I κbα degradation inhibitor and prophylactic or therapeutic agent for diseases caused by NF-κb activation using same

Abstract

Provided is a method for effectively treating or preventing various diseases caused by the activation of the NF-κB pathway. The IκBα degradation inhibitor is characterized by containing indole lactic acid or a salt thereof as an active ingredient. The prophylactic or therapeutic agent for diseases caused by NF-κB activation is characterized by containing the degradation inhibitor.

Description

IκBα degradation inhibitors and disease prevention and treatment agents utilizing them through NF-κB activation

[0001] This invention relates to an IκBα degradation inhibitor and a disease prevention and treatment agent utilizing the same through NF-κB activation.

[0002] Chronic kidney disease (CKD) is a disease characterized by persistent kidney damage and a decline in kidney function, which can progress to end-stage renal failure. It is estimated that there are approximately 13.3 million CKD patients in Japan. Since CKD also increases the risk of cardiovascular disease and death, early diagnosis and appropriate treatment of CKD are crucial.

[0003] Among the uremic toxins that are one of the factors in CKD, indoxyl sulfate (IS), for example, is known to activate the NF-κB (Nuclear factor kappa B) pathway (Non-patent Literature 1), which can lead to various diseases such as barrier dysfunction, inflammation, tumors, and autoimmune diseases.

[0004] Therefore, if there were a technology that could inhibit the NF-κB pathway, it could be an effective treatment and preventive measure for various diseases caused by activation of the NF-κB pathway, but no such technology is known.

[0005] Hidehisa Shimizu, et al., NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells., Am J Physiol Cell Physiol., 2011;301(5): C1201-C1212, (https: / / doi.org / 10.1152 / ajpcell.00471.2010)

[0006] Therefore, the object of the present invention is to provide effective treatment and prevention methods for various diseases caused by activation of the NF-κB pathway.

[0007] As a result of diligent research to solve the above problems, the present inventors discovered that indole-3-lactic acid (ILA) has an inhibitory effect on the degradation of IκBα (inhibitor of κB alpha), thereby enabling the prevention and treatment of diseases through NF-κB activation, and thus completed the present invention.

[0008] In other words, the present invention is as follows: [1] An IκBα degradation inhibitor characterized by comprising indole lactic acid or a salt thereof as an active ingredient. [2] A preventive and therapeutic agent for diseases caused by NF-κB activation, characterized by containing the IκBα degradation inhibitor described in [1]. [3] A preventive and therapeutic agent for diseases caused by NF-κB activation according to [2], wherein the NF-κB activation is due to IκBα degradation. [4] A preventive and therapeutic agent for diseases caused by NF-κB activation according to [3], wherein the disease caused by NF-κB activation is selected from barrier dysfunction, inflammation, tumors, and autoimmune diseases. [5] A preventive and therapeutic agent for diseases caused by NF-κB activation according to [3] or [4], wherein the IκBα degradation is due to uremic substances. [6] A preventive and therapeutic agent for diseases caused by NF-κB activation according to [5], wherein the uremic substance is indoxyl sulfate. [7] A method for inhibiting IκBα degradation, characterized by comprising indole lactic acid or a salt thereof as an active ingredient. A method for preventing and treating diseases caused by NF-κB activation, characterized by containing the IκBα degradation inhibitor described in [8] and [7]. [9] The method for preventing and treating diseases caused by NF-κB activation as described in [8], wherein the NF-κB activation is due to IκBα degradation.

[10] The method for preventing and treating diseases caused by NF-κB activation as described in [9], wherein the disease caused by NF-κB activation is selected from barrier dysfunction, inflammation, tumors, and autoimmune diseases.

[11] The method for preventing and treating diseases caused by NF-κB activation as described in

[10] , wherein the IκBα degradation is due to uremic substances.

[12] The method for preventing and treating diseases caused by NF-κB activation as described in

[11] , wherein the uremic substance is indoxyl sulfate.

[0009] The IκBα degradation inhibitor of the present invention contains indole lactic acid or a salt thereof, which is known to be beneficial to the body, as its active ingredient, and therefore can be safely ingested by mammals, including humans.

[0010] Furthermore, since the NF-κB activation-based disease preventive and therapeutic agent of the present invention contains the above-mentioned IκBα degradation inhibitor as an active ingredient, it can also be safely ingested by mammals, including humans, in the same way as the IκBα degradation inhibitor of the present invention.

[0011] This figure shows the anti-ROS (Reactive Oxygen Species) activity of ILA ((Top panel) shows the results of measuring intracellular ROS in T84 cells treated with ROS inducers (IS; 1000 μM, H2O2; 10 μM) and test substances (ILA; 10 μM, IPA (indole-3-propionic Acid); 10 μM, PDTC (ammonium pyrrolidinedithiocarbamate); 10 μM, NAC (N-acetyl-L-cysteine); 5 μM) for 2 hours (n=3). (Bottom panel) shows the results of quantifying the RNA expression level of the indicated gene by qPCR in T84 cells treated with ILA (500 μM) for 6 hours (n=3). This figure shows the inhibitory effect of ILA on IκBα degradation (T84 cells were treated with IS (1000 μM) and each drug (ILA; 500 μM, CH-223191 (CH); 10 μM, PDTC; 10 μM) for 30 minutes to 2 hours, and the results of detecting intracellular IκBα and β-actin proteins are shown). This figure shows the inhibitory activity of ILA on NF-κB nuclear localization (T84 cells that formed a monolayer membrane were treated with IS (1000 μM) and / or ILA (500 μM) and cultured at 37°C for 72 hours. After culturing, the cells were fixed, stained for NF-κB (green) and occludin (red), and observed by fluorescence microscopy. Each group was performed with n=3, and the figure shows a representative example). This figure shows the effects on NF-κB downstream genes (T84 cells were added with IS (1000 μM) and / or ILA (500 μM) and PDTC (10 μM) and cultured at 37°C for 6 hours. Gene expression in the cells was measured by qPCR and the results are shown (n=3). Ct: control group). This figure shows the site of action of ILA in the IS / ROS / NF-κB pathway. This figure shows the barrier function-enhancing effect of ILA (T84 cells that formed a monolayer membrane were added with ILA (62.5-1000 μM) and cultured at 37°C for 72 hours. TEER values ​​were measured after culture and the tight junction proteins shown in the figure were detected (n=3). Ct: control group, * p<0.05 vs. Ct group, ** p<0.01 vs. Ct group).This figure shows the protective effect of ILA on barrier function against IS-induced barrier dysfunction (T84 cells with a monolayer membrane were treated with ILA (500 μM) and / or IS (1000 μM) and cultured at 37°C for 72 hours. TEER values ​​were measured after culturing to detect the tight junction proteins shown in the figure (n=3). Ct: control group, * p<0.05 vs. Ct group, ** p<0.01 vs. Ct group, †† p<0.01 vs. IS treated group).

[0012] The IκBα degradation inhibitor of the present invention (hereinafter referred to as "the inhibitor of the present invention") is characterized by containing indole lactic acid or a salt thereof as an active ingredient. IκBα is a protein that is a member of the IκB family and inhibits the activation of NF-κB.

[0013] The active ingredient of the inhibitor of the present invention, indole lactic acid, has the molecular formula C １１ H １１ NO ３ Its molecular weight is 205.213. Indole-lactic acid is also called indole-3-lactic acid or ILA.

[0014] Indole lactic acid is an optical isomer. Any of the L-, D-, or DL- forms of indole lactic acid can be used as the inhibitor of the present invention, but the L- and DL- forms of indole lactic acid are preferred.

[0015] The salt of indole-lactic acid, which is the active ingredient of the inhibitor of the present invention, is not particularly limited, but for example, it is a salt of indole-lactic acid and a metal, preferably a salt of indole-lactic acid and an alkali metal or alkaline earth metal, and more preferably a salt of indole-lactic acid and an alkali metal. Among the salts of indole-lactic acid and alkali metals, salts of indole-lactic acid and sodium, potassium, and lithium are preferred.

[0016] Among the above-mentioned indolelactic acid or its salts, indolelactic acid is preferred, and the L- and DL- isomers of indolelactic acid are more preferred.

[0017] The inhibitor of the present invention is not particularly limited as long as it contains indole lactic acid or a salt thereof as an active ingredient and is suitable for administration, and can be, for example, a pharmaceutical product, quasi-drug, food or beverage, supplement, etc. for mammals including humans.

[0018] When the inhibitor of the present invention is formulated in the form of a pharmaceutical or quasi-drug, indole lactic acid or its salt can be formulated as is, or, if necessary, in combination with a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include glucose, lactose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acids, gelatin, albumin, water, physiological saline, etc. Furthermore, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonic agents, and excipients can be added as needed.

[0019] Furthermore, the dosage form of the pharmaceuticals and quasi-drugs is not particularly limited, and examples include liquids, powders, granules, capsules, tablets, patches, and topical preparations, which can be manufactured according to conventional methods. The content of the active ingredient in the inhibitor of the present invention is not particularly limited, for example, preferably 0.00001 to 10% by mass, more preferably 0.0001 to 1% by mass, and particularly preferably 0.001 to 0.1% by mass.

[0020] When the inhibitor of the present invention is provided in the form of food or beverage, it can be prepared by combining indole lactic acid or its salt as is, or, as necessary, with additives, materials, etc., commonly used in food and beverages, according to conventional methods. Examples of food and beverages include starch-based foods such as bread, biscuits, pancakes, noodles, and candy tablets; confectionery such as gum, candy, and Japanese sweets; meat products such as ham and sausages; fish products such as chikuwa and kamaboko; seafood products; seasonings such as dressings, soy sauce, jam, and furikake; and beverages such as tea, juice, soft drinks, and alcoholic beverages.

[0021] When the inhibitor of the present invention is provided in the form of a supplement, it can be prepared by combining indole lactic acid or its salt as is, or, as necessary, with additives, materials, etc., commonly used in supplements, according to conventional methods.

[0022] The inhibitor of the present invention can inhibit the degradation of IκBα when administered to mammals, including humans. The dosage of the inhibitor of the present invention is not particularly limited, but for example, it may be administered in an amount of 0.01 to 10 g per day, preferably 1 to 10 g. The method of administration is not particularly limited and may be oral, transdermal, etc.

[0023] Here, IκBα degradation refers to the ubiquitination and subsequent degradation by the proteasome of IκBα, which has been phosphorylated by stimuli such as inflammatory responses. Inhibiting IκBα degradation means inhibiting the degradation of the IκBα protein by inhibiting phosphorylation, inhibiting ubiquitination, inhibiting the proteasome, or any one or all of these methods. This inhibition of IκBα degradation can be measured and confirmed by known measurement methods or the methods described in the examples below. For example, in methods that detect the expression levels of IκBα protein or its phosphorylated protein using immunoblotting or ELISA with IκBα antibodies or IκBα phosphorylation-specific antibodies, a decrease in IκBα protein expression compared to a control group is judged to indicate IκBα degradation, while an increase in IκBα protein expression is judged to indicate inhibition of IκBα degradation. Other methods include evaluating the nuclear translocation status of NF-κB, which is located downstream of IκBα, and the suppression of downstream gene expression.

[0024] The active ingredient of the inhibitor of the present invention is indole lactic acid or its salt, but instead of indole lactic acid or its salt, microorganisms known to produce indole lactic acid or its salt through metabolism can also be used. Examples of such microorganisms include lactic acid bacteria such as Lactiplantybacillus plantarum and Bifidobacterium bifidum.

[0025] Examples of such *Lactiplantybacillus plantarum* include strain YIT 0132. This strain of Lactobacillus plantarum YIT 0132 was deposited domestically at the Patent Organism Depository Center of the National Institute of Advanced Industrial Science and Technology (AIST) (1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan). A request for transfer to international deposit was made, and it is now internationally deposited as Lactobacillus plantarum YIT 0132 (FERM BP-11349, deposited on February 24, 2010) at the Patent Organism Depository Center of the National Institute of Technology and Evaluation (NITE), the international depositary authority under the Budapest Convention (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba 292-0818, Japan). Previously, the strain Lactobacillus plantarum YIT 0132 was classified as Lactobacillus plantarum, but in recent years, the genus Lactobacillus has been reclassified, and it is now classified as Lactiplantibacillus plantarum (Zheng et al., A taxonomic note on the genus Lactobacillus : Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int. J. Syst. Evol. Microbiol. 2020 Apr; 70(4):2782-2858 DOI 10.1099 / ijsem.0.004107). In this specification, the classification of lactic acid bacteria is described using the new classification system based on the above-mentioned literature.

[0026] Examples of such Bifidobacterium bifidum include strain YIT 10347. This strain of Bifidobacterium bifidum YIT 10347 was deposited domestically at the Patent Organism Depository Center of the National Institute of Advanced Industrial Science and Technology (AIST) (1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan). A request for transfer to international deposit was made, and it is now internationally deposited as Bifidobacterium bifidum YIT 10347 (FERM BP-10613, AIST) (1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan) at the Patent Organism Depository Center of the National Institute of Technology and Evaluation (NITE), the international depositary authority under the Budapest Convention (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba 292-0818, Japan).

[0027] As described above, the inhibitor of the present invention can inhibit the degradation of IκBα, and therefore can prevent and treat diseases caused by NF-κB activation (hereinafter referred to as the "preventive / therapeutic agent of the present invention"). Here, NF-κB activation is a process in which the transcription factor NF-κB translocates from the cytoplasm to the nucleus and promotes the expression of specific genes. This is caused by the release of NF-κB from inhibitory proteins such as IκBα in response to cellular stimulation. Among such NF-κB activations, those caused by IκBα degradation are preferred.

[0028] Diseases in which NF-κB is activated by IκBα degradation include, for example, barrier dysfunction, inflammation, tumors, and autoimmune diseases. Barrier dysfunction is not limited to, but includes, for example, skin barrier dysfunction and intestinal barrier dysfunction. Inflammation is not limited to, but includes, for example, arthritis, atopic dermatitis, and inflammatory bowel disease. Tumors are not limited to, but include, for example, malignant lymphoma, prostate cancer, breast cancer, colorectal cancer, and hepatocellular carcinoma. Autoimmune diseases are not limited to, but include, for example, rheumatoid arthritis and other autoimmune diseases.

[0029] Among the diseases in which NF-κB is activated by the above-mentioned IκBα degradation, diseases in which IκBα degradation is caused by uremic substances are preferred. Known uremic substances include indoxyl sulfate, p-cresol, phenol, phenyl sulfate, urea, hippuric acid, and creatinine, and diseases in which the uremic substance is indoxyl sulfate are more preferred. Examples of conditions in which IκBα degradation is promoted by indoxyl sulfate include CKD and progression to end-stage renal failure.

[0030] Specifically, diseases caused by uremic toxins include, for example, renal fibrosis, cardiovascular disorders, and intestinal barrier function breakdown. In addition to the aforementioned diseases, other conditions caused by uremic toxins, such as the promotion of ovarian cancer metastasis in cisplatin-induced renal dysfunction and CKD complications, can also be prevented and treated in the same way as the aforementioned diseases.

[0031] The preventive and therapeutic agents of the present invention can be prepared as pharmaceuticals, quasi-drugs, foods and beverages, supplements, etc., by means similar to those used for the inhibitors of the present invention, and administered to mammals, including humans, in a similar manner to prevent and treat diseases caused by NF-κB activation.

[0032] The present invention will be described in detail below with reference to examples of the present invention, but the present invention is not limited in any way to these examples.

[0033] Example 1 Confirmation of IκBα degradation inhibitory activity of indole lactate: <Test method> (1) ROS measurement Human colon epithelial cell line T84 cells (ATCC (American Type Culture Collection), CCL-248) 2.5 x 10 4Cells were seeded at 100 μL / well in a Collagen Coated 96-well Black Clear Bottom Plate (Corning, NCO3904). T84 cells were subcultured in DMEM / F-12 medium (Gibco, 10565-018) containing 10% FBS and 100 μg / mL streptomycin at 5% CO2 and 37°C. After overnight incubation, cells were treated for 2 hours in DMEM / F-12 medium supplemented with the test substances (DL-indole-3-lactic acid (ILA); Sigma-Aldrich, #I5508; 10 μM, indole-3-propionic acid (IPA); Sigma-Aldrich, #57400; 10 μM, ammonium pyrrolidine dithiocarbamate (PDTC); Sigma-Aldrich, #P8765; 10 μM, N-acetyl-L-cysteine ​​(NAC); Fujifilm Wako Pure Chemical Industries, #017-05131; 5 μM, indoxyl potassium sulfate (IS); Cayman Chemical, #16926; 1000 μM, H2O2; 10 μM), H2O2; Mitsubishi Gas Chemical Company, #K-8230; 10 μM, and DCFDA / H2DCFDA - Cellular ROS Assay kit (Abcam, Using ab113851, intracellular ROS (O2 - , H2O2, ・OH, 1 O2 was measured. The measurement method followed the kit protocol. For ILA and IPA, a 100 mM solution was prepared using Dimethyl sulfoxide and diluted with the culture medium as appropriate. IS, H2O2, PDTC, and NAC were also diluted with the culture medium as appropriate.

[0034] (2) Immunoblotting T84 cells 5 x 10 5Cells were seeded in a 12-well plate (TPP, #82412) at a concentration of 2 mL / well. After overnight incubation, cells were incubated for 0.5–2 hours in DMEM / F-12 medium supplemented with the test substances (IS; 1000 μM, ILA; 500 μM, specific antagonist of the aryl hydrocarbon receptor (AhR) (CH-223191); MedChemExpress, #HY-12684; 10 μM, PDTC; 10 μM). Cell lysates were then prepared by adding lysis buffer (10 mM Tris-HCl, pH 7.4, 0.1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 1 mM EDTA, 10 mg / mL aprotinin). Cell lysates were subjected to electrophoresis using a 4-15% acrylamide gel, and the proteins in the gel were transferred to an Immobilon PVDF membrane using a semi-dry transfer device. After transfer, the membrane was blocked with 10% BSA solution and immersed overnight in primary antibody solution at 4°C. After further immersion in secondary antibody solution for 1 hour, the target protein was detected using the ECL Prime Western Blotting Detection System (Cytiva). An ImageQuant 800 (Amersham) was used for detection. The primary antibodies used were anti-IkBa antibody (CST, #9242) and anti-b-actin (Merck KGaA, A5316). The secondary antibodies used were peroxidase-labeled anti-rabbit IgG antibody (#NA934V, GE Healthcare Life Sciences) and peroxidase-labeled anti-mouse IgG antibody (#NA931V, GE Healthcare Life Sciences). CH-223191 was prepared as a 100 mM solution using Dimethyl sulfoxide and used after being diluted with the culture medium as appropriate.

[0035] (3) Immunofluorescence staining of T84 monolayer membranes T84 cells 6 x 10 4Cells were seeded at 400 μL / well on the apical side of a Millicell-24 Cell Culture Plate (Millipore, #PSRP010R1). 800 μL of DMEM / F-12 was added to the basal side, and culture was performed at 37°C under 5% CO₂. The medium was replaced every 2 to 3 days. T84 cells were cultured for about 10 days, and it was confirmed that the measured resistance value was 2500-3500 Ω (formation of a monolayer). The T84 monolayer was cultured with test substances (IS; 1000 μM, ILA; 500 μM) for 72 hours. After washing the T84 monolayer twice with PBS, fixation treatment with 10% neutral buffered formalin solution was performed for 15 minutes. After two washing operations, permeabilization treatment with 0.25% Triton X-100 / PBS was performed for 5 minutes. After two more washing operations, blocking with 10% BSA / PBS was performed at 37°C for 60 minutes. After removing this, anti-NF-κB antibody (Santa Cruz, #sc-372) diluted 50-fold with PBS or anti-occludin antibody (Invitrogen, #34-1500) diluted 200-fold was added, and the mixture was incubated overnight at 4°C. Two washing operations were performed, then Alexa 546-labeled anti-rabbit IgG antibody (Invitrogen, #A11010) diluted 500-fold with PBS or Alexa 488-labeled anti-mouse IgG antibody (Invitrogen, #A21202) was added, and the mixture was incubated at 37°C in the dark for 60 minutes. Finally, three washing operations were performed, the membrane of the cell culture insert was cut out and placed on a slide glass, one drop of the mounting medium VECTASHIELD with DAPI (Vector Laboratories) was added, and a cover glass was placed thereon to prepare a slide glass for observation. The cells were observed using an all-in-one fluorescence microscope BZ-X700 (Keyence).

[0036] (4) RNA Extraction and Real-Time PCR T84 cells were cultured at 5 x 10 5Cells were seeded in a 12-well plate (TPP, #82412) at 2 mL / well. After overnight incubation, cells were cultured for 6 hours in DMEM / F-12 medium supplemented with test substances (IS; 1000 μM, ILA; 500 μM, PDTC; 10 μM). Total RNA was then extracted using the PureLink RNA Mini Kit (Life Technologies). Total RNA was reverse transcribed to cDNA using the GoScript Reverse Transcription System (Promega), and qPCR was performed using GoTaq qPCR Master Mix and QuantStudio 12K Flex. Gene expression was standardized using b-actin as the internal standard gene. The PCR reaction was performed for 40 cycles of 95°C_20 sec followed by 95°C_1 sec and 60°C_20 sec. The primer sequences for each gene are shown in Table 1. The primer sequences for each gene are shown in the order of Forward primer and Reverse primer for each gene in Table 1, from Sequence ID No. 1 to 16.

[0037]

[0038] <Results> (1) Anti-ROS effect of ILA The direct anti-ROS effect of ILA was verified. First, the ROS scavenging effect of ILA on IS- and H₂O₂-induced ROS was verified. It was shown that ROS was generated in T84 cells by treating the cells with IS and H₂O₂ (upper panel of Figure 1). No effect of reducing ROS was observed for ILA and IPA, which is the same tryptophan analog as ILA. On the other hand, a decrease in IS- and H₂O₂-induced ROS was observed upon treatment with PDTC and NAC, which are ROS scavengers. Subsequently, the effect of ILA on the gene expression of NQO1 (NAD(P)H quinone dehydrogenase 1) and SOD2 (Superoxide dismutase 2), which are ROS scavenging enzymes, was verified. ILA did not affect the expression of both genes (lower left panel of Figure 1). On the other hand, ILA is present downstream of the AhR pathway and increased the CYP1A1 gene, which is an indicator of AhR activation (lower right panel of Figure 1), indicating that the AhR pathway is activated by ILA treatment.

[0039] (2) Inhibitory effect of ILA on IκBα degradation ROS is known to activate the NF-κB pathway. NF-κB exerts its activity by translocating into the nucleus, but under normal conditions, the nuclear translocation of NF-κB is inhibited by forming a complex with IκBα. It is known that when IκBα is degraded by stimulation such as ROS, NF-κB is released from the complex, translocates into the nucleus, and is activated. Since IS was shown to generate ROS from Figure 1, the effects of IS and ILA on IκBα were examined. Degradation of IκBα was observed when T84 cells were stimulated with IS (Figure 2). The IκBα degrading effect of IS was partially inhibited by simultaneous treatment with ILA. In addition, since the inhibitory effect of ILA on IκBα degradation did not disappear even when CH-223191, which is an AhR inhibitor, was co-treated, it was shown that a pathway other than AhR is involved. PDTC, which is a ROS scavenger, also showed an inhibitory effect on IS-induced IκBα degradation.

[0040] (3) Inhibitory activity of ILA on NF-κB nuclear translocation NF-κB exerts its activity by translocating into the nucleus and inducing the expression of target genes. Therefore, the nuclear translocation of NF-κB is used as an indicator of activation. Accordingly, it was examined whether NF-κB translocates into the nucleus upon IS treatment, and whether ILA inhibits the NF-κB nuclear translocation induced by IS. Cell morphology was observed by staining occludin, a membrane protein. Uniform distribution of NF-κB in the cytoplasm in the control group and localization (nuclear translocation) of NF-κB in the nucleus in the IS treatment group were observed (Figure 3). Inhibition of IS-induced NF-κB nuclear translocation was observed by simultaneous treatment with ILA.

[0041] (4) Inhibitory effect of ILA on the expression of NF-κB downstream genes The effect of ILA on IS-induced NF-κB downstream genes was examined. The gene expression levels of CCL4, CXCL2, IL-6 and TNF-a were found to be increased 1.4 to 1.9 times in the IS treatment group compared with the control group (Figure 4). ILA suppressed the expression of all these genes to a level equal to or lower than that of the control group. On the other hand, PDTC suppressed the gene expression of CXCL2 and TNF-α to a level equal to or lower than that of the control group, but only partially suppressed the gene expression of CCL4 and IL-6.

[0042] From the above results, it was confirmed that one of the mechanisms of the intestinal barrier protective effect of ILA does not involve the AhR pathway like IS, but the ability to inhibit NF-κB activation accompanied by suppression of IκBα degradation (Figure 5). Since the ability of ILA to inhibit the activation of the NF-κB pathway was observed, its application to diseases caused by NF-κB activation was considered.

[0043] Example 2: Barrier function enhancing and barrier function protective effects of ILA: <Test method> (1) Preparation of T84 monolayer membrane The T84 monolayer membrane was prepared using a 24-well cell culture insert plate (Millipore, #PSRP010R1). T84 cells cultured to 50-80% confluence in a 10 cm dish were harvested and seeded on the apical side of the wells in 400 μL of DMEM / F-12 to a concentration of 60,000 cells / well. 800 μL of DMEM / F-12 was added to the basal side, and culture was carried out at 5% CO2 and 37°C. The culture medium was changed every 2-3 days. The T84 cells were cultured for about 10 days, and a measured resistance value of about 2000-4000 Ω was used as the criterion for determining monolayer membrane formation.

[0044] (2) Evaluation of barrier function using T84 monolayer membrane 400 mL of culture medium containing the test substances (ILA; 62.5-1000 μM, IS; 1000 μM, CH-223191; 10 μM, H2O2; 1500 μM, PDTC; 5 μM) was added to the apical side of the T84 monolayer membrane. 800 μL of H2O2-containing medium was added to the basal side. No other test substances were added to the basal side. The measured resistance value (Ω) measured by Millicell MRS-2 (Millipore) 0-72 hours after the addition of the test substances was measured in the culture area (cm²) of the cell culture insert plate. 2 The TEER value (Ω / cm²) is the value obtained by dividing by ). 2 The TEER value of the monolayer film at each time after addition was divided by the TEER value at time 0 (before addition) to calculate the percentage change in barrier function from before addition.

[0045] (3) Immunoblotting A cell lysate was prepared by adding lysis buffer (10 mM Tris-HCl, pH 7.4, 0.1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 1 mM EDTA, 10 mg / mL aprotinin) to a T84 monolayer membrane. Electrophoresis was performed on the cell lysate using a 4-15% acrylamide gel, and the proteins in the gel were transferred to an Immobilon PVDF membrane using a semi-dry transfer apparatus. After transfer, the membrane was blocked with 5% skim milk solution and immersed overnight in primary antibody solution at 4°C. After further immersion in secondary antibody solution for 1 hour, the target protein was detected using the ECL Select Western Blotting Detection System or the ECL Prime Western Blotting Detection System (Cytiva). An ImageQuant 800 (Amersham) was used for detection. Primary antibodies used were anti-Claudin-1 antibody (Invitrogen, #71-7800), anti-Claudin-3 antibody (Invitrogen, #34-1700), anti-occludin antibody (Invitrogen, #33-1500), anti-ZO-1 antibody (abcam, #ab216880), and anti-b-actin (Merck KGaA, A5316). Secondary antibodies used were peroxidase-labeled anti-rabbit IgG antibody (#NA934V, GE Healthcare Life Sciences) and peroxidase-labeled anti-mouse IgG antibody (#NA931V, GE Healthcare Life Sciences).

[0046] <Results> (1) Barrier function enhancing effect of ILA The effect of ILA on barrier function was investigated. Treatment with high concentrations of ILA (500, 1000 μM) increased the TEER value of the T84 monolayer membrane (Figure 6 left). In addition, ILA treatment increased the expression levels of tight junction proteins (Claudin-1, Claudin-3, Occludin, ZO-1) in cells (Figure 6 right).

[0047] (2) Protective effect of ILA on barrier function against IS-induced barrier dysfunction IS treatment significantly reduced the TEER value of the T84 monolayer membrane compared to the control group (Figure 7 left). The TEER value that was reduced by IS treatment was raised to the same level as the ILA-only treatment group by simultaneous treatment with IS and ILA. Increased tight junction protein expression levels (Claudin-1, Occludin, ZO-1) were observed in the ILA and ILA / IS treatment groups (Figure 7 right). On the other hand, no change in tight junction protein expression levels was observed in the IS treatment group.

[0048] From the above results, it was found that indole-lactic acid has a barrier function enhancing effect. Furthermore, it was found that indole-lactic acid can protect against intestinal barrier dysfunction caused by indoxyl sulfate. Taking into account the results of Example 1, it was found that indole-lactic acid can protect against intestinal barrier dysfunction caused by indoxyl sulfate via the NF-κB pathway.

[0049] The IκBα degradation inhibitor of the present invention can prevent and treat diseases caused by NF-κB activation.

Claims

1. An IκBα degradation inhibitor characterized by containing indole lactic acid or a salt thereof as an active ingredient.

2. A preventive and therapeutic agent for diseases caused by NF-κB activation, characterized by containing the IκBα degradation inhibitor described in claim 1.

3. The NF-κB activation-mediated preventive and therapeutic agent for diseases according to claim 2, wherein NF-κB activation is performed by IκBα degradation.

4. The preventive and therapeutic agent for a disease caused by NF-κB activation according to claim 3, wherein the disease caused by NF-κB activation is selected from barrier dysfunction, inflammation, tumors, and autoimmune diseases.

5. The NF-κB activation-mediated preventive and therapeutic agent for diseases according to claim 3, wherein the IκBα degradation is due to uremic substances.

6. The NF-κB activation-mediated preventive and therapeutic agent for diseases according to claim 5, wherein the uremic substance is indoxyl sulfate.

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