Akecman mucophilia and application thereof to prevention of non-alcoholic steatohepatitis

Abstract

The invention relates to an Akecman mucophilic protein strain LWHK0003, which has the effects of preventing non-alcoholic steatohepatitis, resisting obesity, preventing NASH induced by GAN diet, maintaining constant blood sugar and resisting inflammation.

Description

Ekmansia muciniphila and its use in preventing nonalcoholic steatohepatitis

Technical field

[0001] The present disclosure relates to Akkermansia muciniphila and uses thereof, and particularly to Akkermansia muciniphila and use thereof for preventing non-alcoholic steatohepatitis. [Existing Technology]

[0002] Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease. Without proper treatment, it can progress to non-alcoholic steatohepatitis (NASH), leading to irreversible cirrhosis and hepatocellular carcinoma. The progression of NASH is closely linked to metabolic syndrome and gut microbial imbalances caused by an unhealthy diet.

[0003] Probiotics are active microorganisms that live in symbiosis with their hosts. Proper intake can have beneficial effects on the host's health. Although traditional probiotics such as Lactobacillus and Bifidobacterium have been widely used and have good safety and human tolerance, their effects on the human intestinal microbiome remain limited, and the benefits and mechanisms of action vary significantly between different strains.

[0004] Therefore, there is a need to improve existing technologies in order to provide probiotics that can effectively prevent and treat NASH.

[0005] [Summary of the invention]

[0006] One embodiment of the present disclosure provides an Akkermansia muciniphila strain LWHK0003 (Akkermansia muciniphila LWHK0003), which is deposited in the German Collection of Microorganisms DSMZ with a deposit number of DSM 35051.

[0007] Another embodiment of the present disclosure provides a composition for preventing non-alcoholic steatohepatitis, comprising: the aforementioned Ekmansia muciniphila strain LWHK0003; and a pharmaceutically acceptable carrier.

[0008] In some embodiments, the Ekmansia muciniphila strain LWHK0003 is killed.

[0009] In some embodiments, the killed bacteria are pasteurized.

[0010] Another embodiment of the present disclosure provides a use of a strain for preparing a medicine or health food for preventing non-alcoholic steatohepatitis, wherein the strain is the aforementioned Ekmansia muciniphila strain LWHK0003.

[0011] In some embodiments, the strain is live, dead, or a combination thereof.

[0012] In some embodiments, the strain can prevent nonalcoholic steatohepatitis associated with obesity, high body fat, high liver triglyceride content, high nonalcoholic fatty liver disease activity score, high blood triglycerides, or a combination thereof.

[0013] In some embodiments, the strain is capable of preventing high fat content, including body fat or visceral fat, or a combination thereof, associated with nonalcoholic steatohepatitis.

[0014] In some embodiments, the strain is capable of preventing high NAFLD activity scores associated with NAFLD including hepatocyte swelling, hepatitis, or a combination thereof.

[0015] In some embodiments, the drug or health food is in the form of a capsule, tablet, powder or liquid.

[0016] In some embodiments, the drug or nutraceutical is formulated for oral delivery.

[0017]

Brief description of the attached figure

[0018] The various aspects of the present disclosure will be most easily understood when the following detailed description is read in conjunction with the accompanying drawings. It should be noted that, in accordance with industry standard operating procedures, various features may not be drawn to scale. In fact, the dimensions of various features may be arbitrarily increased or decreased for clarity of discussion. To make the above and other objects, features, advantages, and embodiments of the present disclosure more readily apparent, the accompanying drawings are described as follows:

[0019] FIG1A shows the body weight of mice at 8 weeks according to one embodiment of the present disclosure (statistical data were obtained using t-test).

[0020] FIG1B shows the changes in body weight of mice during 8 weeks according to one embodiment of the present disclosure (statistical data were obtained using t-test).

[0021] FIG2A shows the weight gain of mice at week 8 according to one embodiment of the present disclosure (statistical analysis using t-test).

[0022] FIG2B shows the changes in body weight gain of mice during 8 weeks according to one embodiment of the present disclosure (statistical data using t-test).

[0023] FIG3 shows the relative adipose tissue weights of mice according to one embodiment of the present disclosure, including the weights of subcutaneous adipose tissue (SAT), epididymal adipose tissue (EAT), perirenal adipose tissue (PRAT), and brown adipose tissue (BAT) (statistical analysis using t-test).

[0024] FIG4 shows the content of triglycerides (TG) in the liver of mice according to one embodiment of the present disclosure (statistical analysis using t-test).

[0025] 5A to 5C show pathological sections of mouse liver tissue according to one embodiment of the present disclosure.

[0026] FIG6A shows the steatosis score of mouse liver according to one embodiment of the present disclosure (statistical analysis using the Mann-Whitney U test).

[0027] FIG6B shows the ballooning score of mouse hepatocytes according to one embodiment of the present disclosure (statistical analysis using the Mann-Whitney U test).

[0028] FIG6C shows the inflammation score of mouse liver tissue according to one embodiment of the present disclosure (statistical analysis using the Mann-Whitney U test).

[0029] FIG6D shows a histopathological section of mouse liver tissue and the non-alcoholic fatty liver disease activity score (NAS) (statistically using the Mann-Whitney U test) according to one embodiment of the present disclosure.

[0030] FIG. 7A shows the triglyceride level in mouse blood (t-test) according to one embodiment of the present disclosure.

[0031] FIG. 7B shows the blood cholesterol level in mice according to one embodiment of the present disclosure (statistical analysis using t-test).

[0032] FIG8 shows the blood glucose levels of mice after feeding according to one embodiment of the present disclosure (statistical analysis using t-test).

[0033] FIG9A shows an oral glucose tolerance test (OGTT) in mice according to one embodiment of the present disclosure (statistical analysis using t-test).

[0034] FIG9B shows the area under the curve (AUC) of blood glucose in mice according to one embodiment of the present disclosure (statistical data using t-test).

[0035] [Implementation Method]

[0036] To make the description of the present disclosure more detailed and complete, the following provides illustrative descriptions of the implementation aspects and specific embodiments of the present disclosure, but this is not the only form of implementing or using the specific embodiments of the present disclosure. The various embodiments disclosed below can be combined or replaced with each other in beneficial circumstances, and other embodiments can be added to one embodiment without further description or explanation. In the following description, many specific details will be described in detail to enable the reader to fully understand the following embodiments. However, the embodiments of the present disclosure can also be practiced without these specific details.

[0037] In this document, unless the context specifically limits the use of the article, "a," "an," and "the" may refer to one or more. It will be further understood that the use of "comprise," "include," "have," and similar words herein specify the features, regions, integers, steps, operations, elements, and / or components described herein, but do not exclude the presence of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0038] The Akkermansia muciniphila LWHK0003 strain disclosed herein has been deposited with the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute under the accession number BCRC911209. The Akkermansia muciniphila LWHK0003 strain disclosed herein has also been deposited with the German Collection of Microorganisms (DSMZ) under the accession number DSM 35051, with a deposit date of June 14, 2024.

[0039] The Aikmanii muciniphila strain LWHK0003 disclosed herein is an extremely anaerobic bacterium isolated from healthy human feces. The strain is oval in shape, with a diameter of approximately 0.4-0.6 microns and a length of approximately 0.6-1 micron. Live Aikmanii muciniphila strains were cultured in brain heart infusion broth (BHI broth) in an anaerobic environment at 37°C.

[0040] In some embodiments of the present disclosure, the strain is administered to a subject orally or parenterally. In some embodiments of the present disclosure, the strain is formulated into an oral dosage form selected from the group consisting of a solution, a suspension, an emulsion, a powder, a lozenge, a pill, a syrup, a buccal lozenge, a tablet, a chewing gum, and a capsule for administration to a subject.

[0041] In some embodiments, pharmaceutically acceptable carriers include, but are not limited to, water, alcohols, glycols, preservatives, antioxidants, solvents, emulsifiers, suspending agents, decomposers, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, lubricants, absorption enhancers, active agents, humectants, odor absorbers, fragrances, pH adjusting agents, occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, enhancers), anti-irritants, colorants, propellants, surfactants, and other similar or suitable carriers of the present invention.

[0042] Throughout this article, the term "GAN diet" refers to Gubra-Amylin NASH. A high-fat diet known as the ALIOS (American lifestyle-induced obesity syndrome) diet has been shown to promote experimental NASH. Hansen et al. further modified this diet, termed the Amylin liver NASH (AMLN) diet, which reliably induces metabolic biochemical and liver histopathological features of NASH in C57BL / 6J mice (AMLN diet-induced NASH, AMLN DIONASH) and receptor-deficient ob / ob mice (AMLN ob / ob-NASH). However, due to the 2018 FDA ban on trans fats as a food additive, the Gubra Amylin NASH (GAN) diet, similar to the AMLN diet but without trans fats, was developed. Specifically, the GAN diet is diet #D09100310i (Source: Research Diets, Inc.) and contains 40 kcal / % fat, 20 kcal / % fructose, and 2% cholesterol.

[0043] Several examples and experimental examples are listed below to further illustrate the present disclosure of E. muciniphila and its use in preventing non-alcoholic steatohepatitis. However, these examples are for illustrative purposes only and are not intended to limit the present disclosure. The scope of protection of the present disclosure shall be determined by the appended claims.

[0044] Example

[0045] Experimental groups

[0046] C57BL / 6 male mice were divided into 3 groups and all were fed with GAN diet. The positive control group (hereinafter referred to as BAA-835) was fed with 1×10 9 CFU / 200μL (PBS containing 2.5% glycerol) of Ekmansia muciniphila (ATCC BAA-835) Pasteurized. In addition to the GAN diet, the experimental group (hereinafter referred to as LWHK03) was fed with 1×10 9 CFU / 200 μL (PBS containing 2.5% glycerol) of Ekmansia muciniphila (LWHK0003) Pasteurella mucosa. In addition to the GAN diet, the control group (Con) received daily tube-fed 200 μL of PBS containing 2.5% glycerol per mouse, the same volume as the control and experimental groups. Body weight, food intake, and water intake were recorded weekly. An oral glucose tolerance test (OGTT) was performed at week 7. Mice were sacrificed at week 8, and blood, multiple fat compartments, and liver samples were collected.

[0047] Example 1 Weight Gain

[0048] The experimental groups were divided as described above, and body weights were recorded from week 0 to week 8. The results, as shown in Figures 1A and 1B, clearly show that at week 8, while the average body weight of the control group mice was 31.17 grams, the LWHK03 group significantly reduced the average body weight of mice with GAN diet-induced non-alcoholic steatohepatitis (NASH) by 29.36 grams, a significantly greater effect than the BAA-835 group, which had an average weight of 30.16 grams.

[0049] Furthermore, as shown in Figures 2A and 2B, at week 8, the LWHK03 group suppressed the average weight gain of 6.48 grams in mice with GAN diet-induced non-alcoholic steatohepatitis (NASH), a superior effect compared to the BAA-835 group, which saw an average weight gain of 7.32 grams. The control group, however, saw an average weight gain of 8.64 grams.

[0050] Example 2 Fat Weight

[0051] The experimental groups were as described above. Mice were sacrificed at week 8 and fat was collected from multiple sites. The results, shown in Figure 3, show that at week 8, the average subcutaneous fat weight of the control mice was 1.34 grams. The LWHK03 group significantly reduced subcutaneous fat weight to an average of 1.07 grams, a significantly greater effect than the BAA-835 group, which had an average of 1.1 grams. Furthermore, the average perirenal fat weight of the control mice was 0.54 grams. The LWHK03 group significantly reduced perirenal fat weight to an average of 0.41 grams, a significantly greater effect than the BAA-835 group, which had an average of 0.45 grams.

[0052] Example 3 Liver triglyceride content

[0053] The experimental groups were assigned as described above. Mice were sacrificed at week 8 and liver tissue was collected. Triglyceride levels in the mice's livers were divided by liver weight to calculate the triglyceride / liver weight ratio. As shown in Figure 4, after eight weeks of GAN diet administration, the average triglyceride / liver weight ratio in the control group was 81.39 μg / mg. The LWHK03 group significantly reduced the triglyceride / liver weight ratio to an average of 66.6 μg / mg, a significantly greater effect than the BAA-835 group, which had an average of 68.57 μg / mg.

[0054] Example 4 Liver pathology sections and liver pathology scores

[0055] Experimental groups were assigned as described above. Mice were sacrificed at 8 weeks and liver tissue was collected. Sections were stained (Figures 5A to 5C) and interpreted according to the 2005 publication by Kleiner et al. Veterinary pathologists calculated a severity score based on pathological features such as hepatic steatosis, hepatocyte swelling, and inflammation (Figures 6A to 6C). Scores for individual NAS indicators and a total score (Figure 6D) were calculated based on the 2005 publication by Kleiner et al., with a maximum score of 8. A total score of 5 or higher was considered NASH, with higher scores indicating more severe NASH.

[0056] The results showed that after 8 weeks of GAN diet, there was no significant difference in steatosis between the groups (Figure 6A). In the hepatocyte expansion index (Figure 6B), among the 8 mice in the control group, 1 scored 1 and 7 scored 2. Among the 12 mice in the LWHK03 group, 2 scored 0, 5 scored 1, and 5 scored 2. Compared with the control group, the degree of hepatocyte expansion in the LWHK03 group was significantly lower, and the effect was better than that in the BAA-835 group (3 scored 0 and 9 scored 2 among the 12 mice). In the inflammatory response index (Figure 6C), among the 8 mice in the control group, 3 scored 0 and 5 scored 1. Among the 12 mice in the LWHK03 group, 6 scored 0 and 6 scored 1. Compared with the control group, the degree of inflammatory response in the LWHK03 group was significantly lower, and the effect was better than that in the BAA-835 group (1 scored 0, 9 scored 1, and 2 scored 2 among the 12 mice). In the NAS index (Figure 6D), among the 8 mice in the control group, 4 had a total score of 5, and 4 had a total score of 6. Among the 12 mice in the LWHK03 group, 1 had a total score of 3, 3 had a total score of 4, 6 had a total score of 5, and 2 had a total score of 6. Compared with the control group, the NAS index in the LWHK03 group was significantly lower, and the effect was better than that in the BAA-835 group. Among the 12 mice in the LWHK03 group, 1 had a total score of 2, 1 had a total score of 3, 1 had a total score of 4, 7 had a total score of 6, and 2 had a total score of 7.

[0057] Example 5 Blood lipid analysis

[0058] The experimental groups were divided as described above. Mice were sacrificed and blood was collected at week 8. The results, as shown in Figure 7A, show that after eight weeks of GAN diet, blood triglycerides in the control group averaged 126.98 mg / dL. The LWHK03 group significantly reduced blood triglycerides to an average of 94.92 mg / dL, a significantly greater effect than the BAA-835 group, which averaged 131.17 mg / dL. There were no significant differences in blood cholesterol among the three groups (Figure 7B).

[0059] Example 6 Blood Glucose Analysis

[0060] The experimental groups were as described above, and blood samples were collected after a 6-hour fast at week 7. The results, as shown in Figure 8, showed no significant differences in fasting blood glucose levels among the three groups.

[0061] Example 7 Oral glucose tolerance test (OGTT)

[0062] The experimental groups were assigned to an oral glucose tolerance test (OGTT) at week 7, as described previously. The results, as shown in Figure 9A, showed no significant differences in blood glucose levels among the three groups at different time points. Further analysis of the area under the blood glucose curve (AUC) (Figure 9B) revealed that the control group had an average AUC of 28,418, while the LWHK03 group (average AUC of 30,390) exhibited superior glycemic control compared to the BAA-835 group (average AUC of 30,577).

[0063] The Aikmanii muciniphila LWHK0003 strain disclosed herein, when induced by a GAN diet, prevents obesity, reduces weight gain, reduces fat accumulation (such as subcutaneous fat, epididymal fat, and perirenal fat), reduces liver triglyceride levels, prevents GAN diet-induced NASH (such as by reducing hepatocyte swelling and liver inflammation), reduces blood triglycerides, and maintains blood glucose homeostasis. Furthermore, the Aikmanii muciniphila LWHK0003 strain (Pasteurella) disclosed herein exhibits superior effects compared to the Pasteurella strain of ATCC BAA-835.

[0064] Although the present disclosure has been disclosed in the form of implementation methods as described above, it is not intended to limit the present disclosure. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure shall be determined by the appended claims.

[0065]

Biological Material Deposit

[0066] The Akkermansia muciniphila strain LWHK0003 disclosed herein is deposited in the German Collection of Microorganisms DSMZ (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstraβe 7B, 38124 Braunschweig, Germany), with a deposit number of DSM 35051 and a deposit date of June 14, 2024.

Claims

1. An Akkermansia muciniphila strain LWHK0003 (Akkermansia muciniphila LWHK0003), which is deposited in the German Microbiological Culture Collection DSMZ with a deposit number of DSM 35051.

2. A composition for preventing non-alcoholic fatty liver disease, comprising: The Ekmansia muciniphila strain LWHK0003 as claimed in claim 1; and A pharmaceutically acceptable carrier.

3. The composition as claimed in claim 2, wherein the Ekmansia muciniphila strain LWHK0003 is a dead bacterium.

4. The composition as claimed in claim 3, wherein the dead bacteria are sterilized by Pasteurization.

5. Use of a strain for preparing a medicine or health food for preventing non-alcoholic fatty hepatitis, wherein the strain is the Ekmansia muciniphila strain LWHK0003 as claimed in claim 1.

6. The use as claimed in claim 5, wherein the strain is a live bacterium, a dead bacterium or a combination thereof.

7. The use as claimed in claim 5, wherein the strain can prevent non-alcoholic fatty liver disease associated with obesity, high fat, high liver triglycerides, high non-alcoholic fatty liver activity score, high blood triglycerides, or a combination thereof.

8. The use as claimed in claim 7, wherein the strain can prevent high fat including body fat or visceral fat or a combination thereof associated with non-alcoholic steatohepatitis.

9. The use according to claim 7, wherein the strain can prevent high non-alcoholic fatty liver activity scores associated with non-alcoholic steatohepatitis including hepatocyte swelling, liver inflammatory response, or a combination thereof.

10. The use according to claim 5, wherein the medicine or health food is in the form of capsule, tablet, powder or liquid.

11. The use according to claim 5, wherein the medicine or health food is formulated for oral delivery.

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