JP6890798B2 - Composition for prevention or treatment of inflammatory bowel disease - Google Patents

Description

The present invention relates to compositions for the prevention or treatment of inflammatory bowel disease.

Inflammatory bowel disease (IBD), including Crohn's disease (CD), ulcerative colitis (UC) and uncertain colitis (IC), is characterized by chronic, ameliorating and inflammatory disorders of the gastrointestinal tract. The etiology of IBD is not completely understood, but it is gradually becoming clear that it involves the interaction of multiple environmental factors, changes in the gut microbiota, diverse properties of genetic predisposition, and immune system responses. .. There is still no effective treatment to prevent or completely cure IBD, and patients continue to suffer from IBD symptoms and are prone to recurrence. Therefore, it has been desired to develop a composition such as a medicine capable of effectively preventing or treating inflammatory bowel disease.

Therefore, an object of the present invention is to provide a composition capable of effectively preventing or treating inflammatory bowel disease in view of the above-mentioned conventional situations.

As a result of diligent research to solve the above problems, the present inventors have obtained mutual components by ingesting a calcined product of zeolite, a calcined product of shells, and a mixture containing citric acid and red koji as essential components. He found that the action improved the symptoms of inflammatory bowel disease, and completed the invention. That is, the gist of the present invention is as follows.

(1) A composition for preventing or treating inflammatory bowel disease, which comprises a calcined product of zeolite, a calcined product of shell, citric acid and red yeast rice.
(2) In 100% by weight of the composition, the total of the calcined zeolite and the calcined shell is 20% to 80% by weight, citric acid is 20% to 40% by weight, and red koji is 10% to 30% by weight. The composition for preventing or treating inflammatory bowel disease according to (1) above, which contains% by weight.
(3) For prevention or treatment of inflammatory bowel disease according to (1) or (2) above, wherein the compounding ratio of the calcined zeolite product and the calcined shell product is 5:95 to 20:80 (weight ratio). Composition.
(4) The composition for preventing or treating inflammatory bowel disease according to any one of (1) to (3) above, which is a pharmaceutical.
(5) The composition for preventing or treating inflammatory bowel disease according to any one of (1) to (3) above, which is a dietary supplement.
(6) The composition for preventing or treating inflammatory bowel disease according to any one of (1) to (3) above, which is a food product.

The composition of the present invention can be administered to a subject as a medicine or taken as a dietary supplement or food to prevent Crohn's disease (CD), ulcerative colitis (UC), uncertain colitis (IC), etc. Inflammatory bowel disease (IBD) can be effectively prevented or treated.

It is a graph which shows the change of the weight loss rate of each group. It is a graph which shows the change of bloody stool of each group. It is a graph which shows the change of the degree of diarrhea of each group. It is a graph which shows the change of the disease activity index (DAI) value of each group. It is a graph which shows the length of the large intestine of each group. It is a graph which shows the relative mesenteric fat weight of each group. It is a graph which shows the relative retroperitoneal fat weight of each group. It is a graph which shows the large intestine MPO activity of each group. It is a micrograph of a hematoxylin and eosin stained section of the large intestine of each group. It is a graph which shows the expression level of the interleukin-12b gene (Il12b) of each group. It is a graph which shows the expression level of the interleukin 12 receptor subunit beta1 gene (Il12rβ1) of each group. It is a graph which shows the expression level of the signal transducer and the transcription activator 4 gene (Stat4) of each group. It is a graph which shows the expression level of the interferon gamma gene (Ifnγ) of each group. It is a graph which shows the expression level of the Toll-like receptor 5 gene (Tlr5) of each group. It is a graph which shows the expression level of the interleukin 1 beta gene (IL1β) of each group. It is a graph which shows the expression level of the tumor necrosis factor-α gene (Tnfα) of each group. It is a graph which shows the expression level of the interleukin 6 gene (Il6) of each group. It is a graph which shows the expression level of the apoptosis-related tumor suppressor p53 gene (P53) of each group. It is a graph which shows the expression level of the insulin-like growth factor binding protein 3 gene (Igfbp3) of each group. It is a graph which shows the expression level of the insulin-like growth factor 1 gene (Igf1) of each group. It is a schematic diagram of the inflammatory bowel disease pathway changed by the intake of HY in DSS-induced colitis.

Hereinafter, the present invention will be described in detail.
The composition for preventing or treating inflammatory bowel disease of the present invention is characterized by containing a calcined product of zeolite, a calcined product of shells, citric acid and red yeast rice. Since this composition has a function of improving the inflammatory state in the intestine, suppressing apoptosis, and maintaining the epithelial defense function and the barrier function of the large intestine mucosa, it can be used for the prevention or treatment of inflammatory bowel disease. ..

The zeolite may be either a natural product or a synthetic product. Zeolites have various structures, but all known ones are applicable. Examples of this structure include, but are not limited to, A type, X type, Y type, β type, ZSM-5 and the like.

The calcined product of zeolite can be obtained by calcining the above-mentioned zeolite by appropriately adopting a known method. The firing conditions can be, for example, 200 ° C. to 900 ° C. for 10 hours to 20 hours. It is preferably 230 ° C. to 880 ° C. for 15 ± 2 hours. Further, firing is preferably performed using a ceramic furnace.

Zeolites can be milled prior to calcination, if desired. Alternatively, it may be pulverized after firing. The particle size after pulverization is preferably 5 μm to 15 mm, particularly 5 μm to 10 μm so as to ensure sufficient uniformity of the obtained composition for prevention or treatment of inflammatory bowel disease.

Examples of the shellfish used in the composition of the present invention include oysters, scallops, surf clams, clams, clams, abalone, sazae, mussels, surf clams, and freshwater clams. Any one of these can be used alone or in combination of two or more. Among them, from the viewpoint of reducing the environmental load, preserving biodiversity, and effectively utilizing resources, it is easy to obtain because it is generated in large quantities as edible residue, and the shell itself is relatively soft and easy to crush. Oyster shells are preferably used.

The shell can be pretreated to remove deposits, if necessary. This pretreatment step is not particularly limited, and examples thereof include a step of exposing to the sea for one year to release and decompose adhering substances.

The calcined product of the shell can be obtained by appropriately adopting a known method and calcining the shell. The firing conditions are, for example, 200 ° C. to 900 ° C. for 10 hours to 20 hours. It is preferably, but is not limited to, 15 ± 5 hours at 230 ° C to 880 ° C. Further, firing is preferably performed in a ceramic furnace.

If necessary, the shells can be subjected to a pulverization treatment before firing. Alternatively, it may be pulverized after firing. The pulverization step is not particularly limited, but it is preferably performed by a dry pulverization method in order to reduce the environmental load and the manufacturing cost. The particle size after pulverization is preferably 5 μm to 15 mm, particularly 5 μm to 10 μm so as to ensure sufficient uniformity of the obtained composition for prevention or treatment of inflammatory bowel disease.

As citric acid, in addition to general citric acid whose demonstrative formula is represented by H ₃ (C ₆ H ₅ O _{7), citrate can also be applied.} Examples of the citrate include sodium citrate, potassium citrate, calcium citrate and the like. Examples of sodium citrate include monosodium citrate, disodium citrate, trisodium citrate, sodium hydrogen citrate, and the like, and examples of potassium citrate include monopotassium citrate, tripotassium citrate, and citric acid. Examples thereof include potassium hydrogen, and examples of calcium citrate include monocalcium citrate, dicalcium citrate, tricalcium citrate, calcium hydrogen citrate, and the like, and any of them can be used. In addition, the various citric acids described above can be blended alone or in combination of a plurality of types.

Monascus is a rice jiuqu made by growing Monascus spp., Which generally produces red pigment, on steamed rice, and it is appropriately selected from those that have been used as raw materials for red sake, red roasted milk, pickles, etc. for a long time. Can be used.

Further, an anticaking agent may be added to the composition for preventing or treating inflammatory bowel disease of the present invention, if necessary. Examples of anticaking agents include calcium stearate, magnesium stearate and the like. Further, if the amount of the anticaking agent such as calcium stearate in the composition is too large, the relative amount of essential components such as zeolite in the composition decreases, and a preventive or therapeutic effect on inflammatory bowel disease can be obtained. Therefore, it is set appropriately in consideration of this point.

By mixing each of the above components by using known means, a composition for preventing or treating inflammatory bowel disease can be obtained. The compounding ratio of each component is not particularly limited, but the preventive / therapeutic effect on inflammatory bowel disease is due to the synergistic action of each component, so the balance should be considered so that the function of each component is fully exerted. Is set as appropriate. Specifically, in 100% by weight of the composition, the calcined product of zeolite and the calcined product of shell are 20% by weight to 80% by weight, citric acid is 20% to 40% by weight, and red yeast rice is 10% by weight. It is preferably contained in an amount of ~ 30% by weight.

Further, the calcined product of zeolite and the calcined product of shell may be added to the composition separately, or the calcined product of zeolite and the calcined product of shell are mixed together with other components in the state of a mixture. You may. The mixing ratio of the calcined zeolite and the calcined shell is unsuitable because the reduction rate will be low if the calcined zeolite is too small, and the reduction rate will still be high if the calcined shell is too small. Since it becomes low, it is set appropriately in consideration of these balances. For example, the blending ratio of the calcined zeolite product and the calcined shell product is preferably 5:95 to 20:80 (weight ratio).

The composition for preventing or treating inflammatory bowel disease may contain various optional components in addition to the essential components such as a calcined product of zeolite. Such ingredients vary depending on the mode of the composition, but are, for example, excipients, flavoring agents, flavoring agents, binders, sweeteners, acidulants, dispersants, emulsifiers, colorants, preservatives, antioxidants. Agents, stabilizers and the like can be mentioned. The total amount of these optional components to be blended is preferably 3% by weight or less in 100% by weight of the composition.

The composition obtained as described above has an effect of preventing and treating inflammatory bowel disease, and can be used as a medicine. When used as a drug for the prevention or treatment of inflammatory bowel disease, its form is not particularly limited, and tablets, capsules, powders, etc., depending on the conditions such as the administration method and the applicable condition of inflammatory bowel disease. , Granules, drinks and the like can be appropriately selected from various forms. The composition of the present invention can be used as it is as a medicine, or may be used as a medicine in combination with a usual additive such as a carrier, a diluent or an excipient, if necessary.

The ingestion or dose as a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease is a therapeutically effective amount, which varies depending on the symptoms of inflammatory bowel disease and cannot be unequivocally determined, but in the case of an adult weighing 60 kg, The amount of the above composition per day is usually 1440 mg or more, preferably 2880 mg or more. The upper limit of the dose is not particularly limited, but is preferably less than 4000 mg. In addition, the medicament of the present invention can be used in combination with other known therapeutic agents.

In addition to humans, non-human animals such as rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees and other mammals, birds, amphibians, and reptiles are administered. It is applicable to such as.

In addition, the composition of the present invention can be used as a food or dietary supplement having a preventive or therapeutic effect on inflammatory bowel disease. The food is not particularly limited, and for example, various soft drinks, fruit juice drinks, Japanese and Western confectionery, dairy products and other processed livestock products, processed fruit products, processed vegetable products, processed grain products, processed marine products, and seasonings. And many other foods. Further, as the dietary supplement, for example, it can be provided in the form of a liquid preparation or in the form of tablets or the like. In this case, the amount of the composition added to the food or dietary supplement is not particularly limited, but generally, the intake amount of the composition for prevention or treatment of inflammatory bowel disease per day for adults is about 1440 mg to. It is preferable to add it so as to be 2880 mg.

Furthermore, since the composition of the present invention has a preventive or therapeutic effect on inflammatory bowel disease and is excellent in safety, livestock feed, rabbits, etc. used for sheep, pigs, cattle, horses, chickens, etc. It may be added to feed for small animals used for rats, mice and the like, and pet food used for dogs, cats, small birds, squirrels and the like.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to these Examples.

As shown below, using the large intestine mucosal tissue of a mouse model of sodium dextran sulfate (DSS) -induced colitis, the composition according to the present invention was obtained by a neutrigenomix method such as transcriptome and proteome and biochemical analysis. The effect of improving inflammatory bowel disease was investigated.

1. 1. Experiment (1) Preparation of composition Natural zeolite was calcined at 650 ° C. for 15 hours and pulverized to 15 μm or less. Similarly, the oyster shells were calcined at 700 ° C. for 17 hours and pulverized to 15 μm or less. Subsequently, the calcined product of zeolite and the calcined product of oyster shell were mixed, and the obtained mixture was sufficiently ion-exchanged with warm water to remove water at a high temperature. The blending ratio of the calcined zeolite product and the calcined oyster shell product was 1: 9 (weight ratio). Next, 47.6% by weight, citric acid (33.3% by weight), red yeast rice (Monascus) (16.7% by weight), and calcium stearate (2.4% by weight) as an anticaking agent were added to the mixture of the calcined zeolite and the calcined oyster shell. %) Were mixed to prepare the desired composition (hereinafter referred to as "zeolite-containing composition" or "HY").

(2) Animal and prey treatment
7-week-old male C57BL6 mice obtained from Charles River Japan (Tokyo, Japan) were subjected to controlled temperature (23 ± 2 ° C), relative humidity (50% -60%), and 12-hour dark / light cycle throughout the experiment. They were housed in individual cages under lighting conditions. After 3 days of acclimation, each group was assigned to 4 groups of equal average body weight consisting of 6-8 mice. (1) In the HY8 group, AIN-93G basic feed and sterile tap water supplemented with 0.8% by weight zeolite-containing composition (HY) powder were ingested throughout the experiment. (2) In the CON group, AIN-93G basic feed and sterile tap water were ingested throughout the experiment. (3) In the DSS group, after feeding AIN-93G basic feed and sterile tap water for 11 days, sterilized tap water containing 1.5 wt% DSS (DSS molecular weight: 40 kDa; MP Biomedicals, Irvine, CA, USA) was given for 9 days. Gave. (4) The DHY8 group was given AIN-93G basic feed containing 0.8% by weight of HY powder and sterilized tap water for 11 days, and then sterilized tap water containing 1.5% by weight of DSS for 9 days. The composition of the bait is shown in Table 1. The amount of 0.8 wt% HY in this experiment was determined based on preliminary experiments. This experiment was approved by the Animal Care and Use Committee of the University of Tokyo (approval number P13-739).

(3) Evaluation of disease activity index The disease activity index (DAI) is the average value of three scores (weight loss, bloody stool, and diarrhea) after DSS is given to mice. The criteria for the score are: weight loss rate (0: <1%, 1: 1-5%, 2: 5-10%, 3: 10-15%, 4:> 15%), bloody stool ( 0: No bloody stool, 2: ++, 4: +++) and degree of diarrhea (0: normal, 2: soft, 4: diarrhea).

(4) Blood collection and tissue collection At the end of the experiment, all mice were deeply anesthetized with pentobarbital sodium and then bleeding from the carotid artery. The obtained blood was centrifuged at 1000 g for 15 minutes at 4 ° C. to obtain plasma. Colorectal length was measured between the ileum and cecal junction and the proximal rectum, and this measurement was used as one of the criteria for the degree of colitis. Liver, resected large intestine, mesenteric adipose tissue and retroperitoneal adipose tissue were snap frozen in liquid nitrogen and stored at -80 ° C until analysis.

(5) Biochemical test Colonyeloperoxidase (MPO) activity was measured by a colorimetric method using the MPO Activity Colorimetric Assay Kit (BioVision, Palo Alto, CA, USA) according to the manufacturer's manual.

(6) Colon Histology Each colon slice was embedded in an OCT (Optimal Cutting Temperature) compound (Sakura Finetek, Torrance, CA, USA) and then snap frozen in liquid nitrogen. Tissue sections each 5 μm thick were made, stained with hematoxylin and eosin (H & E), and then scanned with an optical microscope (Olympus BX51 microscope, Olympus Optical, Tokyo, Japan).

(7) Total RNA extraction and quality evaluation Using the total RNA separation kit NucleoSpin® RNA plus (Macherey-Nagel, Duren, Germany), total RNA was extracted from the large intestine mucosa according to the manufacturer's instructions. RNA concentration and purity were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

(8) Transcriptome analysis (i) DNA microarray preparation Pool RNA samples from individual mice in each group (n = 6-8), followed by 40,000 probes for profiling genome-wide expression. Microarray analysis was performed using Affymetrix Mouse Genome 430 2.0 Array Genechips (Affymetrix, Santa Clara, CA, USA).
(Ii) Mapping and functional analysis
After scanning the images using the Microarray Suite ver.5.0 software (Affymetrix), the gene expressions of the DSS group and the CON group and the DHY8 group and the DSS group were compared. Expression ratios over 1.5-fold were considered significant expression.

(9) Reverse transcription polymerase chain reaction In order to confirm the expression of the gene, reverse transcription polymerase chain reaction (RT-PCR) was carried out. The primer sequences are shown in Table 2. The expression level of each gene was normalized to the expression of the 60S acidic ribosomal protein p1 (Rplp1) in the colonic mucosa.

(10) Protein preparation for proteomics analysis, iTRAQ labeling and Nano LC-MS / MS analysis Protein was extracted from the colonic mucosa using lysis buffer and then centrifuged at 12,000 g at 4 ° C. for 30 minutes. Protein concentration was measured by the Bradford assay. Pool protein (100 μg) for cysteine blocking and digestion according to the manual of the 4-plex iTRAQ labeling kit (AB Sciex, Framingham, MA, USA), then mass spectrometer (NanoLC-MS / MS; AB Sciex) For further analysis by nanoscale liquid chromatography linked to, labeled with an isobaric tag as follows. CON: 115 tags, DSS: 114 tags, DHY8: 116 tags.

(11) Statistical analysis The data was shown as mean ± standard error (SE) and analyzed by two-way analysis of variance (ANOVA). Significant differences were evaluated at levels of p <0.05 by Tukey's method. In the graph, the signs a, b, and c indicate that there is a statistically significant difference between the different signs, and p ≦ 0.05: there is a significant difference, and 0.05 <p <0.1: there is a tendency.

2. Results (1) General characteristics
There was no significant difference in food intake between the DHY8 group and the DSS group, but there was a significant difference in food intake between the CON group and the DSS group (DHY8: 76.4 ± 3.3 g, DSS: 73.1 ± 1.7 g, CON). : 82.8 ± 3.5g, HY8: 76.7 ± 2.6g). There was a significant difference in water intake between the HY8 group and the DSS group (HY8: 84.8 ± 2.3 ml, DSS: 70.3 ± 2.1 ml, CON: 73.7 ± 1.9 ml, DHY8: 72.2 ± 1.4 ml). After administration of DSS, the degree of weight loss, bloody stool and diarrhea changed, and the DAI value increased significantly compared with the CON group. Ingestion of HY improved weight loss and suppressed the condition of IBD. This was also shown by the DAI values on days 6-9 (FIGS. 1A-1D). DSS-induced shortening of colon length, decrease in mesenteric fat weight, and increase in colon MPO activity tended to be improved by HY intake, but there was no change in the weight of retroperitoneal adipose tissue ( 1E-1H).

(2) Colorectal histology
DSS induces structural damage to mucosal epithelial cells and may increase infiltration of inflammatory cells into mucosal and submucosal tissues. Ingestion of HY reduced infiltration of inflammatory cells and ameliorated structural damage to mucosal epithelial cells (Fig. 1I).

(3) Colon microarray analysis A total of 5,238 genes were significantly expressed, of which 2,300 were significantly up-regulated in DHY8 mice compared to DSS mice and down-regulated in DSS mice compared to CON mice. It was. On the other hand, 2,938 genes were significantly downregulated in the DHY8 group compared to the DSS group and upregulated in the DSS group compared to the CON group.

Ingestion of HY suppresses the expression of genes associated with the inflammatory bowel disease pathway, including interleukin 12b (Il12b) (Fig. 2A), signal transducers and transcriptional activator 4 (Stat4) (Fig. 2C). ), Interferon gamma (Ifnγ) (Fig. 2D), Interleukin 1 beta (IL1β) (Fig. 2F), Tumor necrosis factor-α (Tnfα) (Fig. 2G), Interleukin 6 (Il6) (Fig. 2H), related to apoptosis Tumor necrosis factor p53 (P53) (Fig. 2I), members of the chemokine ligand family (Cxcl): Cxcl1, Cxcl2, Cxcl3, Cxcl10, Cxcl12, and Cxcl17, chemokine (CC motif) ligands (Ccl): Ccl4 and Ccl24, and epithelium It contains the maintenance-related gene, the epidermis growth factor receptor (Egfr). Compared with the DSS group, Trefoil family factor 2 (Tff2) and insulin-like growth factor 1 (Igf1) (Fig. 2K) were up-regulated in the DHY8 group. For reference, the expression levels of the interleukin 12 receptor subunit beta 1 gene (Il12rβ1), Toll-like receptor 5 (Tlr5), and insulin-like growth factor-binding protein 3 (Igfbp3) in each group are shown in FIG. 2B, respectively. It is shown in FIG. 2E and FIG. 2J.

(4) Proteome analysis by iTRAQ
Proteome analysis using iTRAQ confirmed more than 5,000 proteins. Proteins with an expression ratio of more than 1.5 times are considered to be fluctuating proteins, and 668 proteins are fluctuating according to this criterion, of which 332 are upregulated in the DHY8 group compared to the DSS group and compared to CON. It was down-regulated in the DSS group. There were 336 upregulated proteins in DSS mice compared to CON, while downregulated in the DHY8 group compared to the DSS group.

Ingestion of HY up-regulated the following proteins: periostin (Postn) as a protein involved in cancer; plasma cell-induced endoplasmic reticulum protein (Perp1) and galectin 2 (Leg2); BCL2-related atanogen 3 as an anti-apoptotic-related protein (Bag3), RNA-binding motif protein 3 (Rbm3) and periostin heavy chain (Fhc); phosphatase 1 regulation inhibitor subunit 14A (Pp14a) as an intestinal motility-related protein; epithelial cell adhesion as an intestinal mucosal protection and epithelial homeostasis-related protein Isoform 71 kDa (Numb) of molecule (Epcam), tenesin C (Tena) and protein nom homologues.

On the other hand, HY intake down-regulated the following proteins: the inflammatory degree-related protein apoptosis (Hpt), complement component 3 (Co3), myeloperoxidase (Perm), annexin A2 (Anxa2) and the heat shock protein family: A member 4 (Hsp74); apoptotic protein keratin 20 (K1c20), pyrimidine endodeoxyribonuclease 1 (Apex1), caspase-7 (Casp7), and caspase-3 (Casp3); biomarker protein for colorectal cancer Heterologous nuclear ribonuclear protein U (Hnrpu). These results are summarized in Table 3.

3. 3. Discussion Microarray analysis showed that HY intake improved the inflammatory state of mice by suppressing the expression of genes involved in the ^{following inflammatory bowel disease pathways: Il12 pos} Il12rβ1 ^pos Stat4 ^pos Ifng ^pos Tlr5 ^{pos Tnfα / Il6 / Il1β.} It became clear. FIG. 3 is a schematic diagram of the inflammatory bowel disease pathway varied by ingestion of HY in DSS-induced colitis. In this pathway, Stat4 tyrosine phosphorylation is induced by IL12. This is required for T cell-independent induction of the cytokine IFNγ, which allows both lymphoid and non-lymphoid cells to produce the pre-inflammatory cytokine IL6 in response to TLR stimulation. It is believed to be the major pre-inflammatory cytokine, along with TNFα and IL1β. Downregulation of the inflammatory bowel disease pathway primarily results in reduced levels of pre-inflammatory cytokines induced by DSS and reduced inflammatory levels with HY ingestion.

The above-mentioned decrease in pro-inflammatory cytokine levels can be reflected by the expression of the following three marker proteins. (1) Up-regulation of Pp14a (Cpi-17): Pp14a (Cpi-17) is an inhibitor of smooth muscle myosin phosphatase, which is suppressed by up-regulation of IL1β or TNFα induced by chronic inflammatory bowel disease, and then (Ohama, T .; Hori, M .; Momotani, E .; Iwakura, Y .; Guo, F .; Kishi, H .; Kobayashi, S .; Ozaki, H. Intestinal inflammation downregulates smooth muscle cpi-17 through induction of tnf-alpha and causes motility disorders. Am J Physiol Gastrointest Liver Physiol 2007, 292, G1429-1438. And Ohama, T .; Hori, M .; Sato, K .; Ozaki, H. Karaki, H. Chronic treatment with interleukin-1 beta attenuates contractions by decreasing the activities of cpi-17 and mypt-1 in intestinal smooth muscle. J Biol Chem 2003, 278, 48794-48804.). (2) Hpt downregulation: Hpt is an acute phase α-sialoglycoprotein with hemoglobin binding ability that can be induced by pre-inflammatory cytokines such as IL6, IL1β and TNFα (Vanuytsel, T .; Vermeire, S. Cleynen, I. The role of haptoglobin and its related protein, zonulin, in inflammatory bowel disease. Tissue Barriers 2013, 1, e27321.). (3) Downregulation of Hsp74: Hsp74 is induced by inflammation, results in inhibition of apoptosis of inflammatory cells, and enhances immune response by increasing Bcl-2 and Il-12 expression (Adachi, T .; Sakurai, T .; Kashida, H .; Mine, H .; Hagiwara, S .; Matsui, S .; Yoshida, K .; Nishida, N .; Watanabe, T .; Itoh, K., et al. Involvement of heat shock protein a4 / apg-2 in refractory inflammatory bowel disease. Inflamm Bowel Dis 2015, 21, 31-39.).

Fluctuations in the following three proteins showed improvement in inflammation with HY intake. (1) Perp1 upregulation: Perp1 plays an important role in anti-inflammatory by controlling the balance of Th1 / Th2 and reducing the secretion of pre-inflammatory cytokines such as IL6, IL1β and TNFα. Perp1 deficiency damages intestinal epithelial cells because Perp1 is also involved in maintaining the defense mechanism of the intestinal epithelium (Azuma, YT; Hagi, K .; Shintani, N .; Kuwamura, M .; Nakajima, H .; Hashimoto, H .; Baba, A .; Takeuchi, T. Pacap provides colonic protection against dextran sodium sulfate induced epithelium. J Cell Physiol 2008, 216, 111-119.). (2) Co3 downregulation: Plays an important role in activating the complement system. Decreased Co3 expression reduces invasive neutrophils (the major producers of IL1β) and thus functions as tumor suppressors (Ning, C .; Li, YY; Wang, Y .; Han, GC; Wang , RX; Xiao, H .; Li, XY; Hou, CM; Ma, YF; Sheng, DS, et al. Complement activation promotes colitis-associated carcinogenesis through activating intestinal il-1beta / il-17a axis. Mucosal Immunol 2015, 8, 1275-1284.). (3) Decreased Anxa2 expression: Decreased Anxa2 expression can down-regulate the pre-inflammatory cytokine TNFα by preventing TNFα excretion in IBD (Tanida, S .; Mizoshita, T .; Ozeki, K .; Katano, T .; Kataoka, H .; Kamiya, T .; Joh, T. Advances in refractory ulcerative colitis treatment: A new therapeutic target, annexin a2. World J Gastroenterol 2015, 21, 8776-8786. ). Based on the findings on genes significantly altered and induced correlated proteins by HY intake, HY intake causes DSS-induced inflammation primarily by suppressing the inflammatory bowel disease pathway and correlated genes and proteins. It was revealed that it improved and then suppressed the expression of pre-inflammatory cytokine genes Il6, Il1β and Tnfα.

In addition to the anti-inflammatory effect of HY, ingestion of HY improves DSS-induced colitis by improving apoptosis, regulating the cell cycle, maintaining the protective function of epithelial cells, and maintaining the barrier function of the colonic mucosa. Can be relaxed. Microarray analysis showed that apoptosis-related gene P53 was significantly downregulated by HY intake. For the P53 gene, we investigated the ^{P53 pos} Igfbp3 ^{neg Igf1 pathway whose positive expression leads to apoptosis.} However, the expression of the gene Igfbp3 was not significantly different between the DSS group and the DHY8 group. Upregulation of the matrix cell protein Postn revealed by proteome analysis confirmed our hypothesis that its overexpression may cause downregulation of P53 (Li, B .; Wang, L .; Chi). , B. Upregulation of periostin prevents p53-mediated apoptosis in sgc-7901 gastric cancer cells. Mol Biol Rep 2013, 40, 1677-1683.). It is considered that ingestion of HY upregulated Postn, then inhibited the expression of P53, suppressed apoptosis, and improved cell proliferation and cell cycle. In addition, downregulation of P53 reduces the production of DNA damaging molecules and improves cell loss due to inflammation and apoptosis (Spehlmann, ME; Manthey, CF; Dann, SM; Hanson, E .; Sandhu, SS; Liu, LY; Abdelmalak, FK; Diamanti, MA; Retzlaff, K .; Scheller, J., et al. Trp53 deficiency protects against acute intestinal inflammation. J Immunol 2013, 191, 837-847.). It was found that reduced expression of P53 not only exhibits HY's anti-apoptotic function, but can also improve the DSS-induced inflammatory state.

In addition, upregulation of the following three proteins results in improved apoptosis: Bag3 suppresses Bcl-2 apoptosis in vitro, suppresses cell death, and maintains cell survival by regulating gastrointestinal function. (Lee, YD; Yoon, JS; Yoon, HH; Youn, HJ; Kim, J .; Lee, JH Expression of bis in the mouse gastrointestinal system. Anat Cell Biol 2012, 45, 160-169.) .. Rbm3 deficiency can cause mitotic disorders due to a deficiency of important proteins required for cell proliferation (Hjelm, B .; Brennan, DJ; Zendehrokh, N .; Eberhard, J .; Nodin, B .; Gaber, A .; Ponten, F .; Johannesson, H .; Smaragdi, K .; Frantz, C., et al. High nuclear rbm3 expression is associated with an improved prognosis in colorectal cancer. Proteomics Clin Appl 2011, 5, 624-635. And Sureban, SM; Ramalingam, S .; Natarajan, G .; May, R .; Subramaniam, D .; Bishnupuri, KS; Morrison, AR; Dieckgraefe, BK; Brackett, DJ; Postier, RG , et al. Translation regulatory factor rbm3 is a proto-oncogene that prevents mitotic catastrophe. Oncogene 2008, 27, 4544-4556.). Fhc acts as an essential mediator to antagonize apoptosis by inhibiting the activation of mitogen-activated protein kinase 8 (Mapk8) by iron blockade (related to the accumulation of reactive oxygen species), and TNFα-induced apoptosis. Suppress (Pham, CG; Bubici, C .; Zazzeroni, F .; Papa, S .; Jones, J .; Alvarez, K .; Jayawardena, S .; De Smaele, E .; Cong, R .; Beaumont, C., et al. Ferritin heavy chain upregulation by nf-kappab inhibits tnfalpha-induced apoptosis by suppressing reactive oxygen species. Cell 2004, 119, 529-542.).

Based on the results of downregulation of Mapk8 (DSS vs. CON: 1.49, DHY8 vs. DSS: 0.07) by microarray analysis and the results of PCR of Tnfα, ingestion of HY suppresses apoptosis by promoting the ^{pathway Fhc neg} Mapk8 ^{pos Tnfα.} It was speculated. In addition to the three upregulated proteins, the downregulation of the following three other apoptosis-related proteins appears to further support the above speculation. That is, Apex1 may affect the DNA repair process through a diminished ability to repair oxidative damage, increasing the rate of apoptosis, increasing the risk of UC and leading to the collection of damaged DNA. There is (Bardia, A .; Tiwari, SK; Gunisetty, S .; Anjum, F .; Nallari, P .; Habeeb, MA; Khan, AA Functional polymorphisms in xrcc-1 and ape-1 contribute to increased apoptosis and risk of ulcerative colitis. Inflamm Res 2012, 61, 359-365. And Mort, R .; Mo, L .; McEwan, C .; Melton, DW Lack of involvement of nucleotide excision repair gene polymorphisms in colorectal cancer. Br J Cancer 2003, 89, 333-337.). Overexpression of Casp3 has also been reported to result in increased apoptosis and decreased proliferative capacity (Deng, QJ; Deng, DJ; Che, J .; Zhao, HR; Yu, JJ; Lu, YY Hypothalamic paraventricular nucleus. stimulation reduces intestinal injury in rats with ulcerative colitis. World J Gastroenterol 2016, 22, 3769-3776.). K1c20 is a marker for small intestinal goblet cells and can be induced by apoptosis and tissue damage (Zhou, Q .; Cadrin, M .; Herrmann, H .; Chen, CH; Chalkley, RJ; Burlingame, AL; Omary, MB Keratin 20 serine 13 phosphorylation is a stress and intestinal goblet cell marker. J Biol Chem 2006, 281, 16453-16461.).

The expression of the following two other genes may be very important for the maintenance of epithelial cells and barrier function, directly and indirectly. Activation of Egfr, which is upregulated in AOM / DSS-induced colitis, plays a role in improving damaged epithelial cells and maintaining epithelial barrier function in UC (Shimoda, M .; Horiuchi, K .; Sasaki, A. .; Tsukamoto, T .; Okabayashi, K .; Hasegawa, H .; Kitagawa, Y .; Okada, Y. Epithelial cell-derived a disintegrin and metalloproteinase-17 confers resistance to colonic inflammation through egfr activation. 114-124.). In this example, the expression of Egfr was significantly down-regulated by ingestion of HY. Tff2 is expressed and secreted by the gastrointestinal epithelium and plays an important role in maintaining epithelial integrity and mucosal surface, but was upregulated by HY ingestion. In addition, Tff2 is essential for the immune response in inflammatory conditions by controlling the expression of preinflammatory cytokines such as IL6 and IL1β (Kurt-Jones, EA; Cao, L .; Sandor, F .; Rogers). , AB; Whary, MT; Nambiar, PR; Cerny, A .; Bowen, G .; Yan, J .; Takaishi, S., et al. Trefoil family factor 2 is expressed in murine gastric and immune cells and controls both gastrointestinal inflammation and systemic immune responses. Infect Immun 2007, 75, 471-480.).

In this example, the altered Egfr and Tff2 expression caused by HY showed DSS-induced damage to epithelial cells and thereby improved intestinal lesion and barrier cell permeability. The above findings were supported by colonic histological results showing that the state of goblet cells damaged by DSS was alleviated in DHY8 mice compared to DSS mice. In addition, Tff2 deficiency has been reported to result in weight loss (Judd, LM; Chalinor, HV; Walduck, A .; Pavlic, DI; Dabritz, J .; Dubeykovskaya, Z .; Wang, TC; Menheniott, TR. Giraud, AS Tff2 deficiency exacerbates weight loss and alters immune cell and cytokine profiles in dss colitis, and this cannot be rescued by wild-type bone marrow. Am J Physiol Gastrointest Liver Physiol 2015, 308, G12-24.), HY Diet-induced upregulation of Tff2 appears to be consistent with improved weight loss.

Based on the results of the above proteome analysis, it was speculated that HY may have contributed to epithelial protection by upregulating the following proteins. Epcam plays a role in the formation of normal intestinal structure and ^{regulation of Ca 2+} -independent homologous cell adhesion, and in IBD patients, Epcam expression is down-regulated, resulting in loss of contact with lower skin cells. , Increases permeability and increases bacterial products that penetrate (Furth, EE; Li, J .; Purev, E .; Solomon, AC; Rogler, G .; Mick, R .; Putt, M .; Zhang, T. Somasundaram, R .; Swoboda, R., et al. Serum antibodies to epcam in healthy donors but not ulcerative colitis patients. Cancer Immunol Immunother 2006, 55, 528-537. And Balzar, M .; Prins, FA; Bakker, HA; Fleuren, GJ; Warnaar, SO; Litvinov, SV The structural analysis of adhesions mediated by ep-cam. Exp Cell Res 1999, 246, 108-121.). Tena, an extracellular matrix protein, can contribute to intestinal mucosal protection by promoting cell migration and remodeling and accelerating the recovery process in the injured area, and Tena's upregulation inhibits T cell activation. In studies showing that, Tena's anti-inflammatory effect has been reported (Ruegg, CR; Chiquet-Ehrismann, R .; Alkan, SS Tenascin, an extracellular matrix protein, exerts immunomodulatory activities. Proc Natl Acad Sci USA 1989, 86, 7437-7441.). Numb, a membrane-bound protein that is predominantly expressed in the intestinal mucosa and can determine cell fate, is an intestinal epithelial barrier through regulation of paracellular permeability by affecting apical junction complex assembly and myosin light chain phosphorylation. It can bring about functional improvements. Numb deficiency can cause barrier dysfunction (Yang, Y .; Chen, L .; Tian, Y .; Ye, J .; Liu, Y .; Song, L .; Pan, Q .; He , Y .; Chen, W .; Peng, Z., et al. Numb modulates the paracellular permeability of intestinal epithelial cells through regulating apical junctional complex assembly and myosin light chain phosphorylation. Exp Cell Res 2013, 319, 3214-3225.) ..

In this example, upregulation of Epcam, Tena and Numb was shown to enhance the repair or protection of mucosal barriers and ameliorate DSS-induced epithelial adhesion and tight junction dysfunction. The above results are consistent with the observation that (1) epithelial cells were repaired by colon tissue analysis, and (2) clinical symptoms such as bloody stools and diarrhea were improved in DHY8 mice compared with DSS mice.

Regarding the components of HY, RT-PCR results show that zeolite plays a role in reducing P53 expression and improving apoptosis by upregulating Igf1 expression, while citric acid follows the inflammatory bowel disease pathway. It shows that inflammation was significantly suppressed by inhibiting it. From this point of view, the function of HY is considered to be the result of the interaction of various components.

4. CONCLUSIONS: HY suppresses the expression of the inflammatory bowel disease pathway and thus reduces the degree of inflammation in DSS-induced colitis due to downregulation of pre-inflammatory cytokines, increased expression of anti-inflammatory factors by transcriptome and proteomics analysis. It was thought to be. HY inhibits correlated genes such as P53 and significantly alters other epithelial maintenance-related genes and proteins to improve cell cycle disorders caused by DSS to some extent and suppress apoptosis, resulting in mucosal immune homeostasis. Is considered to be maintained. Therefore, the results of integrated omics analysis show that the effect of HY on DSS-induced colitis is primarily based on improved inflammatory status, improved degree of apoptosis, and maintenance of epithelial defense and barrier function of the colonic mucosa. It was suggested that HY is effective in the prevention or treatment of inflammatory bowel disease.

Claims (6)

A composition for preventing or treating inflammatory bowel disease, which comprises a calcined product of zeolite, a calcined product of shell, citric acid and red yeast rice. In 100% by weight of the composition, 20% by weight to 80% by weight of the calcined product of zeolite and the calcined product of shell shell, 20% by weight to 40% by weight of citric acid, and 10% to 30% by weight of red koji are contained. The composition for preventing or treating inflammatory bowel disease according to claim 1. The composition for preventing or treating inflammatory bowel disease according to claim 1 or 2, wherein the blending ratio of the calcined zeolite product and the calcined shell product is 5:95 to 20:80 (weight ratio). The composition for preventing or treating inflammatory bowel disease according to any one of claims 1 to 3, which is a pharmaceutical. The composition for preventing or treating inflammatory bowel disease according to any one of claims 1 to 3, which is a dietary supplement. The composition for preventing or treating inflammatory bowel disease according to any one of claims 1 to 3, which is a food product.

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