KR20230115248A - Prevention or treatment of Western diet-induced inflammatory bowel disease by inhibiting expression and activation of gut-taste receptor TAS1R3 - Google Patents

Abstract

The present invention provides a method for screening a composition for treating inflammatory bowel disease or a drug for treating inflammatory bowel disease comprising a substance that reduces TAS1R3 gene expression or inhibits protein activity as an active ingredient. According to the present invention, when a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor is used, it changes the microbiome in the intestine, strengthens the tight junction between intestinal cells, and suppresses the secretion of inflammatory cytokines, thereby effectively preventing and improving inflammatory bowel disease. Alternatively, it has the advantage of being able to be treated, which can be usefully used for the treatment of inflammatory bowel disease in Asians, who are rapidly increasing due to Western diets for which no appropriate treatment has been developed so far.

Description

Prevention or treatment of Western diet-induced inflammatory bowel disease by inhibiting expression and activation of gut-taste receptor TAS1R3}

The present invention has identified that inflammatory bowel disease caused by Western diet can be prevented or treated by suppressing the expression and activation of the intestinal taste receptor TAS1R3. It provides a method for screening a composition for treating inflammatory bowel disease or a drug for treating inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a disease in which chronic inflammation of unknown cause in the intestinal tract repeatedly improves and recurs, and ulcerative colitis and Crohn's disease are representative examples. According to the US CDC (Centers for Disease Control and Prevention), there are more than 4 million IBD patients worldwide, and it is estimated that 1.4 million people are inflammatory bowel disease patients in the United States alone, and more than 70,000 new patients per year are reported to be constantly occurring. . Accordingly, the global inflammatory bowel disease treatment market reached $11.7 billion as of 2014, and is expected to grow to more than KRW 19 trillion by 2023.

It is noteworthy that the incidence of inflammatory bowel disease in Asian countries was low (in fact, it was classified as a very rare disease in the past), but the number of domestic patients with inflammatory bowel disease has increased rapidly over the past decade. As a result of analyzing the domestic Health Insurance Review and Assessment Service data, in 2015 alone, 18,000 people were treated for Crohn's disease in Korea (Korea), and the total medical cost exceeded 47.3 billion won, and the number of new patients also increased in 2019 compared to 2010. Not only has it more than doubled, but it is estimated that the number of domestic patients will steadily increase in the future due to the nature of the disease in which remission and recurrence are repeated.

As such, the cause of the sudden increase in disease in Asia can be attributed to changes in environmental factors. In particular, changes in Westernized dietary patterns have been reported to be very significant in the occurrence of diseases (The role of diet in the aetiopathogenesis of inflammatory bowel disease. Nature Review Gastroenterology Hepatology , 2018, 15:525-535).

Inflammatory bowel disease, including occasional abdominal pain, is a 'disease directly related to menstrual phenomena', so the emotional burden on patients is very great. Currently, biological cytokine inhibitors such as anti-TNF drugs, anti-integrin drugs, and IL-12/23 inhibitors are used as dominant treatments for the treatment of inflammatory bowel disease, but in the case of anti-TNF drugs, 50-60% of patients There are many cases in which patients who do not respond or the response disappears within 1 year, fail the existing standard drug treatment (5-Aminosalicylic acid (5-ASA) preparation, steroid, immunomodulator, etc.) or undergo surgery due to side effects of treatment. There is a high demand for the development of new therapies.

In addition, research results show that currently marketed treatments and treatment methods are usually targeted to Westerners such as the United States and Europe, so their effectiveness is significantly lower in Asians, whose incidence is rapidly increasing due to Westernized eating habits. Also reported (Treatment of inflammatory bowel disease in Aisa. Intest Res , 2016, 14(3):231-239; Best practices on immunomodulators and biologic agents for ulcerative colitis and Crohn's disease in Asia, Intest Res , 2019, 17 (3):285-310).

Therefore, the present inventors focused on the pathogenesis of inflammatory bowel disease caused by environmental factors such as Western-style diet intake and the fact that there is no separate treatment or treatment method targeted thereto. As a result of diligent efforts to develop a specific therapeutic agent, it was confirmed that long-term consumption of a Western-style diet actually induces severe inflammation in the intestine, and in particular, the intestinal taste receptor TAS1R3 can be an important mediator in mediating such intestinal inflammation. Knockout significantly increases the transcription factor PPAR-γ in enterocytes, i) increases the expression of genes involved in the secretion of tight junction proteins and antimicrobial peptides, The mucosal defense system is increased to strengthen the protective mechanism from western diet-induced intestinal inflammation, and the increased PPAR-γ ii) makes the intestinal lumen state hypoxic, leading to the expansion of anaerobic butyrate bacteria The present invention was completed by verifying that intestinal inflammation can be treated by inducing proliferation of beneficial bacteria and significantly reducing intestinal inflammation indicators.

Nature Review Gastroenterology Hepatology, 2018, 15:525-535 Intest Res, 2016, 14(3):231-239 Intest Res, 2019, 17(3):285-310

An object of the present invention is to provide a therapeutic agent for inflammatory bowel disease caused by a western diet and a method for screening the same.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient.

The present invention also provides a novel use of a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor for preventing or treating inflammatory bowel disease.

The present invention also provides a novel use of a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor for the preparation of a drug for preventing or treating inflammatory bowel disease.

The present invention also provides a method for preventing or treating inflammatory bowel disease comprising administering a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor to a subject in need thereof.

The present invention also provides a food composition for preventing or improving inflammatory bowel disease comprising a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient.

In the present invention, the TAS1R3 expression inhibitor may be a TAS1R3-specific antisense oligonucleotide, siRNA, shRNA or miRNA.

In the present invention, the TAS1R3 protein activity inhibitor may be a TAS1R3-specific antibody, aptamer or antagonist.

In the present invention, the inflammatory bowel disease may be characterized in that it is an inflammatory bowel disease induced by a western diet.

In the present invention, the composition

(i) changes in the gut microbiome;

(ii) strengthening the tight junctions between cells in the intestine; and/or

(iii) an increase in antimicrobial peptide secretion gene expression; may be characterized by preventing, improving or treating inflammatory bowel disease by inducing.

In the present invention, the change in the microbiome in the intestine may be characterized by expansion of anaerobic butyrate bacteria in the intestine and/or suppression of harmful bacteria in the intestine.

The present invention also provides a method for screening a therapeutic agent for inflammatory bowel disease comprising the following steps:

(a) treating cells or tissues in which TAS1R3 is expressed or the TAS1R3 protein is active with a candidate substance;

(b) selecting the candidate substance as a therapeutic agent for inflammatory bowel disease when TAS1R3 expression is inhibited or TAS1R3 protein activity is inhibited.

In the present invention, the inflammatory bowel disease may be characterized in that it is an inflammatory bowel disease caused by a western diet.

According to the present invention, when a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor is used, it changes the microbiome in the intestine, strengthens the tight junction between intestinal cells, and suppresses the secretion of inflammatory cytokines, thereby effectively preventing and improving inflammatory bowel disease. Alternatively, it has the advantage of being able to be treated, which can be usefully used for the treatment of inflammatory bowel disease in Asians, who are rapidly increasing due to Western diets for which no appropriate treatment has been developed so far.

Figure 1a is a result of comparing the degree of intestinal inflammation through tissue staining in normal diet (ND) and western diet (WD) mice.
Figure 1b is the result of calculating the inflammation score (inflammation score) in mice fed a normal diet and a western diet. Levels of inflammation were extremely elevated in mice fed a western diet.
Figure 1c compares the results of RNA sequencing analysis of intestinal tissues extracted from normal and Western diet mice. Intestinal genome expression in Western-fed mice (red squares) was significantly different from that in normal-fed mice (black circles).
Figure 1d is a result of selecting (screening) genes whose expression was statistically significantly changed in the intestinal tissues of mice fed a normal diet and a western diet.
1e is a result of functional classification of genes (Differentially expressed genes, DEGs) showing significant differences between mice fed a normal diet and a western diet. Significantly, changes in the signal transduction system related to taste receptor activity along with an inflammatory response are characteristic of the intestines of mice fed a western diet.
FIG. 1f shows a result confirming that expression of the taste receptor Tas1r3 was significantly increased among various types of taste receptors in mice fed a western diet.
Figure 1g shows the result of confirming the increased protein expression of TAS1R3 in inflammatory intestinal tissues of mice fed a western diet.
1h is a result confirming a significant positive correlation between TAS1R3 and various inflammatory markers. Increased intestinal TAS1R3 expression in mice fed a western diet showed significant positive correlations with several inflammatory markers.
Figure 2a shows the expression of the intestinal taste receptor TAS1R3 and its downstream molecule, GLP-1, when glucose (10 mM), fructose (10 mM), and palmitate (10 μM) were treated in various combinations in the NCI-H716 cell line, which is an enteroendocrine cell line. It is a schematic diagram of an experiment to confirm .
Figure 2b shows the combination shown in Figure 2a, the enteroendocrine cell NCI-H716 cell line was treated with glucose (10mM), fructose (10mM), palmitate (10μM), and 12 hours later, the mRNA expression level of TAS1R3 was measured by GAPDH mRNA It is the result expressed by normalizing with the expression level.
Figure 2c is the combination shown in Figure 2a, the NCI-H716 cell line, which is an enteroendocrine cell, was treated with glucose (10 mM), fructose (10 mM), and palmitate (10 μM), and the mRNA expression level of GCG was compared to the mRNA expression level of GAPDH. This is the result of normalization and observation for 24 hours.
Figure 2d is the combination shown in Figure 2a treated with glucose (10mM), fructose (10mM), palmitate (10μM) to the NCI-H716 cell line, which is an enteroendocrine cell, and observed the secretion of GLP-1 for 24 hours. This is the result. Secretion of GLP-1 means the degree of activation of TAS1R3.
FIG. 2e shows that the NCI-H716 cell line, which is an enteroendocrine cell, was treated with glucose (10 mM), fructose (10 mM), and palmitate (10 μM) in the combination shown in FIG. This is the result of normalization and observation for 24 hours.
Fig. 2f shows the combination shown in Fig. 2a, wherein the NCI-H716 cell line, which is an enteroendocrine cell, was treated with glucose (10 mM), fructose (10 mM), and palmitate (10 μM), and the mRNA expression level of IL6 was expressed as the mRNA expression level of GAPDH. This is the result of normalization and observation for 24 hours.
Figure 2g is the combination shown in Figure 2a, the NCI-H716 cell line, which is an enteroendocrine cell, was treated with glucose (10 mM), fructose (10 mM), and palmitate (10 μM), and the mRNA expression level of TNF was expressed as the mRNA expression level of GAPDH. This is the result of normalization and observation for 24 hours.
Figure 2h is the combination shown in Figure 2a, the NCI-H716 cell line, which is an enteroendocrine cell, was treated with glucose (10 mM), fructose (10 mM), and palmitate (10 μM), and the mRNA expression level of MCP-1 was compared with the mRNA expression of GAPDH. This is the result of observation for 24 hours after normalizing to the amount.
Figure 3a is the result of confirming the amount of TAS1R3 transcript according to TAS1R3 siRNA treatment.
Figure 3b is the result of confirming the TAS1R3 expression inhibition effect according to TAS1R3 siRNA treatment at the protein level.
Figure 3c is the result of confirming the effect of suppressing GCG transcript expression according to TAS1R3 siRNA treatment.
Figure 3d is the result of confirming the active GLP-1 inhibitory effect according to TAS1R3 siRNA treatment.
Figure 3e is the result of confirming the effect of inhibiting IL1B transcript expression according to TAS1R3 siRNA treatment.
Figure 3f is the result of confirming the effect of inhibiting IL6 transcript expression according to TAS1R3 siRNA treatment.
3g shows the result of confirming the effect of inhibiting IL8 transcript expression according to TAS1R3 siRNA treatment.
Figure 3h is the result of confirming the effect of inhibiting IL8 secretion according to TAS1R3 siRNA treatment.
Figure 3i is the result of confirming the effect of TNF transcript expression inhibition according to TAS1R3 siRNA treatment.
Figure 3j is the result of confirming the TNF-α secretion inhibitory effect according to TAS1R3 siRNA treatment.
3k shows the result of confirming the effect of inhibiting GCG transcript expression by treatment with Lactisole, an antagonist for TAS1R3.
Figure 3l is the result of confirming the active GLP-1 inhibitory effect according to lactisole treatment.
3m is a result confirming the effect of inhibiting IL1B transcript expression according to lactisole treatment.
3n shows the result of confirming the effect of inhibiting IL6 transcript expression according to lactisole treatment.
Figure 3o shows the result of confirming the effect of inhibiting IL8 transcript expression according to lactisole treatment.
Figure 3p is the result of confirming the effect of inhibiting IL8 secretion according to lactisole treatment.
Figure 3q is the result of confirming the effect of inhibiting TNF transcript expression according to lactisole treatment.
Figure 3r is a result confirming the effect of inhibiting TNF-α secretion according to lactisole treatment.
4 is a result of confirming non-expression of TAS1R3 in the small intestine and large intestine of the taste receptor Tas1r3 knockout mouse prepared in the present invention.
5a is a result confirming that Tas1r3 transcript expression is suppressed in the small intestine of Tas1r3 knockout mice fed a normal or western diet.
FIG. 5B is a result confirming that Tas1r3 protein expression is suppressed in the small intestine of Tas1r3 knockout mice fed a normal or western diet.
Figure 6a is a result of comparison of spleen weights in wild-type or Tas1r3 knockout mice fed a normal or western diet.
Figure 6b is a result of comparing the length of the small intestine in wild-type or Tas1r3 knockout mice fed a normal or western diet.
Figure 6c shows the results of comparison of colon lengths in wild-type or Tas1r3 knockout mice fed a normal or western diet.
Figure 6d shows the results of comparison of inflammation in intestinal tissue through histological examination in wild-type or Tas1r3 knockout mice fed a normal or western diet.
Figure 6e is a comparison of the intestinal inflammatory index in wild-type or Tas1r3 knockout mice fed a normal or western diet.
Figure 6f compares the pattern of IL-1b transcript expression in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
6g shows the comparison of Tnf transcript expression patterns in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
Figure 6h shows the comparison of IL6 transcript expression patterns in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
7a is a comparison result of protein expression levels of phosphorylated-mTOR (mTOR), mTOR, and PPAR-γ in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
7b is a result of quantifying the protein expression levels of phosphorylated-mTOR (mTOR) protein and PPAR-γ in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
Figure 7c shows tight junction genes (Tjp1, Ocln, Cldn1, Cldn7), which are regulatory genes of the transcription factor PPAR-γ, in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet. This is the result of comparing the expression levels of antimicrobial peptide genes (Reg3g, Lyz1, Defa2, Defa3).
8a is a comparison result of Shannon's diversity index in wild-type or Tas1r3 knockout mice fed a normal or western diet.
8b is a comparison result of Faith's phylogenetic index in wild-type or Tas1r3 knockout mice fed a normal or western diet.
Figure 8c shows the results of comparison of β-dirversity of wild-type or Tas1r3 knockout mice fed a normal or Western-style diet using the Bray-Curtis method.
8d shows the results of comparison of β-diversity of wild-type or Tas1r3 knockout mice fed a normal or Western-style diet using the weighted PCoA method.
FIG. 8E shows the results of comparing the differences in the gut microbiome of wild-type or Tas1r3 knockout mice fed a normal or Western-style diet at the phylum level.
FIG. 8f shows the results of comparing the differences in gut microbial communities at the family (family) and genus (genus) levels in wild-type or Tas1r3 knockout mice fed a western-style diet. Bacteria showing significant differences between the two groups are shown through Cladogram.
8g shows a comparison of the relative abundance of butyrate-producing strains (genus Butyrivibrio , Roseburia , Ruminococcus , Butyricicoccus , Faecalibacterium ) showing the most distinct characteristics in Tas1r3 knockout mice fed a western diet. am.
8h is a result of comparing the amount of butyrate in feces of wild-type or Tas1r3 knockout mice fed a western diet.
9 is a result showing that there is a significant correlation between the PPARγ signaling pathway and anaerobic butyrate-producing bacteria, which are increased by Tas1r3 deficiency.
10a is a result confirming the effect of inhibiting MTOR transcript expression according to TAS1R3 siRNA treatment.
10b is a result confirming the effect of enhancing PPARγ transcript expression according to TAS1R3 siRNA treatment.
10c is a result confirming the effect of enhancing PPARγ protein expression according to TAS1R3 siRNA treatment.
10d is a result confirming the effect of inhibiting MTOR transcript expression according to treatment with Lactisole, a TAS1R3 antagonist.
10e is a result confirming the effect of enhancing PPARγ transcript expression according to lactisole treatment.
10f is a result confirming the effect of enhancing PPARγ protein expression according to Lactisole treatment.
Figure 11 analyzes the difference in expression patterns of transcripts in intestinal biopsy samples of normal control group and experimental group of inflammatory bowel disease patients (11a - 11c), and the expression level of TAS1R3 and downstream molecules (mTOR, PPARγ) (11d - 11f) And it is the result of confirming the association (11g) with related molecules (molecules).
12 is a schematic diagram showing a key mechanism by which the intestinal taste receptor TAS1R3 regulates intestinal inflammation.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

In the present invention, it was confirmed that the expression of TAS1R3, a taste receptor in the intestine, was increased in subjects with inflammatory bowel disease induced by a western diet, and suppressing TAS1R3 expression could treat inflammatory bowel disease induced by a western diet. Under the hypothesis, a TAS1R3 knockout animal model was produced and the experiment was conducted. As a result, when TAS1R3 expression is suppressed, the expression of inflammatory cytokines and chemokines is suppressed, and the signaling pathway centered on mTOR/PPARγ is regulated. It was confirmed that inflammatory bowel disease can be effectively treated by strengthening tight junctions and changing the gut microbiome in favor of intestinal health.

Therefore, in one aspect, the present invention relates to a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient, and in another aspect, a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient. It relates to a composition for food for preventing or improving inflammatory bowel disease comprising a.

In the present invention, the gene sequence encoding the human TAS1R3 may be represented by SEQ ID NO: 1, and the human TAS1R3 protein sequence may be represented by SEQ ID NO: 3, but is not limited thereto.

TAS1R3 gene sequence (Human)

Homo sapiens taste 1 receptor member 3 (TAS1R3), mRNA

> NM_152228.3 Homo sapiens taste 1 receptor member 3 (TAS1R3), mRNA

GCTCACTCCATGTGAGGCCCCAGTCGGGGCAGCCACCTGCCGTGCCTGTTGGAAGTTGCCTCTGCCATGCTGGGCCCTGCTGTCCTGGGCCTCAGCCTCTGGGCTCTCCTGCACCCTGGGACGGGGGCCCCATTGTGCCTGTCACAGCAACTTAGGATGAAGGGGGACTACGTGCTGGGGGGGGCTGTTCCCCCTGGGCGAGGCCGAGGAGGCTGGCCTCCGCAGCCGGACACGGCCCAGCAGCCCT GTGTGCACCAGGTTCTCCTCAAACGGCCTGCTCTGGGCACTGGCCATGAAAATGGCCGTGGAGGAGATCAACAACAAGTCGGATCTGCTGCCCGGGCTGCGCCTGGGCTACGACCTCTTTGATACGTGCTCGGAGCCTGTGGTGGCCATGAAGCCCAGCCTCATGTTCCTGGCCAAGGCAGGCAGCCGCGACATCGCCGCCTACTGCAACTACACGCAGTACCAGCCCCGTGTGCTGGCTGTGT CATCGGGCCCCACTCGTCAGAGCTCGCCATGGTCACCGGCAAGTTCTTCAGCTTCTTCCTCATGCCCCAGGTCAGCTACGGTGCTAGCATGGAGCTGCTGAGCGCCCGGGAGACCTTCCCCTCCTTCTTCCGCACCGTGCCCAGCGACCGTGTGCAGCTGACGGCCGCCGCGGAGCTGCTGCAGGAGTTCGGCTGGAACTGGGTGGCCGCCCTGGGCAGCGACGACGAGTACGGCCG GCAGGGCCTGAGCATCTTCTCGGCCCTGGCCGCGGCACGCGGCATCTGCATCGCGCACGAGGGCCTGGTGCCGCTGCCCCGTGCCGATGACTCGCGGCTGGGGAAGGTGCAGGACGTCCTGCACCAGGTGAACCAGAGCAGCGTGCAGGTGGTGCTGCTGTTCGCCTCCGTGCACGCCGCCCACGCCCTCTTCAACTACAGCATCAGCAGCAGGCTCTCGCCCAAGGTGTGGGTGGCCAGCGA GGCCTGGCTGACCTCTGACCTGGTCATGGGGCTGCCCGGCATGGCCCAGATGGGCACGGTGCTTGGCTTCCTCCAGAGGGGTGCCCAGCTGCACGAGTTCCCCCAGTACGTGAAGACGCACCTGGCCCTGGCCACCGACCCGGCCTTCTGCTCTGCCCTGGGCGAGAGGGAGCAGGGTCTGGAGGAGGACGTGGTGGGCCAGCGCTGCCCGCAGTGTGACTGCATCACGCTGCAGAACGT GAGCGCAGGGCTAAATCACCACCAGACGTTCTCTGTCTACGCAGCTGTGTATAGCGTGGCCCAGGCCCTGCACAACACTCTTCAGTGCAACGCCTCAGGCTGCCCCGCGCAGGACCCCGTGAAGCCCTGGCAGCTCCTGGAGAACATGTACAACCTGACCTTCCACGTGGGCGGGCTGCCGCTGCGGTTCGACAGCAGCGGAAACGTGGACATGGAGTACGACCTGAAGCTGTGGGTGT GGCAGGGCTCAGTGCCCAGGCTCCACGACGTGGGCAGGTTCAACGGCAGCCTCAGGACAGAGCGCCTGAAGATCCGCTGGCACACGTCTGACAACCAGAAGCCCGTGTCCCGGTGCTCGCGGCAGTGCCAGGAGGGCCAGGTGCGCCGGGTCAAGGGGTTCCACTCCTGCTGCTACGACTGTGTGGACTGCGAGGCGGGCAGCTACCGGCAAAACCCAGACGACATCGCCTGCACC TTTTGTGGCCAGGATGAGTGGTCCCCGGAGCGAAGCACACGCTGCTTCCGCCGCAGGTCTCGGTTCCTGGCATGGGGCGAGCCGGCTGTGCTGCTGCTGCTCCTGCTGCTGAGCCTGGCGCTGGGCCTTGTGCTGGCTGCTTTGGGGCTGTTCGTTCACCATCGGGACAGCCCACTGGTTCAGGCCTCGGGGGGGCCCCTGGCCTGCTTTGGCCTGGTGTGCCTGGTCTGCCTC AGCGTCCTCCTGTTCCCTGGCCAGCCCAGCCCTGCCCGATGCCTGGCCCAGCAGCCCTTGTCCCACCTCCCGCTCACGGGCTGCCTGAGCACACTCTTCCTGCAGGCGGCCGAGATCTTCGTGGAGTCAGAACTGCCTCTGAGCTGGGCAGACCGGCTGAGTGGCTGCCTGCGGGGGCCCTGGGCCTGGCTGGTGGTGCTGCTGGCCATGCTGTGGAGGTCGCGTGCACCTGGTACCTGGT GGCCTTCCCGCCGGAGGTGGTGACGGACTGGCACATGCTGCCCACGGAGGCGCTGGTGCACTGCCGCACACGCTCCTGGGTCAGCTTCGGCCTAGCGCACGCCACCAATGCCACGCTGGCCTTTCTCTGCTTCCTGGGCACTTTCCTGGTGCGGAGCCAGCCGGGCTGCTACAACCGTGCCCGTGGCCTCACCTTTGCCATGCTGGCCTACTTCATCACCTGGGTCTCCTTTGTGCCCCTCCTGGC CAATGTGCAGGTGGTCCTCAGGCCCGCCGTGCAGATGGGCGCCCTCCTGCTCTGTGTCCTGGGCATCCTGGCTGCCTTCCACCTGCCCAGGTGTTACCTGCTCATGCGGCAGCCAGGGCTCAACACCCCCGAGTTCTTCCTGGGAGGGGGCCCTGGGGATGCCCAAGGCCAGAATGACGGGAACACAGGAAATCAGGGGAAACATGAGTGACCCAACCCTGTGATTCTCAGCCCCGGTGAACCC AGACTTAGCTGCGATCCCCCCCAAGCCAGCAATGACCCGTGTCTCGCTACAGAGACCCTCCCGCTCTAGGTTCTGACCCCAGGTTGTCTCCTGACCCTGACCCCACAGTGAGCCCTAGGCCTGGAGCACGTGGACACCCCTGTGACCATCTGGGCCCCAGAGCCAAGCTGTGTCCCTGTCCCTCTGTGCCCAGACCAGGCCTGCCCAGGTAACCCAGACCCACTGTTCTGGAAAGAGGCCCGG AGGGCTCCCAGGTACCCGCAACCCACACCGTGAGCTCAGGAAAAGGACGCAGGGAGGCCCCGGCCAGATGGCTGGAAGCCCAAATCAGGCCCTGCCGACCTGACCATGTCCCACCAGGGCCCCCATCCTGCACCCTGCCAGGCACCACAGCAGTGGGAGGCCAGGTGGGGGCACACAGGCATATGCCCAGGGCAGAGCCCGCCGAGGTAGGGGTGGCACCCAGCTTCCTACTCTGCCCTTTGCC CAGTGGGTAGACAGCATCATGACTGTCACCAGTACCAGGGACAGAGCCCAGGTGGGGTGGGGGCGGGGTCCAGCACCACGGCCAGCACCGACCACCAGGACCCCGGAGCCAGCACCATGGACAGAAAACTGCCCACCAGGATCTGACGCCAGCACGCCGCCAGGCCCACACAGGGTCTCCGGTCAGAGTCCCAGGGTCAGCTCCCAGCAGGGCCTAGGGGAGGCTGGACCAGCTCCCTGTGCCT CATTCCAAGGCAGCCCAGCCGGAGAGAAGGGGCACAGGCCACACATCTGTCCCATAAAATTAAACGCTTTTTAGTGTTTAAAATAA

Tas1r3 gene sequence (Mouse)

Mus muscle taste receptor, type 1, member 3 (Tas1r3), mRNA

> NM_031872.2 Mus muscle taste receptor, type 1, member 3 (Tas1r3), mRNA

CTGGAGACTTCTACCTACCATGCCAGCTTTGGCTATCATGGGTCTCAGCCTGGCTGCTTTCCTGGAGCTTGGGATGGGGGCCTCTTTGTGTCTGTCACAGCAATTCAAGGCACAAGGGGACTACATACTGGGCGGGCTATTTCCCCTGGGCTCAACCGAGGAGGCCACTCTCAACCAGAGAACACAACCCAACAGCATCCCGTGCAACAGGTTCTCACCCCTTGGTTTGTTCCTGGCCATGGCTAT GAAGATGGCTGTGGAGGAGATCAACAATGGATCTGCCTTGCTCCCTGGGCTGCGGCTGGGCTATGACCTATTTGACACATGCTCCGAGCCAGTGGTCACCATGAAATCCAGTCTCATGTTCCTGGCCAAGGTGGGCAGTCAAAGCATTGCTGCCTACTGCAACTACACACAGTACCAACCCCGTTGTGCTGGCTGTCATCGGCCCCCACTCATCAGAGCTTGCCCTCATCAGGCAAGTTCTTCAGCT CGCACATGA GGGCCTGGTGCCACAACATGACACTAGTGGCCAACAGTTGGGCAAGGTGCTGGATGTACTACGCCAAGTGAACCAAAGTAAAGTACAAGTGGTGGTGCTGTTTGCCTCTGCCCGTGCTGTCTACTCCCTTTTTAGTTACAGCATCCATCATGGCCTCTCACCCAAGGTATGGGTGGCCAGTGAGTCTTGGCTGACATCTGACCTGGTCATGACACTTCCCAATATTGCCCGTGTGGGCACTGTGCTTGGG TTTTTGCAGCGGGGTGCCCTACTGCCTGAATTTTCCCATTATGTGGAGACTCACCTTGCCCTGGCCGCTGACCCAGCATTCTGTGCCTCACTGAATGCGGAGTTGGATCTGGAGGAACATGTGATGGGGCAACGCTGTCCACGGTGTGACGACATCATGCTGCAGAACCTATCATCTGGGCTGTTGCAGAACCTATCAGCTGGGCAATTGCACCACCAAATATTTGCAACCTATGCAGCTGTGTACAGTG TGGCTCAAGCCCTTCACAACACCCTACAGTGCAATGTCTCACATTGCCACGTATCAGAACATGTTCTACCCTGGCAGCTCCTGGAGAACATGTACAATATGAGTTTCCATGCTCGAGACTTGACACTACAGTTTGATGCTGAAGGGAATGTAGACATGGAATATGACCTGAAGATGTGGGTGTGGCAGAGCCCTACACCTGTATTACATACTGTGGGCACCTTCAACGGCACCCTTCAGCTGCAGCAG TCTAAAATGTACTGGCCAGGCAACCAGGTGCCAGTCTCCCAGTGTTCCCGCCAGTGCAAAGATGGCCAGGTTCGCCGAGTAAAGGGCTTTCATTCCTGCTGCTATGACTGCGTGGACTGCAAGGCGGGCAGCTACCGGAAGCATCCAGATGACTTCACCTGTACTCCATGTAACCAGGACCAGTGGTCCCCAGAGAAAAGCACAGCCTGCTTACCTCGCAGGCCCAAGTTTCTGGCTTGGGGGGAG CCAGTTGTGCTGTCACTCCTCCTGCTGCTTTGCCTGGTGCTGGGTCTAGCACTGGCTGCTCTGGGGCTCTCTGTCCACCACTGGGACAGCCCTCTTGTCCAGGCCTCAGGTGGCTCACAGTTCTGCTTTGGCCTGATCTGCCTAGGCCTCTTCTGCCTCAGTGTCCTTCTGTTCCCAGGGCGGCCAAGCTCTGCCAGCTGCCTTGCACAACAACCAATGGCTCACCTCCCTCACAGGCTGCCT GAGCACACTCTTCCTGCAAGCAGCTGAGACCTTTGTGGAGTCTGAGCTGCCACTGAGCTGGGCAAACTGGCTATGCAGCTACCTTCGGGGACTCTGGGCCTGGCTAGTGGTACTGTTGGCCACTTTTGTGGAGGCAGCACTATGTGCCTGGTATTTGATCGCTTTCCCACCAGAGGTGGTGACAGACTGGTCAGTGCTGCCCACAGAGGTACTGGAGCACTGCCACGTGCGTTCCTGGGTCAGCCTGGGC TTGGTGCACATCACCAATGCAATGTTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACAGAGCCAGCCTGGCCGCTACAACCGTGCCCGTGGTCTCACCTTCGCCATGCTAGCTTATTTCATCACCTGGGTCTCTTTTGTGCCCCTCCTGGCCAATGTGCAGGTGGCCTACCAGCCAGCTGTGCAGATGGGTGCTATCCTAGTCTGTGCCCTGGGCATCCTGGTCACCTTCCACCTGCCCAAGT GCTATGTGCTTCTTTGGCTGCCAAAGCTCAACACCCAGGAGTTCTTCCTGGGAAGGAATGCCAAGAAAGCAGCAGATGAGAACAGTGGCGGTGGTGAGGCAGCTCAGGGACACAATGAATGACCACTGACCCGTGACCTTCCCTTTAGGGAACCTAGCCCTACCAGAAATCTCCTAAGCCAACAAGCCCCGAATAGTACCTCAGCCTGAGACGTGAGACACTTAACTATAGACTTGGACTCCACTGA CCTTAGCCTCACAGTGACCCCTTCCCCAAACCCCCAAGGCCTGCAGTGCACAAGATGGACCCTATGAGCCCACCTATCCTTTCAAAGCAAGATTATCCTTGATCCTATTATGCCCACCTAAGGCCTGCCCAGGTGACCCACAAAAGGTTCTTTGGGACTTCATAGCCATACTTTGAATTCAGAAATTCCCAGGCAGACCATGGGAGACCAGAAGGTACTGCTTGCCTGAACATGCCCAGCCCTGAGCCC TCACTCAGCACCCTGTCCAGGCGTCCCAGGAATAGAAGGCTGGGCATGTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGTACGTATGTATGTATGTATCAGGACAGAACAAGAAAGACATCAGGCAGAGGACACTCAGGAGGTAGGCAACATCCAGCCTTCTCCATCCCTAGCTGAGCCCTAGCCTGTAGGAGAGAACCAGGTCGCCGCCAGCACCTTGGACAGATCA CACACAGGGTGCGGGTCAGCACCACGGCCAGCGCCAGCCACGCGGGACCCCTGGAATCAGCTTCTAGTACCAAGGACAGAAAAGTTGCCGCAAGGCCCCTTACTGGCCAGCACCAGGGACAGAGCCACATGCCTAAGCGGCAAGGGACAAGAGCATCGTCCATCTGCAGGCAGGATCAGACCCGGGTCAGTTCTGGACTGGCCCCCACACCTGAATCCCGGAGCAGCTCAGCTGGAGAAAAG AGAAACAAGCCACACATCAGTCCCATAAAATTAAACGCTTTTTTTAGTGTTTAAAA

TAS1R3 protein sequence (Human)

Taste receptor type 1 member 3 precursor [Homo sapiens]

NCBI Reference Sequence: NP_689414.2

mlgpavlgls lwallhpgtg aplclsqqlr mkgdyvlggl fplgeaeeag lrsrtrpssp

vctrfssngl lwalamkmav eeinnksdll pglrlgydlf dtcsepvvam kpslmflaka

gsrdiaaycn ytqyqprvla vigphssela mvtgkffsff lmpqvsygas mellsaretf

psffrtvpsd rvqltaaael lqefgwnwva algsddeygr qglsifsala aargiciahe

glvplpradd srlgkvqdvl hqvnqssvqv vllfasvhaa halfnysiss rlspkvwvas

eawltsdlvm glpgmaqmgt vlgflqrgaq lhefpqyvkt hlalatdpaf csalgereqg

leedvvgqrc pqcdcitlqn vsaglnhhqt fsvyaavysv aqalhntlqc nasgcpaqdp

vkpwqllenm ynltfhvggl plrfdssgnv dmeydlklwv wqgsvprlhd vgrfngslrt

erlkirwhts dnqkpvsrcs rqcqegqvrr vkgfhsccyd cvdceagsyr qnpddiactf

cgqdewsper strcfrrrsr flawgepavl llllllslal glvlaalglf vhhrdsplvq

asggplacfg lvclglvcls vllfpgqpsp arclaqqpls hlpltgclst lflqaaeifv

eselplswad rlsgclrgpw awlvvllaml vevalctwyl vafppevvtd whmlptealv

hcrtrswvsf glahatnatl aflcflgtfl vrsqpgcynr argltfamla yfitwvsfvp

llanvqvvlr pavqmgalll cvlgilaafh lprcyllmrq pglntpeffl gggpgdaqgq

ndgntgnqgk he

TAS1R3 protein sequence (Mouse)

Taste receptor type 1 member 3 precursor [Mus musculus]

NCBI Reference Sequence: NP_114078.1

mpalaimgls laaflelgmg aslclsqqfk aqgdyilggl fplgsteeat lnqrtqpnsi

pcnrfsplgl flamamkmav eeinngsall pglrlgydlf dtcsepvvtm ksslmflakv

gsqsiaaycn ytqyqprvla vigphssela litgkffsff lmpqvsysas mdrlsdretf

psffrtvpsd rvqlqavvtl lqnfswnwva algsdddygr eglsifssla nargiciahe

glvpqhdtsg qqlgkvldvl rqvnqskvqv vvlfasarav yslfsysihh glspkvwvas

eswltsdlvm tlpniarvgt vlgflqrgal lpefshyvet hlalaadpaf caslnaeldl

eehvmgqrcp rcddimlqnl ssgllqnlsa gqlhhqifat yaavysvaqa lhntlqcnvs

hchvsehvlp wqllenmynm sfhardltlq fdaegnvdme ydlkmwvwqs ptpvlhtvgt

fngtlqlqqs kmywpgnqvp vsqcsrqckd gqvrrvkgfh sccydcvdck agsyrkhpdd

ftctpcnqdq wspekstacl prrpkflawg epvvlsllll lclvlglala alglsvhhwd

splvqasggs qfcfgliclg lfclsvllfp grpssascla qqpmahlplt gclstlflqa

aetfveselp lswanwlcsy lrglwawlvv llatfveaal cawyliafpp evvtdwsvlp

tevlehchvr swvslglvhi tnamlaflcf lgtflvqsqp grynrarglt famlayfitw

vsfvpllanv qvayqpavqm gailvcalgi lvtfhlpkcy vllwlpklnt qefflgrnak

kaadensggg eaaqghne

In the present invention, the TAS1R3 expression inhibitor may be a TAS1R3-specific antisense oligonucleotide, siRNA, shRNA or miRNA, but is not limited thereto.

For example, the TAS1R3 expression inhibitor may be siRNA represented by the following sequence, but is not limited thereto.

TAS1R3 siRNA sequence information

Forward: CUGUCUACGCAGCUGUGUA (dTdT)

Reverse: UACACAGCUGCGUAGACAG (dTdT)

In the present invention, the TAS1R3 protein activity inhibitor may be a TAS1R3-specific antibody aptamer or an antagonist, but is not limited thereto.

In the present invention, the TAS1R3 protein activity inhibitor may be lactisole, but is not limited thereto.

In the present invention, the inflammatory bowel disease may be characterized in that it is an inflammatory bowel disease induced by a western diet.

In the present invention, the composition

(i) changes in the gut microbiome;

(ii) strengthening the tight junctions between cells in the intestine; and/or

(iii) increase in antimicrobial peptide secretion gene expression; may be characterized in that inflammatory bowel disease is prevented or treated by inducing, but the effect is not limited thereto.

In the present invention, the intestinal microbiome change may be characterized by expansion of anaerobic butyrate bacteria in the intestine and/or suppression of harmful bacteria in the intestine, but is not limited thereto.

In the present invention, the anaerobic butyrate bacteria may be characterized in that at least one selected from bacteria consisting of the genus Butyrivibrio , the genus Roseburia , the genus Ruminococcus , the genus Butyricicoccus and the genus Faecalibacterium , but is not limited thereto.

In the present invention, the harmful bacteria may be characterized in that at least one selected from bacteria consisting of the genus Prevotella , the family Paraprevotellaceae, the family Enterobacteriaceae, and the genus Enterobacteriales , but is not limited thereto.

Inhibition of TAS1R3 expression or activity according to the present invention significantly increases PPAR-γ in enterocytes, i) increases expression of genes involved in tight junction protein and antimicrobial peptide secretion Intestinal mucosal defense system is increased to strengthen the protection mechanism from western diet-induced intestinal inflammation, and increased PPAR-γ ii) makes the intestinal lumen state hypoxic, expanding anaerobic butyrate bacteria while inducing the proliferation of beneficial bacteria in the intestine, the intestinal inflammation index is significantly reduced, so intestinal inflammation can be prevented or treated.

In another aspect, the present invention relates to a method for screening a therapeutic agent for inflammatory bowel disease comprising the following steps:

(a) treating cells or tissues in which TAS1R3 is expressed or the TAS1R3 protein is active with a candidate substance;

(b) selecting the candidate substance as a therapeutic agent for inflammatory bowel disease when TAS1R3 expression is inhibited or TAS1R3 protein activity is inhibited.

In the present invention, the inflammatory bowel disease may be characterized in that it is an inflammatory bowel disease caused by a western diet.

The therapeutic agent includes a TAS1R3-specific antisense oligonucleotide, siRNA, shRNA, miRNA or TAS1R3-specific antibody, aptamer or antagonist alone, or is provided as a pharmaceutical composition including one or more pharmaceutically acceptable carriers, excipients or diluents The complex may be included in the pharmaceutical composition in an appropriate pharmaceutically effective amount depending on the disease and its severity, the patient's age, weight, health condition, sex, administration route, and treatment period.

As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions when administered to humans.

The composition according to the present invention may further include a pharmaceutically acceptable excipient. Excipients that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose. , microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The composition of the present invention may be formulated into a formulation for oral administration or a formulation for parenteral administration by a conventional method, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, cryoprotectants It can be prepared using the like.

When the composition of the present invention is formulated as a solid preparation for oral administration, it includes tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate ( Calcium carbonate), sucrose, lactose or gelatin. In addition, in addition to simple excipients, magnesium stearate and lubricants such as talc may be included, but are not limited thereto.

When the composition of the present invention is formulated as a liquid formulation for oral administration, it includes suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents and sweeteners in addition to water and liquid paraffin, which are commonly used simple diluents , fragrances, preservatives, and the like, but are not limited thereto.

When the composition of the present invention is formulated into a formulation for parenteral administration, it may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, and a suppository. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate, but are not limited thereto. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The content of the novel lactic acid bacteria, which is an active ingredient of the pharmaceutical composition of the present invention, may be adjusted in various ranges depending on the specific form, purpose or aspect of the composition. The content of the active ingredient in the pharmaceutical composition according to the present invention is not particularly limited, and may be, for example, 0.01 to 99% by weight, specifically 0.1 to 75% by weight, more specifically 0.5 to 50% by weight based on the total weight of the composition. there is.

The composition for food according to the present invention may be characterized in that it is a composition for food or food additives, but is not limited thereto, and can be easily utilized as, for example, a main ingredient of a food, a supplementary ingredient, a food additive, a health functional food, or a functional beverage. there is.

The food means a natural product or processed product containing one or more nutrients, and preferably means a product that can be eaten directly through a certain degree of processing process, and in a conventional sense, food, food It includes all additives, health functional foods and functional beverages.

Foods to which the food composition according to the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, and functional foods. In addition, foods in the present invention include special nutritional foods (eg, formula milk, infant food, baby food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), breads, health supplements, seasoning Foods (eg, soy sauce, soybean paste, gochujang, mixed paste, etc.), sauces, confectionery (eg, snacks), candies, chocolates, chewing gum, ice cream, dairy products (eg, fermented milk, cheese, etc.), other processed foods, kimchi, It includes, but is not limited to, pickled foods (various types of kimchi, pickled vegetables, etc.), beverages (eg, fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.). The food, beverage or food additive may be prepared by a conventional manufacturing method.

The health functional food refers to a food group or food composition that has added value so that the function of the food acts for a specific purpose by using physical, biochemical, or bioengineering methods, etc. It refers to food designed and processed to sufficiently express the body's regulatory function related to the living body. The functional food may include food additives that are acceptable in food science, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods.

In the present invention, the functional beverage refers to a general term for drinking to quench thirst or enjoy taste, and there are no particular limitations on other ingredients other than including the composition as an essential ingredient in the indicated ratio, and various Flavoring agents or natural carbohydrates may be included as additional ingredients.

Furthermore, foods containing the food composition of the present invention other than those described above are various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Can be used in combination.

In the food containing the composition for food of the present invention, the amount of the composition according to the present invention may be included in 0.001% to 100% by weight of the total weight of the food, preferably 1% to 99% by weight. In the case of beverages, it may be included in a ratio of 0.001g to 10g, preferably 0.01g to 1g, based on 100ml, but in the case of long-term intake for the purpose of health and hygiene or health control It may be less than the above range, and the active ingredient may be used in an amount greater than the above range because there is no problem in terms of safety, so it is not limited to the above range.

In the present invention, antisense oligonucleotides are useful for regulating the expression of nucleic acid molecules (eg, regulating the expression of TAS1R3) through an antisense mechanism. Such modulation can be performed, for example, by providing an oligonucleotide that is complementary to and/or hybridizes to one or more target nucleic acid molecules, such as mRNA. In some embodiments, an oligonucleotide of the invention is complementary to a specific region of a target nucleic acid. In some embodiments, an oligonucleotide of the invention is capable of hybridizing to a specific region of a target nucleic acid. The oligonucleotide compounds of the present invention are complementary to one or more target nucleic acids and interfere with the normal function of the targeted nucleic acids (eg, by an antisense mechanism of action). It interferes with or modulates the function of a target nucleic acid by means of an oligonucleotide of the invention that specifically hybridizes to what is commonly referred to as "antisense". The functions of DNA that are disturbed can include replication and transcription. Functions of the RNA that are hindered may be, for example, translocation of the RNA into a protein translation site, translation of a protein from the RNA, splicing of the RNA to give rise to one or more mRNA species, and participation or promotion by the RNA. functions such as catalytic activity. In some embodiments, the overall effect of interfering with target nucleic acid function is regulation of expression of the product of such target nucleic acid.

In the present invention, siRNA (small interfering RNA) refers to a short double-stranded RNA capable of inducing RNA interference through cleavage of a specific mRNA. siRNA is not limited to complete pairing of double-stranded RNA parts paired with each other, but is not paired by mismatch (corresponding bases are not complementary), bulge (there is no corresponding base on one side of the chain), etc. parts may be included. Paired bases are 15 to 30 bases in length. As the siRNA end structure, either a blunt end or a protruding end may be used as long as the expression of the target gene can be suppressed by the RNA interference effect. The sticky end structure can be both a 3' end protruding structure and a 5' end protruding structure.

The siRNA of the present invention includes variants having one or more substitutions, insertions, deletions, and combinations thereof that do not reduce its activity. Such variants may have 80% or more sequence homology to the siRNA sequence described above, preferably 90% or more, and more preferably 95% or more sequence homology.

The siRNA of the present invention can be directly synthesized chemically (Sui G et al., Proc. Natl. Acad. Sci. USA (2002) 99:5515-5520) or synthesized using in vitro transcription (Brummelkamp TR et al. ., Science (2002) 296:550-553) can be synthesized by various methods known in the art.

In the present invention, shRNA is called short hairpin RNA, and is an RNA molecule having a stem-loop structure so that a part of one strand forms a complementary strand with another region.

In the present invention, miRNA is a single-stranded RNA of about 21 to 25 bases that has an effect of suppressing gene expression. Small RNAs derived from non-coding regions encoded on the genome. Generally, miRNAs are generated from genes transcribed from single or clustered miRNA precursors. That is, pri-miRNA, the primary transcript from the gene, is transcribed, and subsequently, in step-by-step processing from pri-miRNA to mature miRNA, pre-miRNA of about 70 to 80 bases with a characteristic hairpin structure is produced from pri-mRNA. Then, mature miRNAs are produced from pre-miRNAs by Dicer. miRNAs are deeply involved in vital phenomena such as differentiation, cell proliferation, and apoptosis that are indispensable to living organisms.

In the present invention, antibody refers to a protein that binds to other molecules (antigens) through variable regions of light and heavy chains, and includes IgG, IgD, IgA, and IgE-derived proteins. In addition, the antibodies herein include monoclonal antibodies having various types of structures, for example, intact antibodies including two full-length heavy chains and two full-length light chains, as well as containing or not including constant regions. fragments thereof, chimeric antibodies, humanized antibodies, or other genetically engineered antibodies having the characteristics described herein. Antibodies of the present application, including intact antibodies or fragments thereof, may exist as multimers such as dimers, trimers, tetramers, pentamers, etc., including at least a portion of the monomer's antigen-binding ability. Such multimers also include homomultimers or heteromultimers. Since antibody multimers contain multiple antigen-binding sites, they have superior antigen-binding ability compared to monomers. Multimers of antibodies are also easy to construct multifunctional (bifunctional, trifunctional, tetrafunctional) antibodies.

Antibodies that specifically bind to Tas1R3 described herein can be prepared by various methods known in the art (Scheving, L.A. et al., American Journal of Physiology, 306:G370-81, 2014; Kaufmann, A. et al., J. Cell. Biochem. 114:681-69, 2013; :57, 2013; del Blanco, B. et al., J. Immunol, 188:3278-3293, 2012).

In the present invention, an aptamer refers to a small single-stranded oligonucleic acid capable of specifically recognizing a target material with high affinity.

Specifically, a method for selecting nucleic acids that bind to a specific substance is called SELEX (Systematic Evolution of Ligand by Exponential Enrichment), and the nucleic acid selected as a product is generally called an aptamer (Craig et al. , Science, 249:505-510, 1990). Through the SELEX method, molecules capable of binding with very high affinity to various biomolecules including proteins not bound to nucleic acids as well as proteins capable of binding to nucleic acids in nature are selected. The aptamer specifically binding to Egr-1 of the present invention can be prepared by the SELEX standard method (Bock LC et al., Nature, 355:564-6, 1992), a technique known in the art (Mannironi et al. al., Biochemistry 36:9726, 1997;Ellington and Szostak et al., Nature, 346:818, 1990;Tuerk and Gold, et al., Science 249:505, WO 00/20040, WO 99/54506, WO 99 /27133, WO 97/42317). Therefore, it will be apparent to those skilled in the art that an aptamer for Tas1R3 can be easily prepared using known techniques even if it is not described in the examples.

The step of measuring the binding degree of the aptamer that specifically binds to Tas1R3 may be performed using a DNA aptamer binding measurement technique commonly used in the related art, for example, a fluorescent or radioactive material at the end of the aptamer. A method of measuring fluorescence or radioactive intensity by labeling or binding with biotin, imaging, and observation may be used, but is not limited thereto.

In the present invention, an antagonist means, in one aspect, a compound that inhibits or blocks intracellular signal transduction caused by TAS1R3, which can also be referred to as a compound that inhibits the TAS1R3 signal. Such a compound may be a naturally occurring compound or an artificially synthesized compound. It may also be a low molecular weight compound or a high molecular weight compound such as protein.

In the present invention, gene expression is inhibited means that the gene itself is deleted or its expression is reduced, not only that the enzyme or protein produced by the gene is completely removed, but also that the enzyme or protein produced by the gene is endogenous. It includes those modified to be attenuated compared to the activity.

In the present invention, "modified to be attenuated compared to the intrinsic activity" means deletion of a gene showing activity, inactivation of a gene (eg, substitution with a mutant gene), attenuation of gene expression (eg, a weak promoter). substitution, introduction of siRNA, gRNA, sRNA, etc., substitution of the start codon from ATG to GTG, etc.), inhibition of the activity of enzymes expressed by the gene (eg, addition of non-competitive repressors or competitive repressors), etc. It means a state in which the activity after the manipulation is reduced compared to before the manipulation.

As used herein, the term "intrinsic activity" refers to the original active state of an enzyme, such as an enzyme in an unmodified state, and "modified to be weakened compared to the intrinsic activity" means that the activity of the enzyme before modification can be compared. This means that the activity disappears or is further reduced.

In the present invention, "deletion" is a concept encompassing preventing the gene from being expressed through a method of mutating, substituting, or deleting a part or all bases of the gene or preventing the unique function of a protein from being expressed even if expressed. It includes anything that blocks the biosynthetic pathway or signal transduction pathway in which the expressed protein is involved.

In another aspect, the present invention relates to the use of a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor for preventing or treating inflammatory bowel disease.

In another aspect, the present invention relates to the use of the pharmaceutical composition for preventing or treating inflammatory bowel disease.

In another aspect, the present invention relates to the use of a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor for the preparation of a drug for preventing or treating inflammatory bowel disease.

In another aspect, the present invention relates to a novel use of the pharmaceutical composition for the preparation of a drug for preventing or treating inflammatory bowel disease.

In another aspect, the present invention relates to a method for preventing or treating inflammatory bowel disease comprising administering a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor to a subject in need thereof.

In another aspect, the present invention relates to a method for preventing or treating inflammatory bowel disease comprising administering the pharmaceutical composition to a subject in need thereof.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Effect of Western diet on intestinal inflammation

8-10 week old C57BL/6J mice were obtained from the Jackson Laboratory (West Grove, PA, USA), and 10 mice per experimental group were fed either a normal diet or a western diet [sugar solution (30% w/v) + 60% high fat type], and fed free-feeding for 10 weeks. The high-fat diet (60%, D12492) and the normal diet (D12450J), which had the same composition except for the fat content, were all purchased from Research Diets (New Brunswick, NJ, USA).

In order to analyze the degree of intestinal inflammation, histological examination (Pathological examination) and flow cytometry analysis (FACS profiling) were performed. After 10 weeks of diet induction, mice were sacrificed using 20% urethane (U2500, Sigma-Aldrich, St. Louis, MO, USA), and then terminal ileum specimens were obtained. The ileum tissue was fixed in 10% formalin and subjected to H&E staining. Histological analysis followed the previously reported histologic colitis scoring system (Severity of DSS-induced colitis is reduced in Ido1-deficient mice with down-regulation of TLR-MyD88-NF-kB transcriptional networks. Sci Rep , 2015, 5: 17305). The degree of inflammation, the extent of inflammation, the extent of damage to the crypt, and the extent of damage to the entire small intestine were measured, and each variable was added to calculate the inflammation score.

In addition, cells of the lamina propria were analyzed by flow cytometry using Cell Quest software of FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). Antibodies used were CD4 (GK1.5; BioLegend, San Diego, CA, USA), CD8a (53-6.7; BioLegend), CD45 (30-F11; BioLegend), phycoerythrin (PE), allophycocyanin (APC), APC- CyChrome7 (APC-Cy7), Alexa Fluor 700 (AF700).

As a result of histological examination and flow cytometry, it was confirmed that intestinal inflammation was significantly increased in mice by a long-term western diet for 10 weeks (FIGS. 1a and 1b).

In order to confirm the cause of increased intestinal inflammation in mice fed a western diet at the molecular level, mice were sacrificed, and inflamed small intestine tissue was extracted and RNA sequencing was performed in the tissue. After total RNA was extracted from the extracted intestinal tissue (using RNAqueous kit (AM1914, Ambion)), RNA was quantified using a NanoDrop 2000/2000c Spectrophotometer (Thermo Scientific). Intact mRNA was captured with a total of 1μg (1000ng) of total RNA (using Dynabeads mRNA DIRECT Micro Kit (Ambion)), and the captured mRNA was used for library construction through Ion Total RNA Seq Kit v2 (Life Technologies). The prepared library sample was finally loaded into the Ion PI Chip Kit v3 (Life Technologies) and then put into the Ion Proton Sequencer (Life Technologies) and run. All experiments were performed according to the instructions of each kit manufacturer.

As a result, it was confirmed that inflammatory cytokine and chemokine-related signals, which are indicators of inflammation, increased, and at the same time, signal transduction systems related to taste receptor activity significantly increased (FIG. 1c-1e). Among several genes involved in taste receptor activity, it was confirmed that the expression of the taste receptor Tas1r3, in particular, was significantly increased in inflamed intestinal tissues induced by the western diet (Fig. 1f). The protein expression level of TAS1R3 was found to be very high (Fig. 1g).

On the other hand, significant positive correlations were confirmed between TAS1R3 and various markers of inflammation (inflammation score, frequency of CD45+ leukocytes, CD4+ T cells, and CD8+ T cells infiltrating lymph nodes, Il1b mRNA, and Tnf mRNA levels). (Fig. 1h).

These results suggest that long-term consumption of a western-style diet induces severe tissue damage and infiltration of inflammatory cells, thereby causing severe inflammation in the intestine, and that the intestinal taste receptor TAS1R3 may be an important mediator in mediating such intestinal inflammation. suggests

Example 2. Effect of intestinal taste receptor TAS1R3 on western diet-induced intestinal inflammation

Glucose (10 mM), fructose (10 mM), palmitate (10 μM) (all Sigma-Aldrich, St. Louis, MO, USA) were treated with various combinations, and the expression of the intestinal taste receptor TAS1R3 and its downstream molecule, GLP-1, was analyzed at the mRNA and protein levels (FIG. 2a).

For qPCR, first, total RNA in intestinal tissue was extracted using RNAqueous (AM1914, Ambion) kit. The RNA extracted with the kit was synthesized into cDNA using the MML-V reverse transcriptase protocol (11917010, Invitrogen), and the quantitative polymerase reaction was measured to quantitatively express mRNA expression. To 2μl of synthesized cDNA (total volume 10 ng/μl), 5 μl of primers (Forward+Reverse) for each gene and 10 μl of SYBRGreen ^® (4367659, Applied Biosystems) were added to 2μl of synthesized cDNA, and the final volume was increased to 20μl with non-DEPC water. After matching, it was confirmed using StepOnePlus (Real-time PCR System, Applied Biosystems, Foster City, CA, USA) equipment. PCR was performed at 92 ° C for 30 seconds, 60 ° C for 45 seconds, and then 72 ° C for 30 seconds in 40 cycles. Primer information used for qPCR is as follows (Table 1).

Gene Forward primer sequences (5'-3') Reverse primer sequences (5'-3') Human GAPDH CCACTCCTCCACCTTTGACG CCACCACCCTGTTGCTGTAG Human TAS1R3 CACCAGACGTTCTCTGTCTACG CTGAGGCGTTGCACTGAAGA Human GCG CTGAAGGGACCTTTACCAGTGA CCTGGCGGCAAGATTATCAAG Human IL1B ATGATGGCTTATTACAGTGGCAA GTCGGAGATTCGTAGCTGGA Human IL6 CCTGAACCTTCCAAAGATGGC TTCACCAGGCAAGTCTCCTCA Human IL8 ATGACTTCCAAGCTGGCCGTGGCT TCTCAGCCCTCTTCAAAAACTTCTC Human TNF GAGGCCAAGCCCTGGTATG CGGGCCGATTGATCTCAGC Human CCL2 (MCP-1) GCTCAGCCAGATGCAATCA TTTGCTTGTCCAGGTGGTC

As a result, compared to the case of processing glucose, fructose, or palmitate alone, TAS1R3 It was confirmed that the amount of mRNA expression significantly increased (Fig. 2b).

In order to confirm whether TAS1R3, whose expression level was actually increased, even activated the function, the NCI-H716 cell line, an enteroendocrine cell, was treated with glucose (10 mM), fructose (10 mM), and palmitate (10 μM), and the level of GLP-1 secretion was measured. Observed for 24 hours. At this time, the secretion of GLP-1 means the degree of activation of TAS1R3.

To measure GLP-1 secreted from cells, centrifugation was performed at 4°C and 3000 g for 10 minutes, and only supernatants were collected and stored at -80°C. A GLP-1 (Active) ELISA kit (EGLP-35k, Millipore, St. Charles, MI, USA) was used and the experiment was performed according to the manufacturer's protocol.

As a result, compared to the case of processing glucose, fructose, or palmitate alone, when glucose/fructose/palmitate was treated in combination, the level of GLP-1 was increased. It was confirmed that mRNA expression level (GCG) and peptide level (Active GLP-1) were significantly increased (Fig. 2c-2d).

In order to determine whether the intestinal taste receptor TAS1R3 activated by dietary ligand stimulation directly induces the secretion of inflammatory cytokines and chemokines, the main inflammatory cytokines IL1B, IL6, TNF and chemokine MCP-1 in inflammatory bowel disease were investigated. The mRNA level of was confirmed by the qPCR method described above using the primers listed in Table 1.

As a result, the mRNA levels of IL1B, IL6, TNF, and the chemokine MCP-1 at 12 hours after glucose (10 mM), fructose (10 mM), and palmitate (10 μM) alone or in combination were treated with glucose/fructose/palmitate (10 μM) in particular. It was confirmed that a significant increase was observed in the experimental group treated with a combination of palmitate (Fig. 2e - Fig. 2h)

From these results, it can be seen that the results of the animal experiment in Example 1, which were confirmed to exacerbate intestinal inflammation with the intake of a western-style diet and that the taste receptor TAS1R3 induces an inflammatory response at this time, were also verified in an in vitro experiment.

Example 3. Inhibition of Intestinal Cell Inflammatory Mechanism through Inhibition of Intestinal Taste Receptor TAS1R3

In order to inhibit the intestinal taste receptor TAS1R3, siRNA or receptor antagonist lactisole is treated, and expression of GLP-1, a downstream molecule, and cytokine (IL1B, IL6, TNF) and chemokine (MCP-1) expression in the intestine was confirmed by qPCR and ELISA.

To this end, the NCI-H716 cell line was treated with (i) glucose (10 mM), fructose (10 mM), palmitate (10 μM) (control), (ii) glucose (10 mM), fructose (10 mM), palmitate (10 μM), TAS1R3 siRNA treatment (siRNA treatment group), (iii) glucose (10 mM), fructose (10 mM), palmitate (10 μM), and TAS1R3 antagonist (lactisole) treatment (antagonist treatment group), followed by additional incubation.

Human enteroendocrine NCI-H716 cells (CCL-251; American Type Culture Collection, Manassas, VA, USA) were maintained in suspension culture at 37° C. and 5% CO ₂ according to the supplier's protocol. The culture medium was RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin. Two days before the experiment, 1×10 ⁶ cells were seeded in 24-well culture plates pre-coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). On the day of the experiment, the supernatant was replaced with medium containing 10 mM glucose, fructose, glucose + fructose and/or 10 μM palmitate.

siRNA knockdown

siRNA duplex against TAS1R3 was synthesized by Bioneer (Daejeon, South Korea). Scrambled negative control siRNA was also purchased from Bioneer. For knockdown experiments, 5 x 10 ⁵ endocrine differentiated NCI-H716 cells were plated in 6-well culture plates and cultured for 48 hours. TAS1R3 (10 nM) or control (10 nM) siRNA was transfected into cells using Lipofectamine RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA). 48 hours after transfection, cells were incubated for 12 hours with medium containing 10 mM glucose, 10 mM fructose and 10 μM palmitate.

TAS1R3 siRNA sequence information

Forward: CUGUCUACGCAGCUGUGUA (dTdT)

Reverse: UACACAGCUGCGUAGACAG (dTdT)

Treatment with TAS1R3 antagonist lactisole

In the TAS1R3 antagonist experiment, NCI-H716 cells were pretreated with the TAS1R3 antagonist lactisole (2.5 mM) for 30 min and stimulated with a medium containing fructose (10 mM), glucose (10 mM) and palmitate (10 μM) for 12 h. Glucose, fructose, palmitate and lactisol were all purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). After incubation, media was collected and centrifuged at 1,000 x g for 10 min at 4°C to remove floating cells and frozen at -20°C for subsequent biochemical analysis.

qPCR and ELISA were performed in the same manner as in Example 2, IL-8 ELISA kit (Cat# ab46032, Abcam, Cambridge, UK) was used for the protein amount of IL-8, and TNF-α ELISA kit was used for the amount of TNF protein. (Cat# ab181421, Abcam, Cambridge, UK).

As a result, in the control group, TAS1R3 was activated by treatment with glucose (10 mM), fructose (10 mM), and palmitate (10 μM), and the mRNA expression and secretion of GLP-1 increased, whereas in the siRNA treatment group, TAS1R3 mRNA And as a result of suppressing protein expression by about 70%, it was confirmed that secretion of GLP-1 was also inhibited (FIGS. 3a-3d).

In addition, in the control group, the mRNA levels of IL1B, IL6, IL8, and TNF, which are inflammatory cytokines, and the protein levels of IL8 and TNF were significantly increased according to TAS1R3 activation, but in the siRNA-treated group, TAS1R3 expression was suppressed and the level of inflammatory cytokines was lowered. maintained (Figs. 3e-3j).

Similarly, as a result of treatment with lactisole, an antagonist for the TAS1R3 receptor, compared to the control group, the mRNA expression and secretion of GLP-1 could be significantly suppressed (Fig. 3k-3l), and the mRNA of IL1B, IL6, IL8, and TNF It was confirmed that even the expression and secretion protein levels of IL-8 and TNF could be significantly suppressed (FIGS. 3m-3r).

Taken together, the above results indicate that TAS1R3 expressed in the intestine can directly regulate the secretion of inflammatory cytokines induced by diet, and suppressing TAS1R3 in enterocytes can suppress the inflammatory mechanism of enterocytes. could be identified at the cellular level.

Example 4. Functional study of taste receptor TAS1R3 in intestinal inflammation induced by western diet using Tas1r3 knockout mouse model

4-1. Construction of Tas1r3 knockout mice

Tas1r3 ^tm1Csz (JAX013066) breeding pair (purchased frozen sperm) was purchased from The Jackson Lab, and Tas1r3 gene-deficient (knock-out, KO) mice were constructed. Backcrossing with C57BL/6 was performed for at least 7 generations, and the genotype of Tas1r3 knockout mice was confirmed through PCR genotyping. Experiments were conducted according to the Jax protocol, primer information used for genotyping is shown in Table 2, Reaction A information is shown in Table 3, and PCR conditions are shown in Table 4.

PRIMER Sequence (5'->3') reaction 11829 CCA CAG TCA ACA GAA ACC A 11830 CAT CCC AAG CTC CAG GAA A 11831 TAA GGG CCA GCT CAT TCC A

Component Final concentration ddH ₂ O Kapa 2G HS Buffer 1.30Ⅹ MgCl ₂ 2.60 mM dNTPs KAPA 0.26 mM 11829 0.50 uM 11830 0.50 uM 11831 0.50 uM )Glycerol 6.50% Dye 1.00Ⅹ Kapa 2G HS taq polymerase 0.03U/ul DNA

STEP TEMP (℃ NOTE One 94.0 2 94.0 3 65.0 -0.5℃ per cycle decrease 4 68.0 5 Repeat steps 2-4 for 10 cycles (Touchdown) 6 94.0 7 60.0 8 72.0 9 Repeat steps 6-8 for 28 cycles 10 72.0 11 10.0 hold

Wild-type controls (Tas1r3 ^WT ) (Jackson Laboratory (West Grove, PA, USA)) and knockout mice (Tas1r3 ^KO ), matched in age and weight, were bred in SPF (Specific Pathogen Free) animal breeding rooms, and immunohistochemical staining (Immunohistochemistry, Through IHC), it was confirmed at the protein level whether the expression of TAS1R3 was actually lacking in the small intestine and large intestine of each mouse.

For immunohistochemical staining (IHC), sample tissues cut to a thickness of 4-6 μm were fixed in formalin, the tissues were deparaffinized, rehydrated through a series of xylenes and alcohol, and washed in a citrate buffer solution (pH6 .0, Sigma, C9999) and then heated at 100°C for 20 minutes. After washing with phosphate buffered saline, the slides were incubated with 1% bovine serum albumin (Sigma-Aldrich) and Phosphate-buffered saline-tween for 1 hour, followed by anti-TAS1R3 antibody (TAS1R3 antibody (1:100, OSR00184W, Invitrogen, Camarillo) , CA, USA) Mouse and Rabbit-Specific HRP/DAB Detection IHC kit (ab64264, Abcam) was used as a secondary antibody.

As a result, compared to wild-type (Tas1r3 ^WT ) mice as a control group, it was confirmed that the intestinal tissue of knockout mice (Tas1r3 ^KO ) prepared in the present invention lacked expression of TAS1R3 (FIGS. 4, 5a, and 5b).

4-2. Induction of Intestinal Inflammation by Normal Diet and Western Diet in Wild-Type and Tas1r3 Knockout Mice

8-10-week-old wild-type control (Tas1r3 ^WT ) and Tas1r3 knockout mice (Tas1r3 ^KO ) of similar body weight were bred in SPF (Specific Pathogen Free) animal breeding rooms, and 10 animals per experimental group were grouped on a normal diet or Western diet [sugar solution (30% w/v) + 60% high-fat diet], and fed as free food for 10 weeks.

In order to analyze the degree of intestinal inflammation, the mice were sacrificed, and the spleen, small intestine, and colon were removed and histological examination was performed to compare their sizes.

As a result, it was confirmed that the size of the spleen, which was rapidly increased by the western diet in the wild-type control group, was not significantly increased in Tas1r3 knockout mice even when fed the western diet (Fig. 6a), and in the wild-type control group, the Western diet While the size of the small and large intestine was markedly reduced by T., Tas1r3 knockout mice fed a Western-style diet maintained a similar level of small and large intestine to mice fed a normal diet (Fig. 6b, 6c).

In order to analyze the inflammatory cells infiltrating the intestine, the extracted tissues were analyzed using immunofluorescence (IF). The experimental method of immunofluorescence staining (IF) is the same as that of immunohistochemical staining (IHC), and the information of the antibody used is as follows.

Primary antibodies: pan-leukocytes (CD45) (1:100, ab10558, Abcam, Cambridge, UK), anti-CD4 (14-0041-86; eBioscience, San Diego, CA, USA), and rat anti-CD8 (ab22378 , Abcam), and mouse anti-CD11b (14-0112-82, eBioscience)

Secondary and secondary-conjugated primary antibodies: Alexafluor 488 anti-mouse/human CD11b (101219, BioLegend, San Diego, CA, USA), donkey anti-rabbit Alexafluor 594 (A21207, Thermo Fisher Scientific, Waltham, MA, USA), donkey anti-mouse Alexafluor 488 (A21202, Thermo Fisher Scientific), and donkey anti-rat Alexafluor 594 (A21209, Thermo Fisher Scientific).

Immunoreactivity was visualized using a confocal microscope system (LSM 510; Carl Zeiss, Oberkochen, Germany).

As a result, intestinal inflammation was reduced in Tas1r3 knockout mice, and the inflammation level rapidly increased by western diet in wild-type control mice was maintained at the level of normal diet in Tas1r3 knockout mice fed western diet, and inflammatory cytology was reduced. The mRNA expression levels of IL1B, Tnfα, and IL6, which are kines, were also maintained at the levels fed with the normal diet (FIG. 6d-6h), confirming that Tas1r3 knockout mice are protected from intestinal inflammation induced by the western diet.

4-3. Intestinal transcriptome analysis in wild-type and Tas1r3 knockout mice fed a normal and western diet

Since Tas1r3 knockout mice are protected from intestinal inflammation induced by a western diet, transcriptome analysis in the small intestine was performed using the mice of Example 4-2 to investigate the mechanism. The transcriptome experiment is the same as the RNA sequence analysis method of Example 1.

As a result, it was confirmed that the mTOR/PPARγ axis in the intestine was significantly changed in Tas1r3 knockout mice compared to wild-type mice, specifically, mTOR phosphorylation was inhibited and PPARγ expression was increased ( 7a, 7b).

The PPARγ gene in the intestine is known as a transcription factor that regulates the expression of tight junction proteins and antimicrobial peptides that strengthen the intestinal barrier. Accordingly, as a result of further confirming changes in the expression of tight junction proteins and antimicrobial peptides, the expression of tight junction proteins and antimicrobial peptides was also found in Tas1r3 knockout mice. It was confirmed that it was significantly changed (FIG. 7c).

From these results, it was confirmed that the intestinal taste receptor TAS1R3 functions as an intrinsic regulator of the transcription factor PPARγ in enterocytes and can strengthen the barrier by increasing the tight junctions between enterocytes from intestinal inflammation induced by a western diet.

4-4. Analysis of intestinal commensal profiles in wild-type and Tas1r3 knockout mice induced by normal and western-style diets

Tas1r3 knockout mice are protected from western diet-induced intestinal inflammation, and it is known that the intestinal commensal plays an important role in intestinal inflammation. Therefore, the intestinal commensal profile of Tas1r3 knockout mice was analyzed by 16S rRNA sequencing (microbiome). analyzed.

Stool samples for 16S rRNA sequencing (microbiome) analysis were frozen immediately after collection and stored at -80°C. Total bacterial DNA was isolated from stool samples using the QIAamp®Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.

A 16S rRNA sequencing library was constructed according to the 16S metagenomics sequencing library preparation protocol (Illumina, San Diego, CA, USA) targeting the V3 and V4 hypervariable regions of the 16S rRNA gene. A KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, USA) and Agencourt AMPure XP system (Beckman Coulter Genomics, Brea, CA, USA) were used to amplify and purify PCR products, respectively. Final amplicons were sequenced in paired-end mode (PE275) using the MiSeq system (Illumina).

As a result, the proliferation of useful bacteria (obligate anaerobic bacteria) in the intestine, especially butyrate-producing bacteria (Faecalibacterium and Roseburia, etc.), was very significant in Tas1r3 knockout mice fed a western diet compared to wild-type mice fed a western diet. , and the abundance of pathogenic harmful bacteria (Enterobacteriaceae) was confirmed to be significantly low (Figs. 8a -8h).

4-5. Identification of the cause of expansion of butyric acid-producing bacteria in Tas1r3 knockout mice

In a recent study, it was found that the transcription factor PPARγ increases β-oxidation in enterocytes and plays a role in maintaining hypoxia in the intestinal lumen with low oxygen partial pressure (Microbiota-activated PPAR-γ dysbiotic Enterobacteriaceae expansion, Science , 2017, 357:570-575).

It was determined that the reason why the proliferation of butyric acid-producing bacteria, a useful bacterium in the intestine, was increased in Tas1r3 knockout mice fed a Western-style diet could be due to increased expression of the transcription factor PPARγ in enterocytes, and this was analyzed. The correlation between the relative abundance of butyrate-producing bacteria and the transcript expression of PPAR-related molecules was analyzed using Spearman's correlation test.

As a result, as shown in FIG. 9, the PPARγ signaling pathway increased by Tas1r3 knockout showed a very significant positive correlation with anaerobic butyrate-producing bacteria. This suggests that when Tas1r3 deficiency increases the intestinal transcription factor PPARγ, the intestinal lumen becomes hypoxic, induces the expansion of anaerobic butyrate bacteria living in an anaerobic environment, and consequently exerts an effect of protecting against intestinal inflammation. interpreted

Example 5. Regulation of the mTOR/PPARγ axis through inhibition of the intestinal taste receptor TAS1R3

In order to suppress the intestinal taste receptor TAS1R3, we tried to confirm whether the mTOR/PPARγ axis is regulated when treated with siRNA or lactisole, an antagonist of the intestinal taste receptor TAS1R3.

Treatment with siRNA and TAS1R3 antagonist lactisole was performed in the same manner as in Example 3, and the primary antibodies of mTOR, phospho-mTOR, and PPARγ were monoclonal rabbit anti-mTOR (1:1,000) (Cat# 2972S, Cell Signaling Technology (Danvers , MA, USA) and analyzed. As a control, mouse anti-α-tubulin (1:10,000) (Cat# T5168, RRID: AB_477579) was used. Secondary antibodies were Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000) (Cat# 7074S, RRID: AB_2099233) from Cell Signaling Technology (Danvers, MA, USA), goat anti-mouse secondary antibodies (1: 10,000) (Cat# G21040, RRID: AB_2536527) was purchased from Invitrogen (Carlsbad, CA, USA) and used.

As a result, as shown in FIG. 10, when TAS1R3 siRNA or lactisole was treated, mTOR phosphorylation was inhibited and PPARγ expression was increased. It was found that inflammatory bowel disease can actually be treated by regulating the expression of antimicrobial peptides.

Example 6. Expression analysis of TAS1R3 and related molecules (mTOR/PPARγ) in intestinal tissues of patients with inflammatory bowel disease (IBD)

In order to confirm the expression pattern of TAS1R3 and related molecules (mTOR/PPARγ) in the intestinal tissues of patients with inflammatory bowel disease (IBD), we used biopsy mRNA microarray data sets of inflammatory bowel disease patients and healthy controls. It was downloaded from the GEO database (public database, available online: http://www.ncbi.nlm.nih.gov/geo ), and the expression level of each molecule was analyzed.

We screened a dataset that contained both criteria: (i) not cultured cells from adult patients with active IBD, and (ii) samples from patients with IBD who had not received any intervention or treatment. Results, a total of four mRNA expression datasets [GSE160804, GSE126124, GSE95095, GSE75214 and GSE53306] were selected for analysis.

Raw microarray data is downloaded from the GEO database and then subjected to robust multi-chip analysis (RMA) to perform background correction, quantile normalization, and probe summarization. The algorithm was preprocessed using Partek® version 6.6 (Partek®). Differentially expressed genes were performed with R/Bioconductor (available online: http://www.bioconductor.org/), DEG An FDR P < 0.001 was considered the cut off value when selecting .

As a result, as shown in FIG. 11, it was confirmed that the expression level of the taste receptor TAS1R3 in the intestinal tissue was abnormally increased even in patients with inflammatory bowel disease (IBD), consistent with the results analyzed in the Tas1r3 knockout mouse of the present invention. , The elevated expression of TAS1R3 was confirmed to consistently show a significant positive correlation with all inflammatory cytokines IL1B, IL6, and TNF.

As above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

A pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient.
The composition according to claim 1, wherein the TAS1R3 expression inhibitor is a TAS1R3-specific antisense oligonucleotide, siRNA, shRNA or miRNA.
The composition according to claim 1, wherein the TAS1R3 protein activity inhibitor is a TAS1R3-specific antibody, aptamer or antagonist.
The composition according to claim 1, wherein the inflammatory bowel disease is inflammatory bowel disease induced by a western diet.
The method of claim 1, wherein the composition
(i) changes in the gut microbiome;
(ii) strengthening the tight junctions between cells in the intestine; and/or
(iii) an increase in antimicrobial peptide secretion gene expression; characterized in that for preventing or treating inflammatory bowel disease by inducing, the composition.
The composition according to claim 5, wherein the change in the intestinal microbiome is expansion of anaerobic butyrate bacteria in the intestine and/or suppression of harmful bacteria in the intestine.
A food composition for preventing or improving inflammatory bowel disease comprising a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient.
The composition according to claim 7, wherein the TAS1R3 expression inhibitor is a TAS1R3-specific antisense oligonucleotide, siRNA, shRNA or miRNA.
The composition according to claim 7, wherein the TAS1R3 protein activity inhibitor is a TAS1R3-specific antibody, aptamer or antagonist.
The composition according to claim 7, wherein the inflammatory bowel disease is inflammatory bowel disease induced by a western diet.
The method of claim 7, wherein the composition
(i) changes in the gut microbiome;
(ii) strengthening the tight junctions between cells in the intestine; and/or
(iii) an increase in antibacterial peptide secretion gene expression; characterized in that for preventing or improving inflammatory bowel disease by inducing, the composition.
The composition according to claim 11, wherein the change in the intestinal microbiome is expansion of anaerobic butyrate bacteria in the intestine and/or suppression of harmful bacteria in the intestine.
A method for screening for drugs for inflammatory bowel disease comprising the following steps:
(a) treating cells or tissues in which TAS1R3 is expressed or the TAS1R3 protein is active with a candidate substance;
(b) selecting the candidate substance as a therapeutic agent for inflammatory bowel disease when TAS1R3 expression is inhibited or TAS1R3 protein activity is inhibited.
14. The method of claim 13, wherein the inflammatory bowel disease is inflammatory bowel disease caused by a western diet.