JP7805874B2 - Method for evaluating the inflammation-inducing properties of PM2.5 and its use - Google Patents

Description

The present invention relates to a method for evaluating the inflammation-inducing properties of PM2.5 and its use.

In recent years, the problem of air pollution has become more serious, raising concerns about its effects not only on the respiratory system but also on the skin and eyes. PM2.5 (fine particulate matter), a representative air pollutant, is a fine particle derived from automobile exhaust fumes and factory smoke. After coming into contact with the skin and eyes, it is known to penetrate the tissues, inducing inflammation and damaging the cells that make up those tissues.

So far, ingredients have been screened that suppress the exacerbation of skin problems and respiratory diseases induced by PM2.5 (Patent Document 1 and Patent Document 2).

The mechanism by which PM2.5 induces inflammation is known to be that PM2.5 acts on the transcription factor Aryl hydrocarbon receptor (AhR), generating reactive oxygen species, which then induces the production of cytokines (e.g., PGE2 and IL-8) downstream of the signaling pathway of these reactive oxygen species, which then induce inflammation through these cytokines (Non-Patent Document 1).

Re-table 2018/225718 publication Japanese Patent Application Laid-Open No. 2020-125281

Peng F. et al., “Potential role of PM2.5 in melanogenesis” Environment International, 2019 Nov;132:105063

However, the inflammatory properties of PM2.5 are not fully understood, and there is room for further investigation into the inflammatory properties of PM2.5 (e.g., the inflammatory properties of PM2.5 on the skin and eyes).

The present invention has been made in consideration of the above-mentioned problems, and aims to elucidate the unknown mechanism by which PM2.5 induces inflammation, and to provide a novel method for evaluating the inflammation-inducing properties of PM2.5 based on this mechanism, as well as a technology for using the method.

The present invention includes the following inventions:

[1] A method for evaluating the inflammation-inducing properties of PM2.5, comprising:
A method for evaluating the inflammation-inducing ability of PM2.5, comprising: a contacting step of contacting PM2.5 to be evaluated with keratinocyte cells and/or corneal epithelial cells; and a measuring step of measuring the gene expression level and/or protein production level of at least one indicator selected from the group consisting of the following (a) to (d):
(a) CXC chemokines (b) CXC chemokine receptors (c) CC chemokines (d) CC chemokine receptors.

[2] A contacting step of contacting keratinocyte cells and/or corneal epithelial cells with PM2.5 evaluated to have inflammation-inducing properties by the method described in [1] and a candidate substance for an inflammation-suppressing substance, and a measuring step of measuring the gene expression level and/or protein production level of at least one indicator selected from the group consisting of the following (a) to (d) in the cells:
A method for screening for a substance that suppresses inflammation caused by PM2.5, comprising:
(a) CXC chemokines (b) CXC chemokine receptors (c) CC chemokines (d) CC chemokine receptors.

[3] An evaluation kit for evaluating the inflammation-inducing potential of PM2.5, comprising a probe that specifically binds to at least one indicator gene or protein selected from the group consisting of the following (a) to (d):
(a) CXC chemokines (b) CXC chemokine receptors (c) CC chemokines (d) CC chemokine receptors.

The present invention provides a novel method for evaluating the inflammation-inducing properties of PM2.5 and a technology for using the method.

1 is a graph showing the quantification of CXC chemokines and CC chemokines in keratinocyte cells after contact with PM2.5. 1 is a graph showing the quantification results of CXC chemokines and CC chemokines in corneal epithelial cells after contact with PM2.5. 1 is a graph showing the results of quantifying CXC chemokines in three-dimensional skin model cells after contact with PM2.5, which has inflammation-inducing properties, and a test substance.

An example of an embodiment of the present invention will be described in detail below, but the present invention is not limited to this. Various modifications of the present invention are possible within the scope of the claims. Furthermore, embodiments or examples obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included within the technical scope of the present invention. Furthermore, new technical features can be created by combining the technical means disclosed in each embodiment. All academic and patent literature cited in this specification is incorporated herein by reference. Unless otherwise specified in this specification, the expression "X to Y" representing a numerical range means "greater than or equal to X and less than or equal to Y."

1. Method for evaluating the inflammation-inducing properties of PM2.5
A method for evaluating the inflammation-inducing properties of PM2.5 according to one embodiment of the present invention comprises a contacting step of contacting PM2.5 to be evaluated with keratinocyte cells and/or corneal epithelial cells, and a measuring step of measuring the gene expression level and/or protein production level of at least one indicator selected from the group consisting of the following (a) to (d) in the cells: (a) CXC chemokine, (b) CXC chemokine receptor, (c) CC chemokine, and (d) CC chemokine receptor.

PM2.5 is known to contain different compositions of metal ions and other substances depending on the time and/or location at which it is collected. The inventors conducted extensive research into the above-mentioned problem and investigated the effects of PM2.5 collected at different locations on the expression of chemokines and/or chemokine receptors, which are known to strongly induce inflammation. As a result, they discovered that certain types of PM2.5 affect the expression of chemokines and/or chemokine receptors, which are known to strongly induce inflammation, and thus completed the present invention.

In particular, while PM2.5 has previously been known to induce inflammation via cytokines, its association with chemokines and/or chemokine receptors has not been reported, making this a completely novel finding. PM2.5, which induces the production of not only cytokines but also chemokines and/or chemokine receptors, is presumed to be highly toxic. Therefore, the present invention is of great technical significance in that it can evaluate more malignant PM2.5 (which has greater adverse effects on the skin) that induces the production of not only cytokines but also chemokines and/or chemokine receptors. Therefore, the present invention can reduce the threat of PM2.5-related diseases and contribute to the achievement of, for example, Goal 3 of the Sustainable Development Goals (SDGs) (Ensure healthy lives and promote well-being for all at all ages).

The "method for evaluating the inflammation-inducing ability of PM2.5" according to one embodiment of the present invention may be a "method for evaluating the chemokine and/or chemokine receptor expression-inducing ability of PM2.5."

In the present invention, inflammation induced by PM2.5 includes, but is not limited to, inflammation that occurs in organs such as the skin, eyes (e.g., cornea, conjunctiva, eyelids, etc.), and respiratory organs (e.g., lungs, bronchi, etc.). Epithelial cells such as keratinocytes and corneal epithelial cells are abundant in the surface layers of the above-mentioned organs. Therefore, the above-mentioned configuration makes it possible to more accurately evaluate the inflammation-inducing properties of PM2.5.

[1-1. Contact process]
The contacting step is a step of contacting the PM2.5 to be evaluated with keratinocyte cells and/or corneal epithelial cells.

(PM2.5)
PM2.5 refers to particles suspended in the air with a diameter of 2.5 μm or less. More specifically, PM2.5 may be particles collected after removing particles with a diameter larger than 2.5 μm from particles suspended in the air using a particle size separator capable of separating particles with a diameter of 2.5 μm or less.

The PM2.5 to be evaluated may be collected at a desired location, or may be obtained by removing particles larger than 2.5 μm in diameter from particles collected at a desired location, or may be a commercially available product.

(cell)
In one embodiment of the present invention, the cells used are keratinocyte cells and/or corneal epithelial cells.

The cells may be, for example, wild-type cells collected from a living organism, cell lines established from cells collected from a living organism, transformed cells of these, wild-type cells collected from a living organism contained in a tissue model (e.g., a three-dimensional skin model), cell lines established from cells collected from a living organism contained in a tissue model (e.g., a three-dimensional skin model), or transformed cells of these contained in a tissue model (e.g., a three-dimensional skin model). Alternatively, the cells used may be a commercially available three-dimensional model with multiple layers.

The origin of the cells used in one embodiment of the present invention is not particularly limited as long as they are mammalian, and examples include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cows, and horses, with humans being preferred.

(Contact method)
The method for contacting the cells with PM2.5 is not particularly limited and may be a known method, such as adding PM2.5 to a culture medium for culturing the cells and incubating the mixture, or adding PM2.5 directly to the cells.

The method for adding the test substance to the medium and incubating it can be selected appropriately depending on the type of cells used. Such methods include monolayer static culture, suspension culture, rotary culture, and three-dimensional carrier culture.

The medium may contain components suitable for the growth of the cells used (e.g., glucose, amino acids, peptone, vitamins, cell proliferation-promoting factors (e.g., cell growth factors, hormones, binding proteins, cell adhesion factors, lipids), serum (e.g., FBS, FCS, etc.), calcium chloride, magnesium chloride, etc.). The medium may also be commercially available. Examples of media used for cell culture include MEM medium, DMEM medium, and Ham's F-12 medium. When the cells used are keratinocyte cells, EpiLife medium or the like may be used. When the cells used are corneal epithelial cells, EMEM medium or the like may be used.

In addition, conditions such as incubation temperature and carbon dioxide concentration are set appropriately depending on the cells used. For example, when keratinocyte cells are used, the cells are typically incubated in an atmosphere containing 4-6% carbon dioxide by volume at 36.0-38.0°C, preferably 36.5-37.5°C. When corneal epithelial cells are used, the cells are typically incubated in an atmosphere containing 4-6% carbon dioxide by volume at 36.0-38.0°C, preferably 36.5-37.5°C.

The number of cells to be contacted with the test substance is preferably 1 x ¹⁰ cells or more, and more preferably 1 x 10 cells or more, per 100 µL of medium containing PM2.5, from the viewpoint of reliability of test data, and (ii) preferably 1 x ^{10 cells} or less, and more preferably 1 x ¹⁰ cells or less, from the viewpoint of ensuring sufficient spacing between cells and preventing them from becoming too dense.

The method for directly adding PM2.5 to cells is not particularly limited and may include application, patching, dripping, spraying, etc. The amount of PM2.5 to be brought into contact with cells can be appropriately determined depending on the type of PM2.5 and/or the type of cells used.

The cells may be pre-incubated under conditions suitable for the cells in order to maintain the cells in a state suitable for measuring physiological events caused by PM2.5.

In one embodiment of the present invention, the contact time for cells with PM2.5 is not limited. Considering the time required for intracellular signaling, the contact time for cells with PM2.5 is preferably 1 hour or longer, more preferably 6 hours or longer. Furthermore, considering the effects of cytotoxicity, the contact time is preferably 24 hours or shorter, more preferably 12 hours or shorter.

[1-2. Culture process]
If necessary, the evaluation method according to one embodiment of the present invention may further include, after the contact step, a culture step in which the cells contacted with PM2.5 are cultured in a medium not containing PM2.5 for a predetermined period of time.

By contacting cells with PM2.5 in the contact step, changes occur in the production of indicator genes and/or proteins, and it may take some time for these changes to reach measurable levels. In this case, it is preferable to carry out the culture step after the contact step.

The culture method for culturing cells is not particularly limited and can be selected appropriately depending on the type of cells used. Examples of such methods include monolayer static culture, suspension culture, rotary culture, and three-dimensional carrier culture.

The medium can be the same as that described for the contact step above. The incubation temperature, carbon dioxide concentration, and other conditions can be the same as those described for the contact step above.

The culture time can be set appropriately depending on the cells used and the indicator to be measured, but is preferably at least 1 hour, more preferably at least 6 hours, taking into account the time required for transcription initiation, and is preferably within 24 hours, more preferably within 12 hours, taking into account the effects of cytotoxicity.

[1-3. Measurement process]
The measuring step is a step of measuring the gene expression level and/or protein production level of at least one indicator in the cells that have been contacted with PM2.5 in the contacting step.

(index)
The indicator used in one embodiment of the present invention is at least one selected from the group consisting of the following (a) to (d), and in the measurement step, the gene expression level and/or protein production level of the indicator is measured:
(a) CXC chemokines (b) CXC chemokine receptors (c) CC chemokines (d) CC chemokine receptors.

CXC chemokines refer to the CXC chemokine family, also referred to as α chemokines (or their family). Examples of CXC chemokines include CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8 (IL-8), CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, and CXCL17. Of these CXC chemokines, CXCL1, CXCL12, and CXCL8 are particularly preferred as indicators.

The term "CXC chemokine receptor" refers to the CXC chemokine receptor family, including, for example, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, and CXCL6. Among these CXC chemokine receptors, CXCR1, CXCR2, CXCR4, and CXCR7 are particularly preferred as indicators.

CC chemokines refer to the CC chemokine family, also referred to as β-chemokines (or their family). Examples of CC chemokines include CCL1, CCL2, CCL3, CCL3L3, CCL3L1, CCL4L2, CCL4, CCL4L1, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28. Among these CC chemokines, CCL2, CCL3, and CCL4 are particularly preferred as indicators.

The CC chemokine receptor refers to the CC chemokine receptor family, including, for example, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCRL2, and NCCRP1. Among these CC chemokine receptors, CCR2, CCR4, and CCR5 are particularly preferred as indicators.

The accession numbers of the above-mentioned indicator proteins and genes are shown in Tables 1 to 4 below.

(Measurement method)
The method for measuring the expression level of a gene and/or the production level of a protein serving as an indicator is not particularly limited, and may be a known method for measuring the production of a protein serving as an indicator or the expression of a gene. For example, the expression level of a gene can be measured by measuring the expression of mRNA.

Methods for measuring mRNA expression include, for example, extracting total RNA from cells and measuring it using real-time RT-PCR, RNase protection assay, Northern blot analysis, dot blot analysis, RNA sequence analysis, microarray, FISH (fluorescence in situ hybridization), etc.

Methods for measuring protein expression include, for example, Western blotting, immunoprecipitation, ELISA, RIA, EIA, and bioassay.

The above-mentioned measurement methods can be performed in vitro or ex vivo, or can also be performed in vivo. Furthermore, when measuring the expression levels of multiple genes and/or proteins, the expression levels may be measured, for example, individually or collectively at the same time.

1-4. Evaluation process
The evaluation method according to one embodiment of the present invention may further include an evaluation step of evaluating the inflammation-inducing ability of PM2.5 based on the gene expression level and/or protein production level measured in the measurement step.

More specifically, in the evaluation step, the inflammatory inducibility of PM2.5 can be evaluated by comparing the gene expression level and/or protein production level of the indicator in cells after contact with PM2.5 with the gene expression level and/or protein production level of the indicator in control cells.

Controls include, but are not limited to:
Cells before contact with PM2.5 (Control A),
Cells after exposure to PM2.5, which has no known inflammatory properties (Control B);
Cells after exposure to a substance with no known inflammation-inducing properties (Control C),
Cells after exposure to PM2.5, a known inflammatory agent (Control D).
Cells after contact with a known inflammation-inducing agent (Control E);
etc. can be used.

For example, when control A, control B, or control C is used as a control, if the gene expression level or protein production level in cells after contact with PM2.5 is the same as or lower than the gene expression level or protein production level in these controls (controls A to C), it can be evaluated that PM2.5 does not have, or has but a low level of, inflammation-inducing properties. On the other hand, if the gene expression level or protein production level in cells after contact with PM2.5 is higher than the gene expression level or protein production level in these controls (controls A to C), it can be evaluated that PM2.5 has inflammation-inducing properties.

For example, when Control D or Control E is used as a control, if the gene expression level or protein production level in cells after contact with PM2.5 is equal to or higher than the gene expression level or protein production level in these controls (Controls D to E), PM2.5 can be evaluated as having pro-inflammatory properties. On the other hand, if the gene expression level or protein production level in cells after contact with PM2.5 is lower than the gene expression level or protein production level in these controls (Controls D to E), PM2.5 can be evaluated as not having pro-inflammatory properties, or as having low pro-inflammatory properties.

In this specification, "low expression level or production level" means that the level of expression of an indicator gene and/or the level of protein production in cells after contact with PM2.5 is less than 1.0 times, preferably 1/2 or less, more preferably 1/5 or less, and most preferably 1/10 or less, of the expression level or production level in the control.

In addition, as used herein, a high expression level or production level refers to the level of gene expression and/or protein production of an indicator in cells after contact with PM2.5 being more than 1.0-fold, preferably 2.0-fold or more, more preferably 5.0-fold or more, and most preferably 10-fold or more, compared to the level of expression or production in the control.

2. Screening method for substances that suppress inflammation caused by PM2.5
A screening method for a substance that suppresses inflammation caused by PM2.5 according to one embodiment of the present invention comprises the steps of: contacting PM2.5 that has been evaluated as having inflammation-inducing properties by the method for evaluating the inflammation-inducing properties of PM2.5 according to one embodiment of the present invention (the method described in [1] above) (hereinafter in this section referred to as "PM2.5 (X) having inflammation-inducing properties") and a candidate inflammation-inducing substance that is a test substance (hereinafter in this section referred to as "test substance (Y)") with keratinocyte cells and/or corneal epithelial cells; and measuring the gene expression level and/or protein production level of at least one indicator selected from the group consisting of the following (a) to (d) in the cells: (a) CXC chemokine, (b) CXC chemokine receptor, (c) CC chemokine, and (d) CC chemokine receptor.

The screening method for substances that suppress inflammation caused by PM2.5 according to the present invention makes it possible to screen for substances that suppress inflammation caused by PM2.5.

The "method for screening for substances that suppress inflammation caused by PM2.5" according to one embodiment of the present invention may also be a "method for screening for substances that control (e.g., suppress) the induction of expression of chemokines and/or chemokine receptors caused by PM2.5."

The cells, contact method, contact time, indicator, and measurement method used in this screening method are the same as those used in the method for evaluating the inflammation-inducing properties of PM2.5 described above. Therefore, a description of these will be omitted here.

Candidate substances (test substances (Y)) for suppressing inflammation caused by PM2.5 are not particularly limited and may be compounds or compositions. Examples of compositions include cosmetics and pharmaceuticals. Examples of compounds include the components contained in the above-mentioned cosmetics or pharmaceuticals.

Similar to the above-described method for evaluating the inflammation-inducing properties of PM2.5, this screening method may, if necessary, further include a culture step in which, after the contact step in which inflammation-inducing PM2.5 (X) and test substance (Y) are brought into contact with keratinocyte cells and/or corneal epithelial cells, the cells contacted with PM2.5 (X) and test substance (Y) are cultured for a predetermined period of time in a medium that does not contain PM2.5 (X) or test substance (Y). The configuration of this culture step may be similar to that used in the above-described method for evaluating the inflammation-inducing properties of PM2.5, and therefore a description thereof will be omitted here.

This screening method includes a measurement step of measuring the gene expression level and/or protein production level of at least one indicator selected from the group consisting of (a) to (d) above in keratinocyte cells and/or corneal epithelial cells after the contact step or the culture step. This measurement step can be configured in the same way as in the method for evaluating the inflammation-inducing properties of PM2.5 described above, and therefore will not be described here.

The screening method may further include an evaluation step of evaluating whether or not the test substance (Y) is a substance that suppresses inflammation caused by PM2.5, based on the gene expression level and/or protein production level measured in the measurement step.

More specifically, in the evaluation step, by comparing the gene expression level and/or protein production level of the indicator in cells after contact with PM2.5 (X) and test substance (Y) with the gene expression level and/or protein production level of the indicator in control cells, it is possible to evaluate whether test substance (Y) is a substance that suppresses inflammation caused by PM2.5 (X).

Controls include, but are not limited to:
Cells before contact with PM2.5 (X) and test substance (Y) having inflammation-inducing properties (Control A');
Cells after exposure to PM2.5, which has no known inflammatory properties (Control B');
Cells after contact with a substance with no known inflammation-inducing properties (Control C');
Cells after exposure to PM2.5, a known inflammatory agent (Control D').
Cells after contact with a known inflammation-inducing substance (Control E'),
etc. can be used.

For example, when control A', control B', or control C' is used as a control, if the gene expression level or protein production level in cells after contact with PM2.5 (X) and test substance (Y) is equal to or lower than the gene expression level or protein production level in these controls (controls A' to C'), test substance (Y) can be evaluated as a substance that suppresses inflammation caused by PM2.5 (X).

For example, when control D' or control E' is used as a control, if the gene expression level or protein production level in cells after contact with PM2.5 (X) and test substance (Y) is equal to or greater than the gene expression level or protein production level in these controls (controls D' to E'), it can be determined that test substance (Y) is not a substance that suppresses inflammation caused by PM2.5 (X).

In this specification, a low expression or production level means that the level of expression of an indicator gene and/or the level of protein production in cells after contact with PM2.5 (X) and test substance (Y) is less than 1.0 times, preferably 1/2 or less, more preferably 1/5 or less, and most preferably 1/10 or less of the expression or production level in the control.

In addition, as used herein, a high expression level or production level refers to the level of gene expression and/or protein production of an indicator in cells after contact with PM2.5 (X) and test substance (Y) being more than 1.0-fold, preferably 2.0-fold or more, more preferably 5.0-fold or more, and most preferably 10-fold or more, compared to the level of expression or production in the control.

3. Evaluation kit for assessing the inflammation-inducing properties of PM2.5
An evaluation kit for evaluating the inflammation-inducing properties of PM2.5 according to one embodiment of the present invention comprises a probe that specifically binds to at least one indicator gene or protein selected from the group consisting of (a) to (d) below: (a) a CXC chemokine, (b) a CXC chemokine receptor, (c) a CC chemokine, and (d) a CC chemokine receptor.

More specifically, an evaluation kit (e.g., a microarray kit, a PCR kit) for evaluating the inflammation-inducing properties of PM2.5 according to one embodiment of the present invention comprises a probe that specifically binds to at least one indicator mRNA or protein selected from the group consisting of (a) to (d) below: (a) CXC chemokine, (b) CXC chemokine receptor, (c) CC chemokine, and (d) CC chemokine receptor.

In one embodiment of the present invention, the "evaluation kit for evaluating the inflammation-inducing ability of PM2.5" may be an "evaluation kit for evaluating the ability of PM2.5 to induce the expression of chemokines and/or chemokine receptors."

Probes that specifically bind to mRNA include polynucleotides that specifically hybridize to the mRNA. Probes that specifically bind to proteins include antibodies that specifically bind to the protein.

The above probes can also be used by immobilizing them on any solid phase. Therefore, the evaluation kit may be provided as a microarray chip, DNA chip, protein chip, or the like, on which the above probes are immobilized. The solid phase is not particularly limited as long as it can immobilize the probes, and examples of the solid phase include glass plates, nylon membranes, microbeads, silicon chips, capillaries, and other substrates.

The evaluation kit of the present invention may further include a second probe that specifically binds to the mRNA or protein captured by the probe, and that is labeled, in order to quantify the mRNA or protein. Examples of the second probe include a labeled polynucleotide that specifically hybridizes to the mRNA, and a labeled antibody that specifically binds to the protein. The labeling method is not particularly limited, and can be, for example, labeling using a fluorescent dye.

[1: Analysis of protein expression levels in cells upon contact with PM2.5]
(PM2.5)
Four types of PM2.5 (A), (B), (C), and (D) collected at different locations were used as samples to evaluate the inflammation-inducing properties of PM2.5.

Separate analysis has confirmed that PM2.5-(A) and PM2.5-(C) are "weakly inflammatory" PM2.5, with low content of substances that cause inflammation in the skin (e.g., organic carbon), while PM2.5-(B) and PM2.5-(D) are "strongly inflammatory" PM2.5, with high content of substances that cause inflammation in the skin (e.g., organic carbon).

(Cell culture)
A three-dimensional skin model (LabCyte EPI-MODEL, manufactured by Japan Tissue Engineering Co., Ltd., a model with multilayered keratinocyte cells) was cultured in EPI-MODEL medium (manufactured by Japan Tissue Engineering Co., Ltd.) at 37°C in a 5% _CO2 atmosphere for ₂₄ hours. A three-dimensional corneal model (LabCyte CORNEA-MODEL, manufactured by Japan Tissue Engineering Co., Ltd., a model with multilayered corneal epithelial cells) was cultured in CORNEA-MODEL medium (manufactured by Japan Tissue Engineering Co., Ltd.) at 37°C in a 5% CO2 atmosphere for 24 hours.

24 hours after the start of culture, cell supernatants were collected, and the expression levels of indicator proteins (CXCL1, CCL3, CCL4) were analyzed using a commercially available kit (ProcartaPlex Immunoassay Kit, ThermoFisher).

As a control, a three-dimensional skin model or a three-dimensional corneal model was cultured in medium without PM2.5 to obtain an untreated cell sample (shown as "unt" in Figures 1 and 2).

As shown in Figure 1, in a three-dimensional skin model, a significant increase in the expression level of the indicator protein was observed when the skin was exposed to PM2.5-(B) and PM2.5-(D), which have "strong inflammation-inducing properties," compared to when the skin was exposed to PM2.5-(A) and PM2.5-(C), which have "weak inflammation-inducing properties."

As shown in Figure 2, in a three-dimensional corneal model, a significant increase in the expression level of the indicator protein was observed when the model was exposed to PM2.5-(B) and PM2.5-(D), which have "strong inflammation-inducing properties," compared to when the model was exposed to PM2.5-(A) and PM2.5-(C), which have "weak inflammation-inducing properties."

[2: Analysis of changes in mRNA expression levels in cells due to contact with PM2.5 evaluated to have inflammation-inducing properties and test substances]
(Cell culture)
A three-dimensional skin model (LabCyte EPI-MODEL, manufactured by Japan Tissue Engineering Co., Ltd.) was cultured in EPI-MODEL medium (manufactured by Japan Tissue Engineering Co., Ltd.) at 37°C in a 5% CO2 atmosphere for ₂₄ hours.

Aqueous solution 1 containing only water was prepared (corresponding to "untreated" in Figure 3). Aqueous solution 2 containing PM2.5-(B) but not the test substance (specifically, Trolox, which is known to have anti-inflammatory effects) was prepared (corresponding to "PM2.5_B w/o compd" in Figure 3). Aqueous solution 3 containing the test substance (specifically, Trolox, which is known to have anti-inflammatory effects) and PM2.5-(B) was prepared (corresponding to "PM2.5_B w/ compd" in Figure 3).

Each of the aqueous solutions 1 to 3 was added to the medium in the well containing the three-dimensional skin model prepared above, and incubated at 37°C in a 5% CO ₂ atmosphere for 6 hours.

After 6 hours of incubation, the three-dimensional skin model cells were collected from each well, and changes in the mRNA expression levels of the indicator genes (CXCL1, IL-8) were analyzed using real-time RT-PCR. Total RNA was extracted from the cells using a commercially available kit (Maxwell RSC simplyRNA cells kit, manufactured by Promega), and real-time RT-PCR was performed using a commercially available kit (SuperScript RT kit, manufactured by ThermoFisher).

As shown in Figure 3, when the three-dimensional skin model was brought into contact with PM2.5-(B) alone ("PM2.5_B w/o compd" in Figure 3), a significant increase in the mRNA expression level of the indicator gene was observed compared to the untreated cell sample ("untreated" in Figure 3). On the other hand, when the three-dimensional skin model was brought into contact with PM2.5-(B) and the test substance ("PM2.5_B w/ compd" in Figure 3), a significant decrease in the mRNA expression level of the indicator gene was observed compared to the cell sample brought into contact with PM2.5-(B) alone ("PM2.5_B w/o compd" in Figure 3).

This example demonstrates that the screening method for substances that suppress inflammation caused by PM2.5 according to one embodiment of the present invention can accurately screen for substances that suppress inflammation caused by PM2.5.

The present invention can be used in fields related to the evaluation of air pollution, and more specifically, in fields related to the evaluation of PM2.5.

Claims (2)

A method for evaluating the inflammation-inducing properties of PM2.5, comprising:
a contacting step of contacting the PM2.5 to be evaluated with keratinocyte cells and/or corneal epithelial cells;
a measuring step of measuring gene expression levels and/or protein production levels of CXCL1, CCL3, and CCL4 in the cells;
A method for evaluating the inflammation-inducing properties of PM2.5 . a contacting step of contacting PM2.5 evaluated as having inflammation-inducing properties by the method of claim 1 and a candidate inflammation-suppressing substance with keratinocyte cells and/or corneal epithelial cells;
a measuring step of measuring gene expression levels and/or protein production levels of CXCL1, CCL3, and CCL4 in the cells;
A method for screening for a substance that suppresses inflammation caused by PM2.5 .