Real-time quantitative PCR was used to detect the mRNA levels of IL-1β and TNF-α in colon tissue.

[0043] Western blot analysis of Caspase-1 protein expression in colon tissue;

[0044] Western blot was used to detect the expression of intestinal barrier proteins ZO-1 and Claudin5.

[0045] (2) Neuroinflammation and brain barrier related indicators:

[0046] Western blot was used to detect the expression of IL-1β, TNF-α, NLRP3, Caspase-1 and Iba1 proteins in brain tissue;

[0047] Western blot was used to detect the expression of brain barrier-related proteins (such as Claudin5 and ZO-1).

[0048] Immunohistochemical detection of microglia activation status in brain tissue.

[0049] (3) Behavioral testing:

[0050] Elevated Cross Maze (EPM): Records the number of times the open arm enters and the total distance traveled;

[0051] Open field experiment (OFT): Records the exploration time in the central area and the total distance traveled.

[0052] 3. Experimental Results

[0053] (1) EM bacteria improve DSS-induced intestinal inflammation and barrier damage

[0054] Compared with the CON group, mice in the DSS model group showed shortened colon length (p < 0.01), and HE staining of colon tissue revealed extensive inflammatory cell infiltration, mucosal structure destruction, and a significantly increased mucosal injury score (p < 0.001). Simultaneously, IL-1β and TNF-α mRNA levels in colon tissue were significantly upregulated (p < 0.01), and Caspase-1 protein expression was increased, suggesting activation of inflammasome-related pathways. The expression of intestinal barrier proteins ZO-1 and Claudin5 was significantly decreased (p < 0.01), indicating impaired intestinal barrier function.

[0055] Compared with the DSS model group, the colon length of mice in the EM treatment group was significantly restored (p < 0.05), HE staining showed reduced inflammatory cell infiltration, improved mucosal structure, and a significantly lower injury score (p < 0.01). IL-1β and TNF-α mRNA levels and Caspase-1 protein expression were significantly downregulated (p < 0.05), while ZO-1 and Claudin5 expression levels were significantly restored (p < 0.05), indicating that EM has significant anti-inflammatory and barrier repair effects on the intestine (see...). Figure 2 BD, Figure 5 DE Figure 3 CD).

[0056] (2) EM bacteria inhibit DSS-induced central nervous system inflammation and improve brain barrier function

[0057] In the DSS model group of mice, the expression of IL-1β, TNF-α, NLRP3, Caspase-1, and Iba1 proteins in brain tissue were significantly increased (p < 0.01), indicating increased release of pro-inflammatory factors in the brain, activation of the NLRP3 / Caspase-1 inflammasome-related pathway, and microglial activation. Simultaneously, the expression of brain barrier-related proteins was significantly decreased (p < 0.01), suggesting impaired brain barrier function.

[0058] Compared with the DSS model group, the EM bacteria treatment group significantly reduced the expression of the above-mentioned pro-inflammatory factors and inflammasome-related proteins (p < 0.05), the decrease in Iba1 expression indicated that microglial cell activation was inhibited, and the expression of brain barrier proteins was significantly restored (p < 0.05), indicating that EM bacteria can effectively inhibit central nervous system inflammation and improve brain barrier integrity. Figure 5 AC, Figure 3 AB).

[0059] (3) EM bacteria improve DSS-induced anxiety-like behavior

[0060] In the elevated cross maze test, the number of open arm entries and the total distance traveled by mice in the DSS model group were significantly lower than those in the CON group (p < 0.01), exhibiting anxiety-like behavior. In the open field test, the exploration time and distance traveled by mice in the DSS model group were significantly reduced (p < 0.01). After EM intervention, open arm activity increased (p < 0.05), and the exploration time and total distance traveled in the central area significantly recovered (p < 0.05), indicating a significant improvement in anxiety-like behavior (see [link to relevant documentation]). Figure 4 ).

[0061] The above examples demonstrate for the first time that EM (Effective Microorganisms), as a complex live microbial preparation containing both bacteria and fungi, can simultaneously alleviate peripheral intestinal and central nervous system inflammation by repairing the intestinal barrier, inhibiting the NLRP3 / Caspase-1 inflammasome pathway in the intestine and central nervous system, and reducing microglial activation, and significantly improve anxiety-like behaviors induced by enteritis. These results indicate that EM has a clear application prospect in the preparation of drugs for preventing and treating enteritis-related neuroinflammation and improving anxiety-like behaviors.

[0062] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. The application of EM bacteria in the preparation of drugs for the prevention and treatment of enteritis-related neuroinflammation and the improvement of anxiety-like behaviors, characterized in that: The EM bacteria is a compound live microbial preparation composed of fungi and bacteria. Its main components include Lactobacillus lentigines, Hellmann's bacterium, Bacillus subtilis, Lactobacillus, Burkholderia paraknifedipine, Lactobacillus sphaeroides, Methylbazella, Slow-growing rhizobium, Agrobacterium, and Saccharomyces cerevisiae.

2. The application according to claim 1, characterized in that: The concentration of the EM bacteria is 10. 9 CFU / mL, diluted with physiological saline, and used immediately.

3. The application according to claim 1, characterized in that: The enteritis-associated neuroinflammation refers to central nervous system inflammation secondary to ulcerative colitis, Crohn's disease, or other inflammatory bowel diseases.

4. The application according to claim 1, characterized in that: The improvement of anxiety-like behaviors includes increasing the number of open arm entries and the total distance traveled in the elevated cross maze experiment, and increasing the central area exploration time and the total distance traveled in the open field experiment.

5. The application according to claim 1, characterized in that: The EM bacteria reduce intestinal inflammation and repair the intestinal barrier by improving intestinal barrier function, reducing the expression levels of IL-1β, TNF-α, and Caspase-1 in colon tissue, and upregulating the expression of intestinal barrier proteins ZO-1 and Claudin5.

6. The application according to claim 1, characterized in that: The EM bacteria inhibit neuroinflammation and repair the blood-brain barrier by reducing the expression of IL-1β, TNF-α, NLRP3, Caspase-1 and Iba1 proteins in brain tissue and upregulating the expression of brain barrier-related proteins.

7. The application according to claim 1, characterized in that: The EM bacteria are administered in the form of oral live bacterial preparations, including but not limited to gavage solution, lyophilized powder, capsules or emulsion.

8. The application according to claim 1, characterized in that: The EM bacteria simultaneously exert both peripheral and central anti-inflammatory effects.

9. A pharmaceutical composition for preventing and treating enteritis-related neuroinflammation and improving anxiety-like behavior, characterized in that: It contains an effective amount of EM bacteria and a pharmaceutically acceptable carrier or excipient.

Images