Method for evaluating or selecting gastric secretory hormone secretagogues

Abstract

To provide an evaluation or selection method for an agent that promotes the secretion of gastric-secreted hormone.SOLUTION: The present invention provides an evaluation or selection method for an agent that promotes the secretion of gastric-secreted hormone, the method conducting the following steps (1)-(3): (1) a test substance is brought into contact with Hs746T cells or MKN1 cells; (2) intracellular calcium ion concentrations in the cells are measured; and (3), based on the results of measurement in (2), a test substance for raising the intracellular calcium ion concentrations is evaluated as an agent that promotes the secretion of gastric-secreted hormone.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating or selecting gastric secretagogues. [Background technology]

[0002] Hormones such as ghrelin and serotonin released from endocrine cells in the gastrointestinal tract stimulate the vagus nerve via receptors in the nerve (Non-Patent Document 1). Therefore, substances that promote the secretion of such hormones are thought to be useful as substances that promote vagus nerve stimulation. For example, the present applicant has reported that high-density intake of Lactobacillus casei YIT 9029 strain (hereinafter also referred to as "LcS") improves sleep quality and relieves stress, and that this effect is exerted by LcS stimulating the vagus nerve in the stomach (Non-Patent Documents 2 and 3). It is believed that this pharmacological effect is related to hormones secreted from the digestive tract.

[0003] Conventionally, to investigate the effects on the secretion of gastrointestinal endocrine hormones, in vivo tests using humans or animals, in vitro and ex vivo tests using organs or cells collected from the animal stomach, and in vitro tests using already established endocrine cell lines (STC-1 cells (small intestinal cells), RIN-14B cells (pancreatic cells), etc.) have been conducted. However, no established endocrine cell lines are known for the stomach, and only evaluation systems using animals exist (e.g., Patent Documents 1 and 2). Therefore, in vitro evaluation has required searching for cells with endocrine cell-like properties from existing stomach-derived cell lines, or inducing differentiation of pluripotent stem cells into gastric endocrine cell-like cells. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2009-516637 [Patent Document 2] Special Publication No. 2020-513825 [Non-patent literature]

[0005] [Patent Document 1] Berthoud HR. (2008) Vagal and hormonal gut-brain communication: from satiation to satisfaction. Neurogastroenterol Motil. 20 Suppl 1:64-72. [Patent Document 2] Takada, M. et al. (2016) Probiotic Lactobacillus casei strain Shirota relieves stress-associated symptoms by modulating the gut-brain interaction in human and animal models. Neurogastroenterol Motil. 28:1027-1036. [Patent Document 3] Takada, M. et al. (2017) Beneficial effects of Lactobacillus casei strain Shirota on academic stress-induced sleep disturbance healthy in adults: a double-blind, randomized, placebo-controlled trial. Benef Microbes. 26; 8: 153-162. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention relates to providing a method for evaluating or selecting gastric secretory hormone secretagogues. [Means for solving the problem]

[0007] The present inventors have investigated gastric cell lines and found that Hs746T and MKN1 cells, which are established cell lines derived from stomach cancer, have properties similar to gastric endocrine cells, and that the intracellular calcium ion concentration increases when these cells are treated with a gastric secretory hormone secretagogue. Therefore, it is possible to evaluate and select gastric secretory hormone secretagogues using the intracellular calcium ion concentration in these cells as an index.

[0008] That is, the present invention relates to the following 1) to 3). 1) A method for evaluating or selecting a gastric secretory hormone secretagogue, which comprises carrying out the following steps (1) to (3): (1) contacting a test substance with Hs746T cells or MKN1 cells (2) measuring the intracellular calcium ion concentration in the cells; (3) A step of evaluating a test substance that increases the intracellular calcium ion concentration as a gastric secretory hormone secretagogue based on the results of the measurement in (2). 2) The method according to 1), wherein the gastric secretory hormone is serotonin, ghrelin, gastrin or somatostatin. 3) The method of 1) or 2), in which the test substance is a microorganism. [Effects of the Invention]

[0009] According to the present invention, secretagogues for gastric secretory hormones contained in gastric endocrine cells, such as ghrelin and serotonin, can be easily searched for or evaluated in vitro. [Brief explanation of the drawings]

[0010] [Figure 1] Changes in intracellular calcium ion concentration in six gastric cancer cell lines following hormone release-inducing treatment. The test substance was treated at the time indicated by the arrow in the graph. [Figure 2] Chromogranin A (CgA) immunocytochemical staining of Hs746T cells. Blue indicates DAPI (nuclei), and red indicates CgA. Scale bar = 100 μm. [Figure 3] Multiple staining of calcium ion-increasing cells and CgA-positive cells in Hs746T cells. (A) Fluorescence (F495) image at 510 nm when excited at 495 nm wavelength 28 seconds (s) after LcS treatment. (B) CgA immunostained image of the same field. (C) F495 image 28 seconds after denatonium benzoate (DB) treatment. (D) CgA immunostained image of the same field. Blue represents DAPI (nuclei), and red represents CgA. Scale bar = 50 μm. [Figure 4] Expression of gastric endocrine hormones in Hs746T and MKN1 cells. DETAILED DESCRIPTION OF THE INVENTION

[0011] In the present invention, the term "gastric secretory hormone" refers to a hormone contained in gastric endocrine cells, which is stimulated by food or its digestive products passing through the gastric lumen and released into the submucosa of the stomach. Specific examples include serotonin, ghrelin, gastrin, somatostatin, histamine, pancreastatin, etc., of which serotonin, ghrelin, gastrin, and somatostatin are preferred.

[0012] As shown in the Examples below, gastric cancer cells Hs746T and MKN1 cells express endocrine cell markers and have the ability to secrete gastric secretory hormones such as ghrelin and serotonin (Examples 2 and 3). When these cells are treated with DB, a gastric secretory hormone secretion inducer, or LcS (FERM BP-1366), which has a vagus nerve stimulating effect, the intracellular calcium ion concentration increases (Example 1). LcS is a strain that was known as Lactobacillus casei YIT 9029 (FERM BP-1366) before the reclassification of Lactobacillus bacteria in 2020 (Zheng J et al. Int J Syst Evol Microbiol. 2020 Apr; 70(4): 2782-2858). It was deposited at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary (currently the National Institute of Technology and Evaluation Patent Microorganism Depositary) on January 12, 1981. However, it is currently classified as Lacticaseibacillus paracasei and is therefore Lacticaseibacillus paracasei YIT 9029. These results suggest that test substances that promote the secretion of gastric secretory hormones can be evaluated or selected by measuring the intracellular calcium ion concentration using Hs746T cells or MKN1 cells.

[0013] The method for evaluating or selecting a gastric secretory hormone secretagogue is characterized by carrying out the following steps (1) to (3). (1) contacting a test substance with Hs746T cells or MKN1 cells (2) measuring the intracellular calcium ion concentration in the cells; (3) A step of evaluating a test substance that increases the intracellular calcium ion concentration as a gastric secretory hormone secretagogue based on the results of the measurement in (2).

[0014] In the present invention, Hs746T cells are an established cell line derived from human stomach cancer and can be purchased from ATCC. Hs746T cells were cultured in, for example, Dulbecco's Modified Eagle Medium (DMEM), 10% FBS, 10 5 DMEM is a medium containing 1000 U / L penicillin, 0.1 g / L streptomycin, and 1 mM sodium pyruvate, and can be cultured at 37°C in 5% CO2 for subculture when the cells are semi-confluent. DMEM is a versatile medium for culturing mammalian cells and can be purchased from reagent manufacturers. Hs746T cells express the endocrine cell marker CgA and the gastric secretory hormones serotonin and ghrelin.

[0015] In the present invention, MKN1 cells are an established cell line derived from human gastric adenosquamous carcinoma, and can be purchased from the Japan Human Science Resources Foundation. MKN1 cells were cultured in RPMI1640, 10% FBS, 10 5 Cells can be passaged at semi-confluent conditions by culturing them at 37°C in 5% CO2 using a medium containing 1000 U / L penicillin, 0.1 g / L streptomycin, and 1 mM sodium pyruvate. RPMI1640 is a versatile medium for culturing mammalian cells and can be purchased from reagent manufacturers. MKN1 cells express the endocrine cell markers CgA, neural cell adhesion molecule 1 (NCAM1), and enolase 2 (ENO2), and also express the gastric secretory hormones serotonin, ghrelin, gastrin, and somatostatin.

[0016] The test substance to be contacted with the above cells is not particularly limited as long as it is a substance desired to be used as a gastric secretory hormone secretagogue, and examples include animals, plants, marine organisms, microorganisms, etc. and extracts thereof; natural components derived therefrom; synthetic compounds; and mixtures and compositions thereof.

[0017] The means for contacting the cells with the test substance may be any known means in the art, including, for example, adding the test substance to a cell culture medium or buffer solution, or directly adding the test substance to the cells (e.g., dripping, applying, spraying, atomizing, patching, etc.). The concentration and contact amount of the test substance may be appropriately determined based on the form, chemical properties, cytotoxicity, etc. of the test substance. For example, a predetermined amount of the test substance diluted to an appropriate concentration may be exposed to the cells at room temperature to 37°C. It is recommended that the cells used in the contact step be loaded with a calcium-sensitive dye before contacting them with the test substance in order to measure intracellular calcium ion concentration, as described below.

[0018] Measurement of intracellular calcium ion concentration may be performed by an intracellular calcium amount measurement method. For example, fluorescence intensity measurement using a calcium-sensitive dye or the like can be used. This method allows measurement of changes in intracellular calcium ion concentration by measuring changes in fluorescence intensity when a test substance is contacted with Hs746T cells or MKN1 cells transfected with a calcium-sensitive dye (e.g., Fura-2, Fluo-3, Fluo-4, etc.) for a certain period of time. For example, changes in intracellular calcium ion concentration can be measured by measuring changes in fluorescence intensity immediately after contact at room temperature to 37°C for 160 seconds. The calcium-sensitive dye can be introduced into Hs746T cells or MKN1 cells by contacting them with a buffer solution containing the calcium-sensitive dye and leaving them to stand for a certain period of time, for example, 30 to 60 minutes at room temperature to 37° C. Examples of buffer solutions include Hanks' HEPES buffer, HBSS buffer, and Krebs Ringer HEPES buffer. The step of contacting the cells with the test substance described above is carried out by washing and replacing the cells with a buffer solution that does not contain a calcium-sensitive dye. The buffer solution used for washing and replacement is not particularly limited as long as it does not contain a calcium-sensitive dye, but an inhibitor such as Probenecid that prevents the calcium-sensitive dye from leaving the cells may be added. Buffer solutions for measuring intracellular calcium ion concentrations are also commercially available as kits; for example, the Calcium Kit-Fura-2 kit manufactured by Dojindo Laboratories may be used.

[0019] Fluorescence intensity can be measured using a fluorescent plate reader such as the infinite M200 PRO (TECAN) at a temperature of room temperature to 37°C, a measurement time of 25 seconds before injection of the test substance and 160 seconds after injection, and excitation and detection wavelengths set to the specified wavelengths of the indicator. For example, when Fura-2 is used as the calcium-sensitive dye, changes in the ratio of fluorescence intensity at a fluorescence wavelength of 510 nm (F340 / F380) when excited at wavelengths of 340 nm and 380 nm represent changes in intracellular calcium ion concentration.

[0020] The effect of the test substance on promoting secretion of gastric secretory hormones is then evaluated based on the changes in the intracellular calcium ion concentration measured as described above. Such evaluation is carried out, for example, by comparing the results before and after the addition of the test substance, or by comparing a group with the test substance added with a group without the test substance or a control substance added. Alternatively, evaluation can be carried out by comparing the measurement results between various concentrations of the test substance. A test substance that increases intracellular calcium ion concentration is then identified as a gastric secretory hormone secretagogue.

[0021] The gastric secretory hormone secretagogue thus obtained can be used as a gastric secretory hormone secretagogue for promoting the secretion of gastric secretory hormones, as a food or pharmaceutical product, or by being incorporated therein. [Example]

[0022] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0023] Example 1 Increase in intracellular calcium ion concentration DB is a gastrointestinal endocrine hormone secretion inducer that induces the secretion of ghrelin and serotonin. [1) Rozengurt E. (2006) Taste receptors in the gastrointestinal tract. I. Bitter taste receptors and alpha-gustducin in the mammalian gut. Am J Physiol Gastrointest Liver Physiol. 291: G171-7., 2) Janssen S. et al. (2011) Bitter taste receptors and alpha-gustducin regulate the secretion of ghrelin with functional effects on food intake and gastric emptying. Proc Natl Acad Sci US A. 108(5):2094-9, 3) Folia Pharmacol. Jpn., 124, 210-218 (2004)] and LcS, which is thought to have a vagus nerve stimulating effect (Non-Patent Documents 2 and 3) and induce hormone secretion from gastrointestinal endocrine cells in the stomach, were prepared. The six gastric cancer-derived cell lines shown in (1) below were treated with these gastrointestinal endocrine hormone secretion inducers, and the intracellular calcium ion concentrations in Table 1 were measured using the fluorescent indicator shown in (2).

[0024] (1)Cell culture Six gastric cancer cell lines were prepared as stomach-derived cell lines. The source of purchase and culture medium for each gastric cancer cell line are shown in Table 1. Cells were cultured in 100 mm dishes using various media at 37°C and 5% CO2, and maintained by passage at semi-confluent conditions. All media reagents (RPMI1640, DMEM, MEM, FBS, sodium pyruvate, and penicillin / streptomycin) were purchased from GIBCO.

[0025] [Table 1]

[0026] (2) Measurement of intracellular calcium ion concentration using Fura-2 fluorescent indicator Appropriate cell numbers for each cell line were seeded into 96-well plates (Thermo Fisher Scientific) and used after 2–3 days when the cells were semi-confluent. The medium was replaced with Hanks' HEPES buffer (20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 13.8 mM glucose, pH 7.4, 100 μL / well) and incubated at 37°C for 1.5 hours. 100 μL of Hanks' HEPES buffer containing 5 μM Fura-2-acetoxymethyl ester (Dojindo Laboratories), 2.5 mM Probenecid (Dojindo Laboratories), and 0.02% Pluronic F127 (Dojindo Laboratories) was added and incubated at 37°C for 30 minutes. After washing once with Hanks' HEPES buffer, the buffer was replaced with Hanks' HEPES buffer containing 1.25 mM Probenecid and left to stand at 37°C for 30 minutes or more. After that, DB (Sigma-Aldrich) or LcS treatment was performed, and the fluorescence wavelength of Fura-2 was measured. For DB treatment, DB was dissolved in Dulbecco's Phosphate-Buffered Saline (DPBS) to a concentration of 100 mM and used as the test substance. For LcS treatment, LcS was inoculated into lactose-ILS medium at 1% and cultured at 37°C for 20 hours. After washing three times with DPBS, 1 × 10 10 The test substance was suspended in DPBS to a concentration of cfu / mL. The infinite M200 PRO has a reagent injection function, and the above test substances were automatically injected (100 μL / sec) in an amount of 1 / 10 of the buffer during continuous measurement of the fluorescence wavelength. Therefore, DB was at a final concentration of 10 mM, and LcS was at a final concentration of 1 × 10 9 The density is expressed as cfu / mL. The control group was treated with the same amount of DPBS. Measurements were started approximately 25 seconds before DB or LcS treatment and continued until approximately 160 seconds after treatment. For measuring the fluorescence wavelength, infinite M200 PRO was used, and the intensity of the fluorescence wavelength of 510 nm when excited with wavelengths of 340 nm and 380 nm was measured.

[0027] (3) Results DB (10 mM), LcS (1 × 10 9 The F340 / F380 ratio was measured over time from the time point when each cell was treated with 1000 μg of ... ** P<0.01, * P<0.05 vs cont(Dunnet post hoc test)).

[0028] As a result, in MKN1 and Hs746T cells, DB and LcS treatment significantly increased the fluorescence intensity ratio (Fig. 1(A) and (F)). On the other hand, DB treatment significantly decreased the fluorescence intensity ratio in MKN45 and KATOIII cells (Fig. 1(B) and (D)). In MKN74 and NUGC-4, DB and LcS treatment did not significantly change the fluorescence intensity ratio (Fig. 1(C) and (E)).

[0029] Example 2 Physiological properties of Hs746T cells (1) Immune cell staining 1) Method Hs746T cells were cultured in an 8-well chamber slide (IWAKI) at 1.0 × 10 5Cells were seeded at 1000 x 1000 cells / well, and after 1-2 days, when semi-confluent, they were fixed in 4% paraformaldehyde / 0.1M phosphate buffer (PB). After washing three times with DPBS for 5 minutes, they were blocked with 5% normal donkey serum (Jackson Immuno Research) / DPBS with 0.3% Triton X100 (PBST) for 1 hour at room temperature. The primary antibody was rabbit anti-CgA antibody (1 / 300, Abcam) overnight at 4°C. After washing three times with PBST for 5 minutes, the secondary antibody was donkey anti-rabbit IgG Alexa Fluor 546 (1 / 1,000, Molecular Probes) for 1 hour at room temperature. After washing three times with PBST for 5 minutes, the cells were treated with DPBS containing 0.1% DAPI for 30 minutes. The chamber was removed, the cells were mounted in VECTASHIELD (Vector Laboratories), and then examined under a confocal microscope (TCS SP8, Leica Microsystems). The percentage of positive cells was calculated by averaging the percentage of DAPI-positive cells in two fields of view at ×400 magnification. 2) Results CgA-positive cells were confirmed as shown in Figure 2. The percentage of CgA-positive cells relative to the number of DAPI-positive cells was 85.4±0.8%.

[0030] (2) CgA expression ratio in cells with elevated calcium ion concentration 1) Method 1.0 × 10 Hs746T cells 5 The cells were seeded at 1000 cells / well in a 96-well glass-bottom plate (IWAKI). Treatment was performed in the same manner as in Example 1(2), except that 5 μM Fluo-4-acetoxymethyl ester (Dojindo Laboratories) was used instead of Fura-2. Fluo-4 fluorescence wavelength was measured using a BZ-X710 fluorescence microscope (KEYENCE), with 495 nm excitation and 510 nm fluorescence. In this study, fluorescence intensity was quantified by measuring the average brightness across the entire field of view. Measurements were initiated 8 seconds before DB or LcS treatment and continued for 64 seconds after treatment. After measurement, cells were fixed in 4% paraformaldehyde / 0.1M PB and immunocytochemically stained for CgA as described in (1). After DAPI treatment, wells were filled with Hanks' HEPES buffer without mounting, and the cells were observed under a BZ-X710 fluorescence microscope. Images were taken from the same field of view as used for Fluo-4 measurement and superimposed on the Fluo-4 microscope image. The number of cells showing Fluo-4 fluorescence, the number of CgA-positive cells, and the number of CgA-positive cells with increased Fluo-4 fluorescence intensity were counted. The number of positive cells was calculated by averaging the cell counts in three fields of view at ×200 magnification. 2) Results Figure 3(A) shows a Fluo-4 fluorescence image of the LcS-treated group (at 28 seconds), and Figure 3(B) shows a CgA immunostained image of the same field. At 28 seconds, the number of cells with elevated Fluo-4 fluorescence intensity, the number of CgA-positive cells, and the number of CgA-positive cells with elevated Fluo-4 fluorescence intensity were counted and calculated. The percentage of CgA-positive cells among cells with elevated Fluo-4 fluorescence intensity was 94.17 ± 1.58% (n = 3), confirming that most cells with elevated intracellular calcium ion concentrations were CgA-positive cells. Furthermore, the percentage of CgA-positive cells with elevated Fluo-4 fluorescence intensity was 40.41 ± 3.34% (n = 3). Furthermore, in the DB-treated group (at 28 seconds), most cells with elevated Fluo-4 fluorescence intensity were confirmed to be CgA-positive cells (Figures 3(C) and (D)).

[0031] Example 3 Physiological properties of MKN1 cells (1)Cell culture MKN1 cells were cultured in RPMI1640, 10% FBS, 10 5 The cells were cultured in a petri dish at 37°C under 5% CO2 conditions using a medium containing 1000 U / L penicillin, 0.1 g / L streptomycin, and 1 mM sodium pyruvate, and were maintained by subculture in a semi-confluent state.

[0032] (2) Expression of endocrine cell markers The expression of endocrine cell markers was examined by RT-PCR. CgA was selected as the primary endocrine cell marker, and NCAM1 and ENO2 were selected as complementary markers. After culturing, the cells were washed with DPBS and then suspended in TRIzol reagent (Invitrogen) to recover RNA. 1,250 ng of RNA was reverse transcribed using the PrimeScript RT reagent Kit (TaKaRa) to produce cDNA, which was then subjected to PCR. PCR was performed using 0.5 μM primers, 250 ng cDNA, and TaKaRa Ex Taq (TaKaRa). After denaturation at 95°C for 5 minutes, the following cycle was repeated 35 times: denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 1 minute, followed by a final extension at 72°C for 7 minutes. The primer sequences are shown in Table 2. After PCR, electrophoresis was performed on a 2% agarose gel (Kanto Chemical), stained with GelRed nucleic acid gel stain (Biotium), and photographed with a UV transilluminator. As a result, as shown in Table 3, the expression of CgA, NCAM1 and ENO2 was confirmed.

[0033] [Table 2]

[0034] [Table 3]

[0035] (3) Expression of gastric endocrine hormones MKN1 cells were seeded in 8-well chamber slides and fixed in 4% paraformaldehyde / 0.1M PB at semi-confluence. After washing three times with DPBS, cells were blocked with 5% normal donkey serum / PBST at room temperature for 30 minutes, followed by treatment with primary antibodies overnight at 4°C. After washing three times with PBST, cells were treated with secondary antibodies at room temperature for 1 hour. After washing three times with PBST, cells were treated with DPBS containing 0.1% DAPI at room temperature for 30 minutes, and then mounted with VECTASHIELD. All samples were analyzed using a confocal microscope. The primary antibodies targeted the endocrine hormones serotonin (5-HT), ghrelin (Ghrl), gastrin (Gast), and somatostatin (SST) as specific antigens. The primary and secondary antibodies used are summarized in Table 4.

[0036] [Table 4]

[0037] As shown in FIG. 4, the expression of serotonin (5-HT), ghrelin (Ghrl), gastrin (Gast), and somatostatin (SST) was confirmed.

[0038] Example 4 Physiological properties of Hs746T cells 2 The MKN1 cell line of Example 3 was replaced with Hs746T cells, and similar treatment was carried out to confirm the expression of serotonin (5-HT) and ghrelin (Ghrl).

[0039] As shown in Figure 4, the expression of serotonin (5-HT) and ghrelin (Ghrl) was confirmed.

Claims

1. A method for evaluating or selecting a gastric secretory hormone secretagogue, comprising carrying out the following steps (1) to (3), wherein the gastric secretory hormone is serotonin, ghrelin, gastrin, or somatostatin: (1) contacting a test substance with Hs746T cells or MKN1 cells (2) measuring the intracellular calcium ion concentration in the cells; (3) A step of evaluating a test substance that increases the intracellular calcium ion concentration as a gastric secretory hormone secretagogue based on the results of the measurement in (2).

2. The method of claim 1, wherein the test substance is a microorganism.

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