KR102359437B1 - A method for screening anti-acne meterials - Google Patents

Abstract

Disclosed herein is a method for screening an anti-acne substance through the amount of exosome secretion derived from the acne-causing bacteria. The method includes treating the acne-causing bacteria with a test substance, then separating the exosomes from the acne-causing bacteria and confirming the amount of the separated exosomes, and the acne-causing bacteria may be Propionibacterium acnes . . The method is effective in screening anti-acne substances that reduce the secretion of exosomes from acne-causing bacteria.

Description

A METHOD FOR SCREENING ANTI-ACNE METERIALS

The present specification discloses a method for screening an anti-acne substance through the amount of exosome secretion derived from the bacteria causing acne.

Most animal cells have the ability to secrete extracellular vesicles of intracellular origin of various sizes and components, and these extracellular vesicles contain all biological vesicles, including blood, urine, saliva, and cell cultures. found in biological fluids (Loyer X, Vion AC, Tedgui A, Boulanger CM. Microvesicles as cell-cell messengers in cardiovascular diseases. Circ Res 2014;114:345-53) (Ohno S, Ishikawa A, Kuroda M) (Roles of exosomes and microvesicles in disease pathogenesis. Adv Drug Deliv Rev 2013;65:398-401).

Extracellular vesicles are membrane-structured vesicles having a size of about 20 nm in diameter to about 5 μm in diameter as large as possible, and are heterogeneous in size and composition, and include exosomes (about 30-100 nm) and ectosomes. (ectosome), microvesicles (about 100-1,000 nm), microparticles, apoptotic bodies (about 1-5 μm), and the like, and many different species.

The different types of extracellular vesicles differ in their origin, diameter, density in sucrose, shape, sedimentation rate, lipid composition, protein marker or mode of secretion (ie, whether by signal (inducible) or spontaneous (constitutive). ) and so on. For example, microvesicles are membrane vesicles having an irregular shape of about 100 to 1,000 nm that germinate toward the outside of the plasma membrane (originating from the plasma membrane), and have phospholipids including integrin, selectin, CD40 ligand, and phosphatidylserine. it is known On the other hand, exosomes are the smallest membrane vesicles having a cup shape of about 30 to 100 nm (<200 nm) and germinate from the inside of late endosomes (originating from endosomes), CD63, CD9 tetraspanin, TSG101, It is known to have lipids including markers including ESCRT, cholesterol, sphingomyelin, ceramides, and phosphatidylserine.

These extracellular vesicles reflect the state of the secreting cell of origin (donor cell), exhibit various biological activities depending on which cell they are secreted from, and play an important role in cell-to-cell interactions by transferring genetic material and proteins between cells. carry out

Prokaryotic or eukaryotic cells are also known to secrete extracellular vesicles (Camussi, G., Deregibus, MC, Bruno, S., Cantaluppi, V., & Biancone, L. (2010). Exosomes/microvesicles as a mechanism Kidney international, 78(9), 838-848, Bang, Claudia, and Thomas Thum. "Exosomes: New players in cell-cell communication." The international journal of biochemistry & cell biology 44.11 ( 2012): 2060-2064. Kim, DK, Lee, J., Simpson, RJ, Lotvall, J., & Gho, YS (2015, April), EVpedia: A community web resource for prokaryotic and eukaryotic extracellular vesicles research. Seminars in cell & developmental biology (Vol. 40, pp. 4-7). Academic Press, Kim, JH, Lee, J., Park, J., & Gho, YS (2015, April). Gram-negative and Gram -positive bacterial extracellular vesicles.In Seminars in cell & developmental biology (Vol. 40, pp. 97-104). Academic Press).

On the other hand, acne is an inflammatory disease of the sebaceous glands in the hair follicles, which mainly occurs on the face, chest, neck, and shoulders of the skin. It is known as a complex action by acne bacteria. Abnormal hyperkeratinization of the follicular wall in the sebaceous gland thickens the lining of the sebaceous duct and clogs the duct that secretes sebum. As acne bacteria actively inhabit here, it develops into purulent acne accompanied by pain.

Conventionally, the importance of acne-causing bacteria has been recognized in the onset of acne, and a method of screening anti-acne substances has been developed based on whether or not the growth-inhibiting activity of acne-causing bacteria has been developed. There is no disclosure on a method for screening an anti-acne substance through the

Korean Patent Publication No. 10-2012-0015021

In one aspect, an object of the present specification is to provide a method for screening an anti-acne substance using an exosome derived from an acne causative organism.

In one aspect, the technology disclosed herein comprises the steps of treating a test substance to an acne causative agent; Separating the exosomes from the acne-causing bacteria treated with the test substance; and confirming the amount of the isolated exosome; provides a method of screening an anti-acne substance comprising.

In an exemplary embodiment, the acne-causing bacterium may be Propionibacterium acnes .

In an exemplary embodiment, it may include culturing the acne-causing bacteria for 72 to 96 hours after treating the acne-causing bacteria with the test substance.

In an exemplary embodiment, in the step of isolating the exosome, the culture solution of the acne-causing bacteria treated with the test substance is centrifuged at 2,000 to 20,000 × g, and then the supernatant is collected and ultracentrifuged at 100,000 to 200,000 × g may include the step of

In an exemplary embodiment, the centrifugation may include centrifugation at 2,000 to 5,000 × g, followed by centrifugation at 8,000 to 20,000 × g.

In an exemplary embodiment, the step of isolating the exosomes may include centrifuging the culture solution of the acne causative bacteria and then filtering it with a filter of 0.2 to 0.5 μm, followed by ultracentrifugation.

In an exemplary embodiment, the isolated exosome may have a diameter of 20 to 500 nm.

In an exemplary embodiment, the isolated exosomes may be sedimented in ultracentrifugation of 100,000 to 200,000 × g of the acne-causing bacteria culture medium.

In an exemplary embodiment, it may include comparing the amount of the isolated exosomes with the amount of exosomes isolated from acne-causing bacteria that have not been treated with the test substance.

In an exemplary embodiment, when the amount of the isolated exosomes is less than the amount of exosomes isolated from acne-causing bacteria that have not been treated with the test substance, it may include evaluating the test substance as an anti-acne substance.

In one aspect, the technology disclosed herein has the effect of providing a method for screening an anti-acne substance by utilizing the secretion amount of exosomes secreted to the extracellular by the acne-causing bacteria as a marker for screening of the anti-acne substance.

1 shows the size (FIG. 1a) and transmission electron micrograph (FIG. 1b) of the exosomes isolated from the acne-causing bacteria in one test example of the present specification.
2 shows an increase in the expression of inflammatory cytokine genes (FIG. 2a) and protein (FIG. 2b) when exosomes (1 to 10 μg/ml) are treated in keratinocytes (NHEK) in one test example of the present specification.
3 shows an increase in cell proliferation upon treatment with exosomes (1 to 20 μg/ml) in keratinocytes (HaCaT cells) in one test example of the present specification.
Figure 4 shows a decrease in the expression of cell differentiation marker genes (Figure 4a) and protein (Figure 4b) when exosomes (1 to 10 μg/ml) treatment in keratinocytes (NHEK) in one test example of the present specification .

Hereinafter, the present invention will be described in detail.

In one aspect, the technology disclosed herein comprises the steps of treating a test substance to an acne causative agent; Separating the exosomes from the acne-causing bacteria treated with the test substance; and confirming the amount of the isolated exosome; provides a method of screening an anti-acne substance comprising.

In an exemplary embodiment, the acne-causing bacterium may be Propionibacterium acnes .

In an exemplary embodiment, it may include culturing the acne-causing bacteria for 72 to 96 hours after treating the acne-causing bacteria with the test substance.

In an exemplary embodiment, the test substance may be inoculated into a medium by diluting the acne-causing bacteria that have undergone pre-cultivation, and then treated with the diluted acne-causing bacteria.

As used herein, "exosome" refers to nano-sized extracellular vesicles secreted from cells and released into the extracellular space, that is, extracellular vesicles. It is classified and has bacterial cell membrane lipids (plasma membrane lipid), plasma membrane protein (plasma membrane protein), nucleic acid (nucleic acid), and cytoplasmic components. In addition, exosomes bind to other cells and deliver membrane components, mRNAs, miRNAs, etc., and deliver these messengers to recipient cells, thereby acting as an important extracellular transporter mediating cell-cell communication.

In one aspect, the exosome may be an extracellular vesicle having a diameter of 20 to 500 nm. In another aspect, the exosome is 20 nm or more, 30 nm or more, 40 nm or more, or 50 nm or more, and 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 100 nm or less, 90 nm or less, 80 It may be an extracellular vesicle having a diameter of less than or equal to 70 nm, or less than or equal to 60 nm.

In an exemplary embodiment, the exosomes are centrifuged (centrifugation), ultracentrifugation (ultracentrifugation), differential centrifugation (Differential Centrifugation), equal density centrifugation (Equilibrium Density Centrifugation), density gradient (density gradient), filtration Separation may be performed using one or more methods selected from the group consisting of filtration, dialysis, and free-flow electrophoresis, but the separation method of exosomes is not limited thereto.

The density gradient is the most used method to distinguish substances with different densities, and specific examples of this method include ficoll, glycerol, sucrose, cesium chloride, iodixanol. (iodixanol) may be carried out using a density gradient separation material such as, but is not limited thereto. In one aspect, density gradients can be used in conjunction with ultracentrifugation and the like. In another aspect, gel filtration or ultrafiltration may be used to select exosomes. In another aspect, dialysis can be used instead of filtration to remove small molecules. In another aspect, free-flowing electrophoresis can be used.

In an exemplary embodiment, the step of isolating the exosome is 2,000 to 20,000 × g, or 2,000 to 15,000 × g, or 2,000 to 10,000 × g, or 3,000 to 10,000, of the culture medium of the acne-causing bacteria treated with the test substance After centrifugation at × g, the supernatant is collected and ultracentrifuged at 100,000 to 200,000 × g, or 120,000 to 180,000 × g, or 130,000 to 170,000 × g, or 100,000 to 150,000 × g, or 150,000 to 200,000 × g may include steps.

In an exemplary embodiment, the centrifugation may be performed for 10 to 50 minutes, and ultracentrifugation may be performed for 2 to 4 hours.

In addition, the centrifugation may be performed in stages by changing the speed or time. For example, the centrifugation is 2,000 to 5,000 × g, or 2,000 to 4,000 × g, or 3,000 to 4,000 × g, after centrifugation for 10 to 30 minutes, 8,000 to 20,000 × g, or 8,000 to 15,000 × g, or 8,000 to 12,000 x g, or 9,000 to 12,000 x g, for 10 to 30 minutes.

In an exemplary embodiment, the step of separating the exosomes is ultracentrifugation after centrifuging the culture solution of the acne-causing bacteria and then filtering with a filter of 0.2 to 0.5 μm, or 0.3 to 0.5 μm, or 0.4 to 0.5 μm. may include the step of It is possible to increase the purity of the centrifuged culture medium through the filtration process.

In an exemplary embodiment, the isolated exosomes may be sedimented in ultracentrifugation of 100,000 to 200,000 × g of the acne-causing bacteria culture medium. At this time. The acne causative bacteria culture medium may be a supernatant obtained by centrifuging the culture medium at 2,000 to 20,000 × g.

In an exemplary embodiment, the secretion amount of the exosome is, for example, the amount of exosomes separated by the same separation method from the same cell number by using a quantitative analysis equipment Q-Nano or NanoSight device, nanoparticle tracking analysis (NTA) of the particle It can be confirmed through counting or quantification of proteins, lipids on the surface of exosomes, or proteins, peptides, RNA, miRNA (microRNA), lncRNA (long noncoding RNA), metabolites inside the exosome, and the target to be quantified Western blot, dot blot, ELISA, northern blot, PCR, RT-qPCR, GC-MS, LC-MS, NMR, radioimmunoassay, immunoprecipitation Methods such as immunoprecipitation assay, radioimmunodiffusion, FACS, and protein chip may be appropriately selected by those skilled in the art.

In an exemplary embodiment, the method may include comparing the amount of the isolated exosomes with the amount of exosomes isolated from acne-causing bacteria that have not been treated with the test substance.

In an exemplary embodiment, the method may include evaluating the test substance as an anti-acne substance when the amount of the isolated exosomes is less than the amount of the exosomes isolated from acne-causing bacteria that have not been treated with the test substance. .

In an exemplary embodiment, when the amount of the isolated exosomes is reduced by 5% or more based on the amount of exosomes isolated from acne-causing bacteria that are not treated with the test substance, the test substance can be evaluated as an anti-acne substance. Specifically, when the amount of the isolated exosomes is reduced to 5% or more to 100% or less based on the amount of exosomes isolated from acne-causing bacteria that are not treated with the test substance, more specifically 10% or more, 20% The test substance can be evaluated as an anti-acne substance when it decreases by more than, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

Extracellular vesicles, more specifically exosomes, which are complex physiologically active substances secreted out of cells by acne-causing bacteria, increase the expression of inflammatory cytokine genes and proteins in keratinocytes and promote cell proliferation. By reducing the expression of genes and proteins of cell differentiation markers, the skin barrier is damaged and keratin differentiation is inhibited, which directly causes acne. Accordingly, by treating the acne-causing bacteria with the test substance, substances that reduce the amount of exosome secretion of the acne-causing bacteria can be screened as anti-acne substances.

The technology disclosed herein provides a method of screening an anti-acne agent by comparing the increase or decrease in the amount of exosomes derived from an acne-causing bacterium in the presence and absence of an anti-acne candidate substance, that is, a test substance. Specifically, a substance that indirectly or directly reduces the amount of exosome secretion may be selected as an anti-acne substance. That is, after comparing the amount of exosome secretion in the absence of the test substance and the amount of exosome secretion in the presence of the test substance, a substance that reduces the amount of exosome secretion in the presence of the test substance compared to the amount of exosome secretion in the absence of the test substance is an anti-acne substance can be selected as

Conventional anti-acne substances by inhibiting the growth of acne-causing bacteria have side effects other than anti-acne efficacy by releasing a large amount of side-effect-causing substances including exosomes in the process of killing the bacteria. On the other hand, the anti-acne substance that reduces the amount of exosome secretion disclosed herein suppresses the secretion of the exosome itself causing acne, thereby reducing side effects compared to conventional antibacterial agents.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

test example 1. From acne-causing bacteria of exosomes separation

Propionibacterium acnes (ATCC 6919), the causative agent of acne, was inoculated in Brain Heart Infusion (BHI) medium, pre-cultured at 35-37 °C anaerobic conditions for 72 hours, and the bacteria were collected by centrifugation. The collected bacteria were inoculated at a 1/100 dilution in BHI medium, and then cultured at 35-37° C. in anaerobic conditions for 72 to 96 hours to collect the culture medium. The collected culture solution was centrifuged at 4 °C, 3,000 × g for 10 minutes to precipitate bacteria, and the supernatant was collected, and then centrifuged at 4 °C and 10,000 × g for 20 minutes to collect the supernatant. Thereafter, the remaining bacteria were removed using a 0.45 μm bottle-top filter, and the solution passed through the filter was collected and ultracentrifuged at 4° C. and 150,000 × g for 3 hours. The precipitated exosomes were dissolved in HEPES-buffered saline, the size of the exosomes was measured using dynamic light scattering analysis, and the shape was observed with a transmission electron microscope, which is shown in FIG. 1 .

Test Example 2. Increased expression of inflammatory cytokine genes and proteins upon exosome treatment in keratinocytes

Human keratinocytes (Normal Human Epidermal Keratinocyte, NHEK) were cultured in a 6-well plate incubator, and after 24 hours, a medium containing 1, 5, and 10 μg/ml of exosomes derived from the acne causative bacteria isolated in Test Example 1 above. was replaced with After 24 hours, the cells are harvested, RNA is isolated, cDNA is synthesized through RT-PCR (reverse transcriptional polymerase chain reaction), and Taqman real-time PCR is performed using the synthesized cDNA to perform inflammatory cytokines IL-8, IL -6, the gene expression level of TNF-alpha was confirmed (see Fig. 2a). In addition, the culture medium of keratinocytes treated with the exosomes derived from the acne-causing bacteria was collected after 24 hours, and the corresponding cytokines were quantified using an IL-8 and IL-6 quantitative ELISA kit (see FIG. 2b ). As a result, it was confirmed that exosomes derived from the bacteria causing acne increase the expression of genes and proteins of inflammatory cytokines in keratinocytes.

Test Example 3. Promotion of cell proliferation when keratinocytes are treated with exosomes

Human keratinocytes (HaCaT cells) were cultured in a 6-well plate incubator and replaced with a medium containing 1, 5, 10, and 20 μg/ml of exosomes derived from the acne causative bacteria isolated in Test Example 1 after 24 hours. did After 24 hours of incubation, CCK-8 solution was added, and the absorbance was measured at 540 nm after incubation for 2 hours (see FIG. 3). As a result, it was confirmed that exosomes derived from the bacteria causing acne increase cell proliferation in keratinocytes.

Test Example 4. Reduction of the expression of genes and proteins of cell differentiation markers upon exosome treatment on keratinocytes

Human keratinocytes (NHEK) were cultured in a 6-well plate incubator, and after 24 hours, it was replaced with a medium containing 1, 5, and 10 μg/ml of exosomes derived from the acne causative bacteria isolated in Test Example 1. After 4 days of culture, cells are harvested, RNA is isolated, cDNA is synthesized through RT-PCR (reverse transcriptional polymerase chain reaction), and Taqman real-time PCR is performed using the synthesized cDNA to perform keratinocyte differentiation marker (KRT1). , KRT10) was measured (see Fig. 4a). In addition, protein expression of keratinocyte differentiation markers (KRT1, KRT10) was confirmed through Western blotting by extracting keratinocyte proteins (see FIG. 4b ). As a result, it was confirmed that the exosomes derived from the acne causative bacteria reduced the expression of genes and proteins of cell differentiation markers in keratinocytes.

test example 5. anti-acne screening of substances

Propionibacterium acnes (ATCC 6919), the causative agent of acne, was inoculated in Brain Heart Infusion (BHI) medium, pre-cultured at 35-37 °C anaerobic conditions for 72 hours, and the bacteria were collected by centrifugation. The collected bacteria were inoculated at a 1/100 dilution in BHI medium, and then the test material was treated, and the culture medium was collected and the number of bacteria was counted by culturing for 72 to 96 hours in an anaerobic condition at 35 to 37°C. The collected culture solution was centrifuged at 4 °C, 3,000 × g for 10 minutes to precipitate bacteria, and the supernatant was collected, and then centrifuged at 4 °C and 10,000 × g for 20 minutes to collect the supernatant. Thereafter, the remaining bacteria were removed using a 0.45 μm bottle-top filter, and the solution passed through the filter was collected and ultracentrifuged at 4° C. and 150,000 × g for 3 hours. Dissolve the precipitated exosomes in HEPES-buffered saline to check the amount of exosome secretion, but the amount of exosome secretion (secreted from the same number of bacteria) derived from Propionibacterium acnes (ATCC 6919) cultured in the same manner as above without treatment with the test substance The amount of exosomes) was compared to evaluate whether the test substance was an anti-acne substance. The amount of exosome secretion was confirmed by quantification of exosome protein using Bradford protein assay.

As mentioned above, specific parts of the present invention have been described in detail, for those of ordinary skill in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be obvious. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (10)

treating the test substance to acne-causing bacteria;
Separating the exosomes from the acne-causing bacteria treated with the test substance; and
A method of screening an anti-acne substance comprising a; confirming the amount of the isolated exosome.
The method of claim 1,
The acne-causing bacterium is Propionibacterium acnes ( Propionibacterium acnes ), a method of screening an anti-acne substance.
The method of claim 1,
A method for screening an anti-acne substance, comprising the step of culturing the acne-causing bacteria for 72 to 96 hours after treating the acne-causing bacteria with the test substance.
The method of claim 1,
The step of separating the exosomes comprises centrifuging the culture solution of the acne-causing bacteria treated with the test substance at 2,000 to 20,000 × g, then collecting the supernatant and ultracentrifuging at 100,000 to 200,000 × g, anti- How to screen for acne substances.
5. The method of claim 4,
The centrifugation is a method of screening an anti-acne substance, comprising centrifuging at 2,000 to 5,000 × g and then centrifuging at 8,000 to 20,000 × g.
5. The method of claim 4,
Separating the exosomes, centrifuging the culture solution of the acne-causing bacteria, and then filtering with a filter of 0.2 to 0.5 μm, followed by ultracentrifugation, a method for screening anti-acne substances.
The method of claim 1,
The isolated exosomes will have a diameter of 20 to 500 nm, a method of screening an anti-acne substance.
The method of claim 1,
The separated exosomes are sedimented in ultracentrifugation of 100,000 to 200,000 × g of the acne causative bacteria culture medium, the method of screening for anti-acne substances.
The method of claim 1,
A method for screening an anti-acne substance, comprising comparing the amount of the isolated exosomes with the amount of exosomes isolated from acne-causing bacteria that have not been treated with the test substance.
10. The method of claim 9,
A method for screening an anti-acne substance, comprising the step of evaluating the test substance as an anti-acne substance when the amount of the isolated exosome is less than the amount of the exosome isolated from the acne-causing bacteria that has not been treated with the test substance.

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