Screening method for anti-inflammatory agents to be used on aging tissues

Abstract

The inventors' primary objective was to provide a technology for screening materials that can suppress inflammation, particularly in aging tissues. [Solution] The above problem can be solved by a method that includes step B: contacting periodontal ligament cells that have been passaged 15 or more times with a test substance, and step C: evaluating the expression level of at least one specific inflammation-related factor in the periodontal ligament cells that have been contacted with the test substance in step B.

Description

Technical Field

[0004] , , , , ,

[0005] , , ,

[0001] The present disclosure relates to a method for screening anti-inflammatory agents for use against aged tissues, etc.

Background Art

[0002] Periodontal disease is an inflammatory disease including gingivitis and periodontitis. Since the progression of the pathological condition of periodontal disease may lead to tooth loss, prevention and / or improvement of inflammation in oral tissues can contribute to the extension of healthy life expectancy.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

[0005]

Non-Patent Document 2

[0006] It is known that the risk of periodontal disease increases with age, but its essential mechanism remains unclear. Therefore, conventional technologies cannot necessarily be said to effectively suppress inflammation in aging tissues. Furthermore, in recent years, there has been a growing demand for personalized medicine and personalized supplements, which are tailored to each individual's age, gender, lifestyle, health status, and genetic predispositions. In light of these circumstances, the inventors of this invention primarily aimed to provide a technology for screening materials that can suppress inflammation, particularly in aging tissues within the oral cavity. [Means for solving the problem]

[0007] The inventors of this invention, Step B: A step of contacting periodontal ligament cells that have been passaged 15 or more times with the test substance, and Step C: A method comprising the step of evaluating the expression level of at least one specific inflammation-related factor in periodontal ligament cells that were contacted with the test substance in Step B. According to this research, it was found that materials capable of suppressing inflammation, particularly in aging tissues, could be screened. Further improvements were then made to complete this disclosure.

[0008] This disclosure includes, for example, the following subjects: Section 1. A screening method for anti-inflammatory agents to be used on aging tissues, Step B: A step of contacting periodontal ligament cells that have been passaged 15 or more times with the test substance, and A screening method comprising step C: evaluating the expression level of at least one inflammation-related factor selected from the group consisting of IL-6, CXCL3, MMP-3, ICAM-1, CCL2, and CCL5 in periodontal ligament cells that have been contacted with the test substance in step B. Section 2. A screening method for anti-inflammatory agents to be used on aging tissues, Step B': A step of contacting periodontal ligament cells that have been passaged 15 or more times with the test substance and lipopolysaccharide, and A screening method comprising step C: evaluating the expression level of at least one inflammation-related factor selected from the group consisting of IL-6, IL-8, CXCL1, CXCL2, CXCL3, CXCL10, MMP-1, MMP-3, ICAM-1, CCL2, and CCL5 in periodontal ligament cells that have been contacted with the test substance in step B'. Section 3. Before process B or process B', Process A: A process of subculturing periodontal ligament cells 15 or more times. The screening method according to item 1 or 2, further comprising: Section 4. The screening method according to any one of items 1 to 3, wherein the periodontal ligament cells are human periodontal ligament fibroblasts. Section 5. The screening method described in any of items 1 to 4, wherein the aging tissue is the tissue of a subject aged 40 years or older. [Effects of the Invention]

[0009] This disclosure provides a technology for screening materials that can suppress inflammation, particularly in aging tissues. [Brief explanation of the drawing]

[0010] [Figure 1]Test 1: The left figure shows a fluorescence microscope image of SA-β-gal positive cells detected using the β-galactosidase detection fluorescent reagent SPiDER-βGal, along with the fluorescence intensity (relative value with fluorescence intensity at p.4 set to 1, right figure). In the fluorescence microscope image, SA-β-gal is stained red, and the nucleus is stained blue. In the figure, the top of the bar graph shows the mean value, and the error bars show the standard deviation (the same applies hereafter). **p<0.01, n=4. [Figure 2] Test 1: Shows gene expression levels for p16, p21, and p53. **p<0.01, *p<0.05, n=5. [Figure 3] Test 1: Shows the gene expression levels of CDK2 and CDK4. **p<0.01, *p<0.05, n=5. [Figure 4] Test 1: Shows gene expression levels of IL-6, CCL2, and ICAM-1. **p<0.01, n=5. [Figure 5] Test 1: Shows gene expression levels of IL-1β and MMP-2. n=3. [Figure 6] Experiment 1: Results of analysis of p16 and ICAM-1 protein expression levels by Western blotting are shown. The upper part of the figure shows the quantification results of band intensity performed using ImageJ. **p<0.01, n=3. [Figure 7] Experiment 2: The results of analyzing the expression levels of IL-6, IL-8, CXCL1, and CXCL2 in p.4 and p.18 cells with or without the addition of Porphyromonas gingivalis (hereinafter, Pg LPS) (indicated as "+ Pg LPS" in the figure) are shown. The results represent relative values ​​with the gene expression level when p.4 cells were cultured without Pg LPS set to 1. *p<0.05, **p<0.01, n=2-5. [Figure 8]Experiment 2: The results of analyzing the expression levels of CXCL3, CXCL10, MMP-1, and MMP-3 in each cell of p.4 and p.18 with or without the addition of Pg LPS (indicated as “+ Pg LPS” in the figure or “- Pg LPS” in the figure) are shown. The results represent the relative values when the gene expression level when culturing p.4 without the addition of Pg LPS is set to 1. **p<0.01, n = 2 - 5. [Figure 9] Experiment 2: The results of analyzing the expression levels of ICAM-1, CCL2, CCL5, IL-11, and E-selectin in each cell of p.4 and p.18 with or without the addition of Pg LPS (indicated as “+ Pg LPS” in the figure or “- Pg LPS” in the figure) are shown. The results represent the relative values when the gene expression level when culturing p.4 without the addition of Pg LPS is set to 1. *p<0.05, **p<0.01, n = 2 - 5. [Figure 10] Experiment 3: For p.22 transfected with each siRNA shown in the figure, the results of analyzing the expression levels of IL-6, CXCL2, and CXCL3 with or without the addition of Pg LPS (indicated as “+ Pg LPS” in the figure or “- Pg LPS”) are shown. The results represent the relative values when the gene expression level when culturing cells transfected with the siRNA of the negative control without the addition of Pg LPS is set to 1. n = 2. [Figure 11] Experiment 3: For p.22 transfected with each siRNA shown in the figure, the results of analyzing the expression levels of CXCL10, ICAM-1, and CCL2 with or without the addition of Pg LPS (indicated as “+ Pg LPS” in the figure or “- Pg LPS”) are shown. The results represent the relative values when the gene expression level when culturing cells transfected with the siRNA of the negative control without the addition of Pg LPS is set to 1. n = 2.

Modes for Carrying Out the Invention

[0011] The embodiments included in this disclosure will be described in more detail below. This disclosure preferably includes, but is not limited to, methods for screening anti-inflammatory agents for use in aging tissues, and encompasses everything disclosed herein and recognizable to those skilled in the art.

[0012] The screening methods included in this disclosure are: Step B: A step of contacting periodontal ligament cells that have been passaged 15 or more times with the test substance, and Step C: A step to evaluate the expression level of at least one specific inflammation-related factor in periodontal ligament cells that were exposed to the test substance in Step B. This includes the following. Hereinafter, the screening method included in this disclosure may be referred to as the "Screening Method of this Disclosure."

[0013] I. Processes B and B' In step B, the test substance is brought into contact with periodontal ligament cells that have been passaged 15 or more times. The lower limit of the number of passages may be 15, 16, 17, or 18 times. The number of passages is more preferably 16 or more, even more preferably 17 or more, and particularly preferably 18 or more. Cellular senescence tends to progress as the number of passages increases. The upper limit of the number of passages is not particularly limited and may be 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, or 25 or less. The upper limit of the number of passages may be 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 times. The number of subculturing cycles may be, for example, 15 to 50, preferably 16 to 45, more preferably 17 to 40, even more preferably 17 to 35, and particularly preferably 18 to 30.

[0014] The timing of subculturing is not particularly limited. For example, subculturing may be performed at 50-100% confluence. The upper or lower limits of the range may be 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% confluence. Subculturing is preferably performed at 60-100% confluence, more preferably at 65-95% confluence, and even more preferably at 70-90% confluence.

[0015] Subculturing (also called "cell subculturing") is generally a procedure performed to prevent overcrowding of cultured cells and to maintain their proliferation. The general method for subculturing adherent cells is described below: First, the culture medium is removed from the culture vessel, and the cells are washed with PBS (phosphate-buffered saline) to remove any remaining serum or culture medium components. Next, trypsin or EDTA solution is added to detach the cells from each other and from the vessel surface. Then, the enzyme activity is stopped to prepare a cell suspension. The cell suspension is centrifuged to collect the cells, and they are seeded in a new culture vessel with new culture medium at an appropriate density. The subculturing method in the art of this disclosure is not particularly limited and can be carried out in accordance with the general method described above.

[0016] The specific types of periodontal ligament cells used in the screening method of this disclosure are not particularly limited as long as they originate from the periodontal ligament. Specific examples of periodontal ligament cells include periodontal ligament fibroblasts, periodontal ligament stem cells, cementoblasts, osteoblasts, osteoclasts, etc. The periodontal ligament cells used in the screening method of this disclosure are preferably periodontal ligament fibroblasts or periodontal ligament stem cells, and are particularly preferably human periodontal ligament fibroblasts.

[0017] Step B is preferably Step B': a step in which the test substance and lipopolysaccharide are brought into contact with periodontal ligament cells that have been passaged 15 or more times. The specific method for bringing the test substance into contact with periodontal ligament cells that have been passaged 15 or more times is not particularly limited. For example, the test substance may be added to a culture medium containing periodontal ligament cells. After addition, stirring or shaking may be performed as desired, or the mixture may be left to stand. After addition, the next step may be performed after 20 seconds to 96 hours, 3 minutes to 72 hours, 1 hour to 48 hours, or 12 hours to 36 hours, as desired. The upper or lower limit of the aforementioned time may be 10, 20, 30, 40, or 50 seconds, 1, 2, 3, 4, 5, 10, 15, 20, 30, or 45 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 72, 84, or 96 hours.

[0018] Lipopolysaccharide (LPS), also known as endotoxin, is a pathogenic factor and a component of the outer cell wall membrane of Gram-negative bacteria such as Porphyromonas gingivalis, Fusobacterium nuclatum, Aggregatibacter actinomycetemcomitans, and Escherichia coli. In the technology disclosed herein, LPS from any bacterium can be used, but it is preferable to use LPS derived from P. gingivalis and / or F. nuclatum. As shown in the examples described later, contact with periodontal ligament cells that have been passaged 15 or more times with LPS significantly induces the expression of specific inflammation-related factors, making it easier to evaluate whether the test substance is a candidate anti-inflammatory agent.

[0019] There are no particular limitations on the specific method for contacting periodontal ligament cells that have been passaged 15 or more times with LPS. For example, LPS may be added to a culture medium containing periodontal ligament cells. After addition, stirring or shaking may be performed as desired, or the medium may be left to stand. After addition, the next step may be performed after 1 minute to 24 hours, 10 minutes to 18 hours, 30 minutes to 12 hours, or 1 hour to 9 hours, as desired. The upper or lower limit of the above time may be 1, 5, 10, 15, 20, 30, or 45 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours.

[0020] There are no particular restrictions on the timing of contacting periodontal ligament cells that have been passaged 15 or more times with LPS; it may be before, during, or after contact with the test substance.

[0021] II. Processes C and C' In step C, the expression levels of at least one (preferably at least two, three, four, or five, or all) inflammation-related factors selected from the group consisting of IL-6 (Interleukin-6), CXCL3 (CXC motif chemokine ligand 1), MMP-3 (Matrix metalloproteinase-3), ICAM-1 (Intercellular adhesion molecule-1), CCL2 (CC motif chemokine ligand 2), and CCL5 are evaluated in periodontal ligament cells that were contacted with the test substance in step B.

[0022] In step C', the expression levels of at least one inflammation-related factor selected from the group consisting of IL-6, IL-8, CXCL1, CXCL2, CXCL3, CXCL10, MMP-1, MMP-3, ICAM-1 (Intercellular adhesion molecule-1), CCL2, and CCL5 (preferably at least two, three, four, five, six, seven, eight, nine, or ten, or all of them) in periodontal ligament cells that were contacted with the test substance and lipopolysaccharide in step B' are evaluated.

[0023] The inflammation-related factors described above may be collectively referred to as the "inflammation-related factors of this disclosure." In this disclosure, the term "factor" is used to encompass both genes and proteins.

[0024] Specific methods for evaluating gene expression levels include quantitative PCR (qPCR), digital droplet PCR (ddPCR), RNA sequencing (RNA-seq), and microarray analysis. Specific methods for evaluating protein expression levels include Western blotting, ELISA (enzyme-linked immunosorbent assay), and flow cytometry. The expression levels of inflammation-related factors described herein can be evaluated using these evaluation methods individually or in combination of two or more.

[0025] In the screening method of this disclosure, if the expression level of at least one of the inflammation-related factors of this disclosure in periodontal ligament cells contacted with the test substance in step B is lower than the expression level of the same factor in periodontal ligament cells cultured in the same manner except that they are not contacted with the test substance, then it is determined that the test substance can suppress inflammation in aging tissue. Similarly, if the expression level of at least one of the inflammation-related factors of this disclosure in periodontal ligament cells contacted with the test substance and lipopolysaccharide in step B' is lower than the expression level of the same factor in periodontal ligament cells cultured in the same manner except that they are not contacted with the test substance, then it is determined that the test substance can suppress inflammation in aging tissue. Such test substances can be considered promising candidates for anti-inflammatory agents to be used on aging tissue.

[0026] While not particularly limited, in a preferred embodiment, the aging tissue is the tissue of a subject aged 40 years or older (preferably the periodontal ligament). More preferably, the aging tissue is the tissue of a subject aged 45 years or older, even more preferably the tissue of a subject aged 50 years or older or 55 years or older, and particularly preferably the tissue of a subject aged 60 years or older. The lower limit of the range may be 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 years.

[0027] In this disclosure, senescent tissue refers to tissue containing senescent cells (preferably the periodontal ligament). Senescent cells are known to exhibit several characteristics that differ from normal cells. One such characteristic of senescent cells is that they are SA-β-gal (senescence-associated beta galactosidase) positive.

[0028] The anti-inflammatory agents selected by the screening method of this disclosure can be incorporated into, for example, an oral composition. In this case, the dosage form of the oral composition is not particularly limited and may be a solid composition, a semi-solid composition, a liquid composition, etc. More specifically, it may be in the form of an ointment, paste, paste, gel, liquid, spray, mouthwash, liquid toothpaste, toothpaste, gum, tablet, drop, etc., and among these, ointments, pastes, gels, liquids, mouthwashes, liquid toothpaste, and toothpaste are preferred.

[0029] The target population for which the anti-inflammatory agents selected by the screening method of this disclosure can be applied is not particularly limited, and may include, for example, humans and non-human mammals. Examples of non-human mammals include rats, mice, rabbits, cattle, pigs, dogs, cats, sheep, and monkeys.

[0030] While not particularly limited, anti-inflammatory agents selected by the screening method of this disclosure are preferably applied to persons aged 40 years or older, more preferably to persons aged 45 years or older, even more preferably to persons aged 50 years or older or 55 years or older, and particularly preferably to persons aged 60 years or older. The lower limit of the range may be 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 years.

[0031] The target population for anti-inflammatory agents selected by the screening method of this disclosure is preferably people who want to prevent aging, people who want to suppress aging, people who want to improve aging, people who want to prevent aging in the oral cavity (preferably the periodontal ligament), people who want to suppress aging in the oral cavity (preferably the periodontal ligament), people who want to improve aging in the oral cavity (preferably the periodontal ligament), people who want to maintain and / or improve oral health, people who want to prevent inflammation in the oral cavity (preferably inflammation in the periodontal ligament), people who want to suppress inflammation in the oral cavity (preferably inflammation in the periodontal ligament), and people who want to improve inflammation in the oral cavity (preferably inflammation in the periodontal ligament).

[0032] The periodontal ligament is connective tissue that surrounds the tooth root, located between the alveolar bone and cementum, and plays a role in fixing the tooth within the jawbone. Furthermore, in the progression of periodontal disease, the periodontal ligament fibers rupture, forming a periodontal pocket. Therefore, the periodontal ligament is considered to play a very important role in the transition from gingivitis to periodontitis and in the progression of periodontitis. Accordingly, the anti-inflammatory agents selected by the screening method of this disclosure can be preferably used in people who wish to prevent and / or improve periodontal disease.

[0033] III. Process A While not particularly limited, the screening method of this disclosure may optionally further include step A: passage of periodontal ligament cells 15 or more times, before step B or step B'.

[0034] In step A, the lower limit of the number of passages may be 15, 16, 17, or 18. The number of passages is more preferably 16 or more, even more preferably 17 or more, and particularly preferably 18 or more. Cellular senescence tends to progress as the number of passages increases. The upper limit of the number of passages is not particularly limited and may be 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, or 25 or less. The upper limit of the number of passages may be 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18. The number of subculturing cycles may be, for example, 15 to 50, preferably 16 to 45, more preferably 17 to 35, even more preferably 17 to 25, and particularly preferably 18 to 30.

[0035] In process A, the timing of subculturing is not particularly limited. For example, subculturing may be performed at a timing of 50-100% confluence. The upper or lower limit of the above range may be 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% confluence. Subculturing is preferably performed at a timing of 60-100% confluence, more preferably at a timing of 65-95% confluence, and even more preferably at a timing of 70-90% confluence.

[0036] In this specification, the term “comprising” includes not only “containing” but also “essentially consisting of” and “consisting of.” Furthermore, this disclosure encompasses all combinations of the constituent elements described herein.

[0037] Furthermore, the various characteristics (properties, structure, numerical values, functions, etc.) described for each embodiment of this disclosure described above may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinable characteristics described herein. [Examples]

[0038] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below. The density of the culture medium used for cell culture was approximately 1.0 g / mL in all cases.

[0039] Experiment 1. Induction of aging in human periodontal ligament cells and confirmation of aging characteristics. 1-1. Method 1-1-1. Evaluation of SA-β-gal-positive cells (Staining - Microscope) Human periodontal ligament fibroblasts were subcultured until they reached passage 3 (Passage 3, hereafter p.3) or passage 17 (Passage 17, hereafter p.17). Subsequently, cells from p.3 and p.17 were seeded into 12-well plates, designated as p.4 and p.18, respectively, and cultured in DMEM medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest) and 1% Antibiotics (Gibco) (hereafter simply referred to as "medium") until approximately 80% confluence was reached. After washing once with PBS(-), Bafilomycin A1 (Focus Biomolecules) was added to a concentration of 100 nM and incubated for 1 hour. After 1 hour, the cells were washed with PBS(-) and fixed with 4% paraformaldehyde-phosphate buffer (FUJIFILM Wako Pure Chemical) for 3 minutes. After washing with PBS(-), SPiDER-βGal (Dojindo) was added and incubated for 30 minutes. Then, Hoechst 33342 (Thermo Scientific), diluted in PBS(-) containing 0.3% Triton X-100 (Sigma-Aldrich), was added and incubated for 15 minutes. After washing with PBS(-), fluorescence images were acquired using the EVOS M7000 Imaging System (Thermo Scientific). Fluorescence intensity was quantified by processing the fluorescence images using Celleste 5 Image Analysis Software (Thermo Scientific).

[0040] 1-1-2. Evaluation of the expression of inflammation-related genes and cell cycle regulatory genes (qPCR) Human periodontal ligament fibroblasts were subcultured until they reached passage 3 (Passage 3, hereafter p.3) or passage 17 (Passage 17, hereafter p.17). Subsequently, cells from p.3 and p.17 were seeded into 12-well plates, designated as p.4 and p.18, respectively, and cultured in culture medium until approximately 80% confluence was reached.

[0041] Total RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the extracted total RNA using the PrimeScript RT reagent Kit (Takara Bio). Using the synthesized cDNA as a template, gene expression was quantified using QuantStudio 5 Real-Time PCR (Thermo Fisher Scientific) with primers specific to each gene (p16, p21, p53, CDK2, CDK4, IL-6, CCL2, ICAM-1, IL-1β, MMP-2, and 18S rRNA) and an intercalator method using TB Green Premix Ex Taq II FAST qPCR (Takara Bio). The expression levels of each gene were corrected to the expression levels of 18S rRNA. The primer sets used to quantify each gene expression in this example are described in "4. Appendix".

[0042] 1-1-3. Evaluation of ICAM-1 protein expression on p16 (Western Blotting) Human periodontal ligament fibroblasts were subcultured until they reached passage 3 (Passage 3, hereafter p.3) or passage 17 (Passage 17, hereafter p.17). Then, cells seeded in 6-well plates from p.3 and p.17 were designated as p.4 and p.18, respectively, and cultured until confluence was achieved. Afterward, the cells were washed with PBS and harvested. Proteins were extracted from the harvested cells using a standard method, and the total protein concentration of the resulting protein extract was measured by a BCA assay (Thermo Scientific). After standardizing the protein concentration, Laemmli Sample Buffer (BIO-RAD) was added, and the proteins were denatured by boiling. The denatured proteins were separated by SDS-PAGE and transferred to a PVDF membrane. The membranes with the transferred proteins were blocked with PBS containing 5% skim milk and 0.05% Tween-20. After blocking, the film was sequentially reacted with primary and secondary antibodies, followed by the addition of substrate to induce chemiluminescence. The emission intensity was detected using an Amersham Imager 680 RGB (Cytiva) system. Band intensity was quantified using ImageJ.

[0043] 1-2. Results Senescent cells are known to be positive for SA-β-gal (senescence-associated beta galactosidase). Figure 1 shows fluorescence microscopy images (left) and fluorescence intensity (relative value with fluorescence intensity at p.4 set to 1, right) when SA-β-gal positive cells were detected using the β-galactosidase detection fluorescent reagent SPiDER-βGal. In the fluorescence microscopy image on the left, SA-β-gal is stained red and the nucleus is stained blue. As shown in Figure 1, the expression of SA-β-gal, a characteristic of aging, is elevated at p.18.

[0044] The expression levels of each gene are shown in Figures 2-5. Note that all the genes evaluated are those reported to be associated with aging (aging-related genes). Figure 2 shows p16, p21, and p53, genes known to halt the cell cycle, and all showed significantly increased expression at p.18. This result is consistent with previous findings that the expression of these genes is induced in senescent cells. Figure 3 shows CDK2 and CDK4, genes known to advance the cell cycle, and both showed significantly decreased expression. This result is consistent with previous findings that the expression of these genes is decreased in senescent cells.

[0045] Figures 4 and 5 show IL-6, CCL2, ICAM-1, IL-1β, and MMP-2, which are inflammation-related genes. Previous findings predicted that the expression of all these genes would increase at p.18; however, contrary to expectations, IL-1β and MMP-2 showed a tendency towards decreased expression at p.18.

[0046] Figure 6 shows the results of analyzing the protein expression levels of p16 and ICAM-1 by Western blotting. Consistent with the gene expression levels shown in Figures 2 and 4, the protein expression levels of p16 and ICAM-1 were significantly increased in p.18.

[0047] These results confirm that senescence is induced in human periodontal ligament fibroblasts that have undergone repeated passage. Furthermore, it was revealed that among inflammation-related factors generally known to have increased expression in senescent cells, there are some whose increased expression was not observed in human periodontal ligament fibroblasts that had undergone repeated passage.

[0048] Study 2. Evaluation of inflammation-related gene responses to P. gingivalis LPS. 2-1. Method Human periodontal ligament fibroblasts were subcultured until they reached passage 3 (Passage 3, hereafter p.3) or passage 17 (Passage 17, hereafter p.17). Subsequently, cells from p.3 and p.17 were seeded into 12-well plates, designated as p.4 and p.18, respectively, and cultured in culture medium until approximately 80% confluence was reached. After that, 100 ng / mL of Porphyromonas gingivalis-derived lipopolysaccharides (Sigma-aldrich, hereafter Pg LPS) was added, or not added, and incubated for 6 hours.

[0049] Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the extracted total RNA using the PrimeScript RT reagent Kit (Takara Bio). Using the synthesized cDNA as a template, gene expression was quantified using QuantStudio 5 Real-Time PCR (Thermo Fisher Scientific) with primers specific to each gene: IL-6, IL-8, CXCL1, CXCL2, CXCL3, CXCL10, MMP-1, MMP-3, ICAM-1, CCL2, CCL5, IL-11, E-selectin, and 18S rRNA, as well as an intercalator method using TB Green Premix Ex Taq II FAST qPCR (Takara Bio). The expression levels of each gene were corrected to the expression level of 18S rRNA. Relative values ​​were calculated with the gene expression level when p.4 was cultured in a medium without Pg LPS added set to 1. The tests were conducted with n=5 for IL-8, CXCL1, MMP-1, ICAM-1, and CCL2, with n=3 for IL-6, and n=2 for all others.

[0050] 2-2. Results The results are shown in the table and figures 7-9 below. In the table and figures, "- Pg LPS" indicates the case without Pg LPS addition, and "+ Pg LPS" indicates the case with Pg LPS addition.

[0051] [Table 1]

[0052] [Table 2]

[0053] As shown in the table and Figures 7-9, the expression levels of IL-6, IL-8, CXCL1, CXCL2, CXCL3, CXCL10, MMP-1, MMP-3, ICAM-1, CCL2, and CCL5 were higher in p.18 than in p.4 when Pg LPS was added. In particular, although the expression levels of IL-8, CXCL1, MMP-1, and CXCL10 were lower in p.18 than in p.4 when Pg LPS was not added, the expression levels were higher in p.18 when Pg LPS was added. On the other hand, there was no significant difference in the expression levels of IL-11 and E-selectin between p.4 and p.18 when Pg LPS was added.

[0054] 3. Analysis of the induction pathways for inflammation-related factor expression 3-1. Method The main inflammatory response pathways that have been studied to date include those involving the transcription factor NF-κB (nuclear factor-κB) and those involving the transcription factor AP-1 (activator protein-1). The inventors of this invention analyzed which pathway induces the expression of inflammation-related factors that were observed to be upregulated in periodontal ligament cells after repeated passage (in other words, which of the NF-κB and AP-1 pathways they are located downstream of).

[0055] Specifically, human periodontal ligament fibroblasts were subcultured until they reached passage 21 (Passage 21, hereafter p.21). Then, cells seeded from p.21 onto 12-well plates were designated as p.22 and cultured in medium until approximately 80% confluence was reached. Using Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific) and Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific), siRNA against p65 (referred to as "p65 siRNA" in Figures 10 and 11), siRNA against AP-1 (referred to as "AP-1 siRNA" in Figures 10 and 11), and a negative control siRNA (referred to as "control siRNA" in Figures 10 and 11) were introduced into these cells, respectively. Note that p65 is the gene for a protein that constitutes the protein complex NF-κB. After washing the cells with PBS 4 hours after siRNA introduction, the medium was replaced with serum-free medium and cultured for 44 hours. Subsequently, 100 ng / mL of Pg LPS was added, or the mixture was incubated for 6 hours without adding Pg LPS.

[0056] Total RNA was extracted from the above cells using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the extracted total RNA using the PrimeScript RT reagent Kit (Takara Bio). Using the synthesized cDNA as a template, gene expression was quantified using QuantStudio 5 Real-Time PCR (Thermo Fisher Scientific) with primers specific to each gene (IL-6, CXCL2, CXCL3, CXCL10, ICAM-1, CCL2, and 18S rRNA) and an intercalator method using TB Green Premix Ex Taq II FAST qPCR (Takara Bio). The expression levels of each gene were corrected for the expression level of 18S rRNA. Relative values ​​were calculated by setting the expression level of each gene to 1 when cells introduced with negative control siRNA were cultured without the addition of Pg LPS. The experiment was performed with n=2.

[0057] 3-2. Results The results are shown in Figures 10 and 11. As shown in Figures 10 and 11, in cells into which siRNA against p65 was introduced (i.e., p65 was knocked down and the NF-κB protein complex was reduced), the expression levels of all genes, IL-6, CXCL2, CXCL3, CXCL10, ICAM-1, and CCL2, were significantly lower than those of the negative control when stimulated with Pg LPS. On the other hand, no such decrease in expression was observed in cells with AP-1 knocked down. These results suggest that the inflammation-related factors whose expression was increased by the addition of Pg LPS in Experiment 2 were induced via the NF-κB pathway.

[0058] As described in Non-Patent Documents 1 and 2, IL-1β and MMP-2 are also located downstream of NF-κB and their expression is induced via NF-κB activation. As shown in Test 1 of this example, IL-6, ICAM-1, and CCL2 showed increased expression in human periodontal ligament fibroblasts that had undergone repeated passage, while IL-1β and MMP-2 did not show increased expression in human periodontal ligament fibroblasts that had undergone repeated passage. In other words, the technology of this disclosure suggests that by using the expression levels of specific factors among the inflammation-related factors located downstream of NF-κB as indicators, it is possible to evaluate whether a test substance is a candidate anti-inflammatory agent or not.

[0059] 4. Appendix The primer sets used to quantify each gene expression in the examples of this disclosure are shown in the table below.

[0060] [Table 3]

Claims

1. A screening method for anti-inflammatory agents to be used on aging tissues, Step B: A step of contacting periodontal ligament cells that have been passaged 15 or more times with the test substance, and A screening method comprising step C: evaluating the expression level of at least one inflammation-related factor selected from the group consisting of IL-6, CXCL3, MMP-3, ICAM-1, CCL2, and CCL5 in periodontal ligament cells that have been contacted with the test substance in step B.

2. A screening method for anti-inflammatory agents to be used on aging tissues, Step B': A step of contacting periodontal ligament cells that have been passaged 15 or more times with the test substance and lipopolysaccharide, and A screening method comprising step C: evaluating the expression level of at least one inflammation-related factor selected from the group consisting of IL-6, IL-8, CXCL1, CXCL2, CXCL3, CXCL10, MMP-1, MMP-3, ICAM-1, CCL2, and CCL5 in periodontal ligament cells that have been contacted with the test substance in step B'.

3. Before process B or process B', Process A: A process of subculturing periodontal ligament cells 15 or more times. The screening method according to claim 1 or 2, further comprising:

4. The screening method according to claim 1 or 2, wherein the periodontal ligament cells are human periodontal ligament fibroblasts.

5. The screening method according to claim 1 or 2, wherein the aging tissue is tissue from a subject aged 40 years or older.

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