Microbiome-based sitz bath method for patients with bowel disease

Abstract

The present invention relates to a microbiome-based sitz bath method for patients with bowel disease. By periodically performing sitz baths for a certain period of time on mice induced with inflammatory bowel disease using sitz bath water containing an industrial lactic acid bacteria product as a microbial agent, it was confirmed that not only were symptoms of inflammatory bowel disease alleviated, but also the level of inflammatory cytokines was significantly reduced, compared to mouse groups orally or rectally administered with the lactic acid bacteria product. Accordingly, the present invention demonstrates that inflammatory bowel disease in the actual intestine is alleviated when a microbial agent such as a lactic acid bacteria product is included in sitz bath water for sitz bathing, and is thus provided as a microbiome-based sitz bath method for patients with bowel disease.

Description

Microbiome-based sitz bath method for patients with intestinal disease

[0001] The present invention relates to a microbiome-based sitz bath method for patients with intestinal disease.

[0002] Inflammatory bowel disease (IBD) is a disease characterized by abnormal chronic inflammation in the intestinal tract that recurs and improves repeatedly. While ulcerative colitis and Crohn's disease are well-known examples, the exact mechanism of its onset has not yet been clearly elucidated. It is known to be associated with genetic and immunological abnormalities, as well as environmental factors such as stress or drugs. It typically begins in young adults and is accompanied by symptoms such as abdominal pain, fever, diarrhea, and rectal bleeding.

[0003] Although the incidence rate in Asian countries was previously reported to be relatively lower compared to the West, the global prevalence of inflammatory bowel disease is on the rise, with the rate of increase appearing higher in Asian countries than in the West. Furthermore, interest within the Korean medical community is growing due to the fact that the disease epidemiology and clinical characteristics in Asian countries differ somewhat from those in the West. Additionally, research on this disease is gradually increasing as concerns regarding the risk of developing Crohn's disease in young men are also rising.

[0004] Inflammatory bowel disease (IBD) is a chronic condition requiring lifelong management. The goal of treatment is to improve symptoms to induce remission and maintain it for an extended period. The optimal treatment method is selected by considering the extent of the lesions, severity, clinical manifestations, and complications. Currently, there are four classes of drugs representative for the treatment of IBD: 5-ASA (5-Aminosalicylic acid), corticosteroids, immunomodulators, and anti-tumor necrosis factor-α agents (anti-TNF-α). Since these drugs exert their effects through different mechanisms of action, they are used in a stepwise manner according to sophisticated clinical guidelines; in particular, anti-TNF-α is recommended as a second-line treatment for patients who have not responded to or have failed first-line medications. However, there are limitations to overcoming inflammatory bowel disease of unknown mechanistic etiology using the drugs currently used in clinical practice. Therefore, there is an urgent need to develop new targets that can effectively treat inflammatory bowel disease and new drugs that regulate them.

[0005] Meanwhile, research on the microbiome is actively underway as recent studies have revealed that microorganisms residing within the human body have a significant impact on human health. The microbiome refers to the entire genetic information of microorganisms coexisting within the human body, allowing for the analysis of the principles behind the generation of beneficial and harmful bacteria as well as their correlations with diseases. In particular, cholesterol, allergies, rhinitis, atopic dermatitis, various metabolic diseases related to obesity, immune diseases, enteritis, and heart disease are reported to be linked to the microbiome.

[0006] As research on the microbiome progresses, studies on probiotics and prebiotics within the human body are also actively underway. Probiotics refer to components that inhibit harmful bacteria in the gut, while prebiotics refer to components that promote or activate the growth of beneficial bacteria; the health-promoting effects of probiotics and prebiotics are currently being confirmed through various related papers. However, most existing probiotic products are manufactured for oral consumption in the form of fermented milk, granules, or powders. It has been found that when these products are consumed orally, the number of bacteria reaching the intestines to produce actual effects is very small, and their efficacy varies from person to person, indicating limited effectiveness. Therefore, efforts are being made to develop new types of products that utilize probiotics to improve intestinal inflammation through various pathways, rather than through conventional methods of consumption.

[0007] The objective of the present invention is to provide a microbiome-based sitz bath method for patients with intestinal disease.

[0008] The present invention provides a sitz bath method for a patient with intestinal disease, comprising the steps of: preparing a sitz bath solution by mixing a microbial preparation in powder form with lukewarm water; and immersing the anus in the sitz bath solution to perform a sitz bath.

[0009] In addition, the present invention provides a microbial preparation for the above-mentioned sitz bath method.

[0010] According to the present invention, when mice with induced inflammatory bowel disease were periodically subjected to sitz baths for a certain period in water containing an industrial lactic acid bacteria product, which is a microbial preparation, it was confirmed that not only were the symptoms of inflammatory bowel disease improved compared to mouse groups that received oral or rectal administration of the lactic acid bacteria product, but the levels of inflammatory cytokines were also significantly reduced. This demonstrates that sitz bath treatment with a microbial preparation, such as a lactic acid bacteria product, improves inflammatory bowel disease in the actual intestine, thereby providing a microbiome-based sitz bath method for patients with intestinal disease.

[0011] Figure 1 shows the results of observing changes in body weight for about 9 days after inducing intestinal inflammation in mice with 3% DSS and treating them with microbial agents via oral administration, rectal administration, and sitz bath methods, respectively.

[0012] Figure 2 shows the results of measuring the length of the large intestine after inducing intestinal inflammation in mice with 3% DSS and treating the mice with microbial agents by oral administration, rectal administration, and sitz bath for 8 days, respectively, sacrificing the mice, extracting the large intestine, and measuring the length of the large intestine.

[0013] Figure 3 shows the results of inducing intestinal inflammation in mice with 3% DSS, treating mice with microbial agents orally, rectalally, and by sitz bath for 8 days, sacrificing the mice, extracting the colon, and performing a pathological analysis of the colon tissue through H&E staining.

[0014] Figure 4 shows the results of measuring the length of the large intestine stained with methylene blue after treating mice with a sitz bath for 15 minutes and 30 minutes, respectively, using methylene blue.

[0015] Figure 5 shows the results of measuring the amounts of cytokines and chemokines in colon tissue after inducing intestinal inflammation in mice with 3% DSS and treating the mice with microbial agents by oral administration, rectal administration, and sitz bath methods for 8 days, respectively, sacrificing the mice, extracting the colon, and measuring the amounts of cytokines and chemokines in the colon tissue.

[0016] The terms used in this specification have been selected based on currently widely used general terms whenever possible, taking into account their functions in the present invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0017] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0018] The present invention will be described in more detail below.

[0019] The present invention provides a sitz bath method for a patient with intestinal disease, comprising the steps of: preparing a sitz bath solution by mixing a microbial preparation in powder form with lukewarm water; and immersing the anus in the sitz bath solution to perform a sitz bath.

[0020] The above microbial preparation comprises live bacteria, dead bacteria, culture medium, fermented medium, or a mixture thereof, and the lactic acid bacteria may include one or more strains selected from the group consisting of the genus Streptococcus, the genus Bifidus, the genus Lactobacillus, the genus Lactiplantibacillus, and the genus Lacticaseibacillus.

[0021] The above microbial preparation may be contained in lukewarm water at a concentration of 50 mg / mL to 150 mg / mL.

[0022] The above intestinal disease is a disease selected from the group consisting of constipation, diarrhea, irritable bowel syndrome, Crohn's disease, ulcerative colitis, intestinal Behcet's disease, infectious enteritis, ischemic bowel disease, and radiation enteritis.

[0023] In addition, the present invention provides a microbial agent for the above-mentioned sitz bath method.

[0024] Hereinafter, experimental examples and embodiments will be described in detail to aid in understanding the present invention. However, the following experimental examples and embodiments are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following experimental examples and embodiments. The experimental examples and embodiments of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0025]

[0026] [Example]

[0027] 1. Experimental Animals and Rearing Conditions

[0028] All animal experiments were conducted after obtaining approval from the Animal Ethics Committee of Pusan ​​National University (PNU-2022-0125), and the entire process was performed by experienced researchers to minimize unnecessary suffering to the mice. The 7-week-old female C57BL / 6 mice used in the experiments were purchased from Samtaco, a company specializing in laboratory animal production, and were housed under standard conditions (temperature 24℃–26℃, humidity 45%–55%, 12-hour day / night cycle). Prior to the experiment, all mice underwent a one-week acclimatization period provided with sterilized feed and water to adapt to the rearing environment.

[0029]

[0030] 2. Application of Intestinal Disease Animal Models and Sitz Bath Systems

[0031] An animal model of intestinal disease was constructed to evaluate the effects of a sitz bath system using microbial preparations on intestinal disease. In this embodiment, microbial preparations comprising Streptococcus thermophilus, Bifidus breve, Bifidus animalis, Lactobacillus acidophilus, Lactobacillus helveticus, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei were used. The degree of improvement in induced intestinal inflammation was evaluated by administering the above microbial preparations to mice in various ways. First, mice were classified into a total of four experimental groups based on the method of administration of the microbial agent: a control group (Nil), an oral administration group (Oral gavage), an intrarectal administration group (Intrarectal), and a sitz bath treatment group (Hip bath). To create an intestinal disease model in mice, intestinal inflammation was induced by feeding drinking water containing 3% DSS (Dextran Sodium Sulfate) (MP Biomedicals, CA, USA) for 8 days, followed by feeding water for 1 day. For the oral administration group (Oral gavage) and the intrarectal administration group (Intrarectal), 125 mg of the microbial agent was diluted (625 mg / mL) in 200 μL of distilled water and administered. For the sitz bath treatment group (Hip bath) to which the sitz bath system of the present invention was applied, 2.5 g of the microbial agent was diluted (approx. 83.3 mg / mL) in 30 mL of lukewarm water (35°C to 42°C) and used. Mice in the hip bath treatment group were placed in a 100 mL experimental glass jar with their heads facing upward, and a microbial agent was placed in the jar (about 25–30 ml) to sufficiently submerge the mouse's anus. The hip bath was conducted for 15 or 30 minutes.After the sitz bath, the mice were thoroughly dried on dry, sterile bedding and then returned to their original rearing cages. The administration of microbial agents and the application of the sitz bath system were carried out once daily for 8 days during the supply of DSS.

[0032]

[0033] 3. Histological analysis of intestinal tissue

[0034] Colon tissue was collected from mice after the experiment was completed. The feces and contents of the colon tissue were thoroughly washed with physiological saline, and the entire intestine was sampled in a Swiss roll form and fixed in 4% formaldehyde for 3 hours. The fixed tissue was dehydrated by slowly immersing it sequentially in 70%, 80%, 90%, 95%, and 100% ethanol for 1 hour each, and then prepared into paraffin blocks. The paraffin blocks were sectioned to a thickness of 6 mm and stained with hematoxylin and eosin (H&E). The dihydrate-stained samples were observed under a microscope.

[0035] 4. Cytokine and Chemokine Analysis

[0036] Colon tissue was collected from mice after the experiment was completed. The feces and contents of the colon tissue were thoroughly washed with physiological saline, and the colon was divided into proximal (near the cecum) and distal (near the anus) sections, each of which was homogenized. After obtaining the protein supernatant by centrifugation at 2000 rpm at 4°C for 10 minutes, inflammatory cytokines (TNF-α, IL-6) and chemokines (KC, RANTES, MIP-α, MCP1) were quantitatively analyzed using the Luminex Mouse Discovery Assay kit.

[0037]

[0038] 5. Evaluation Results

[0039] 5-1. Changes in mouse body weight

[0040] To confirm the effect of microbial agents on improving intestinal inflammation according to treatment methods in a mouse model of intestinal inflammation induced by DSS, mice were treated with 3% DSS as drinking water, and microbial agents were administered orally, rectally, and in sitz baths once daily. After tracking changes in mouse body weight for about 9 days, the rectal administration group showed no significant difference compared to the control group, but the oral administration group and the sitz bath group showed significantly improved body weight loss compared to the control group.

[0041]

[0042] 5-2. Changes in the Length of the Large Intestine

[0043] To investigate the effects of microbial agents on improving intestinal inflammation according to treatment methods in a mouse model of DSS-induced intestinal inflammation, mice were treated with 3% DSS as drinking water, while microbial agents were administered orally, rectalally, or via sitz bath once daily. On the 9th day of the experiment, the mice were sacrificed and their colons were excised, after which the colon lengths of the mice in each group were measured. As a result, it was confirmed that the oral administration group and the sitz bath treatment group showed a significant improvement in the reduction of colon length, with the sitz bath treatment group exhibiting the most significant improvement.

[0044]

[0045] 5-3. Histological Analysis

[0046] To confirm the pathological improvement effects of microbial agents on intestinal colon tissue according to treatment methods in a mouse model of intestinal inflammation induced by DSS, the colon was sampled in Swiss roll form and the results were confirmed by H&E staining. As a result, in the control group, epithelial cells of the colon were destroyed and immune cell infiltration increased rapidly due to inflammation caused by DSS, leading to the loss of the protective barrier of the colon's epithelial cells. However, it was confirmed that these pathological symptoms of the colon tissue were alleviated in all three groups treated with microbial agents; among them, the sitz bath treatment group was found to improve colon inflammation much more effectively compared to the oral administration group and the rectal administration group.

[0047]

[0048] 5-4. Analysis of the Influence of Sitz Bath Systems on Treatment Time

[0049] To measure the areas of the intestine where sitz bath treatment can actually affect using methylene blue, mice were sitz bathed with methylene blue for 15 and 30 minutes, respectively. Afterward, the mice were sacrificed and their colons were excised. Upon examining the areas of the colon stained with methylene blue, it was confirmed that the area of ​​the intestine stained with methylene blue significantly increased with the duration of the sitz bath treatment. In particular, when treated for 15 minutes or longer, methylene blue staining occurred in more than 40% of the colon, suggesting that sitz bath treatment may have a significant overall effect on the colon.

[0050]

[0051] 5-5. Analysis of Cytokines and Chemokines in the Colon Following Sitz Bath Treatment

[0052] To analyze intestinal inflammatory cytokines and chemokines according to the treatment method of microbial agents in a mouse model of intestinal inflammation induced by DSS, mouse colons were excised, divided into proximal (near the cecum) and distal (near the anus), and homogenized to obtain protein samples. Subsequently, inflammatory cytokines and chemokines were quantitatively analyzed using the Luminex MAGPIX system. As a result, it was confirmed that the inflammatory cytokine TNF-α and the chemokine RANTES in the colon were significantly reduced in the sitz bath treatment group. Since this phenomenon was observed in the distal portion of the colon, which is actually affected by the sitz bath treatment, it was confirmed that actual sitz bath treatment can improve the inflammatory response induced by DSS at a molecular level.

[0053]

[0054] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

[0055] Numerical ranges include the values ​​defined in the above ranges. All maximum numerical limits given throughout this specification include all lower numerical limits as clearly written. All minimum numerical limits given throughout this specification include all higher numerical limits as clearly written. All numerical limits given throughout this specification will include all better numerical ranges within a wider numerical range, as clearly written.

Claims

1. A step of preparing sitz bath water by mixing a powdered microbial preparation with lukewarm water; and A sitz bath method for a patient with intestinal disease, comprising the step of immersing the anus in the above-mentioned sitz bath water.

2. A sitz bath method for a patient with intestinal disease according to claim 1, characterized in that the microbial preparation comprises live bacteria, dead bacteria, culture medium, fermented liquid, or a mixture thereof.

3. A sitz bath method for a patient with intestinal disease according to claim 2, characterized in that the lactic acid bacteria comprises one or more strains selected from the group consisting of the genus Streptococcus, the genus Bifidus, the genus Lactobacillus, the genus Lactiplantibacillus, and the genus Lacticaseibacillus.

4. A sitz bath method for a patient with intestinal disease according to claim 1, characterized in that the microbial preparation is contained in lukewarm water at a concentration of 50 mg / mL to 150 mg / mL.

5. A sitz bath method for a patient with an intestinal disease according to claim 1, characterized in that the intestinal disease is a disease selected from the group consisting of constipation, diarrhea, irritable bowel syndrome, Crohn's disease, ulcerative colitis, intestinal Behcet's disease, infectious enteritis, ischemic intestinal disease, and radiation enteritis.

6. A microbial preparation for the sitz bath method of any one of paragraphs 1 to 5.

7. The microbial preparation according to claim 6, characterized in that the microbial preparation comprises one or more strains selected from the group consisting of the genus Streptococcus, the genus Bifidus, the genus Lactobacillus, the genus Lactiplantibacillus, and the genus Lacticaseibacillus.

8. A microbial preparation according to claim 6, characterized in that the microbial preparation comprises Streptococcus thermophilus, Bifidus breve, Bifidus animalis, Lactobacillus acidophilus, Lactobacillus helveticus, Lactiplantibacillus plantarum, and Lacticaseibacillus paracasei in the examples.

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