Lactobacillus reuteri with colitis-myocardial infarction dual-relieving effect and application thereof

Abstract

The invention relates to lactobacillus reuteri CAU 806 and application of the lactobacillus reuteri CAU 806 in relieving chronic colitis and myocardial infarction, and belongs to the technical field of microorganisms. The invention provides a strain of lactobacillus reuteri CAU 806, the preservation number of the lactobacillus reuteri CAU 806 is CGMCC No.34092, and the lactobacillus reuteri CAU 806 aims at defining the effect of the lactobacillus reuteri CAU 806 on the aspects of relieving colitis and myocardial infarction, and particularly, the effect of the lactobacillus reuteri CAU 806 on the aspects of relieving colitis and myocardial infarction caused by sodium dextran (DSS) induced colitis and pseudosurgery is further verified, and the lactobacillus reuteri CAU 806 has the advantages that the lactobacillus reuteri CAU 806 can be used for treating colitis and myocardial infarction caused by pseudosurgery; therefore, a targeted treatment scheme which is efficient and safe and has dual effects of relieving colitis and myocardial infarction at the same time is provided.

Description

Technical fields:

[0001] This invention relates to *Lactobacillus reuteri* (… Limosilactobacillus reuteri CAU 806 and its application in alleviating chronic colitis and myocardial infarction belong to the field of microbial technology. Background technology: Colitis, particularly ulcerative colitis and Crohn's disease, both forms of inflammatory bowel disease (IBD), is a chronic, relapsing inflammatory bowel disease with a continuously rising incidence rate worldwide. In Europe and the United States, the incidence of IBD has reached 100-200 per 100,000 people, with a particularly significant increase in Asia. It severely impacts patients' quality of life and exacerbates the social healthcare burden. Its pathogenesis is complex, involving the combined effects of genetic susceptibility, abnormal immune regulation, and environmental factors, with gut microbiota imbalance being a key contributing factor—the presence of Bifidobacteria (Bifidobacteria) in the patient's gut is a significant contributing factor. Bifidobacterium ), Lactobacillus ( Lactobacillus The number of beneficial bacteria such as Escherichia coli (E. coli) has decreased sharply. Escherichia coli Clostridium ( Clostridium Excessive proliferation of harmful bacteria such as [unspecified bacteria] leads to impaired intestinal barrier function, abnormal activation of immune cells, and persistent inflammation. Currently, clinical treatments include aminosalicylic acid derivatives, glucocorticoids, immunosuppressants, and biologics, but all have limitations: aminosalicylic acid derivatives are not very effective for moderate to severe cases; long-term use of glucocorticoids causes serious side effects; immunosuppressants have a slow onset of action and may suppress immune function; biologics are not only expensive, but some patients may also develop drug resistance and adverse reactions. Therefore, the development of safe and effective new treatment strategies for colitis is urgently needed. Myocardial infarction, a fatal cardiovascular disease caused by acute coronary ischemia and hypoxia, accounts for a significant proportion of the more than 17 million cardiovascular deaths worldwide each year. In my country, its incidence continues to rise due to population aging and lifestyle changes, posing a serious threat to public health. Its pathogenesis mainly involves thrombus formation after the rupture of atherosclerotic plaques in the coronary arteries, leading to acute vascular occlusion and myocardial ischemia and necrosis. Simultaneously, the inflammatory response exacerbates myocardial damage and cardiac dysfunction. Current clinical treatment primarily focuses on early reperfusion (thrombolysis, interventional therapy) and subsequent drug intervention (antiplatelet drugs, statins, etc.). However, the ischemia-reperfusion injury associated with reperfusion therapy can worsen myocardial cell death and cardiac function deterioration. Drug therapy has limited effectiveness in repairing damaged myocardium, and patients still face the risk of serious complications such as heart failure. Therefore, there is an urgent need to explore new therapeutic targets and strategies to improve prognosis.

[0002] Lactobacillus reuteri ( Lactobacillus reuteriAs a Gram-positive probiotic, *Lactobacillus reuteri* (L.) possesses the ability to colonize and adhere to the gastrointestinal tract, forming a biological barrier with intestinal epithelial cells to resist the invasion of harmful bacteria. Simultaneously, its metabolites (short-chain fatty acids, bacteriocins, vitamins, etc.) can regulate intestinal pH, inhibit the growth of harmful bacteria, and participate in human metabolism. In the field of gut health, *Lactobacillus reuteri* can effectively regulate the balance of gut microbiota, increase the abundance of beneficial bacteria, enhance intestinal barrier function by promoting mucus secretion and strengthening epithelial cell junctions, and also regulate immune cell activity and inhibit the release of inflammatory factors to alleviate intestinal inflammation. In cardiovascular health, studies have confirmed that it can intervene in cardiovascular diseases such as atherosclerosis by regulating lipid metabolism, lowering blood pressure, and reducing vascular endothelial inflammation; its metabolites can also directly protect the heart and blood vessels through blood circulation.

[0003] Currently, there are various interventions and treatments for colitis and myocardial infarction in clinical practice, but all have certain limitations. While existing studies have shown that *Lactobacillus reuteri* has certain benefits for gut health and cardiovascular health, its application in alleviating colitis and myocardial infarction remains insufficient. On the one hand, most studies focus only on the effects of *Lactobacillus reuteri* on a single disease, with limited research on its dual efficacy in alleviating both colitis and myocardial infarction, lacking systematic exploration. On the other hand, existing research mainly focuses on the general functions and mechanisms of action of *Lactobacillus reuteri*, while the unique properties and advantages of specific strains, such as *Lactobacillus reuteri* CAU 806, have not been thoroughly explored, and its efficacy and safety in vivo lack sufficient verification. Summary of the Invention: The present invention aims to clarify the efficacy of *Lactobacillus reuteri* CAU 806 in alleviating colitis and myocardial infarction, and to further verify this efficacy through dextran sulfate sodium salt (DSS)-induced colitis and sham surgery-induced myocardial infarction, thereby providing a highly effective, safe and targeted treatment option.

[0004] One of the technical solutions provided by this invention is a strain of *Lactobacillus reuteri*, specifically *Lactobacillus reuteri* (… Limosilactobacillus reuteri CAU 806, this strain was deposited on April 3, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 34092.

[0005] The second technical solution provided by this invention is the application of *Lactobacillus reuteri* CAU 806 described in the first technical solution, particularly in the preparation of functional bacterial agents for treating or improving colitis and / or myocardial infarction; The active ingredient of the functional microbial agent includes Lactobacillus reuteri CAU 806, and the microbial agent is a liquid microbial agent or a solid microbial agent; Furthermore, one of the technical solutions involves the application of *Lactobacillus reuteri* CAU 806 in the preparation of products for treating or improving colitis and / or myocardial infarction; the products include, but are not limited to, food, health products, or pharmaceuticals. Furthermore, treatment or improvement of colitis includes, but is not limited to, the following: (1) DAI reduction: improves weight loss, loose stools and rectal bleeding caused by colitis; (2) Improves colon condition; inhibits colonic shortening, eliminates edema, eliminates inflammatory cell infiltration, and restores the integrity of the mucus layer; (3) Repairing the intestinal barrier: It can significantly upregulate the expression of MUC2, ZO-1 and Occludin proteins to repair the intestinal barrier and reduce intestinal permeability and inflammatory cell infiltration; (4) Relief of inflammatory state: The levels of anti-inflammatory factors IL-4 and TGF-β were significantly increased, while the level of pro-inflammatory factor IL-6 was significantly decreased; (5) Regulation of gut microbiota disorder: Shannon index and Simposn index were significantly improved; gut microbiota community was restored.

[0006] Furthermore, the treatment or improvement of myocardial infarction includes, but is not limited to, the following: (1) Improve myocardial fibrosis and myocardial damage caused by myocardial infarction: reduce the degree of myocardial fibrosis and reduce the area of ​​myocardial infarction; reduce edema, slow down myocardial cell damage, and improve cell arrangement; (2) Reduce neutrophil infiltration and alleviate inflammatory response; (3) Reduce intestinal damage caused by myocardial infarction; restore the expression of Occludin and ZO-1.

[0007] Beneficial effects: 1. The *Lactobacillus reuteri* CAU 806 provided by this invention can significantly alleviate colitis symptoms: This has been verified through animal experiments, as detailed below: (1) The disease activity index (DAI) of mice in the *Lactobacillus reuteri* CAU 806 treatment group was significantly reduced. Specifically, the trend of weight loss in mice was significantly curbed, returning to near-normal levels; fecal characteristics gradually changed from diarrhea and soft stools to normal formed stools; and fecal bleeding was significantly reduced or even disappeared. This indicates that *Lactobacillus reuteri* CAU 806 can effectively alleviate the clinical symptoms of colitis, improve the quality of life of affected mice, and provide a new and effective approach for the clinical treatment of colitis.

[0008] (2) Optimizing the gut microbiota: High-throughput sequencing analysis showed that *Lactobacillus reuteri* CAU 806 could reshape the DSS-induced gut microbiota imbalance. Increasing beneficial bacteria such as... Xylanibacter , Duncaniella The abundance of bacteria such as Dubstein can inhibit harmful bacteria such as Dubsteinella. Dubosiella Bacteroides ( Bacteroids ), genus *Alternaria* ( Alistipes The growth and reproduction of these microorganisms help to bring the gut microbiota structure into a healthy state.

[0009] (3) Precisely regulate immune response: reduce the levels of inflammatory factor IL-6 in serum and colon tissue, upregulate the levels of anti-inflammatory cytokines IL-4 and transforming growth factor-β (TGF-β), effectively reduce intestinal inflammatory response, and alleviate colitis from an immune perspective.

[0010] 2. The *Lactobacillus reuteri* CAU 806 provided by this invention has a targeted repair effect on myocardial infarction: (1) In terms of cardioprotection, *Lactobacillus reuteri* CAU 806 can effectively reduce cardiac fibrosis and significantly slow down the progression of pathological fibrosis. Comparison of pathological changes in mouse heart tissue revealed that it significantly improves myocardial fibrosis in mice with myocardial infarction, reducing the cross-sectional area of ​​cardiomyocytes, alleviating edema and cell damage after myocardial infarction, and optimizing cell arrangement. Mechanistically, *Lactobacillus reuteri* CAU 806 may alleviate cardiac inflammation by regulating the immune response and inhibiting neutrophil infiltration. (2) Multidimensional protective effect against myocardial infarction-related intestinal damage: Lactobacillus reuteri CAU 806 has a potential protective effect against intestinal damage caused by myocardial infarction, promotes the expression of intestinal epithelial cells, and repairs the intestinal barrier.

[0011] 3. Broad Potential Applications: *Lactobacillus reuteri* CAU 806 is easily culturable, has a clear genetic background, well-defined growth conditions, and relatively low cost, making large-scale acquisition of this strain possible. This technology holds promise for developing novel probiotic preparations for the prevention and adjunctive treatment of colitis and cardiomyocyte damage. Compared to traditional treatments, *Lactobacillus reuteri* CAU 806, as a natural probiotic, boasts high safety and is included in the list of strains permitted for use in food, demonstrating excellent market application prospects. Attached image description: Figure 1 Lactobacillus reuteri ( Limosilactobacillus reuteri CAU806 phylogenetic tree.

[0012] Figure 2 The colon length of mice in each group is shown.

[0013] Figure 3 Pathological sections of mice in each group Among them, (A) HE staining; (B) pathological score; (C) AB-PAS staining; (D) mucus content in colonic goblet cells.

[0014] Figure 4 Effects of Lactobacillus reuteri CAU 806 on colonic barrier function in DSS-induced colitis mice Among them, (A) immunohistochemical results of MUC2; (B) positive expression area of ​​MUC2 in the colon; (C) immunohistochemical results of ZO-1; (D) positive expression area of ​​ZO-1 in the colon; (E) immunohistochemical results of Occludin in the colon; (F) positive expression area of ​​Occludin in the colon.

[0015] Figure 5 Effects of Lactobacillus reuteri CAU 806 on inflammatory cytokine levels in mice with DSS-induced colitis Among them, (A) IL-4; (B) IL-6; (C) TGF-β.

[0016] Figure 6 Effects of *Lactobacillus reuteri* CAU 806 on the β- and β-diversity of gut microbiota in DSS-induced colitis mice. Among them, (A) Shannon index; (B) Simpson index; (C) PCoA analysis.

[0017] Figure 7 Barplots of community at the genus level and analysis of differential abundance of bacterial genera.

[0018] Figure 8 Lactobacillus reuteri CAU 806 improves myocardial fibrosis in mice with myocardial infarction. Among them, (A) representative Masson trichrome staining of each group of heart slices; (B) myocardial infarction area.

[0019] Figure 9 Pathological changes in myocardial tissue of mice with myocardial infarction treated with Lactobacillus reuteri CAU 806 Among them, (A) representative HE staining of each group of heart sections; (B) cross-sectional area of ​​cardiomyocytes.

[0020] Figure 10 Effects of Lactobacillus reuteri CAU 806 on neutrophils in mice with myocardial infarction Among them, (A) representative Ly6G immunofluorescence staining of heart sections in each group; (B) relative positive expression of Ly6G in the heart.

[0021] Figure 11 Representative HE staining of colon sections from various groups of mice after intervention with Lactobacillus reuteri CAU 806 following myocardial infarction.

[0022] Figure 12 Effects of Lactobacillus reuteri CAU 806 on colonic barrier function in mice with myocardial infarction Among them, (A) Immunofluorescence results of Occludin and ZO-1 in the colon; (B) Positive relative expression levels of Occludin and ZO-1 in the colon. (Note: Figures 2-12 middle:****: P <0.0001; ***: P <0.001;**: P <0.01; *: P <0.05). Detailed implementation method: The present invention will now be described through specific embodiments. All technical means not specifically described herein are methods well-known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of the present invention.

[0023] The present invention will be further explained and described below through specific embodiments.

[0024] Example 1: Isolation, purification and identification of *Lactobacillus reuteri* (1) Sample collection: Kimchi is rich in a variety of microorganisms and is a potential source of Lactobacillus reuteri. Kimchi from different regions and with different production processes were carefully selected as samples. Kimchi juice or kimchi tissue was collected under aseptic conditions to ensure that the samples were not contaminated by external bacteria. (2) Enrichment culture: The collected samples were inoculated into MRS medium rich in nutrients, which provides a suitable environment for the growth of lactic acid bacteria. The culture conditions were adjusted, with the temperature controlled at around 37℃ and the pH maintained at 5.5-6.5, and anaerobic culture was carried out for 48 h. During the culture process, *Lactobacillus reuteri* gradually accumulated and increased in number under this environment.

[0025] (3) Isolation and purification: The enriched bacterial culture was inoculated onto MRS solid medium plates using the dilution plating method or streak plating method. After a period of incubation, colonies of different morphologies will appear on the plates. Based on the typical characteristics of *Lactobacillus reuteri* colonies, such as round shape, neat edges, and moist and smooth surface, a suspected colony was selected for repeated purification until a pure culture was obtained.

[0026] (4) Select a single colony, extract the genome, amplify the 16S rDNA sequence, and sequence it. Compare the obtained gene sequence with the sequence of known strains in the NCBI database to construct a phylogenetic tree. See [link to phylogenetic tree]. Figure 1 It was identified as *Lactobacillus reuteri* (…). Limosilactobacillus reuteri ), named Lactobacillus reuteri ( Limosilactobacillus reuteri CAU806.

[0027] The 16S rDNA sequence of *Lactobacillus reuteri* CAU806 is as follows: Example 2: Acid and bile salt resistance test of Lactobacillus reuteri CAU 806 The environmental tolerance of *Lactobacillus reuteri* CAU 806 in Example 1 to acid and bile salts was evaluated.

[0028] (1) Acid resistance test: After the strain was activated for 24 h, it was washed twice with 0.9% physiological saline. The bacterial suspension was resuspended in equal volumes in MRS liquid medium with pH 1.0, 2.0 and 3.0 respectively and cultured for 1-3 h before the number of viable bacteria was determined. (2) Bile salt tolerance test: After the strain was activated for 24 h, it was washed twice with 0.9% physiological saline. The bacterial suspension was resuspended in equal volumes in MRS liquid medium with pH 2.0 and 0.3%-1% bile salt concentration, respectively. After anaerobic culture at 37℃ for 2 and 4 h, the viable number was measured.

[0029] Calculate the survival rate separately. The formula is:

[0030] In the formula: Nt is the number of viable bacteria after treatment; N0 is the number of viable bacteria at 0 h.

[0031] (3) As shown in Table 1, Lactobacillus reuteri can tolerate treatment conditions with pH 1.0-3.0. At pH 1.0, after 3 hours of treatment, the survival rate can still reach 89%, and at pH 2.0-3.0, after 3 hours of treatment, the survival rate reaches 99%, showing high acid resistance.

[0032] (4) As shown in Table 2, the bile salt tolerance of Lactobacillus reuteri is high. After treatment with 1% bile salt for 4 hours, the bacterial survival rate of Lactobacillus reuteri is still above 81%.

[0033] Table 1. Survival rates of *Lactobacillus reuteri* CAU 806 under different treatment times and pH values.

[0034] Table 2. Bile salt tolerance test of Lactobacillus reuteri CAU 806

[0035] Example 3: Animal experiment on the relief of DSS-induced colitis by Lactobacillus reuteri CAU 806 1. Establishment and grouping of experimental animal models Twenty-four 6-week-old SPF-grade male C57Bl / 6J mice (weighing 18-22g) were housed in a standard SPF barrier environment at the Department of Experimental Animal Science, China Agricultural University, with constant temperature (22℃), constant humidity (55%), 12-hour day-night cycle, and free access to food and water.

[0036] The mice were randomly divided into 3 groups: (1) Control group: 8 animals, which were fed and drank normally during the modeling period, and were given 0.2 mL of 0.9% sterile saline by gavage every day; (2) DSS group: 8 animals, normal diet during the modeling period, with a cycle of 7 days. For the first 4 days, they drank DSS water with a concentration of 1.5% and were given 0.2 mL of sterile physiological saline with a concentration of 0.9% by gavage every day. For the next 3 days, they drank water normally. This cycle continued for 49 days. (3) DSS+CAU 806 group: 8 mice. During the modeling period, they were fed a normal diet. The cycle was 7 days. For the first 4 days, they drank water with a concentration of 1.5% DSS and were also given 200 μL of 1×10⁻⁶ DSS solution by gavage every day. 9 The patient was given a CFU / mL Lactobacillus reuteri CAU 806 bacterial culture, followed by 3 days of normal drinking water; this cycle was continued for 49 days. During the colitis modeling period, the following were recorded daily: mouse weight, stool characteristics, occult blood, etc. (scores were calculated using the standard Disease Activity Index (DAI) scoring system to assess the severity of colitis; specific criteria are shown in Table 3). The presence of blood in the anus and the general condition of the mice (mental state, fur, presence of arched back, etc.) were also recorded. Mice were fasted for 12 hours on day 48 and sacrificed on day 49. A section of colonic tissue 1 cm above the anus was cryopreserved in liquid nitrogen, 0.5 cm was fixed in 4% formaldehyde solution for later use, and single cells from the lamina propria of the remaining intestinal segment were extracted for later use.

[0037] Table 3 Disease Activity Index (DAI) Scoring Criteria

[0038] 2. Collection of tissue specimens Before the end of day 49 of the experiment, fecal samples were collected from each group of mice. Under aseptic conditions, the mice were fixed in place, and the skin around the anus was disinfected with alcohol swabs. The anus was then stimulated to promote defecation. Fresh feces were collected and stored in sterile cryovials. The feces from each group were divided into two groups and immediately frozen in liquid nitrogen and stored at -80°C for later use. Blood was collected from the orbital veins of the mice. After coagulation at room temperature for 10-20 minutes, the blood was centrifuged at 3000 rpm for 20 minutes at 4°C. The supernatant was collected and placed in EP tubes and stored at -80°C for later use.

[0039] 3. Effects on the morphology of mouse organ tissues Colonic tissue fixed in formalin solution was sequentially dehydrated, cleared, embedded in paraffin, sectioned, dewaxed, and stained with water and hematoxylin and eosin (H&E), AB-PAS, and immunohistochemically. Inflammatory cell infiltration and mucosal tissue damage were observed under a microscope.

[0040] 4. ELISA test The levels of IL-4, IL-6, and TGF-β in mouse serum and colon tissue were determined using the double-antibody sandwich method provided by Shanghai Enzyme-Link Biotechnology Co., Ltd. The experimental procedures were performed according to the kit instructions.

[0041] 5. High-throughput sequencing analysis of colon contents microorganisms Microbial DNA was extracted from the contents of mouse colon, and high-throughput sequencing technology was used to analyze the composition and structural changes of the gut microbiota, exploring the effects of Lactobacillus reuteri CAU 806 on the gut microbiota.

[0042] 6. The experimental results are as follows: (1) Changes in DAI in mice with chronic experimental colitis The results, as shown in Table 4, indicate that compared to the PBS group, the rate of change in body weight in the DSS group mice was significantly lower at day 43. P <0.05%, and the feces became loose and watery, with visible fresh blood. After ingestion of *Lactobacillus reuteri* CAU 806, the mice experienced reduced weight loss, and the diarrhea and bloody stools were alleviated. P <0.05). This indicates that treatment with *Lactobacillus reuteri* CAU806 significantly reduced disease activity caused by DSS-induced ulcerative colitis.

[0043] Table 4. Effects of *Lactobacillus reuteri* CAU 806 on changes in DAI in mice with chronic experimental colitis.

[0044] Note: PBS, normal control group; DSS, model group; DSS+CAU806, intervention group; Ratio (%) is the percentage of body weight on the day of the experiment to the starting body weight (D0); the scores for stool characteristics and rectal bleeding were calculated on day 48 of the experiment. * indicates a comparison between the PBS group and the DSS group. P <0.05; # indicates that other groups are compared with the DSS group. P <0.05.

[0045] (2) Lactobacillus reuteri CAU 806 inhibited colonic shortening in colitis mice and improved pathological conditions. After dissecting the mice, we measured the length of their colon. Figure 2 This is a comparison of colon length among the experimental groups. In the modeling group, due to DSS-induced intestinal damage, the colon was significantly shortened, accompanied by colonic redness and swelling. However, after ingestion of *Lactobacillus reuteri* CAU 806, both the length and appearance of the colon were altered, indicating that the intervention with *Lactobacillus reuteri* CAU 806 effectively alleviated the colonic shortening in mice.

[0046] We also performed a comparative analysis of colon tissue sections, and the results are as follows: Figure 3 As shown, HE staining revealed that the control group exhibited a normal morphology without colitis, with well-shaped, compact columnar epithelium, clear separation between the mucosa and submucosa, intact deep and narrow-septated intestinal crypts, and abundant goblet cells. Mice in the DSS group showed severe histopathological lesions and inflammatory infiltration, with significant loss of goblet cells and superficial epithelial cells, extensive crypt destruction, and increased inflammatory cell infiltration in the lamina propria and submucosa, resulting in the highest histopathological score. AB-PAS analysis showed a significant decrease in mucus content in the colonic goblet cells of the DSS group mice, and poorer mucus layer integrity. In contrast, the pathological damage in the *Lactobacillus reuteri* CAU 806 treatment group was significantly reduced, including elimination of edema, disappearance of inflammatory cell infiltration, restoration of mucus layer integrity, and only a mild inflammatory response. These results indicate that *Lactobacillus reuteri* CAU 806 has a protective effect on the colon of mice with colitis.

[0047] (3) Lactobacillus reuteri CAU 806 repairs the intestinal barrier in mice with colitis The content and distribution of mucin MUC2, closed band-1 (ZO-1), and transmembrane protein Occludin in the colon of colitis mice were observed and analyzed by immunohistochemical staining. The results are as follows: Figure 4 As shown, DSS group MUC2 ( Figure 4 A), ZO-1 ( Figure 4 C) and Occludin Figure 4 The positive expression area of ​​E protein was significantly lower than that in the control group, and its distribution in the inner lining of the colonic epithelium was discontinuous, indicating significant depletion. P< 0.0001, P <0.001, P< 0.0001), while after ingestion of Lactobacillus reuteri CAU 806, the expression of MUC2, ZO-1 and Occludin proteins was significantly upregulated, repairing the intestinal barrier ( P <0.01, P <0.001, P< 0.0001) Figure 4 B, 4D and 4F) reduce intestinal permeability and inflammatory cell infiltration, thereby alleviating colonic tissue damage and repairing the intestinal barrier in DSS-induced colitis mice.

[0048] (4) Lactobacillus reuteri CAU 806 alleviated the inflammatory state in mice with colitis. The levels of inflammatory factors IL-4, IL-6, and TGF-β in the serum of mice with colitis were detected by ELISA to assess their inflammation levels. The results are as follows: Figure 5 As shown. Compared with the PBS group, the DSS group had significantly lower levels of the anti-inflammatory factors IL-4 and TGF-β. P <0.0001, P <0.0001, after ingestion of *Lactobacillus reuteri* CAU 806, this trend was reversed, and its content increased significantly ( P <0.0001, P <0.01). Similarly, the level of the pro-inflammatory factor IL-6 in the DSS group was significantly higher than that in the PBS group ( P <0.0001), the content of *Lactobacillus reuteri* CAU 806 was significantly reduced after intervention ( P <0.01). In summary, intake of *Lactobacillus reuteri* CAU 806 can significantly promote the level of anti-inflammatory factors, reduce the level of pro-inflammatory factors, and alleviate intestinal inflammation.

[0049] (5) Lactobacillus reuteri CAU 806 alleviates DSS-induced intestinal flora disorder in mice like Figure 6 As shown, the Shannon index of mice in the DSS group ( Figure 6 A) and the Simposn index ( Figure 6 B) Both indices were lower than those in the PBS group. After ingestion of *Lactobacillus reuteri* CAU 806, both indices were significantly improved. Meanwhile, PCoA analysis ( Figure 6 C) shows that after DSS intervention, the distance between each group and the PBS group changed and was well distinguished, indicating that the composition of the gut microbiota community in mice may have changed more significantly after DSS treatment. After treatment with Lactobacillus reuteri CAU 806, the bacteria gradually separated from the DSS group and moved closer to the PBS group, indicating that the intake of Lactobacillus reuteri CAU 806 changed the similarity and stability of the gut microbiota community structure of DSS mice and restored the gut microbiota community of DSS mice.

[0050] like Figure 7 As shown, after DSS treatment, *Duboscirella* spp. ( Dubosiella Bacteroides ( Bacteroids ), genus *Alternaria* ( Alistipes) The abundance of harmful bacteria was significantly increased, while the abundance of beneficial bacteria decreased after ingestion of *Lactobacillus reuteri* CAU 806. Xylanibacter genus, DuncaniellaThe abundance of the genera indicates that intake of *Lactobacillus reuteri* CAU 806 can effectively alleviate DSS-induced intestinal microecological dysbiosis.

[0051] Example 4: Animal experiment in mice showing that *Lactobacillus reuteri* CAU 806 alleviates cardiomyocyte damage caused by myocardial infarction. 1. Establishment and grouping of experimental animal models Eight-week-old SPF-grade male C57Bl / 6J mice (weighing 18–22 g) were housed in a standard SPF barrier environment at the Department of Experimental Animal Science, China Agricultural University, under constant temperature (22°C), constant humidity (55%), 12-hour day-night cycle, and free access to food and water. All mice were allowed to acclimatize to the environment for one week before the experiment to ensure their health.

[0052] Thirty male C57BL / 6J mice were then randomly divided into three groups: (1) 10 animals in the normal control group (sham surgery group, Sham group); (2) Myocardial infarction group (MI group): 10 animals; (3) 10 animals in the myocardial infarction + Lactobacillus reuteri CAU 806 intervention group (Group B).

[0053] Before modeling, mice in the Sham and MI groups were administered 200 μL of physiological saline by gavage every two days; mice in the myocardial infarction + Lactobacillus reuteri CAU 806 intervention group were administered 200 μL of Lactobacillus reuteri CAU 806 bacterial suspension (concentration 10) by gavage every two days. 9 (CFU / mL); continued for three weeks. Afterwards, all mice underwent either left anterior descending coronary artery ligation or sham surgery. Following surgery, mice in the Sham and MI groups were administered 200 μL of normal saline via gavage daily, while mice in group B were administered 200 μL of 10% saline via gavage daily. 9 Mice were given a CFU / mL solution of Lactobacillus reuteri CAU 806 for one week. After the fourth week, all mice were fasted for 12 hours and then sacrificed.

[0054] In the MI and B groups, the left anterior descending coronary artery was ligated to establish the model: mice were lightly anesthetized with 1.0% to 1.5% isoflurane gas, and after endotracheal intubation, the heart was exposed via left thoracotomy. The left anterior descending coronary artery was ligated using 7-0 sterile silk sutures. After the heart was repositioned into the pleural cavity, the gas was quickly expelled and the sutures were immediately closed. The Sham group mice underwent the same surgical procedures as the MI group, except that ligation was not performed. All surgeries were performed by experienced technicians to ensure the safety and accuracy of the procedures.

[0055] 2. Postoperative Observation, Intervention, and Pre-specimen Collection Processing: Postoperatively, the establishment of the myocardial infarction model was assessed by observing the physiological manifestations of the mice (such as activity level, respiratory rate, and hair growth). Mice in the myocardial infarction group and the treatment group were observed postoperatively and received corresponding treatments according to the experimental design. Mice in the treatment group received the designed drug treatment or intervention, while mice in the control group maintained normal water intake.

[0056] During the experiment, the mice's activity was observed daily, with particular attention paid to whether they exhibited obvious symptoms of heart failure (such as shortness of breath and limited activity). After the fourth week, all mice were fasted for 12 hours and then sacrificed.

[0057] 3. Effects on the morphology of mouse organ tissues Heart and colon tissues from mice were collected and cryopreserved in liquid nitrogen for subsequent experiments. A portion of the heart tissue was placed in 4% formaldehyde fixative for histological section analysis. Inflammatory cell infiltration and mucosal tissue damage in the colon tissue were observed under a microscope.

[0058] 4. Experimental Results (1) Lactobacillus reuteri CAU 806 improves myocardial fibrosis and myocardial injury in MI mice. Masson staining in heart sections from mice with myocardial infarction is primarily used to observe and assess collagen fiber deposition in the cardiac tissue. Specifically, Masson staining effectively distinguishes between myocardial tissue and collagen fibers; collagen fibers appear blue, while myocardial cells appear red. The different staining colors reveal pathological changes in the cardiac tissue, particularly the degree of fibrosis. Results are as follows: Figure 8 As shown in Figure A, collagen deposition in the heart tissue of the Sham group (sham surgery group) was extremely low, with collagen fibers only distributed around blood vessels or subendocardially. In the MI group (myocardial infarction model group), the myocardial infarction area was significantly increased, indicating extensive cardiac fibrosis. The fibrosis in the infarcted area was very dense, and the fibrosis in the surrounding interstitium also extended, indicating severe structural damage and fibrotic response after myocardial injury. In Group B (treated with *Lactobacillus reuteri* CAU 806), collagen fibers were reduced, and the myocardial infarction area decreased (…). P <0.01)( Figure 8 (B) indicates that *Lactobacillus reuteri* CAU 806 can effectively reduce cardiac fibrosis and slow the progression of pathological fibrosis. Comparison of cardiac tissue pathological manifestations in different groups of mice revealed that *Lactobacillus reuteri* CAU 806 has a significant ameliorative effect on myocardial fibrosis in MI mice.

[0059] HE staining is an effective method for assessing myocardial injury in mice with myocardial infarction, reflecting the remodeling process and repair status of cardiac tissue. Figure 9As shown in Figure A, the Sham group (sham-operated group) had normal cardiac structure and function. The MI group (myocardial infarction model group) showed typical post-myocardial infarction changes, including cardiomyocyte necrosis, swelling, edema, and inflammatory response, reflecting significant cardiac damage. Group B (treated with *Lactobacillus reuteri* CAU 806) showed a decrease in the cross-sectional area of ​​cardiomyocytes (…). P <0.01)( Figure 9 B) It showed good protective effects after myocardial infarction, reducing edema, slowing myocardial cell damage, and improving cell arrangement. Overall, *Lactobacillus reuteri* CAU 806 can play a certain protective role in the repair of myocardial infarction, effectively alleviating damage and edema, and may become a potential treatment strategy for intervention after myocardial infarction.

[0060] (2) Lactobacillus reuteri CAU 806 inhibits neutrophil infiltration and reduces inflammatory response in MI mice. Ly6G is a specific marker for neutrophils and is commonly used to detect and quantify neutrophil infiltration. Neutrophils are important effector cells in the inflammatory response after myocardial infarction, playing a crucial role in both acute and chronic inflammatory responses. Figure 10 As shown in Figure A, the Sham group (sham-operated group) had a lower number of Ly6G-positive cells, and the healthy cardiac tissue showed no significant inflammatory response. The Ly6G staining intensity was generally weak, with almost no neutrophil infiltration. In the MI group (myocardial infarction group), due to ischemia and necrosis of the heart, the acute inflammatory response led to the recruitment of a large number of neutrophils to the infarct area. Ly6G immunofluorescence staining showed a significant accumulation of Ly6G-positive cells in the cardiac tissue within the infarct area, with a substantial increase in both the number of Ly6G-positive cells and the fluorescence intensity. P <0.0001 indicates that neutrophils extensively infiltrate the heart and participate in the inflammatory response. The number of Ly6G-positive cells was reduced in group B (Lactobacillus reuteri CAU 806 group). P <0.0001)( Figure 10 (B) This indicates that it alleviates the inflammatory response in the heart to some extent. The results suggest that *Lactobacillus reuteri* CAU 806 may reduce the inflammatory response in the heart after myocardial infarction by modulating the immune response and reducing neutrophil infiltration.

[0061] (3) Lactobacillus reuteri CAU 806 improves the intestinal barrier in MI mice HE staining of colonic tissue primarily reflects the indirect effects of myocardial infarction on the intestines. Myocardial infarction not only affects cardiac function but also triggers a series of systemic reactions, particularly significant changes in the structure and function of the intestines, which can be observed through HE staining. Figure 11As shown, in the Sham group (sham-operated group), the villi were intact, regularly arranged, and of normal length; the glandular structure was clear, without degeneration or collapse. There was no edema or inflammatory cell infiltration in the submucosa; the overall tissue was healthy, with no obvious pathological features, indicating that the intestinal tissue was normal without modeling. In the MI group (myocardial infarction model group), the villi were broken, shrunken, short, or completely detached, with disordered structure; the glands were disordered, partially collapsed, and had irregular cavities; the mucosa was thinned, with tissue necrosis; there was significant inflammatory cell infiltration and edema; the myocardial infarction model caused severe pathological damage to the distal intestine, consistent with an inflammatory response. In the B group (treated with *Lactobacillus reuteri* CAU 806), the villi structure was nearly normal, with consistent length and arrangement; the glandular structure was regular and relatively intact; the mucosa was thicker, with a small amount of inflammatory cell infiltration and almost no edema. The results showed that treatment with *Lactobacillus reuteri* CAU 806 could effectively reduce intestinal damage caused by myocardial infarction and had a potential protective effect on the gut, possibly by regulating the gut microbiota, inhibiting inflammatory responses and promoting tissue repair.

[0062] Occludin and ZO-1 are important components of tight junction proteins, playing a crucial role in maintaining tight junctions between intestinal epithelial cells and barrier function. Normal expression of Occludin and ZO-1 helps maintain the integrity of the intestinal barrier. Figure 12 As shown in Figure A, in healthy mice, the expression of Occludin and ZO-1 was generally high and uniformly distributed, indicating normal intestinal barrier function, good tight junctions between cells, and effective maintenance of intestinal barrier integrity. In the MI group (myocardial infarction group), the expression of Occludin and ZO-1 decreased, and the positive expression levels in immunostaining were significantly reduced. P <0.0001 indicates that myocardial infarction triggers intestinal barrier disruption. The reduction in tight junction proteins signifies increased intercellular permeability and impaired barrier function, potentially leading to intestinal leakage. In group B (Lactobacillus reuteri CAU 806 group), the expression of Occludin and ZO-1 in the mouse colon was restored, and the relative positive expression levels of Occludin and ZO-1 were significantly increased. P <0.0001)( Figure 12 (B) indicates that *Lactobacillus reuteri* CAU 806 may help restore colonic barrier function by inhibiting inflammation, regulating the gut microbiota, or directly affecting epithelial cell function. This suggests that *Lactobacillus reuteri* CAU 806 alleviates the damage to the intestinal barrier caused by myocardial infarction to some extent.

[0063] In summary, *Lactobacillus reuteri* CAU 806 alleviates myocardial infarction damage through a multi-target mechanism: at the cardiac level, it inhibits fibrosis, reduces myocardial edema and cell damage, and improves myocardial structure (e.g., reducing the cross-sectional area of ​​cardiomyocytes); its mechanism involves immunomodulation—reducing cardiac inflammation by inhibiting neutrophil infiltration. Simultaneously, CAU 806 can repair intestinal damage caused by myocardial infarction and promote the recovery of intestinal barrier function (e.g., upregulating tight junction protein expression), thereby synergistically maintaining cardio-gut axis homeostasis and achieving comprehensive intervention for myocardial infarction.

[0064] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A strain of *Lactobacillus reuteri*, characterized in that, Specifically, *Lactobacillus reuteri* ( Limosilactobacillus reuteri CAU 806, accession number CGMCC No. 34092.

2. The application of *Lactobacillus reuteri* CAU 806 as described in claim 1, characterized in that, It is used in the preparation of functional bacterial agents for the treatment or improvement of colitis and / or myocardial infarction.

3. The application as described in claim 2, characterized in that, The active ingredient of the functional microbial agent includes Lactobacillus reuteri CAU 806; the microbial agent is a liquid microbial agent or a solid microbial agent.

4. The application of *Lactobacillus reuteri* CAU 806 as described in claim 1, characterized in that, It is used in the preparation of products for the treatment or improvement of colitis and / or myocardial infarction.

5. The application of *Lactobacillus reuteri* CAU 806 as described in claim 4, characterized in that, The products mentioned include, but are not limited to: food, health products, or medicines.

6. The application of *Lactobacillus reuteri* CAU 806 as described in claim 4, characterized in that, Treatment or improvement of colitis includes, but is not limited to, the following: (1) DAI reduction: improves weight loss, loose stools and rectal bleeding caused by colitis; (2) Improves colon condition; inhibits colonic shortening, eliminates edema, eliminates inflammatory cell infiltration, and restores the integrity of the mucus layer; (3) Repairing the intestinal barrier: It can significantly upregulate the expression of MUC2, ZO-1 and Occludin proteins to repair the intestinal barrier and reduce intestinal permeability and inflammatory cell infiltration; (4) Relief of inflammatory state: The levels of anti-inflammatory factors IL-4 and TGF-β were significantly increased, while the level of pro-inflammatory factor IL-6 was significantly decreased; (5) Regulation of gut microbiota disorder: Shannon index and Simposn index were significantly improved; gut microbiota community was restored.

7. The application of *Lactobacillus reuteri* CAU 806 as described in claim 4, characterized in that, Treatment or improvement of myocardial infarction includes, but is not limited to, the following: (1) Improve myocardial fibrosis and myocardial damage caused by myocardial infarction: reduce the degree of myocardial fibrosis and reduce the area of ​​myocardial infarction; reduce edema, slow down myocardial cell damage, and improve cell arrangement; (2) Reduce neutrophil infiltration and alleviate inflammatory response; (3) Reduce intestinal damage caused by myocardial infarction; restore the expression of Occludin and ZO-1.

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