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 vascular endothelial cells that have been passaged 15 or more times with a test substance, and step C: evaluating the expression level of at least one inflammation-related factor selected from the group consisting of IL-6, IL-8, CXCL1, PAI-1, MMP-1, ICAM-1, VCAM-1, E-selectin, and CCL2 in the vascular endothelial cells that were contacted with the test substance in step B.

Description

[Technical Field]

[0001] This disclosure relates to a screening method for anti-inflammatory agents for use in aging tissues, etc. [Background technology]

[0002] As we age, the structure and function of blood vessels deteriorate, such as arteriosclerosis and decreased endothelial function, which is known to increase the risk of various diseases. For example, stroke, vascular dementia, myocardial infarction, hypertension, diabetes, and renal failure have been reported to be closely related to vascular aging (Non-Patent Literature 1). Since these diseases are factors that cause a decline in quality of life and an increase in mortality, preventing and / or improving vascular aging is important from the perspective of extending healthy life expectancy.

[0003] In recent years, research has focused particularly on vascular endothelial cells within blood vessels. For example, Non-Patent Literature 2 reports that in transgenic mice (E-DNIκB mice) in which functional NF-κB signaling in vascular endothelial cells was inhibited, inflammation and oxidative stress in vascular endothelial cells were reduced, the progression of vascular aging was suppressed, and lifespan was extended. Furthermore, Non-Patent Literature 3 shows that in control mice fed a high-calorie diet, p53 expression in vascular endothelial cells was significantly increased, leading to metabolic abnormalities, while in vascular endothelial cell-specific p53-deficient mice (EC-p53KO), insulin sensitivity was improved and fat accumulation decreased. It should be noted that p53 is a factor known to be involved in aging.

[0004] These studies suggest that inhibiting vascular aging may contribute to reducing disease risk and extending healthy life expectancy. Approaches to prevent or delay vascular aging include the use of antioxidants and anti-inflammatory drugs, and lifestyle improvements (moderate exercise, balanced diet, smoking cessation, etc.). In recent years, it has also been reported that certain flavonoids and polyphenols may have anti-vascular aging effects. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Han Y, Kim SY. Endothelial senescence in vascular diseases: current understanding and future opportunities in senotherapeutics. Exp Mol Med. 2023;55(1):1-12. doi:10.1038 / s12276-022-00906-w [Non-Patent Document 2] Hasegawa Y, Saito T, Ogihara T, et al. Blockade of the nuclear factor-κB pathway in the endothelium prevents insulin resistance and prolongs life spans. Circulation. 2012;125(9):1122-1133. doi:10.1161 / CIRCULATIONAHA.111.054346 [Non-Patent Document 3] Yokoyama M, Okada S, Nakagomi A, et al. Inhibition of endothelial p53 improves metabolic abnormalities related to dietary obesity. Cell Rep. 2014;7(5):1691-1703. doi:10.1016 / j.celrep.2014.04.046 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] In recent years, there has been a growing demand for personalized medicine and personalized supplements, which are health management tailored to each individual's age, gender, lifestyle, health condition, genetic predisposition, etc. 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. [Means for solving the problem]

[0007] The inventors of this invention, Step B: A step of contacting vascular endothelial 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 inflammation-related factor selected from the group consisting of IL-6, IL-8, CXCL1, PAI-1, MMP-1, ICAM-1, VCAM-1, E-selectin, and CCL2 in vascular endothelial 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 vascular endothelial 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, IL-8, CXCL1, PAI-1, MMP-1, ICAM-1, VCAM-1, E-selectin, and CCL2 in vascular endothelial cells that were contacted with the test substance in step B. Section 2. Process B is Step B': A step in which the test substance and lipopolysaccharide are brought into contact with vascular endothelial cells that have been passaged 15 or more times. The screening method described in item 1. Section 3. Before process B, Step A: Step of subculturing vascular endothelial cells 15 times or more The screening method according to claim 1 or 2, further comprising . Item 4. The screening method according to any one of claims 1 to 3, wherein the vascular endothelial cells are human umbilical vein endothelial cells (HUVEC). Item 5. The screening method according to any one of claims 1 to 4, wherein the aging tissue is a tissue of a subject 40 years old or older.

Advantages of the Invention

[0009] According to the present disclosure, a technique for screening a material capable of suppressing inflammation, particularly in an aging tissue, is provided.

Brief Description of the Drawings

[0010] [Figure 1] Test 1: Fluorescence microscope images (left in the figure) and fluorescence intensities (relative values with the fluorescence intensity at p.4 set to 1, right in the figure) when detecting SA-β-gal positive cells using the β-galactosidase detection fluorescent reagent SPiDER-βGal. In the figure, the upper end of the bar graph indicates the average value, and the error bar indicates the standard deviation (the same applies hereinafter). **p<0.01, n = 6. [Figure 2] Test 1: Results of analyzing SA-β-gal positive cells by flow cytometry are shown. **p<0.01, n = 5. [Figure 3] Test 1: Results of analyzing cells highly expressing SA-β-gal by flow cytometry are shown. **p<0.01, n = 4. [Figure 4] Test 1: Results of analyzing the cell cycles of cells at p.4 and p.21 by flow cytometry are shown. The vertical axis of the graph indicates the percentage (% cell) in all cells. n = 2. [Figure 5] Test 1: Gene expression levels of p16, p21, and p53 are shown. **p<0.01, *p<0.05, n = 6. [Figure 6]Test 1: Shows the gene expression levels of SIRT1, NRF1, and TFAM. **p<0.01, *p<0.05, n=6. [Figure 7] Test 1: Shows the results of analyzing the protein expression levels of p16, p21, and SIRT1 by Western blotting. Above the figure, the quantitative results of band intensities performed using Image J are shown. *p<0.05, **p<0.01, n=5-6 each. [Figure 8] Test 1: Shows the gene expression levels of IL-8, CXCL1, and ICAM-1. **p<0.01, n=6. [Figure 9] Test 1: Shows the gene expression levels of IL-6, PAI-1, MMP-1, and CXCL11. **p<0.01. For IL-6, PAI-1, and MMP-1, n=6. For CXCL11, n=3. [Figure 10] Test 2: Shows the results of analyzing the gene expression levels of IL-6, IL-8, CXCL1, and PAI-1 in each of the p.4 and p.21 cells with (in the figure "E.coli LPS") or without (in the figure "control") the addition of E.coli LPS. The results represent relative values when the gene expression level when culturing p.4 without the addition of E.coli LPS is set to 1. **p<0.01, n=3. [Figure 11] Test 2: Shows the results of analyzing the gene expression levels of MMP-1, ICAM-1, VCAM-1, and E-selectin in each of the p. (should be p.4) and p.21 cells with (in the figure "E.coli LPS") or without (in the figure "control") the addition of E.coli LPS. The results represent relative values when the gene expression level when culturing p.4 without the addition of E.coli LPS is set to 1. *p<0.05, **p<0.01, n=3. [Figure 12]Experiment 2: The results of analyzing the gene expression levels of CCL2 and CXCL11 in p.4 and p.21 cells with or without E. coli LPS (indicated as "E. coli LPS" in the figure). The results represent relative values ​​with the gene expression level when p.4 cells were cultured without E. coli LPS set to 1. *p<0.05, n=3. [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 vascular endothelial 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 inflammation-related factor selected from the group consisting of IL-6, IL-8, CXCL1, PAI-1, MMP-1, ICAM-1, VCAM-1, E-selectin, and CCL2, in vascular endothelial 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. Process B In step B, the test substance is brought into contact with vascular endothelial cells that have been passaged 15 or more times. The lower limit of the number of passages may be 15, 16, 17, 18, 19, 20, or 21 times. The number of passages may be 16 or more, preferably 18 or more, more preferably 19 or more, even more preferably 20 or more, and particularly preferably 21 or more. Cellular senescence tends to progress as the number of passages increases. There is no particular upper limit to the number of passages, and it may be 50 or less, 45 or less, 40 or less, 30 or less, or 25 or less. The upper limit of the number of subculturing cycles 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, or 22. The number of subculturing cycles may be, for example, 15 to 50, preferably 16 to 45, more preferably 18 to 40, even more preferably 19 to 30, and particularly preferably 20 to 25.

[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 above range may be 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% confluence. Subculturing is preferably performed at 55-100% confluence, more preferably at 60-90% confluence, and even more preferably at 65-85% 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 origin of the vascular endothelial cells used in the screening method of this disclosure is not particularly limited. Examples of vascular endothelial cells include human umbilical vein endothelial cells (HUVEC), human aortic endothelial cells (HAoEC), human coronary artery endothelial cells (HCAEC), human microvascular endothelial cells, HMVEC), Human Dermal Microvascular Endothelial Cells (HDMEC), Human Cardiac Microvascular Endothelial Cells (HCMEC), Bovine Aortic Endothelial Cells (BAEC), Porcine Aortic Endothelial Cells, PAoEC), and iPSC-Derived endothelial cells (iPSC-Derived Examples include Endothelial Cells. In particular, it is preferable that the vascular endothelial cells used in the screening method of this disclosure be human umbilical vein endothelial cells (HUVECs).

[0017] There are no particular limitations on the specific method for contacting vascular endothelial cells that have been passaged 15 or more times with the test substance. For example, the test substance may be added to a culture medium containing vascular endothelial 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 any interval of 1 minute to 96 hours, 30 minutes to 72 hours, 1 hour to 48 hours, or 12 hours to 36 hours. The upper or lower limit of the aforementioned 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, 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] Step B is preferably Step B': a step in which the test substance and lipopolysaccharide are brought into contact with vascular endothelial cells that have been passaged 15 or more times. Lipopolysaccharide (LPS) is a pathogenic factor also called an endotoxin, and is a component of the outer membrane of the cell wall of Gram-negative bacteria such as Porphyromonas gingivalis 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 E. coli. As shown in the examples described later, when LPS is brought into contact with vascular endothelial cells that have been passaged 15 or more times, the expression of specific inflammation-related factors is significantly induced, making it easier to evaluate whether the test substance is a candidate anti-inflammatory agent or not.

[0019] There are no particular limitations on the specific method for contacting vascular endothelial cells that have been passaged 15 or more times with LPS. For example, LPS may be added to a culture medium containing vascular endothelial 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 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 vascular endothelial 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. Process C In step C, the expression levels of at least one (preferably at least two, three, four, five, six, seven, or eight, or all) inflammation-related factors selected from the group consisting of IL-6 (Interleukin-6), IL-8 (Interleukin-8), CXCL1 (CXC motif chemokine ligand 1), PAI-1 (Plasminogen activator inhibitor-1), MMP-1 (Matrix metalloproteinase-1), ICAM-1 (Intercellular adhesion molecule-1), VCAM-1 (Vascular cell adhesion molecule-1), E-selectin (Endothelial-selectin), and CCL2 (CC motif chemokine ligand 2) are evaluated in vascular endothelial cells that were contacted with the test substance in step B. These inflammation-related factors may be collectively referred to as "inflammation-related factors of this disclosure." In this disclosure, the term "factor" is used to include both genes and proteins.

[0022] 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.

[0023] In the screening method of this disclosure, if the expression level of at least one of the inflammation-related factors of this disclosure in vascular endothelial cells that have been contacted with the test substance in step B is lower than the expression level of the same factor in vascular endothelial cells that have been cultured in the same manner except that they have not been contacted with the test substance, then the test substance is determined to be able to suppress inflammation in aging tissue. Such a test substance can be said to be a promising candidate for an anti-inflammatory agent to be used on aging tissue.

[0024] While not particularly limited, in a preferred embodiment, the aging tissue is tissue from a subject aged 40 years or older (preferably vascular endothelial tissue). More preferably, the aging tissue is tissue from a subject aged 45 years or older, even more preferably from a subject aged 50 years or older or 55 years or older, and particularly preferably from 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.

[0025] In this disclosure, senescent tissue refers to tissue containing senescent cells (preferably vascular endothelial tissue). 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 galactsidase) positive.

[0026] The method of administering the anti-inflammatory agents selected by the screening method of this disclosure to the target is not particularly limited. For example, they may be administered orally or parenterally. Parenteral administration includes, for example, intravenous, arterial, intramuscular, subcutaneous, abdominal, rectal, and topical administration, as well as transdermal administration. Although not particularly limited, it is preferable that the anti-inflammatory agents selected by the screening method of this disclosure be administered orally.

[0027] The subjects to whom the anti-inflammatory agents selected by the screening method of this disclosure are administered or ingested are 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.

[0028] 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.

[0029] The anti-inflammatory agents selected by the screening method of this disclosure are preferably applied to people who want to prevent aging, people who want to suppress aging, people who want to improve aging, people who want to prevent aging of blood vessels (especially vascular endothelium), people who want to suppress aging of blood vessels (especially vascular endothelium), people who want to improve aging of blood vessels (especially vascular endothelium), people who want to maintain and / or improve vascular function, people who want to maintain and / or improve the flexibility and suppleness of blood vessels that decline with age, people who want to prevent inflammation (especially inflammation in the vascular endothelium), people who want to suppress inflammation (especially inflammation in the vascular endothelium), and people who want to improve inflammation (especially inflammation in the vascular endothelium).

[0030] III. Process A While not particularly limited, the screening method of this disclosure may optionally further include step A: passage of vascular endothelial cells 15 or more times, prior to step B.

[0031] In step A, the lower limit of the number of passages may be 15, 16, 17, 18, 19, 20, or 21. The number of passages may be 16 or more, preferably 18 or more, more preferably 19 or more, even more preferably 20 or more, and particularly preferably 21 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, 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, or 22. The number of subculturing cycles may be, for example, 15 to 50, preferably 16 to 45, more preferably 18 to 40, even more preferably 19 to 30, and particularly preferably 20 to 25.

[0032] 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 limits 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 55-100% confluence, more preferably at a timing of 60-90% confluence, and even more preferably at a timing of 65-85% confluence.

[0033] 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.

[0034] 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]

[0035] 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.

[0036] Experiment 1. Induction of aging in human vascular endothelial cells and confirmation of aging characteristics. 1-1. Method 1-1-1. Evaluation of SA-β-gal-positive cells (Staining - Microscope) Human umbilical vein endothelial cells (HUVEC, Lonza) were subcultured until they reached passage 4 (Passage 4, hereafter p.4) or passage 21 (Passage 21, hereafter p.21). Each subculturing was performed when the cells reached 70-80% confluence (the same procedure was followed thereafter). Subsequently, p.4 cells were 2.0 × 10⁶ 4 At a density of cells / 1 mL / well, P.21 is 2.5 × 10⁻⁶ 4Cells were seeded at a density of 1 mL / well into 12-well plates and cultured for 3 days in growth medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation), and 10 ng / mL FGF (Kaken Pharmaceutical Co., Ltd)). After washing once with PBS(-), Bafilomycin A1 (Focus Biomolecules), diluted to 100 nM in evaluation medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), and 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation)), was added and incubated for 1 hour.

[0037] After 1 hour, the cells were washed twice with PBS(-) and fixed for 3 minutes using 4% paraformaldehyde-phosphate buffer (FUJIFILM Wako Pure Chemical). After washing three times with PBS(-), SPiDER-βGal (Dojindo), dissolved in DMSO and diluted with McIlvaine buffer (pH 6.0), was added and incubated for 30 minutes. Then, Hoechst 33342 (Thermo Scientific), diluted with PBS(-) containing 0.3% Triton X-100 (Sigma-Aldrich), was added and incubated for 15 minutes. After washing three times 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).

[0038] 1-1-2. Evaluation of SA-β-gal-positive cells (Staining-Flow cytometry) Human umbilical vein endothelial cells (HUVEC, Lonza) were subcultured until they reached passage 4 (Passage 4, hereafter p.4) or passage 21 (Passage 21, hereafter p.21). Subsequently, p.4 cells measured 2.0 × 10⁶ cells. 4 At a density of cells / 1 mL / well, P.21 is 2.5 × 10⁻⁶ 4 Cells were seeded at a density of 1 mL / well into 12-well plates and cultured for 3 days in growth medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation), and 10 ng / mL FGF (Kaken Pharmaceutical Co., Ltd)). After washing once with HBSS, Bafilomycin A1 (Focus Biomolecules), diluted to 100 nM in evaluation medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), and 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation)), was added and incubated for 1 hour.

[0039] After 1 hour, SPiDER-βGal (Dojindo), dissolved in DMSO and diluted in evaluation medium, was added and incubated for 30 minutes. After washing once with HBSS, the cells were detached from the plate using trypsin and collected. The collected cells were centrifuged at 4°C (1,000 rpm, 5 minutes) and the supernatant was removed. The cells were washed again with HBSS, centrifuged at 4°C (1,000 rpm, 5 minutes), and the supernatant was removed. Subsequently, the precipitated cells were dispersed in HBSS, and then dispersed individually using a cell strainer (40 μm, Corning). The dispersed cells were analyzed by fluorescence measurement using a BD Accuri C6 Plus Flow Cytometer (BD Biosciences).

[0040] 1-1-3. Cell cycle evaluation (Staining-flow cytometry) Human umbilical vein endothelial cells (HUVEC, Lonza) were subcultured until they reached passage 4 (Passage 4, hereafter p.4) or passage 21 (Passage 21, hereafter p.21). Subsequently, p.4 cells measured 2.0 × 10⁶ cells. 4 At a density of cells / 1 mL / well, P.21 is 2.5 × 10⁻⁶ 4Cells were seeded at a density of 1 mL / well into 12-well plates and cultured for 3 days in growth medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation), and 10 ng / mL FGF (Kaken Pharmaceutical Co., Ltd)). After reaching confluence, the cells were washed once with PBS(-), detached from the plate using trypsin, and collected. The collected cells were centrifuged at 20°C (300 g, 5 min), the supernatant was removed, and the cells were dispersed in PBS(-). A predetermined number of cells were mixed with Cell Cycle Assay Solution Deep Red (Dojindo) and incubated at 37°C under light-shielded conditions for 15 minutes. After dispersing the cells individually using a cell strainer (40 μm, Corning), fluorescence measurements were performed and analyzed using a BD Accuri C6 Plus Flow Cytometer (BD Biosciences).

[0041] 1-1-4. Evaluation of the expression of inflammation-related genes, cell cycle regulatory genes, and mitochondrial function-related genes (qPCR) Human umbilical vein endothelial cells (HUVEC, Lonza) were subcultured until they reached passage 4 (Passage 4, hereafter p.4) or passage 21 (Passage 21, hereafter p.21). Subsequently, p.4 cells measured 2.0 × 10⁶ cells. 4 At a density of cells / 1 mL / well, P.21 is 2.5 × 10⁻⁶ 4Cells were seeded at a density of 1 mL / well into 12-well plates and cultured for 3 days in growth medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation), and 10 ng / mL FGF (Kaken Pharmaceutical Co., Ltd)). After reaching confluence, the cells were switched to evaluation medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), and 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation)) and cultured for 30 hours.

[0042] 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, SIRT1, NRF-1, TFAM, IL-8, CXCL1, ICAM-1, IL-6, PAI-1, MMP-1, CXCL11, and RPS18) 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 level of RPS18, and the relative value at p.21 was determined when the expression at p.4 was set to 1. The primer sets used to quantify each gene expression in this example are described in "4. Appendix".

[0043] 1-1-5. Evaluation of the expression levels of inflammation-related proteins, cell cycle regulatory proteins, and mitochondrial function-related proteins (Western Blotting) Human umbilical vein endothelial cells (HUVEC, Lonza) were subcultured until they reached passage 4 (Passage4, hereinafter p.4) or passage 21 (Passage21, hereinafter p.21). Thereafter, p.4 was seeded at a density of 2.0 × 10 4 cells / 1 mL / well, and P.21 was seeded at a density of 2.5 × 10 4 cells / 1 mL / well into 12-well plates, respectively, and cultured in growth medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation), and 10 ng / mL FGF (Kaken Pharmaceutical Co., Ltd)) for 3 days. After reaching confluence, the cells were washed twice with cold PBS(-) and harvested on ice with M-PER buffer (Thermo Scientific) supplemented with protease inhibitor (Thermo Scientific) and phosphatase inhibitor (Thermo Scientific). The harvested cells were centrifuged at 4°C (15,000 g, 20 minutes), and the supernatant was collected.

[0044] The total protein concentration of the obtained protein extract was measured by 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 transferred proteins were blocked with PBS containing 5% skim milk and 0.05% Tween-20. After blocking, the membranes were sequentially reacted with primary and secondary antibodies, and then chemiluminescent membranes were induced using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and SuperSignal West Atto Ultimate Sensitivity Substrate (Thermo Scientific) as substrates. The emission intensity was detected using an Amersham Imager 680 RGB (Cytiva) system. Band intensity quantification was performed using Image J.

[0045] 1-2. Results Senescent cells are known to be SA-β-gal (senescence-associated beta galactosidase) positive. The results regarding SA-β-gal positive cells are shown in Figures 1-3. Figure 1 shows fluorescence microscope 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. Figure 2 shows the results of flow cytometry analysis of SA-β-gal positive cells. Figure 3 shows the results of flow cytometry analysis of cells that highly express SA-β-gal. As shown in Figures 1-3, it was shown that SA-β-gal expression, a characteristic of aging, is upregulated at p.21.

[0046] Figure 4 shows the results of flow cytometry analysis of the cell cycles of cells p.4 and p.21. As shown in Figure 4, p.21 showed a decrease in G0 / G1 and S phase cells and an increase in G2 phase cells.

[0047] Figure 5 shows the gene expression levels of the cell cycle-related factors p16, p21, and p53. p16, p21, and p53 are all genes reported to be associated with aging (aging-related genes). All of the genes shown in Figure 5 showed significantly increased expression in p.21. This result is consistent with previous findings that the expression of these genes is induced in senescent cells. Figure 6 shows the gene expression levels of the mitochondrial function-related factors SIRT1, NRF1, and TFAM. All of the genes shown in Figure 6 showed significantly decreased expression in p.21. This result is consistent with previous findings that mitochondrial function is impaired in senescent cells.

[0048] Figure 7 shows the results of analyzing the protein expression levels of p16, p21, and SIRT1 by Western blotting. Consistent with the gene expression levels shown in Figures 5 and 6, the protein expression levels of p16 and p21 were significantly increased in p.21, while the protein expression level of SIRT1 was significantly decreased.

[0049] The gene expression levels of inflammation-related factors IL-8, CXCL1, ICAM-1, IL-6, PAI-1, MMP-1, and CXCL11 are shown in Figures 8 and 9. The gene expression levels of IL-8, CXCL1, ICAM-1, IL-6, PAI-1, and MMP-1 were significantly elevated at p.21 compared to p.4. However, there was no significant difference in CXCL11 gene expression levels between p.21 and p.4.

[0050] Study 2. Evaluation of inflammation-related gene responses to E. coli LPS. 2-1. Method Human umbilical vein endothelial cells (HUVEC, Lonza) were subcultured until they reached passage 4 (Passage 4, hereafter p.4) or passage 21 (Passage 21, hereafter p.21). Subsequently, p.4 cells measured 2.0 × 10⁶ cells. 4 At a density of cells / 1 mL / well, P.21 is 2.5 × 10⁻⁶ 4 Cells were seeded at a density of 1 mL / well into 12-well plates and cultured for 3 days in growth medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation), and 10 ng / mL FGF (Kaken Pharmaceutical Co., Ltd)). After reaching confluence, the cells were switched to evaluation medium (MCDB 131 medium (Thermo Fisher Scientific) containing 10% FBS (Fetal Bovine Serum, Biowest), 1% Antibiotics (Gibco), and 2 mM L-Glutamine (Fujifilm Wako Pure Chemical Corporation)) and cultured for 24 hours. After 24 hours, 100 ng / mL of Escherichia coli-derived Lipopolysaccharides (hereinafter referred to as E. coli LPS) diluted in evaluation medium was added, or not added, and the cells were incubated for 6 hours.

[0051] 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, PAI-1, MMP-1, ICAM-1, VCAM-1, E-selectin, CCL2, and CXCL11) 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 RPS18, and the relative values ​​were determined with the gene expression level when p.4 was cultured in evaluation medium without E. coli LPS added set to 1.

[0052] 2-2. Results The results are shown in Figures 10-12. The gene expression levels of IL-6, IL-8, CXCL1, PAI-1, MMP-1, ICAM-1, VCAM-1, E-selectin, and CCL2 were significantly elevated in p.21 compared to p.4, particularly under E. coli LPS supplementation conditions. However, there was no significant difference in CXCL11 gene expression levels between p.21 and p.4, regardless of whether E. coli LPS was added or not.

[0053] 3. Discussion The results suggest that by using the expression levels of IL-6, IL-8, CXCL1, ICAM-1, VCAM-1, E-selectin, and CCL2, among other inflammation-related factors, it may be possible to evaluate whether a test substance can suppress inflammation, particularly in aging tissues. Furthermore, contact with senescent vascular endothelial cells with LPS significantly induced the expression of IL-6, IL-8, CXCL1, ICAM-1, VCAM-1, E-selectin, and CCL2, suggesting that it may be easier to evaluate whether a test substance is a candidate anti-inflammatory agent.

[0054] 4. Appendix The primer sets used to quantify each gene expression in the examples of this disclosure are shown in the table below.

[0055] [Table 1]

Claims

1. A screening method for anti-inflammatory agents to be used on aging tissues, Step B: A step of contacting vascular endothelial 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, IL-8, CXCL1, PAI-1, MMP-1, ICAM-1, VCAM-1, E-selectin, and CCL2 in vascular endothelial cells that were contacted with the test substance in step B.

2. Process B is Step B': A step in which the test substance and lipopolysaccharide are brought into contact with vascular endothelial cells that have been passaged 15 or more times. The screening method according to claim 1.

3. Before process B, Process A: A process of subculturing vascular endothelial 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 vascular endothelial cells are human umbilical vein endothelial cells (HUVEC).

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

Images