WO2022265151A1 - Composition containing bifidobacterium bifidum bgn4 for relieving, treating, or preventing renal failure - Google Patents
Composition containing bifidobacterium bifidum bgn4 for relieving, treating, or preventing renal failure Download PDFInfo
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- WO2022265151A1 WO2022265151A1 PCT/KR2021/010563 KR2021010563W WO2022265151A1 WO 2022265151 A1 WO2022265151 A1 WO 2022265151A1 KR 2021010563 W KR2021010563 W KR 2021010563W WO 2022265151 A1 WO2022265151 A1 WO 2022265151A1
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- renal failure
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
Definitions
- the present invention relates to a composition for improving, treating or preventing renal failure containing Bifidobacterium bifidum BGN4, and more specifically, to improving renal failure containing Bifidobacterium bifidum BGN4. It relates to a food composition for renal failure or a pharmaceutical composition for preventing or treating renal failure.
- Acute kidney injury is one of the most common complications in the treatment of critically ill patients, and patients with underlying diseases are known to be a particularly high-risk group.
- AKI contributes to increased length of stay, increased mortality and increased health care costs.
- the prevalence of AKI as a complication of COVID-19 was 8.4%, 3.6% of patients required dialysis, and mortality increased 13-fold.
- Probiotics are "living organisms" that exert beneficial effects in a variety of chronic inflammatory situations.
- Probiotics are "living organisms" that exert beneficial effects in a variety of chronic inflammatory situations.
- several studies have been published proving the beneficial effects and potential of probiotics or bacterial products, little progress has been made in the application of pre/probiotics in AKI.
- Bifidobacterium BGN4 Bifidobacterium BGN4 (BGN4) was selected as the probiotic. Bifidobacterium , a major component of the gut microbiome, has multiple functions including carbohydrate fermentation, vitamin synthesis or immune modulation. The effects of BGN4 on the intestinal microbial environment as well as the severity of AKI and distant organ damage were investigated.
- the present invention provides a food composition for improving renal failure comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
- Bifidobacterium bifidum BGN4 KCCM12754P
- KCCM12754P Bifidobacterium bifidum BGN4
- the renal failure may be preferably acute renal failure.
- the present invention provides a pharmaceutical composition for preventing or treating renal failure comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
- Bifidobacterium bifidum BGN4 KCCM12754P
- KCCM12754P Bifidobacterium bifidum BGN4
- the renal failure may be preferably acute renal failure.
- the present invention provides an animal food composition for improving renal failure in animals, comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
- Bifidobacterium bifidum BGN4 KCCM12754P
- KCCM12754P Bifidobacterium bifidum BGN4
- the renal failure may preferably be acute renal failure.
- the present invention provides an animal pharmaceutical composition for preventing or treating renal failure in animals, comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
- Bifidobacterium bifidum BGN4 KCCM12754P
- KCCM12754P Bifidobacterium bifidum BGN4
- the renal failure may be acute renal failure.
- the present invention provides a feed composition comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
- a healthy microbiome helps maintain intestinal barriers and mucosal immune tolerance.
- Previous studies have confirmed that acute kidney injury (AKI) induces dysbiosis, inflammatory response, and increased permeability in the intestine.
- AKI acute kidney injury
- IRI bilateral ischemic reperfusion injury
- BGN4 administration greatly increased the diversity of the microbiome, and in particular, suppressed the increase of Enterobacteria and Bacteroides, which are characteristics of AKI.
- BGN4 administration markedly improved the colonic microenvironment, including intestinal wall permeability, apoptosis of colonic epithelial cells, and infiltration of neutrophils and inflammatory macrophages.
- Immune cells co-cultured with BGN4 showed an increase in CD103+CD11c+ dendritic cells and CD4+CD25+ regulatory T cells, suggesting a direct immunomodulatory effect.
- BGN4 treatment directly increased regulatory T cells in the colon, mesenteric lymph nodes (MNL) and kidney after acute renal injury, whereas suppressed the Th17 pathway in the small intestine, and these changes in the intestinal immune response were associated with AKI. It was also accompanied by a reduction in severity, renal IL-6 expression, and remote organ damage (liver damage) caused by AKI.
- Figure 3 is the microbiome diversity index Simpson Richness and Simpson Evenness measurement results of the experimental group and the control group .
- Figure 4 is a result showing the relative distribution of microbiome at the genus level of the experimental group and the control group.
- 9 and 10 show changes in the number of infiltrating F4/80 staining positive macrophages and Ly6G staining positive neutrophils in the experimental group and the control group.
- 16 and 17 show changes in AST and ALT of the experimental group and the control group.
- Figure 18 shows the LDH change of the experimental group and the control group
- 19 and 20 are results confirming an increase in the fraction of CD11c+CD103+ regulatory dendritic cells and CD4+CD25+ regulatory T cells in CD11c+CD103+ splenocytes co-cultured with the strain BGN4 of the present invention for 72 hours.
- 21 and 22 show changes in Foxp3-positive cells in the colon and kidney.
- Figure 23 shows changes in Foxp3 mRNA expression level in the colon and kidney.
- 26 and 27 are comparison results of flow cytometry analysis of small intestine IL-17A+ cells in an experimental group and a control group.
- the present inventors studied the effects of probiotics on kidney damage and primary organs in AKI, especially changes in the intestinal environment.
- intestinal wall permeability was enhanced and neutrophils, inflammatory macrophages, and Th17 pathway activation were decreased, while regulatory T cells with immunomodulatory functions were increased. It led to a reduction in organ damage.
- Bifidobacterium is a microorganism that accounts for more than 80% of the intestinal microflora of healthy breastfeeding infants.
- Bifidobacterium bifidum ( B. bifidum ) is the second most abundant species among bifidobacterium, and BGN4 used in the present invention is is one of the strains.
- formula-fed infants have significantly lower proportions of bifidobacterial species in the gut microbiome, and the abundance of bifidobacteria decreases with age.
- BGN4 is a probiotic microorganism obtained from the intestines of breastfed infants.
- the supply of BGN4 increased the uniformity in terms of the diversity of intestinal microorganisms.
- the Simpson-diversity index is one of the indicators that present microbiome characteristics by considering the number and abundance of microbiome components at the same time. Changes in the intestinal microenvironment observed along with changes in the microbiome led to protective effects against renal damage caused by ischemia/reperfusion. BGN4 supplementation could help maintain colonic barrier function by enhancing strict junction protein levels, reducing colonic epithelial cell apoptosis, and preventing mucosal disruption.
- the present invention confirmed that the beneficial effect of BGN4 was to directly enhance the barrier function as well as prevent the spread of pathogenic bacteria.
- BGN4 pretreatment could create a solid colonic environment and prevent primary organ damage from AKI.
- BGN4 showed colonic immune modulating effects.
- the present inventors discovered a specific immunomodulatory effect of BGN4, the expansion of regulatory T cells in the intestine. It has been well established in several studies that increased regulatory T cells have systemic beneficial functions.
- BGN4 exerts renoprotective effects by reducing inflammation and inhibiting colon apoptosis. Intestinal permeability decreased in IRI after administration of BGN4, indicating that probiotics enhance colonic mucosal function. Systemic inflammation and aggravating effects were blocked as the colonic barrier function was strengthened with the supply of BGN4.
- Serum LDH levels are used to detect tissue damage and have been reported to correlate with cardiovascular and all-cause mortality in patients with metabolic syndrome. Serum LDH level elevation of AKI was also reduced by BGN4 pretreatment. This means that BGN4 shows a beneficial protective effect in reducing systemic inflammation and may exert anti-inflammatory effects.
- IRI-associated liver damage could be reduced along with the renal protective effect through the supply of BGN4.
- Liver histology using H&E staining showed no clear histological differences, but when compared with IRI, the elevation of liver enzymes AST and ALT was suppressed in IRI administered with BGN4.
- the ability of probiotics to strengthen the colonic barrier and modulate the immune system could play a role in protecting against distant organ damage caused by AKI.
- the present invention provides Bifidobacterium bifidum BGN4, which is effective in preventing or treating renal failure.
- the renal failure may preferably be acute renal failure.
- Bifidobacterium bifidum BGN4 is a well-known microorganism that can be purchased from Bifido Co., Ltd. (688-1 Sangoan-ri, Hongcheon-eup, Hongcheon-gun, Gangwon-do) and deposited with the Korea Microorganism Conservation Center (foreign) as KCCM12754P.
- the present invention provides a food composition for improving renal failure or an animal food composition for improving renal failure in animals, comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
- the renal failure may be acute renal failure.
- the Bifidobacterium bifidum BGN4 is preferably included in an amount of 0.00001 to 50% by weight compared to the food composition for improving renal failure. If it is less than 0.00001% by weight, the effect is insufficient, and if it exceeds 50% by weight, the increase in effect compared to the amount used is insignificant, which is uneconomical.
- the food composition of the present invention is, for example, meat, grains, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, noodles, chewing gum, dairy products, ice cream, alcoholic beverages, alcohol, vitamin complexes And it may be any one selected from other health supplements, but is not necessarily limited thereto.
- the present invention provides a pharmaceutical composition for preventing or treating renal failure or an animal pharmaceutical composition for preventing or treating renal failure in animals, comprising Bifidobacterium bifidum BGN4 or a culture solution thereof or a dry powder thereof.
- the renal failure may be acute renal failure.
- the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, diluent or excipient.
- Usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, There are microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and one or more selected from among them may be used.
- the pharmaceutical composition of the present invention may further include at least one selected from among fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifiers, or preservatives.
- the pharmaceutical composition dosage form of the present invention may be in a preferred form depending on the method of use, and in particular, formulated by adopting a method known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It is good.
- Examples of specific formulations include PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, Syrups ( SYRUPS), OPHTHALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS ), SUSPENSIONS, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, SUPPOSITORIES, INJECTIONS, SPIRITS, CATAPLSMA ), capsules (CAPSULES), creams (CREAMS), troches (TROCHES), tinctures (TINCTURES), pastas (PASTES), pills (PILLS), soft or hard gelatin capsules.
- the dosage of the pharmaceutical composition for preventing or treating renal failure of the present invention is preferably determined in consideration of the administration method, the age, sex, weight, and severity of the disease of the user.
- the pharmaceutical composition for preventing or treating renal failure of the present invention can be administered once or more at 0.00001 to 100 mg/kg (body weight) per day based on the active ingredient.
- the above dosage is only an example for illustration, and may be changed according to the doctor's prescription according to the condition of the user.
- the present invention provides a feed composition comprising Bifidobacterium bifidum BGN4 or a culture solution thereof or a dry powder thereof.
- 0.001 to 1.0% by weight of the Bifidobacterium bifidum BGN4 may be contained based on the total weight of the composition. This is because when the content is less than 0.001% by weight based on the total weight of the composition, the effect of improving, preventing or treating renal failure cannot be sufficiently exerted, and when it exceeds 1.0% by weight, stability and safety of the formulation are problematic.
- composition of the present invention is fed by oral administration by mixing it with a conventional formulated feed, and there is little tolerance or side effects such as immunosuppression even when continuously administered or over-administered.
- the animal may be, for example, a companion animal, and may specifically be a dog or cat.
- the composition of the present invention can be said to be more effective because cats suffer from renal failure and have a high mortality rate.
- mice Male C57BL/6 mice, 6 weeks of age, were purchased from Orient Bio. Animals were housed in a specific pathogen free 1 facility with free access to water and feed. They were randomized into probiotics supplementation and IRI trials. Probiotics were supplied and used from Bifido, and BGN4 was individually orally administered at a concentration of 2x10 9 once a day for 5 days a week (FIG. 1). 1 shows the experimental scheme of the present invention.
- Acute kidney injury was induced through bilateral ischemic reperfusion injury (IRI). Two weeks later, rats were subjected to IRI. The contents of the operation were as follows. After intraperitoneal anesthesia, both renal vessels were ligated through a back incision for 24.5 minutes. After surgery, they were monitored on a heating pad until waking up. This study was approved by the Institutional Review Board of the Center for Animal Research, Korea University College of Medicine (IRB number: KOREA-2016-0260).
- Plasma creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactite dehydrogenase (LDH) were measured using Beckman AU ® 5821 Beckman (Beckman Coulter, USA). Tissues were excised after intraperitoneal anesthesia, and liver and colon tissues were fixed with 4% paraformaldehyde and paraffin. Kidney tissue was evaluated using standard light microscopy. Kidney damage was evaluated in darkness by evaluating the tubular damage site in 5 random areas ( ⁇ 100) throughout the tissue using PAS staining.
- the degree of damage was evaluated semi-quantitatively, and if there is no visible necrosis, it is grade 0, 0 to 25% (grade 1), 25 to 50% (grade 2), 50 to 75% (grade 3), and 75 to 100%. (grade 4).
- F4/80 (1:100; mch-497-GA; Bio-Rad Laboratories, Hercules, CA, USA) and Ly6G (1:200; 14-59-85; eBioscience) were used to evaluate the level of macrophage/neutrophil infiltration. Kidney and colon tissues were stained with monoclonal antibodies against each other.
- Proteins were extracted from whole colon tissue samples using the bicinchoninic acid method, and mouse antibodies against Claudin-1 (1:200; ab15098; abcam) and Occludin (1:200; ab168986; abcam) were used to detect cell junction proteins. expression was investigated. Band intensities were analyzed using Image Studio TM (Lite LI-COR Biosciences, Lincoln, NE, USA) software. Target protein concentration was quantified as ⁇ -actin concentration and compared.
- Splenocytes were cultured with BGN4 in 2% FBS at 37°C for 72 hours in a 5% CO2 incubator, and the CD103+CD11c+ or CD4+CD25+ cell fractions were compared and analyzed.
- Fluorescein isothiocyanate (FITC)-conjugated dextran (FITC-dextran; catalog number FD4; Sigma-Aldrich, St. Louis, MO, USA) was incubated in PBS (100 mg/mL) with limited drinking water overnight. After oral administration, fluorescence was extracted from the blood after 4 hours.
- Flow cytometric analysis was performed on immune cells isolated from the mesenteric lymph node (MNL), spleen, kidney and intestine. Cells were stained with fluorescently labeled monoclonal antibodies (anti-CD4, anti-CD25, Fixable viability Dye, anti-Foxp3, anti-CD45, anti-CD11c, anti-CD103, anti-CX3CR1, anti-Ly6C, eBioscience, BioLegend and BD Biosciences) and analyzed using FlowJo software (Tree Star Inc.; Ashland, CA, USA) after measurement using a 4-channel flow cytometer (FACSCantoII TM , BD Bioscience).
- FACSCantoII TM 4-channel flow cytometer
- RT-PCR Real-time reverse transcription-polymerase chain reaction
- RNA was purified using TRIzol extraction reagent (Thermo Fisher Scientific, US) according to the manufacturer's protocol and cDNA was synthesized using standard procedures.
- Foxp3 RT-PCR was performed using iCycler IQ Real-Time PCR Detection System (Bio-Rad Laboratories) and iQ TM SYBR ® Green Supermix (Bio-Rad Laboratories). 18S rRNA was used as a reference gene reference (RT2 PCR Primer Set; Applied Biosystems, Foster City, CA, USA), and the results of the control group were compared by fold difference.
- At least two stool pellets per mouse were collected and stored at -70 °C for batch analysis.
- 16S rRNA pyrosequencing analysis was used.
- a next-generation microbiome information platform, QIIME 2 TM was used.
- Data are expressed as mean ⁇ standard error of the mean (SEM). Unpaired t-test and one-way analysis of variance (using Bonferroni post hoc tests) were used to compare groups of two or more and groups of three or more, respectively. A P-value ⁇ 0.05 was considered statistically significant. Data were analyzed using GraphPad Prism version 8.5 (GraphPad Software Inc., La Jolla, Calif.).
- the experimental group and the control group showed different characteristics in microbiome composition in principal coordinate analysis (PCoA) (FIG. 2).
- PCoA principal coordinate analysis
- 2 is a result of principal coordinate analysis of component 1 and component 2 of the experimental group and the control group.
- Component 1 and component 2 constituting the x-axis and y-axis in the coordinates are two vectors, and are values representing relative dispersion indicating how close and how far apart the distributions of the intestinal microbiome of the experimental group and the control group are from each other.
- the results of this experiment showing different distributions in the coordinates mean that the characteristics of the two groups are not the same, suggesting that the distribution of microorganisms in the intestine is changed by IRI.
- Microbiome diversity indices are one of several ways to characterize the distribution of gut microbes.
- the Simpsons-Evenness index in terms of uniformity increased, but the Simpson richness index in terms of diversity did not increase (FIG. 3).
- Figure 3 is the Simpson Richness and Simpson Evenness measurement results of the experimental group and the control group.
- FIG. 4 shows the relative distribution of microbiome at the genus level of the experimental group and the control group.
- Claudin-1 a splicing protein that enhances the barrier function, decreased during IRI, but was normalized by BGN4 administration, and there was no difference in occludin, another splicing protein (FIGS. 5 and 6). 5 and 6 show changes in Claudin-1 and Occludin in the experimental group and the control group.
- Intestinal epithelial cell death was increased in the IRI group and decreased in the BGN4 administration group (FIGS. 7 and 8).
- 7 and 8 show the degree of apoptosis of intestinal epithelial cells in the experimental group and the control group.
- the graph of FIG. 8 quantifies the number of cells showing each staining positive in a high power field (HPF).
- FIG. 11 shows changes in intestinal permeability between the experimental group and the control group.
- the ATN score (Acute Tubular Necrosis, acute tubular necrosis) is evaluated for blindness by evaluating the tubular damage site in 5 random areas ( ⁇ 100) throughout the tissue using PAS staining, a tissue staining method to determine the degree of tubular necrosis. did The degree of damage was evaluated semi-quantitatively, and if there is no visible necrosis, it is grade 0, 0 to 25% (grade 1), 25 to 50% (grade 2), 50 to 75% (grade 3), and 75 to 100%. (grade 4).
- the Kidney ATN score in the graph of FIG. 13 quantifies and displays the degree of tubular damage in a high power field (HPF), and Kidney Ly6G and Kidney F4/80 quantify the number of cells showing staining positivity. .
- IL-6 is a representative of cytokines that can determine the level of inflammation in the kidney, and damage to the kidney tissue occurs after IRI, and shows a decrease in inflammation after IRI in the experimental group compared to the control group.
- Serum creatinine showed that BGN4 supplementation reduced the severity of IRI (FIG. 15). 15 shows changes in blood creatine in the experimental group and the control group.
- FIGS. 16 and 17 show changes in AST and ALT of the experimental group and the control group. Elevations of liver enzymes AST and ALT were suppressed in IRI administered with BGN4.
- the graph of FIG. 17 quantifies the number of cells showing each staining positive in a high power field (HPF).
- HPF high power field
- 18 shows LDH changes in the experimental group and the control group. Serum LDH levels are used to detect tissue damage and have been reported to correlate with cardiovascular and all-cause mortality in patients with metabolic syndrome. Serum LDH level elevation of IRI was also reduced by BGN4 pretreatment.
- FSC forward scatter
- SSC side scatter
- FIG. 19 the cells widely distributed along the long axis of the ellipse in the fourth quadrant are macrophages, and the cells showing clustered characteristics on the left under the third quadrant are lymphocytes.
- the graph of FIG. 20 quantitatively indicated the increase in the fraction of CD11c+CD103+ regulatory dendritic cells and CD4+CD25+ regulatory T cells.
- Vehicle was a control group not treated with BGN4, and when co-cultured with BGN4, CD11c+CD103+ regulatory dendritic cells and CD4+CD25+ regulatory T cells were significantly expanded and differentiated.
- FIGS. 21 and 22 show changes in Foxp3-positive cells in the colon and kidney, and Figure 22 shows the quantification of the number of cells showing each staining positive in a high power field (HPF).
- Figure 23 shows changes in the expression of Foxp3 mRNA in the colon and kidney, and shows the quantification of the number of cells showing each staining positive in a high power field (HPF).
- Foxp3+ regulatory T cells (Foxp3+ Treg cells) were consistently increased in Kidney, mesenteric lymph nodes (MNL), and colon, and CX 3 CR 1 intermediate Ly 6 C high The effect of suppressing the infiltration of monocytes was also shown together (FIG. 24, FIG. 25).
- 24 and 25 are flow cytometry analysis results of the experimental group and the control group. Flow cytometry antibody staining showed that the fraction of Foxp3-positive regulatory T cells (Foxp3+ Treg cells) increased in IRI, and the number of regulatory T cells (Foxp3+ Treg cells) was significant in the colon, mesenteric lymph nodes, and kidneys of the BGN4-treated experimental group. expanded to one level.
- FIGS. 26 and 27 are comparison results of flow cytometry analysis of small intestine IL-17A+ cells in an experimental group and a control group.
- the cells showing clustering characteristics were lymphocytes, and after confirming that they were negative by fixable viability dye (FVD) antibody staining, whether or not the positive fraction of CD4+IL-17A+ helper T 17 cells increased was confirmed.
- FVD fixable viability dye
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Abstract
The present invention relates to a food composition for relieving renal failure, or a pharmaceutical composition for treating and preventing renal failure, the composition containing Bifidobacterium bifidum BGN4. The Bifidobacterium bifidum BGN4 according to the present invention exhibits the effects of relieving or treating acute renal failure, and thus can be effectively used as a probiotic material.
Description
본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4를 함유한 신부전 개선용, 치료용 또는 예방용 조성물에 관한 것으로, 더욱 구체적으로 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4를 함유하는 신부전 개선용 식품 조성물 또는 신부전 치료, 예방용 약학 조성물에 관한 것이다.The present invention relates to a composition for improving, treating or preventing renal failure containing Bifidobacterium bifidum BGN4, and more specifically, to improving renal failure containing Bifidobacterium bifidum BGN4. It relates to a food composition for renal failure or a pharmaceutical composition for preventing or treating renal failure.
급성 콩팥 손상(Acute kidney injury, AKI)는 중환자 치료에서 흔히 발생하는 합병증 중 하나이며 기저질환이 있는 환자들은 특히 고위험군으로 알려져 있다. AKI는 재원 기간 증가, 사망률 증가 및 의료비 증가에 기여한다. 최근의 메타 분석에 따르면 COVID-19 관련 합병증으로 나타난 AKI의 유병률은 8.4%이고, 3.6%의 환자들은 투석이 필요했으며, 사망률은 13배 증가하였다.Acute kidney injury (AKI) is one of the most common complications in the treatment of critically ill patients, and patients with underlying diseases are known to be a particularly high-risk group. AKI contributes to increased length of stay, increased mortality and increased health care costs. According to a recent meta-analysis, the prevalence of AKI as a complication of COVID-19 was 8.4%, 3.6% of patients required dialysis, and mortality increased 13-fold.
한편, 인간은 장 내에 숙주와 완전한 공생을 유지하고 면역체계의 발달과 형성에 중요한 역할을 하는 100조 이상의 미생물 세포들을 가지고 있다. 이 공생관계는 다양한 질병 상황에서 변화하며, 장내 불균형은 병태 생리 기전에서 중요한 한 요인이 될 수 있다. AKI가 다른 장기에도 손상을 일으키면서 심한 전신 염증을 유발한다는 점은 의미있는 시사점을 제공한다. AKI에서 신장-장 누화 현상 (Kidney-gut crosstalk)이 존재할 가능성이 있다는 것이다. 다만 당뇨병, 비만, 염증성 장질환과는 달리 AKI 분야에서 신장과 내장의 연관성을 입증한 연구는 소수에 불과하다. On the other hand, humans have more than 100 trillion microbial cells in the intestine that play an important role in the development and formation of the immune system and maintain complete symbiosis with the host. This symbiotic relationship changes in various disease situations, and intestinal imbalance can be an important factor in the pathophysiological mechanism. The fact that AKI causes severe systemic inflammation while causing damage to other organs provides a meaningful implication. It is possible that kidney-gut crosstalk exists in AKI. However, unlike diabetes, obesity, and inflammatory bowel disease, only a handful of studies have demonstrated the relationship between kidneys and intestines in the field of AKI.
최근에 본 발명자들은 AKI에서 신장과 장의 독특한 양방향성 관계를 보여주었다. 우리는 선행 연구에서 AKI후 초래된 장 내 불균형이 박테리아 전위(translocation), 장 염증, 단쇄 지방산(Short chain fatty acid, SCFA) 감소와 함께 장벽 투과성 증가와 관련이 있다는 것을 보여준 바 있다. 경구 항생제를 사용하여 마이크로바이옴을 완전히 고갈시키면 신기능 손상이 완화되었고, 이러한 신장 보호 효과는 조절 T 세포와 M2 대식세포의 증가와 함께 장내 Th17, Th1 반응 감소와 관련이 있었다. 또한, AKI를 유발한 쥐의 분변을 무균쥐에게 이식하면, 정상쥐 분변을 이식한 경우에 비해 AKI가 악화됨을 보여줌으로서 장 마이크로바이옴이 AKI 치료의 새로운 치료 목표가 될 수 있음을 제시하였다.Recently, we have shown a unique bidirectional relationship between kidney and intestine in AKI. Previous studies have shown that intestinal imbalances after AKI are associated with increased barrier permeability with reduced bacterial translocation, intestinal inflammation, and short chain fatty acids (SCFA). Complete depletion of the microbiome using oral antibiotics ameliorated renal function impairment, and this renoprotective effect was associated with reduced intestinal Th17 and Th1 responses along with increases in regulatory T cells and M2 macrophages. In addition, it was suggested that the intestinal microbiome could be a new therapeutic target for AKI treatment by showing that transplantation of AKI-induced mouse feces into germ-free mice worsened AKI compared to transplantation of normal mouse feces.
프로바이오틱스는 다양한 만성 염증 상황에서 유익한 효과를 나타내는 "생존 미생물(living organism)"이다. 그러나, 프로바이오틱스 또는 균제품의 이로운 효과와 가능성을 입증한 몇몇 연구가 발표되었음에도 불구하고, AKI에서 프리/프로바이오틱스를 응용하는 연구는 거의 진전이 없었다. Probiotics are "living organisms" that exert beneficial effects in a variety of chronic inflammatory situations. However, although several studies have been published proving the beneficial effects and potential of probiotics or bacterial products, little progress has been made in the application of pre/probiotics in AKI.
본 발명에서는 양측 허혈 재관류 손상(ischemic reperfusion injury, IRI)을 통해 AKI 동물 모델에서 프로바이오틱스 투여가 신장 보호 효과가 있는지를 실험을 통해 확인하고자 하였다. 프로바이오틱스로는 비피도박테리움 BGN4(BGN4)를 선택했다. 장내 미생물군 중 주요 구성 요소인 비피도박테리움속 (Bifidobacterium)은 탄수화물 발효, 비타민 합성 또는 면역 변조를 포함한 다양한 기능을 가지고 있다. BGN4가 장내 미생물 환경뿐만 아니라 AKI 중증도, 원거리 장기손상 등에 미치는 영향에 대하여 살펴보았다.In the present invention, it was attempted to confirm through experiments whether probiotics administration has a renal protective effect in an AKI animal model through bilateral ischemic reperfusion injury (IRI). Bifidobacterium BGN4 (BGN4) was selected as the probiotic. Bifidobacterium , a major component of the gut microbiome, has multiple functions including carbohydrate fermentation, vitamin synthesis or immune modulation. The effects of BGN4 on the intestinal microbial environment as well as the severity of AKI and distant organ damage were investigated.
본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4 (KCCM12754P) 또는 그의 배양액 또는 그 건조 분말을 포함하는 것을 특징으로 하는 신부전 개선용 식품 조성물을 제공한다.The present invention provides a food composition for improving renal failure comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
본 발명의 신부전 개선용 식품 조성물에 있어서, 상기 신부전은, 바람직하게 급성신부전일 수 있다.In the food composition for improving renal failure of the present invention, the renal failure may be preferably acute renal failure.
본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4 (KCCM12754P) 또는 그의 배양액 또는 그 건조 분말을 포함하는 것을 특징으로 하는 신부전 예방 또는 치료용 약학 조성물을 제공한다. The present invention provides a pharmaceutical composition for preventing or treating renal failure comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
본 발명의 신부전 예방 또는 치료용 약학 조성물에 있어서, 상기 신부전은, 바람직하게 급성신부전일 수 있다. In the pharmaceutical composition for preventing or treating renal failure of the present invention, the renal failure may be preferably acute renal failure.
본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4 (KCCM12754P) 또는 그의 배양액 또는 그 건조 분말을 포함하는 것을 특징으로 하는 동물 신부전 개선용 동물 식품 조성물을 제공한다.The present invention provides an animal food composition for improving renal failure in animals, comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
본 발명의 동물 신부전 개선용 동물 식품 조성물에 있어서, 상기 신부전은, 바람직하게 급성신부전일 수 있다. In the animal food composition for improving renal failure in animals of the present invention, the renal failure may preferably be acute renal failure.
본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4 (KCCM12754P) 또는 그의 배양액 또는 그 건조 분말을 포함하는 것을 특징으로 하는 동물 신부전 예방 또는 치료용 동물 약학 조성물을 제공한다. The present invention provides an animal pharmaceutical composition for preventing or treating renal failure in animals, comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
본 발명의 동물 신부전 예방 또는 치료용 동물 약학 조성물에 있어서, 상기 신부전은, 바람직하게 급성신부전일 수 있다. In the animal pharmaceutical composition for preventing or treating renal failure in animals of the present invention, the renal failure may be acute renal failure.
본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4 (KCCM12754P) 또는 그의 배양액 또는 그 건조 분말을 포함하는 것을 특징으로 하는 사료조성물을 제공한다. The present invention provides a feed composition comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
건강한 마이크로바이옴은 장 내 방어막과 점막 면역 내성을 유지하는데 도움을 준다. 선행 연구에서 급성 콩팥 손상(Acute kidney injury, AKI)이 장 내 미생물 불균형 (Dysbiosis), 염증 반응 및 투과성 증가를 유발한다는 것을 확인한 바 있다. 본 발명에서는 프로바이오틱스 비피도박테리움 BGN4이 상기와 같은 급성 콩팥 손상 모델의 개선, 예방 또는 치료 효능이 있는지를 살펴보았다. A healthy microbiome helps maintain intestinal barriers and mucosal immune tolerance. Previous studies have confirmed that acute kidney injury (AKI) induces dysbiosis, inflammatory response, and increased permeability in the intestine. In the present invention, it was investigated whether the probiotics Bifidobacterium BGN4 has an improvement, preventive or therapeutic effect on the acute kidney injury model as described above.
하기 실험에서는 C57BL/6 생쥐를 이용하여 양측 허혈 재관류 손상 (bilateral ischemic reperfusion injury, IRI)을 진행하고 대조군과 비교하였다. 실험군은 IRI 진행 2주 전부터 매일 한 번씩 경구로 프로바이오틱스를 투여하였다.In the following experiment, bilateral ischemic reperfusion injury (IRI) was performed using C57BL/6 mice and compared with a control group. The experimental group was orally administered probiotics once daily for 2 weeks prior to IRI.
실험 결과, BGN4 투여는 마이크로바이옴의 다양성을 크게 증가시켰고, 특히 AKI의 특징인 엔테로박테리아와 박테로이데스의 증가을 억제하였다. 이외에도 BGN4 투여에 의해 장 벽 투과성, 대장 상피세포의 세포 사멸, 중성구와 염증성 대식세포 침윤을 포함한 대장 미세환경이 현저하게 개선되었다. BGN4와 공동 배양된 면역세포는 CD103+ CD11c+ 수지상세포와 CD4+ CD25+ 조절 T 세포의 증가를 보여 직접적인 면역 조절 효과를 시사했다. 또한, BGN4 치료는 급성신손상 후 대장, 장간막 림프절(mesenteric lymph nodes: MNL) 및 신장에서 직접적으로 조절 T 세포의 증가를 일으킨 반면, 소장 내 Th17 pathway 를 억제하였으며, 이러한 장내 면역반응의 변화는 AKI 중증도의 감소, 신장 IL-6 발현 및 AKI에 의한 원격장기손상(간손상)의 경감도 동반되었다. As a result of the experiment, BGN4 administration greatly increased the diversity of the microbiome, and in particular, suppressed the increase of Enterobacteria and Bacteroides, which are characteristics of AKI. In addition, BGN4 administration markedly improved the colonic microenvironment, including intestinal wall permeability, apoptosis of colonic epithelial cells, and infiltration of neutrophils and inflammatory macrophages. Immune cells co-cultured with BGN4 showed an increase in CD103+CD11c+ dendritic cells and CD4+CD25+ regulatory T cells, suggesting a direct immunomodulatory effect. In addition, BGN4 treatment directly increased regulatory T cells in the colon, mesenteric lymph nodes (MNL) and kidney after acute renal injury, whereas suppressed the Th17 pathway in the small intestine, and these changes in the intestinal immune response were associated with AKI. It was also accompanied by a reduction in severity, renal IL-6 expression, and remote organ damage (liver damage) caused by AKI.
결론적으로 선제적으로 BGN4를 투여함으로써 AKI의 중증도, 동반된 간손상이 완화되었다. 이러한 신장 보호 효과는 대장, 장간막 림프절 및 신장 조절 T 세포의 증가와 동시에 소장의 Th 17 pathway 억제와 함께 나타났으며, BGN4가 면역 조절 효과를 일으켜 신장 보호 효과에 기여할 수 있음을 시사한다. 이를 통해 본 발명 프로바이오틱스 BGN4는 AKI의 심각성이나 원격 장기 손상을 줄일 수 있는 것으로 판단되었다. In conclusion, preemptive administration of BGN4 alleviated the severity of AKI and accompanying liver damage. This renoprotective effect was accompanied by an increase in colon, mesenteric lymph node, and renal regulatory T cells and suppression of the Th 17 pathway in the small intestine, suggesting that BGN4 may contribute to the renoprotective effect by causing immunomodulatory effects. Through this, it was determined that the probiotics BGN4 of the present invention can reduce the severity of AKI or remote organ damage.
도 1은 본 발명의 실험 계획을 보여준다.1 shows the experimental scheme of the present invention.
도 2는 실험군과 대조군의 component 1 및 component 2 주좌표분석 결과이다. 2 is a result of principal coordinate analysis of component 1 and component 2 of the experimental group and the control group.
도 3은 실험군과 대조군의 의 마이크로바이옴 다양성 지수인 Simpson Richness 및 Simpson Evenness 측정결과이다.
Figure 3 is the microbiome diversity index Simpson Richness and Simpson Evenness measurement results of the experimental group and the control group .
도 4는 실험군과 대조군의 속(Genus) 수준의 마이크로바이옴 상대적분포도를 보여주는 결과이다.Figure 4 is a result showing the relative distribution of microbiome at the genus level of the experimental group and the control group.
도 5 및 도 6은 실험군과 대조군의 Claudin-1 및 Occludin의 변화를 보여준다. 5 and 6 show changes in Claudin-1 and Occludin in the experimental group and the control group.
도 7 및 도 8은 실험군과 대조군의 장 상피세포 사멸 정도를 보여준다. 7 and 8 show the degree of apoptosis of intestinal epithelial cells in the experimental group and the control group.
도 9 및 도 10은 실험군과 대조군의 F4/80 염색 양성 대식세포 및 Ly6G 염색 양성 중성구 침윤의 수의 변화를 보여준다.9 and 10 show changes in the number of infiltrating F4/80 staining positive macrophages and Ly6G staining positive neutrophils in the experimental group and the control group.
도 11은 실험군과 대조군의 장 투과도 변화를 보여준다. 11 shows changes in intestinal permeability between the experimental group and the control group.
도 12 및 도 13은 실험군과 대조군의 신장 기능 지표 변화를 보여준다12 and 13 show changes in kidney function indicators in the experimental group and the control group
도 14는 실험군과 대조군의 신장 IL-6 발현 변화를 보여준다14 shows changes in renal IL-6 expression in the experimental group and the control group.
도 15는 실험군과 대조군의 혈중 크레아틴 변화를 보여준다. 15 shows changes in blood creatine in the experimental group and the control group.
도 16 및 도 17은 실험군과 대조군의 AST 및 ALT 변화를 보여준다. 16 and 17 show changes in AST and ALT of the experimental group and the control group.
도 18은 실험군과 대조군의 LDH 변화를 보여준다Figure 18 shows the LDH change of the experimental group and the control group
도 19 및 도 20은 72시간 동안 본 발명 균주 BGN4와 공동배양된 CD11c+CD103+ 비장세포의 조절 수지상 세포와 CD4+CD25+ 조절 T 세포의 분율 증가를 확인한 결과이다19 and 20 are results confirming an increase in the fraction of CD11c+CD103+ regulatory dendritic cells and CD4+CD25+ regulatory T cells in CD11c+CD103+ splenocytes co-cultured with the strain BGN4 of the present invention for 72 hours.
도 21 및 도 22는 대장, 신장에서 Foxp3 양성세포의 변화를 보여준다. 21 and 22 show changes in Foxp3-positive cells in the colon and kidney.
도 23은 대장, 신장에서 Foxp3 mRNA 발현량 변화를 보여준다.Figure 23 shows changes in Foxp3 mRNA expression level in the colon and kidney.
도 24 및 도 25는 실험군과 대조군의 유세포분석 결과이다. 24 and 25 are flow cytometry analysis results of the experimental group and the control group.
도 26 및 도 27은 실험군과 대조군의 소장 IL-17A+ 세포의 유세포분석 비교 결과이다. 26 and 27 are comparison results of flow cytometry analysis of small intestine IL-17A+ cells in an experimental group and a control group.
장 마이크로바이옴과 대사물들이 염증, 산화 스트레스, 섬유화를 조절해 신장-장 축(kidney-gut axis)의 활성화를 통해 신장 질환의 병인과 생리기전에 중추적인 역할을 한다는 연구 결과가 축적되고 있다. Research is accumulating that the gut microbiome and metabolites play a pivotal role in the pathogenesis and physiological mechanisms of kidney disease through activation of the kidney-gut axis by regulating inflammation, oxidative stress, and fibrosis. .
본 발명자들은 AKI에서 신장 손상 및 원발 장기에 미치는 프로바이오틱스의 효과 특히 장내 환경의 변화에 대해 연구하였다. 프로바이오틱스 BGN4를 전처치 하였을 때, 장 벽 투과성을 강화하고 중성구, 염증성 대식세포 및 Th17 pathway 활성화를 감소시키는 반면 면역조절기능을 가지는 조절 T 세포를 증가시켰으며 이러한 장 환경의 변화는 급성신손상 및 원격장기 손상의 감소로 이어졌다. The present inventors studied the effects of probiotics on kidney damage and primary organs in AKI, especially changes in the intestinal environment. When pretreated with probiotics BGN4, intestinal wall permeability was enhanced and neutrophils, inflammatory macrophages, and Th17 pathway activation were decreased, while regulatory T cells with immunomodulatory functions were increased. It led to a reduction in organ damage.
비피도박테리움은 건강한 모유수유아 장내 미생물 중 80% 이상을 차지하는 미생물로, 비피도박테리움 비피둠(B. bifidum)은 비피도박테리움 중 두 번째로 풍부한 종이며 본 발명에서 사용한 BGN4는 해당 균주 중 한가지이다. 모유 수유아와 달리 분유 수유아는 장내 마이크로바이옴 중 비피도박테리아 종의 비율이 현저하게 낮고, 비피도박테리아의 풍부함은 나이가 들수록 감소한다. Bifidobacterium is a microorganism that accounts for more than 80% of the intestinal microflora of healthy breastfeeding infants. Bifidobacterium bifidum ( B. bifidum ) is the second most abundant species among bifidobacterium, and BGN4 used in the present invention is is one of the strains. Unlike breast-fed infants, formula-fed infants have significantly lower proportions of bifidobacterial species in the gut microbiome, and the abundance of bifidobacteria decreases with age.
BGN4는 모유수유아의 장에서 얻은 프로바이오틱스 미생물이다. 본 발명에서 BGN4 공급은 장내 미생물의 다양성 측면에서 균일성을 높였다. Simpson-diversity index는 마이크로바이옴 구성 요소의 수와 풍부함을 동시에 고려해 마이크로바이옴 특성을 제시하는 지표 중 하나이다. 마이크로바이옴의 변화와 함께 관찰된 장내 미세환경의 변화는 허혈/재관류에 의한 신장손상의 보호 효과로 이어졌다. BGN4 공급은 엄격한 접합 단백질 수준을 강화하고, 대장 상피세포 세포 사멸을 줄이고, 점막 파괴를 방지함으로써 대장 장벽 기능을 유지하는 데 도움이 될 수 있었다.BGN4 is a probiotic microorganism obtained from the intestines of breastfed infants. In the present invention, the supply of BGN4 increased the uniformity in terms of the diversity of intestinal microorganisms. The Simpson-diversity index is one of the indicators that present microbiome characteristics by considering the number and abundance of microbiome components at the same time. Changes in the intestinal microenvironment observed along with changes in the microbiome led to protective effects against renal damage caused by ischemia/reperfusion. BGN4 supplementation could help maintain colonic barrier function by enhancing strict junction protein levels, reducing colonic epithelial cell apoptosis, and preventing mucosal disruption.
본 발명은 BGN4의 유익한 효과가 병원성 세균 확산 방지 뿐만 아니라 장벽 기능을 직접 강화하는 것임을 확인했다. BGN4 선처치는 단단한 대장 환경을 만들 수 있고, AKI로부터 원발 장기 손상을 예방할 수 있었다. 점막 장벽 기능의 직접적인 강화 외에도, BGN4는 대장 면역 변조 효과를 보여주었다. 본 발명자들은 BGN4의 특별한 면역 조절 효과를 발견했는데, 장 내 조절 T 세포의 확장이다. 조절 T 세포가 증가하면 전신적으로 유익한 기능을 한다는 것은 여러 연구에서 이미 잘 밝혀진 사실이다. The present invention confirmed that the beneficial effect of BGN4 was to directly enhance the barrier function as well as prevent the spread of pathogenic bacteria. BGN4 pretreatment could create a solid colonic environment and prevent primary organ damage from AKI. In addition to direct enhancement of mucosal barrier function, BGN4 showed colonic immune modulating effects. The present inventors discovered a specific immunomodulatory effect of BGN4, the expansion of regulatory T cells in the intestine. It has been well established in several studies that increased regulatory T cells have systemic beneficial functions.
본 발명자들은 BGN4가 염증을 줄이고 대장 세포 사멸을 억제함으로써 신장 보호 효과를 발휘한다는 것을 발견했다. BGN4를 투여한 뒤 IRI에서 장의 투과성이 감소했는데, 이는 프로바이오틱스가 대장 점막 기능을 강화한다는 것을 의미한다. BGN4 공급으로 대장 장벽 기능이 강화되면서 조직적인 염증과 악화 효과가 차단됐다. We found that BGN4 exerts renoprotective effects by reducing inflammation and inhibiting colon apoptosis. Intestinal permeability decreased in IRI after administration of BGN4, indicating that probiotics enhance colonic mucosal function. Systemic inflammation and aggravating effects were blocked as the colonic barrier function was strengthened with the supply of BGN4.
혈청 LDH 수준은 조직 손상 탐지에 사용되며, 대사 증후군 환자의 심혈관 사망률과 전원인 사망률과 상관관계가 있는 것으로 보고되었다. AKI의 혈청 LDH 레벨 상승도 BGN4 전처치를 통해 감소하였다. 이는 BGN4가 전신 염증을 감소시키는 데 유익한 보호 효과를 보여주며 항염증 효과를 나타낼 수 있음을 의미한다. Serum LDH levels are used to detect tissue damage and have been reported to correlate with cardiovascular and all-cause mortality in patients with metabolic syndrome. Serum LDH level elevation of AKI was also reduced by BGN4 pretreatment. This means that BGN4 shows a beneficial protective effect in reducing systemic inflammation and may exert anti-inflammatory effects.
본 발명에서는 BGN4 공급을 통하여 신장 보호 효과와 더불어 IRI연관 간 손상도 줄일 수 있었다. H&E 염색을 이용한 간 조직학에서는 뚜렷한 조직학적 차이를 보이지 않았지만, IRI와 비교하였을 때 BGN4를 투여한 IRI에서 간 효소 AST, ALT 상승이 억제되었다. 대장 장벽을 강화하고 면역 체계를 조절하는 프로바이오틱스 기능이 AKI에 의한 원거리 장기 손상으로부터 보호하는 역할을 할 수 있었다. In the present invention, IRI-associated liver damage could be reduced along with the renal protective effect through the supply of BGN4. Liver histology using H&E staining showed no clear histological differences, but when compared with IRI, the elevation of liver enzymes AST and ALT was suppressed in IRI administered with BGN4. The ability of probiotics to strengthen the colonic barrier and modulate the immune system could play a role in protecting against distant organ damage caused by AKI.
본 발명은 신부전 예방 또는 치료에 효능을 보이는 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4를 제공한다. 이때, 상기 신부전은 바람직하게 급성신부전일 수 있다.The present invention provides Bifidobacterium bifidum BGN4, which is effective in preventing or treating renal failure. At this time, the renal failure may preferably be acute renal failure.
비피도박테리움 비피덤 BGN4 (Bifidobacterium bifidum BGN4)는 공지된 것으로, 주식회사 비피도(강원도 홍천군 홍천읍 상오안리 688-1)로부터 구매가능하고, 한국미생물보존센터(국외)에 KCCM12754P로 기탁된 미생물이다. Bifidobacterium bifidum BGN4 is a well-known microorganism that can be purchased from Bifido Co., Ltd. (688-1 Sangoan-ri, Hongcheon-eup, Hongcheon-gun, Gangwon-do) and deposited with the Korea Microorganism Conservation Center (foreign) as KCCM12754P.
본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4 (KCCM12754P) 또는 그의 배양액 또는 그 건조 분말을 포함하는 것을 특징으로 하는 신부전 개선용 식품 조성물 또는 동물 신부전 개선용 동물 식품 조성물을 제공한다. 이때, 상기 신부전은 급성신부전일 수 있다. 본 발명의 식품 조성물에 있어, 상기 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4는 바람직하게 신부전 개선용 식품 조성물 대비 0.00001~50 중량% 포함되는 것이 좋다. 0.00001 중량% 미만일 경우에는 그 효과가 미비하고, 50 중량%를 초과하는 경우에는 사용량 대비 효과 증가가 미미하여 비경제적이다.The present invention provides a food composition for improving renal failure or an animal food composition for improving renal failure in animals, comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof. At this time, the renal failure may be acute renal failure. In the food composition of the present invention, the Bifidobacterium bifidum BGN4 is preferably included in an amount of 0.00001 to 50% by weight compared to the food composition for improving renal failure. If it is less than 0.00001% by weight, the effect is insufficient, and if it exceeds 50% by weight, the increase in effect compared to the amount used is insignificant, which is uneconomical.
본 발명의 식품 조성물은 일 예로, 육류, 곡류, 카페인 음료, 일반음료, 초콜릿, 빵류, 스낵류, 과자류, 사탕, 피자, 젤리, 면류, 껌류, 유제품류, 아이스크림류, 알코올성 음료, 술, 비타민 복합제 및 그 밖의 건강보조식품류 중 선택되는 어느 하나일 수 있으나, 반드시 이에 한정되는 것은 아니다.The food composition of the present invention is, for example, meat, grains, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, noodles, chewing gum, dairy products, ice cream, alcoholic beverages, alcohol, vitamin complexes And it may be any one selected from other health supplements, but is not necessarily limited thereto.
한편, 본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4 또는 그의 배양액 또는 그 건조 분말을 포함하는 것을 특징으로 하는 신부전 예방 또는 치료용 약학 조성물 또는 동물 신부전 예방 또는 치료용 동물 약학 조성물을 제공한다. 이때, 상기 신부전은 급성신부전일 수 있다. On the other hand, the present invention provides a pharmaceutical composition for preventing or treating renal failure or an animal pharmaceutical composition for preventing or treating renal failure in animals, comprising Bifidobacterium bifidum BGN4 or a culture solution thereof or a dry powder thereof. . At this time, the renal failure may be acute renal failure.
본 발명의 약학 조성물은 약제학적으로 허용 가능한 담체, 희석제 또는 부형제를 더욱 포함할 수 있다. 사용 가능한 담체, 부형제 또는 희석제로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자이리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유가 있으며, 이중 선택되는 하나 이상을 사용할 수 있다. 또한, 본 발명의 약학 조성물은 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 또는 방부제 중 선택되는 하나 이상을 추가로 포함할 수 있다.The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, diluent or excipient. Usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, There are microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and one or more selected from among them may be used. In addition, the pharmaceutical composition of the present invention may further include at least one selected from among fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifiers, or preservatives.
본 발명의 약학 조성물 제형은 사용방법에 따라 바람직한 형태일 수 있으며, 특히 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 채택하여 제형화 하는 것이 좋다. 구체적인 제형의 예로는 경고제(PLASTERS), 과립제(GRANULES), 로션제(LOTIONS), 리니멘트제(LINIMENTS), 리모나데제(LEMONADES), 방향수제(AROMATIC WATERS), 산제(POWDERS), 시럽제(SYRUPS), 안연고제(OPHTHALMIC OINTMENTS), 액제(LIQUIDS AND SOLUTIONS), 에어로솔제(AEROSOLS), 엑스제(EXTRACTS), 엘릭실제(ELIXIRS), 연고제(OINTMENTS), 유동엑스제(FLUIDEXTRACTS), 유제(EMULSIONS), 현탁제(SUSPENSIONS), 전제(DECOCTIONS), 침제(INFUSIONS), 점안제(OPHTHALMIC SOLUTIONS), 정제(TABLETS), 좌제(SUPPOSITORIES), 주사제(INJECTIONS), 주정제(SPIRITS), 카타플라스마제(CATAPLSMA), 캅셀제(CAPSULES), 크림제(CREAMS), 트로키제(TROCHES), 틴크제(TINCTURES), 파스타제(PASTES), 환제(PILLS), 연질 또는 경질 젤라틴캅셀 중 선택되는 어느 하나일 수 있다.The pharmaceutical composition dosage form of the present invention may be in a preferred form depending on the method of use, and in particular, formulated by adopting a method known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It is good. Examples of specific formulations include PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, AROMATIC WATERS, POWDERS, Syrups ( SYRUPS), OPHTHALMIC OINTMENTS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS ), SUSPENSIONS, DECOCTIONS, INFUSIONS, OPHTHALMIC SOLUTIONS, TABLETS, SUPPOSITORIES, INJECTIONS, SPIRITS, CATAPLSMA ), capsules (CAPSULES), creams (CREAMS), troches (TROCHES), tinctures (TINCTURES), pastas (PASTES), pills (PILLS), soft or hard gelatin capsules.
본 발명의 신부전 예방 또는 치료용 약학 조성물의 투여량은 투여방법, 복용자의 연령, 성별, 체중 및 질환의 중증도 등을 고려하여 결정하는 것이 좋다. 일 예로, 본 발명의 신부전 예방 또는 치료용 약학 조성물은 유효성분을 기준으로 하였을 때 1일 0.00001 내지 100㎎/㎏(체중)으로 1회 이상 투여 가능하다. 그러나 상기의 투여량은 예시하기 위한 일 예에 불과하며, 복용자의 상태에 따라 의사의 처방에 의해 변화될 수 있다.The dosage of the pharmaceutical composition for preventing or treating renal failure of the present invention is preferably determined in consideration of the administration method, the age, sex, weight, and severity of the disease of the user. For example, the pharmaceutical composition for preventing or treating renal failure of the present invention can be administered once or more at 0.00001 to 100 mg/kg (body weight) per day based on the active ingredient. However, the above dosage is only an example for illustration, and may be changed according to the doctor's prescription according to the condition of the user.
한편, 본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4 또는 그의 배양액 또는 그 건조 분말을 포함하는 것을 특징으로 하는 사료조성물을 제공한다.On the other hand, the present invention provides a feed composition comprising Bifidobacterium bifidum BGN4 or a culture solution thereof or a dry powder thereof.
상기 사료 조성물에 있어, 조성물 총 중량에 대하여 상기 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4를 0.001 ~ 1.0중량% 함유할 수 있다. 이는 그 함량이 조성물 총 중량에 대해 0.001중량% 미만이면 그 신부전 개선, 예방 또는 치료 효과를 충분히 발휘할 수 없으며, 1.0중량%를 초과하는 경우에는 제형의 안정성 및 안전성에 문제를 야기하기 때문이다.In the feed composition, 0.001 to 1.0% by weight of the Bifidobacterium bifidum BGN4 may be contained based on the total weight of the composition. This is because when the content is less than 0.001% by weight based on the total weight of the composition, the effect of improving, preventing or treating renal failure cannot be sufficiently exerted, and when it exceeds 1.0% by weight, stability and safety of the formulation are problematic.
본 발명의 상기 조성물은 통상의 배합사료에 섞어서 경구투여의 방법으로 급여하며, 계속 투여 또는 과다 투여시에도 면역저하 등의 내성이나 부작용의 문제가 거의 없다.The composition of the present invention is fed by oral administration by mixing it with a conventional formulated feed, and there is little tolerance or side effects such as immunosuppression even when continuously administered or over-administered.
본 발명에서 동물은 일 예로, 반려 동물일 수 있는데, 구체적으로는 개 또는 고양이일 수 있다. 특히, 고양이는 신부전 질환이 많아, 폐사율이 높으므로 본 발명의 조성물이 더욱 효과적이라 할 수 있다.In the present invention, the animal may be, for example, a companion animal, and may specifically be a dog or cat. In particular, the composition of the present invention can be said to be more effective because cats suffer from renal failure and have a high mortality rate.
이하, 본 발명의 내용에 대해 하기 실시예 및 실험예를 통해 더욱 상세히 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예 및 실험예에만 한정되는 것은 아니고, 그와 등가의 기술적 사상의 변형까지를 포함한다. Hereinafter, the contents of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited only to the following examples and experimental examples, and includes modifications of equivalent technical ideas.
[실시예 1: 본 발명 BGN4 균주의 신장 보호 효과 확인] [Example 1: Confirmation of the renal protective effect of the BGN4 strain of the present invention]
1. 실험 목적 1. Experiment purpose
본 실험에서는 급성 콩팥병에서 면역 조절 작용을 통한 BGN4 프로바이오틱스의 신장 보호 효과를 확인하고자 하였다. In this experiment, we tried to confirm the renoprotective effect of BGN4 probiotics through immunomodulatory action in acute kidney disease.
2. 실험 방법2. Experimental method
(1) 실험동물모델 (1) Experimental animal model
생후 6주령의 수컷 C57BL/6 쥐를 오리엔트 바이오사에서 구매하였다. 동물들은 특정 병원체가 없는 환경 (specific pathogen free 1 facility)에서 물과 사료를 자유롭게 공급받으면서 사육되었다. 프로바이오틱스 공급과 IRI 실험으로 무작위 배정되었다. 프로바이오틱스는 비피도사에서 공급받아 사용하였고, BGN4는 1일 1회 2x109의 농도로 1주일에 5일간 개별적으로 경구 투여하였다 (도 1). 도 1은 본 발명의 실험 계획을 보여준다. Male C57BL/6 mice, 6 weeks of age, were purchased from Orient Bio. Animals were housed in a specific pathogen free 1 facility with free access to water and feed. They were randomized into probiotics supplementation and IRI trials. Probiotics were supplied and used from Bifido, and BGN4 was individually orally administered at a concentration of 2x10 9 once a day for 5 days a week (FIG. 1). 1 shows the experimental scheme of the present invention.
급성신장손상 (AKI)은 양측 허혈 재관류 손상(ischemic reperfusion injury, IRI)을 통해 유도하였다. 2주 뒤 쥐들에게 IRI를 진행하였다. 수술 내용은 다음과 같았다. 복강 내 마취 후 24.5분간 등 절개를 통해 양쪽 신장 혈관을 결찰했다. 수술 후 깨어날 때까지 온열패드 위에서 감시하였다. 이 연구는 고려대학교 의과대학 동물연구센터 기관심사위원회의 승인을 받았다(IRB 번호: KOREA-2016-0260). Acute kidney injury (AKI) was induced through bilateral ischemic reperfusion injury (IRI). Two weeks later, rats were subjected to IRI. The contents of the operation were as follows. After intraperitoneal anesthesia, both renal vessels were ligated through a back incision for 24.5 minutes. After surgery, they were monitored on a heating pad until waking up. This study was approved by the Institutional Review Board of the Center for Animal Research, Korea University College of Medicine (IRB number: KOREA-2016-0260).
(2) 혈청화학 및 조직학적 분석 (2) Serum chemistry and histological analysis
혈장 크레아티닌, 아스파르테이트 아미노전달효소(AST), 알라닌 아미노전달효소(ALT), 락타이트 탈수소효소(LDH) 측정은 Beckman AU® 5821 Beckman (Beckman Coulter, USA)을 이용하였다. 복강 내 마취 후 조직을 적출하여 간, 대장 조직은 4% 파라포름알데히드 및 파라핀 함유로 고정했다. 신장 조직은 표준 광 현미경을 사용하여 평가되었다. 신장손상은 PAS 염색을 이용하여 조직 전체에 걸쳐 5개의 무작위 영역(Х100)에서 요세관 손상 부위를 평가하여 암맹평가 하였다. 손상 정도는 반정량적으로 평가되었으며, 눈에 보이는 괴사수준이 없으면 0등급, 0~25%(1등급), 25~50%(2등급), 50~75%(3등급), 75~100%(4등급)으로 하였다. 대식세포/중성구 침윤수준 평가를 위해 F4/80 (1:100; mch-497-GA; Bio-Rad Laboratories, Hercules, CA, USA) 와 Ly6G (1:200; 14-59-85; eBioscience)에 대항하는 단일 항체로 신장 및 대장 조직을 염색했다. 조절 T 세포의 검출을 위해 FOXP3(1:1000, ab215206, abcam)를 사용하여 신장 및 대장 면역화학염색을 진행하여 10개의 고배율 시야 당 평균 양성 세포 수를 비교했다. 대장 상피세포의 세포사멸은 8-10개의 고배율 시야(Х200)에서 TUNEL(terminal deoxynucleotidyl transferase dUTP nick end labeling) 양성 상피세포를 계산하여 정량화 하였다. 각 그룹에서 얻은 간 조직 분석은 H&E 염색을 사용하여 수행되었다. 자동 이미지 캡처 시스템인 슬라이드 스캐너(Axio Scan Z1, Zeiss Korea, Seoul)를 사용하여 이미지를 20배 확대했다.Plasma creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactite dehydrogenase (LDH) were measured using Beckman AU ® 5821 Beckman (Beckman Coulter, USA). Tissues were excised after intraperitoneal anesthesia, and liver and colon tissues were fixed with 4% paraformaldehyde and paraffin. Kidney tissue was evaluated using standard light microscopy. Kidney damage was evaluated in darkness by evaluating the tubular damage site in 5 random areas (Х100) throughout the tissue using PAS staining. The degree of damage was evaluated semi-quantitatively, and if there is no visible necrosis, it is grade 0, 0 to 25% (grade 1), 25 to 50% (grade 2), 50 to 75% (grade 3), and 75 to 100%. (grade 4). F4/80 (1:100; mch-497-GA; Bio-Rad Laboratories, Hercules, CA, USA) and Ly6G (1:200; 14-59-85; eBioscience) were used to evaluate the level of macrophage/neutrophil infiltration. Kidney and colon tissues were stained with monoclonal antibodies against each other. To detect regulatory T cells, immunochemical staining of the kidney and colon was performed using FOXP3 (1:1000, ab215206, abcam), and the average number of positive cells per 10 high-magnification fields was compared. Apoptosis of colon epithelial cells was quantified by counting TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) positive epithelial cells in 8-10 high-magnification fields (Х200). Liver tissue analysis obtained from each group was performed using H&E staining. Images were magnified 20x using an automatic image capture system, slide scanner (Axio Scan Z1, Zeiss Korea, Seoul).
(3) 웨스턴 블롯 분석 (3) Western blot analysis
Bicinchoninic acid 방법을 사용하여 전체 대장 조직 샘플에서 단백질을 추출했으며, Claudin-1 (1:200; ab15098; abcam)와 Occludin(1:200; ab168986; abcam)에 대한 마우스 항체를 사용하여 세포 접합 단백질의 발현을 조사했다. 밴드 강도는 Image StudioTM (Lite LI-COR Biosciences, Lincoln, NE, USA) 소프트웨어를 이용하여 분석하였다. 타겟 단백질 농도는 β-actin 농도로 정량 후 비교하였다.Proteins were extracted from whole colon tissue samples using the bicinchoninic acid method, and mouse antibodies against Claudin-1 (1:200; ab15098; abcam) and Occludin (1:200; ab168986; abcam) were used to detect cell junction proteins. expression was investigated. Band intensities were analyzed using Image Studio TM (Lite LI-COR Biosciences, Lincoln, NE, USA) software. Target protein concentration was quantified as β-actin concentration and compared.
(4) (4)
In vitroIn vitro
분석 analyze
비장세포는 BGN4와 함께 37℃에서 2% FBS에서 72시간 동안 5% CO2 인큐베이터에서 배양되었으며, CD103+ CD11c+ 또는 CD4+ CD25+ 세포 분획을 비교 분석하였다.Splenocytes were cultured with BGN4 in 2% FBS at 37°C for 72 hours in a 5% CO2 incubator, and the CD103+CD11c+ or CD4+CD25+ cell fractions were compared and analyzed.
(5) 장 투과도 분석(5) Intestinal permeability analysis
하룻밤 음수 공급을 제한한 상태에서 형광이 붙은 덱스트란 Fluorescein isothiocyanate (FITC)-conjugated dextran (FITC-dextran; catalog number FD4; Sigma-Aldrich, St. Louis, MO, USA)을 PBS (100 mg/mL)에 녹여 경구 투여한 뒤 4시간 후 혈액에서 형광을 추출하였다. Fluorescein isothiocyanate (FITC)-conjugated dextran (FITC-dextran; catalog number FD4; Sigma-Aldrich, St. Louis, MO, USA) was incubated in PBS (100 mg/mL) with limited drinking water overnight. After oral administration, fluorescence was extracted from the blood after 4 hours.
(6) 유세포 분석 (6) flow cytometry
장간막 림프절 (mesenteric lymph node, MNL), 비장, 신장 및 장에서 분리한 면역세포에 대한 유세포 분석을 진행하였다. 세포는 형광 라벨 단일 클론 항체로 염색하여 (anti-CD4, anti-CD25, Fixable viability Dye, anti-Foxp3, anti-CD45, anti-CD11c, anti-CD103, anti-CX3CR1, anti-Ly6C, eBioscience, BioLegend 및 BD Biosciences) 4 채널 유세포분석기 (FACSCantoIITM, BD Bioscience)를 사용하여 측정 후 FlowJo 소프트웨어 (Tree Star Inc.; Ashland, CA, USA)로 분석하였다. Flow cytometric analysis was performed on immune cells isolated from the mesenteric lymph node (MNL), spleen, kidney and intestine. Cells were stained with fluorescently labeled monoclonal antibodies (anti-CD4, anti-CD25, Fixable viability Dye, anti-Foxp3, anti-CD45, anti-CD11c, anti-CD103, anti-CX3CR1, anti-Ly6C, eBioscience, BioLegend and BD Biosciences) and analyzed using FlowJo software (Tree Star Inc.; Ashland, CA, USA) after measurement using a 4-channel flow cytometer (FACSCantoII ™ , BD Bioscience).
(7) 실시간 역 전사-폴리머레이즈 연쇄 반응 (RT-PCR) (7) Real-time reverse transcription-polymerase chain reaction (RT-PCR)
Foxp3 mRNA 발현 검출을 위해 제조사의 규약에 따라 TRIzol 추출 시약(Thermo Fisher Scientific, US)을 이용해 총 RNA를 정제하고 표준시술을 이용해 cDNA를 합성했다. Foxp3 RT-PCR은 iCycler IQ Real-Time PCR Detection System (Bio-Rad Laboratories)과 iQTM SYBR® Green Supermix (Bio-Rad Laboratories)을 이용하여 진행하였다. 18S rRNA를 기준유전자 레퍼런스로 사용하였고 (RT2 PCR Primer Set; Applied Biosystems, Foster City, CA, USA), 대조군의 결과와 배수의 차이로 비교하였다.For detection of Foxp3 mRNA expression, total RNA was purified using TRIzol extraction reagent (Thermo Fisher Scientific, US) according to the manufacturer's protocol and cDNA was synthesized using standard procedures. Foxp3 RT-PCR was performed using iCycler IQ Real-Time PCR Detection System (Bio-Rad Laboratories) and iQ TM SYBR ® Green Supermix (Bio-Rad Laboratories). 18S rRNA was used as a reference gene reference (RT2 PCR Primer Set; Applied Biosystems, Foster City, CA, USA), and the results of the control group were compared by fold difference.
(8) 대변 마이크로바이옴 분석 (8) Stool microbiome analysis
마우스 당 2개 이상의 대변 펠렛을 모아 -70℃로 보관하여 일괄 분석하였다. 정량적 마이크로바이옴을 위하여 16S rRNA 파이로시퀀싱 분석을 이용하였다. 차세대 마이크로바이옴 정보 플랫폼인 QIIME 2TM 이 이용되었다.At least two stool pellets per mouse were collected and stored at -70 °C for batch analysis. For quantitative microbiome, 16S rRNA pyrosequencing analysis was used. A next-generation microbiome information platform, QIIME 2 TM , was used.
(9) 통계 분석 (9) Statistical analysis
데이터는 평균±표준 오차(standard error of the mean, SEM)로 표시하였다. Unpaired t-test 및 일원 배치 분산 분석(Bonferroni 사후 검정을 사용함)은 각각 두 개 이상의 그룹과 세 개 이상의 그룹을 비교하는데 사용되었다. P-value < 0.05는 통계적으로 유의한 것으로 간주되었다. 데이터는 GraphPad Prism 버전 8.5(GraphPad Software Inc., La Jolla, CA)를 사용하여 분석하였다.Data are expressed as mean ± standard error of the mean (SEM). Unpaired t-test and one-way analysis of variance (using Bonferroni post hoc tests) were used to compare groups of two or more and groups of three or more, respectively. A P-value < 0.05 was considered statistically significant. Data were analyzed using GraphPad Prism version 8.5 (GraphPad Software Inc., La Jolla, Calif.).
3. 실험 결과 3. Experimental results
(1) BGN4의 AKI에 의한 장내 불균형을 개선 및 점막 장벽 기능 강화 확인 (1) Improvement of intestinal imbalance caused by AKI of BGN4 and confirmation of enhancement of mucosal barrier function
실험군과 대조군은 주좌표분석 (Principal coordinate analysis, PCoA)에서 마이크로바이옴 구성은 서로 다른 특성을 보였다 (도 2). 도 2는 실험군과 대조군의 component 1 및 component 2 주좌표분석 결과이다. 좌표에서 x축과 y축을 구성하는 component 1, component 2는 두 개의 벡터로서 실험군과 대조군의 장내 마이크로바이옴의 분포가 서로 얼마나 가깝고 멀리 떨어져있는지를 나타내는 상대적 분산을 나타내는 값이다. 좌표에서 서로 다른 분포를 보이는 본 실험 결과는 두 그룹간의 특성이 같지 아니하다는 것을 의미하며, IRI에 의하여 장 내 미생물의 분포가 변화하였다는 것을 시사한다.The experimental group and the control group showed different characteristics in microbiome composition in principal coordinate analysis (PCoA) (FIG. 2). 2 is a result of principal coordinate analysis of component 1 and component 2 of the experimental group and the control group. Component 1 and component 2 constituting the x-axis and y-axis in the coordinates are two vectors, and are values representing relative dispersion indicating how close and how far apart the distributions of the intestinal microbiome of the experimental group and the control group are from each other. The results of this experiment showing different distributions in the coordinates mean that the characteristics of the two groups are not the same, suggesting that the distribution of microorganisms in the intestine is changed by IRI.
마이크로바이옴 다양성 지수인 Simpson richness, Simpson evneness는 장내 미생물 분포의 특성을 제시하는 여러 가지 방법 중 하나이다. BGN4를 미리 공급했던 그룹에서 균일성 측면에서 Simpsons-Evenness 지수는 증가하였지만 다양성 측면의 Simpson richness 지수는 늘어나지 않았다 (도 3). 이를 통해 특정 미생물 종류 혹은 개체 수가 증가하지는 않았으나, 한 구성 집단의 풍부화 정도가 개선되었다고 파악할 수 있었다. 도 3은 실험군과 대조군의 Simpson Richness 및 Simpson Evenness 측정결과이다. Microbiome diversity indices, Simpson richness and Simpson evneness, are one of several ways to characterize the distribution of gut microbes. In the group that supplied BGN4 in advance, the Simpsons-Evenness index in terms of uniformity increased, but the Simpson richness index in terms of diversity did not increase (FIG. 3). Through this, it was found that the number of specific microbial species or individuals did not increase, but the degree of enrichment of one constituent group was improved. Figure 3 is the Simpson Richness and Simpson Evenness measurement results of the experimental group and the control group.
IRI 후 1일째에 마이크로바이옴은 다양성 지수는 비슷했지만 Enterobacteriaceae 와 Bacteroidaceae 가 대조군 대비 늘어났다 (도 4). 도 4는 실험군과 대조군의 속(Genus) 수준의 마이크로바이옴 상대적분포도를 보여준다. On day 1 after IRI, the microbiome had similar diversity indices, but Enterobacteriaceae and Bacteroidaceae increased compared to the control group (FIG. 4). Figure 4 shows the relative distribution of microbiome at the genus level of the experimental group and the control group.
장벽 기능을 강화하는 접합 단백인 Claudin-1 은 IRI 때 감소하였으나 BGN4 투여를 통해 정상화되었고 다른 접합 단백인 Occludin은 차이가 없었다(도 5, 도 6). 도 5 및 도 6은 실험군과 대조군의 Claudin-1 및 Occludin의 변화를 보여준다. Claudin-1, a splicing protein that enhances the barrier function, decreased during IRI, but was normalized by BGN4 administration, and there was no difference in occludin, another splicing protein (FIGS. 5 and 6). 5 and 6 show changes in Claudin-1 and Occludin in the experimental group and the control group.
장 상피세포 사멸은 IRI 군에서 증가하였다가 BGN4 투여군에서 감소하였다 (도 7, 도 8). 도 7 및 도 8은 실험군과 대조군의 장 상피세포 사멸 정도를 보여준다. 도 8의 그래프는 고배율 시야(high power field, HPF)에서 각 염색 양성을 나타내는 세포의 개수를 정량화하여 나타낸 것이다. Intestinal epithelial cell death was increased in the IRI group and decreased in the BGN4 administration group (FIGS. 7 and 8). 7 and 8 show the degree of apoptosis of intestinal epithelial cells in the experimental group and the control group. The graph of FIG. 8 quantifies the number of cells showing each staining positive in a high power field (HPF).
또한, IRI에서 증가했던 F4/80 염색 양성 대식세포 뿐만 아니라 Ly6G 염색 양성 중성구 침윤의 수도 BGN4 투여군에서 의미있게 감소했다(도 9, 도10). 도 9 및 도10은 실험군과 대조군의 F4/80 염색 양성 대식세포 및 Ly6G 염색 양성 중성구의 침윤 수 변화를 보여준다. 도 10의 그래프는 고배율 시야(high power field, HPF)에서 각 염색 양성을 나타내는 세포의 개수를 정량화하여 나타낸 것이다.In addition, the number of infiltrating Ly6G staining positive neutrophils as well as F4/80 staining positive macrophages, which increased in IRI, were significantly decreased in the BGN4 administration group (Figs. 9 and 10). 9 and 10 show changes in the infiltration numbers of F4/80 staining-positive macrophages and Ly6G staining-positive neutrophils in the experimental group and the control group. The graph of FIG. 10 quantifies the number of cells showing each staining positive in a high power field (HPF).
마지막으로, IRI에서 장 투과도가 증가하였다가 BGN4 투여를 통하여 부분적으로 회복되었다(도 11). 도 11은 실험군과 대조군의 장 투과도 변화를 보여준다. Finally, intestinal permeability increased in IRI and was partially recovered through BGN4 administration (FIG. 11). 11 shows changes in intestinal permeability between the experimental group and the control group.
(2) BGN4의 IRI 중증도와 원발 장기 손상 완화 (2) IRI severity of BGN4 and alleviation of primary organ damage
IRI 유발 2주 전부터 BGN4를 투여하면 신장 기능 손상이 완화되었다. BGN4 투여 쥐에서 요세관손상 점수, 중성구(Ly6G 염색 양성)와 대식세포(F4/80 염색 양성) 침윤이 호전되었고 (도 12, 도 13), 신장 IL-6 발현도 호전되었다 (도 14). 도 12 및 도 13은 실험군과 대조군의 신장 기능 지표 (ATN score, Ly6G, F4/80) 변화를 부여준다. ATN score (Acute Tubular Necrosis, 급성세뇨관괴사)는 세뇨관 괴사의 정도를 파악하기 위한 조직염색방법인 PAS 염색을 이용하여 조직 전체에 걸쳐 5개의 무작위 영역(Х100)에서 요세관 손상 부위를 평가하여 암맹평가 하였다. 손상 정도는 반정량적으로 평가되었으며, 눈에 보이는 괴사수준이 없으면 0등급, 0~25%(1등급), 25~50%(2등급), 50~75%(3등급), 75~100%(4등급)으로 하였다. 도 13의 그래프의 Kidney ATN score는 고배율 시야(high power field, HPF)에서 세뇨관 손상의 정도를 정량화하여 표시한 것이며, Kideny Ly6G 와 Kidney F4/80는 염색 양성을 나타내는 세포의 개수를 정량화하여 나타낸 것이다. 도 14는 실험군과 대조군의 신장 IL-6 발현 변화를 보여준다. IL-6는 신장의 염증수치를 파악할 수 있는 사이토카인 중 대표적인 것으로 IRI 후 신장 조직의 손상이 발생하며, 대조군 대비 실험군에서 IRI 후 염증의 감소를 보여준다.Administration of BGN4 2 weeks prior to induction of IRI ameliorated the impairment of renal function. The urinary tubular injury score, neutrophil (Ly6G staining positive) and macrophage (F4/80 staining positive) infiltration were improved in BGN4-administered rats (FIGS. 12 and 13), and renal IL-6 expression was also improved (FIG. 14). 12 and 13 show changes in renal function indices (ATN score, Ly6G, F4/80) of the experimental group and the control group. The ATN score (Acute Tubular Necrosis, acute tubular necrosis) is evaluated for blindness by evaluating the tubular damage site in 5 random areas (Х100) throughout the tissue using PAS staining, a tissue staining method to determine the degree of tubular necrosis. did The degree of damage was evaluated semi-quantitatively, and if there is no visible necrosis, it is grade 0, 0 to 25% (grade 1), 25 to 50% (grade 2), 50 to 75% (grade 3), and 75 to 100%. (grade 4). The Kidney ATN score in the graph of FIG. 13 quantifies and displays the degree of tubular damage in a high power field (HPF), and Kidney Ly6G and Kidney F4/80 quantify the number of cells showing staining positivity. . 14 shows changes in renal IL-6 expression in the experimental group and the control group. IL-6 is a representative of cytokines that can determine the level of inflammation in the kidney, and damage to the kidney tissue occurs after IRI, and shows a decrease in inflammation after IRI in the experimental group compared to the control group.
혈청 크레아티닌은 BGN4 공급이 IRI의 중증도를 떨어뜨렸다는 것을 보여주었다 (도 15). 도 15는 실험군과 대조군의 혈중 크레아틴 변화를 보여준다. Serum creatinine showed that BGN4 supplementation reduced the severity of IRI (FIG. 15). 15 shows changes in blood creatine in the experimental group and the control group.
또한 BGN4 공급이 IRI에서 동반된 간손상을 완화함을 비록 조직학적인 차이는 없었지만 AST, ALT 그리고 LDH 수치의 감소를 통해 확인할 수 있었다 (도 16, 도 17). 도 16 및 도 17은 실험군과 대조군의 AST 및 ALT 변화를 보여준다. BGN4를 투여한 IRI에서 간 효소 AST, ALT 상승이 억제되었다. 도 17의 그래프는 고배율 시야(high power field, HPF)에서 각 염색 양성을 나타내는 세포의 개수를 정량화하여 나타낸 것이다. 도 18은 실험군과 대조군의 LDH 변화를 보여준다. 혈청 LDH 수준은 조직 손상 탐지에 사용되며, 대사 증후군 환자의 심혈관 사망률과 전원인 사망률과 상관관계가 있는 것으로 보고되었다. IRI의 혈청 LDH 레벨 상승도 BGN4 전처치를 통해 감소하였다.In addition, although there was no histological difference, it was confirmed that BGN4 supply alleviated liver damage accompanying IRI through reductions in AST, ALT, and LDH levels (FIGS. 16 and 17). 16 and 17 show changes in AST and ALT of the experimental group and the control group. Elevations of liver enzymes AST and ALT were suppressed in IRI administered with BGN4. The graph of FIG. 17 quantifies the number of cells showing each staining positive in a high power field (HPF). 18 shows LDH changes in the experimental group and the control group. Serum LDH levels are used to detect tissue damage and have been reported to correlate with cardiovascular and all-cause mortality in patients with metabolic syndrome. Serum LDH level elevation of IRI was also reduced by BGN4 pretreatment.
(3) BGN4의 면역조절효과로 말미암은 신기능 보호효과 확인(3) Confirmation of renal function protective effect due to immunomodulatory effect of BGN4
신장 보호 효과의 기초가 되는 기저 메커니즘을 밝히기 위하여 BGN4와 비장세포를 공동 배양하였을 때 면역 세포 기능 변화를 직접적으로 일으키는지 실험하였다. 이 in vitro 실험을 통해 BGN4가 CD103+CD11C+ 조절수지상세포와 CD4+CD25+ 조절T세포를 증가시키는 것을 확인하였다 (도 19, 도 20). 도 19 및 도 20은 72시간 동안 본 발명 균주 BGN4와 공동배양된 비장세포의 CD11c+CD103+ 조절 수지상 세포 (CD11c+CD103+ regulatory dendritic cell)와 CD4+CD25+ 조절 T 세포 (CD4+CD25+ regulatory T cell)의 분율 증가를 확인한 결과이다. 도 19는 Flow cytometry 즉 유세포분석의 결과인데. 세포의 과립화 정도와 크기에 따라 forward scatter(FSC), side scatter(SSC)에서 세포의 특성을 먼저 확인하였다. 도 19의 첫번째 이미지에서 4사분면 타원의 장축으로 넓게 분포하는 세포는 대식세포이며 3사분면 아래 좌측의 모여있는 특성을 보이는 세포들은 림프구이다. 도 20의 그래프는 CD11c+CD103+ 조절 수지상 세포 (CD11c+CD103+ regulatory dendritic cell)와 CD4+CD25+ 조절 T 세포 (CD4+CD25+ regulatory T cell)의 분율 증가를 정량적으로 표시하였다. Vehicle은 BGN4 처리를 하지 않은 대조군이고, BGN4와 공동 배양하였을 때, CD11c+CD103+ 조절 수지상세포와 CD4+CD25+ 조절 T세포가 유의한 수준으로 확대 분화되었다.In order to elucidate the underlying mechanism underlying the renal protective effect, we tested whether the co-culture of BGN4 and splenocytes directly causes changes in immune cell function. Through this in vitro experiment, it was confirmed that BGN4 increased CD103+CD11C+ regulatory dendritic cells and CD4+CD25+ regulatory T cells (FIGS. 19 and 20). 19 and 20 are CD11c + CD103 + regulatory dendritic cells and CD4 + CD25 + regulatory T cells of splenocytes co-cultured with the strain BGN4 of the present invention for 72 hours. This is the result of confirming the fraction increase. 19 is the result of flow cytometry, that is, flow cytometry. Cell characteristics were first confirmed in forward scatter (FSC) and side scatter (SSC) according to the granulation degree and size of cells. In the first image of FIG. 19 , the cells widely distributed along the long axis of the ellipse in the fourth quadrant are macrophages, and the cells showing clustered characteristics on the left under the third quadrant are lymphocytes. The graph of FIG. 20 quantitatively indicated the increase in the fraction of CD11c+CD103+ regulatory dendritic cells and CD4+CD25+ regulatory T cells. Vehicle was a control group not treated with BGN4, and when co-cultured with BGN4, CD11c+CD103+ regulatory dendritic cells and CD4+CD25+ regulatory T cells were significantly expanded and differentiated.
또한, BGN4 투여군의 대장, 신장에서 모두 Foxp3 염색 양성 세포 및 Foxp3 mRNA 발현이 증가됨을 확인하였으며 이는 IRI 로 인한 신손상을 경감시키는 효과를 가질 것으로 생각되었다 (도 21, 도 22). 도 21 및 도 22는 대장, 신장에서 Foxp3 양성세포의 변화를 보여두는 것이고, 도 22는 고배율 시야(high power field, HPF)에서 각 염색 양성을 나타내는 세포의 개수를 정량화하여 나타낸 것이다. 도 23은 대장, 신장에서 Foxp3 mRNA 발현양 변화를 보여주는 것으로 고배율 시야(high power field, HPF)에서 각 염색 양성을 나타내는 세포의 개수를 정량화하여 나타낸 것이다.In addition, it was confirmed that Foxp3 staining-positive cells and Foxp3 mRNA expression were increased in both the colon and kidney of the BGN4-administered group, which was thought to have an effect of reducing renal damage caused by IRI (FIGS. 21 and 22). Figures 21 and 22 show changes in Foxp3-positive cells in the colon and kidney, and Figure 22 shows the quantification of the number of cells showing each staining positive in a high power field (HPF). Figure 23 shows changes in the expression of Foxp3 mRNA in the colon and kidney, and shows the quantification of the number of cells showing each staining positive in a high power field (HPF).
한편, 유세포분석을 통하여 확인하였을 때, Foxp3+ 조절 T 세포 (Foxp3+ Treg cell)는 신장(Kidney), 장간막 림프절(MNL), 대장(Colon)에서 모두 일관되게 증가하였고 CX3CR1
intermediateLy6Chigh 단핵구의 침윤을 억제하는 효과도 동반하여 보여주었다 (도 24, 도 25). 도 24 및 도 25는 실험군과 대조군의 유세포분석 결과이다. 유세포분석 항체염색을 진행하여 Foxp3양성을 나타내는 조절 T 세포 (Foxp3+ Treg cell)의 분율이 IRI에서 상승하는데 BGN4 처리를 한 실험군의 대장, 장간막 림프절, 신장에서 모두 조절 T세포(Foxp3+ Treg cell)가 유의한 수준으로 확대 분화되었다. On the other hand, when confirmed through flow cytometry, Foxp3+ regulatory T cells (Foxp3+ Treg cells) were consistently increased in Kidney, mesenteric lymph nodes (MNL), and colon, and CX 3 CR 1 intermediate Ly 6 C high The effect of suppressing the infiltration of monocytes was also shown together (FIG. 24, FIG. 25). 24 and 25 are flow cytometry analysis results of the experimental group and the control group. Flow cytometry antibody staining showed that the fraction of Foxp3-positive regulatory T cells (Foxp3+ Treg cells) increased in IRI, and the number of regulatory T cells (Foxp3+ Treg cells) was significant in the colon, mesenteric lymph nodes, and kidneys of the BGN4-treated experimental group. expanded to one level.
소장 면역 세포의 특성을 살펴보면 IRI 군에서 IL-17A+ CD4+ 분획이 증가하며 BGN4 투여군에서는 감소되었으며 이는 BGN4가 직접적으로 소장의 Th 17 pathway를 억제하는 기능을 통해 장 염증을 조절하는 기능을 가지고 있다는 것을 제시하였다 (도 26, 도 27). 도 26 및 도 27은 실험군과 대조군의 소장 IL-17A+ 세포의 유세포분석 비교 결과이다. 모여있는 특성을 보이는 세포들은 림프구이며 이들의 활성화 여부를 fixable viability dye(FVD) 항체염색으로 음성인 것을 확인 후, 다시 CD4+IL-17A+ helper T 17세포 양성 분율이 증가하는지 확인하였다. BGN4 실험군에서 CD4+IL-17A+ helper T 17세포 분율이 유의한 수준으로 증가하였다.Looking at the characteristics of small intestine immune cells, the IL-17A+ CD4+ fraction increased in the IRI group and decreased in the BGN4-administered group, suggesting that BGN4 has a function of regulating intestinal inflammation through the function of directly inhibiting the Th 17 pathway in the small intestine. (Figs. 26 and 27). 26 and 27 are comparison results of flow cytometry analysis of small intestine IL-17A+ cells in an experimental group and a control group. The cells showing clustering characteristics were lymphocytes, and after confirming that they were negative by fixable viability dye (FVD) antibody staining, whether or not the positive fraction of CD4+IL-17A+ helper T 17 cells increased was confirmed. In the BGN4 experimental group, the fraction of CD4+IL-17A+ helper T 17 cells significantly increased.
Claims (9)
- 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4 (KCCM12754P) 또는 그의 배양액 또는 그 건조 분말을 포함하는 것을 특징으로 하는 신부전 개선용 식품 조성물.Bifidobacterium bifidum ( Bifidobacterium bifidum ) BGN4 (KCCM12754P) or a culture solution thereof or a food composition for improving renal failure comprising a dry powder thereof.
- 제1항에 있어서, According to claim 1,상기 신부전은, The renal failure,급성신부전인 것을 특징으로 하는 신부전 개선용 식품 조성물.A food composition for improving renal failure, characterized in that acute renal failure.
- 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4 (KCCM12754P) 또는 그의 배양액 또는 그 건조 분말을 포함하는 것을 특징으로 하는 신부전 예방 또는 치료용 약학 조성물.Bifidobacterium bifidum ( Bifidobacterium bifidum ) BGN4 (KCCM12754P) or a pharmaceutical composition for preventing or treating renal failure, characterized in that it comprises a culture solution or dry powder thereof.
- 제3항에 있어서, According to claim 3,상기 신부전은, The renal failure,급성신부전인 것을 특징으로 하는 신부전 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating renal failure, characterized in that acute renal failure.
- 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4 (KCCM12754P) 또는 그의 배양액 또는 그 건조 분말을 포함하는 것을 특징으로 하는 동물 신부전 개선용 동물 식품 조성물.An animal food composition for improving animal renal failure, comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
- 제5항에 있어서, According to claim 5,상기 신부전은, The renal failure,급성신부전인 것을 특징으로 하는 동물 신부전 개선용 동물 식품 조성물.An animal food composition for improving animal renal failure, characterized in that acute renal failure.
- 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4 (KCCM12754P) 또는 그의 배양액 또는 그 건조 분말을 포함하는 것을 특징으로 하는 동물 신부전 예방 또는 치료용 동물 약학 조성물.An animal pharmaceutical composition for preventing or treating renal failure in animals, comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
- 제7항에 있어서, According to claim 7,상기 신부전은, The renal failure,급성신부전인 것을 특징으로 하는 동물 신부전 예방 또는 치료용 동물 약학 조성물.An animal pharmaceutical composition for the prevention or treatment of animal renal failure, characterized in that the acute renal failure.
- 비피도박테리움 비피덤(Bifidobacterium bifidum) BGN4 (KCCM12754P) 또는 그의 배양액 또는 그 건조 분말을 포함하는 것을 특징으로 하는 사료조성물.A feed composition comprising Bifidobacterium bifidum BGN4 (KCCM12754P) or a culture solution thereof or a dry powder thereof.
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| PCT/KR2021/010563 WO2022265151A1 (en) | 2021-06-17 | 2021-08-10 | Composition containing bifidobacterium bifidum bgn4 for relieving, treating, or preventing renal failure |
Country Status (3)
| Country | Link |
|---|---|
| KR (1) | KR102362828B1 (en) |
| CN (1) | CN117425411A (en) |
| WO (1) | WO2022265151A1 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007507526A (en) * | 2003-09-30 | 2007-03-29 | キボー バイオテック、インク | Compositions and methods for increasing kidney function |
| WO2018074514A1 (en) * | 2016-10-20 | 2018-04-26 | ビオフェルミン製薬株式会社 | Agent acting on transcellular ion transporter in intestinal tract, chloride channel activator, prophylactic or therapeutic agent for kidney disease, or defecation promoter |
| KR20180065570A (en) * | 2016-12-08 | 2018-06-18 | 주식회사 비피도 | Composition comprising bifidobacteria for preventing or treating of fatty liver |
| KR20180102923A (en) * | 2017-03-08 | 2018-09-18 | 서울대학교산학협력단 | Composition comprising bifidobacteria for preventing or treating of obesity |
| KR102253283B1 (en) * | 2020-12-21 | 2021-05-18 | 주식회사 비피도 | Method for production of human EGF with Bifidobacterium bifidum and composition thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9980988B2 (en) | 2002-03-13 | 2018-05-29 | Kibow Biotech, Inc. | Compositions and methods for augmenting kidney function |
| KR100635354B1 (en) * | 2005-02-01 | 2006-10-17 | 주식회사 비피도 | Composition for Prevention and Treatment of Food Allergy containing Bifidobacterium bifidum BGN4 as Active Ingredient |
-
2021
- 2021-06-17 KR KR1020210078585A patent/KR102362828B1/en active IP Right Grant
- 2021-08-10 WO PCT/KR2021/010563 patent/WO2022265151A1/en active Application Filing
- 2021-08-10 CN CN202180099048.0A patent/CN117425411A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007507526A (en) * | 2003-09-30 | 2007-03-29 | キボー バイオテック、インク | Compositions and methods for increasing kidney function |
| WO2018074514A1 (en) * | 2016-10-20 | 2018-04-26 | ビオフェルミン製薬株式会社 | Agent acting on transcellular ion transporter in intestinal tract, chloride channel activator, prophylactic or therapeutic agent for kidney disease, or defecation promoter |
| KR20180065570A (en) * | 2016-12-08 | 2018-06-18 | 주식회사 비피도 | Composition comprising bifidobacteria for preventing or treating of fatty liver |
| KR20180102923A (en) * | 2017-03-08 | 2018-09-18 | 서울대학교산학협력단 | Composition comprising bifidobacteria for preventing or treating of obesity |
| KR102253283B1 (en) * | 2020-12-21 | 2021-05-18 | 주식회사 비피도 | Method for production of human EGF with Bifidobacterium bifidum and composition thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117425411A (en) | 2024-01-19 |
| KR102362828B1 (en) | 2022-02-15 |
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