JP5513028B2 - Method for evaluating or selecting skin photodamage prevention or healing agent - Google Patents

Description

  The present invention relates to a method for evaluating or selecting a skin photodamage prevention or healing agent.

Skin photodamage is known to cause quantitative and qualitative changes in both the epidermis and dermis, but the changes that occur in the dermis are widely known. That is, the cells that make up the dermis fibers become smaller and fewer with chronic exposure to sunlight, especially the collagen fibers are greatly lost, and the elasticity of the skin is lost due to degeneration of the dermis, reduction of subcutaneous fat tissue, etc. Changes in shape, such as the skin becoming loose or the surface of the skin becoming rough.
In order to elucidate the mechanism of such photodamaged skin and to evaluate pharmaceuticals useful for the prevention and healing of skin troubles caused by photodamage, there is a system that can more accurately reflect human photodamaged skin. Useful.

  Photoaging is the result of photodamage, but as a model skin or model animal, a system that is exposed to ultraviolet rays is most commonly used. For example, UV rays are applied daily to hairless mouse dorsal skin and rat sole skin. There are known methods for continuous irradiation for several weeks (Non-Patent Documents 1 and 2). However, it is considered more desirable to use human skin itself that reflects the dynamics in the subcutaneous tissue or transplanted human skin.

  As a model using human skin, there is known a method in which a nude mouse (SCID mouse) into which human skin is transplanted is subjected to a single ultraviolet irradiation (Non-patent Document 3). Because of UV irradiation in a short period rather than a model system that continuously irradiates UV, it is not possible to sufficiently observe the photodamaged state of human skin due to UVB long-term irradiation. Therefore, a model animal having human photodamaged skin that is very close to photodamage has been created by continuously irradiating a specific amount of UVB for a specific time (Non-Patent Document 4).

  On the other hand, matrix metalloproteinase (MMP) is an enzyme that degrades extracellular matrix such as collagen, elastin, and proteoglycan, and has been reported to be involved in photodamage. In light-damaged skin, it has been reported that an imbalance of MMP-1 simultaneously causes a decrease in collagen I and III due to a decrease in procollagen synthesis and a degradation of collagen in the dermis (Non-patent Document 5). Moreover, in the skin where photodamage is conspicuous, decrease in collagen VII and IV at the epidermal dermis junction was observed, and in experiments using hairless mice, repeated UVB irradiation induced the production of MMP-2 and MMP-9. It has been reported that the degradation of collagen VII and IV is increased (Non-patent Document 6).

It is known that the activity of MMP is inhibited by TIMP (tissue inhibitor of metalloproteinases), and TIMP exerts an inhibitory action by forming a complex with active MMP at a ratio of 1: 1. Therefore, normalizing the balance between MMP activity and TIMP activity is thought to be useful for preventing skin aging, and so far, for example, Murasaki cultured cell extract and caffeic acid polymer have been found to have MMP inhibitory activity. (Patent Document 1). Currently, TIMP-1, TIMP-2, TIMP-3, and TIMP-4 are known as TIMP types. In the above-mentioned patent documents, TIMP-2 activity enhancing action is found in lysospermic acid.
Patent Document 2 describes that by combining retinol and sitosterol, the contents of MMP-1 and MMP-3 related to intrinsic aging are reduced, and the content of TIMP-1 is increased. However, in the data shown in the examples, the contents of MMP-1 and MMP-3 are not necessarily decreased by the combination, and similarly, the content of TIMP-1 is not increased. Further, Patent Document 2 is not related to optical damage.

Regarding TIMP-1, further, the expression level of TIMP-1 mRNA is increased by a single irradiation of UVB (Non-patent Document 7). On the other hand, the expression level of TIMP-1 protein is changed by a single irradiation of UVB. Not reported (Non-Patent Document 8).
However, the influence of long-term UVB irradiation on TIMP-1 has not been known so far, and the relationship between skin photodamage and TIMP-1 has not been known.

JP 2008-31088 A JP 2000-86489 A

Bissett DL, Hannon DP, Orr TV, Photochem Photobiol. 46 (3), 367-78 (1987) Imayama S, Nakamura K, Takeuchi M, Hori Y, Takema Y, Sakaino Y, Imokawa G, J Dermatol Sci. 7 (1), 32-8 (1994) Del Bino S, Vioux C, Rossio-Pasquier P, Jomard A, Demarchez M, Asselineau D, Bernerd F, Br J Dermatol. 150 (4), 658-67 (2004) Hachiya A, et al., Am J Pathol, 174, 401 (2009) Brennan M, et al., Photochem Photobiol 78, 43-48 (2003) Contet-Audonneau JL, et al., Br J Dermatol 140, 1038-1047 (1999) Brenneisen P, et al., Photochem Photobiol 64, 877-885 (1996) Brenneisen P, et al., Biochem J 365, 31-40 (2002)

  The present invention relates to providing a method for evaluating or selecting a substance that prevents or cures skin photodamage.

  The present inventors showed that TIMP-2 expression of TIMPs was low in the inner skin of the upper arm where the exposure to ultraviolet rays was low, whereas in the skin where long-term UVB irradiation was performed on human transplanted skin, photodamage was induced. No changes were observed in the expression of TIMP-2 and TIMP-3, and only the expression of TIMP-1 was found to be significantly reduced. Furthermore, it was found that the surface roughness of the human transplanted skin increases when the TIMP-1 neutralizing antibody is intradermally administered immediately after UVB is irradiated to the human transplanted skin. Therefore, TIMP-1 is involved in the prevention and healing of surface shape change in photodamage, and a substance that can prevent or cure photodamage of skin by using the TIMP-1 production increasing action as an index Has been found to be more accurately evaluated or selected.

That is, the present invention includes the following steps (A) to (D):
(A) a step of bringing a test substance into contact with a cell capable of expressing a TIMP-1 gene or TIMP-1 protein;
(B) a step of measuring the expression level of TIMP-1 gene or TIMP-1 protein in the cell,
(C) Expression of TIMP-1 gene or TIMP-1 protein in a control group in which the expression level calculated in (B) above is not brought into contact with a cell capable of expressing TIMP-1 gene or TIMP-1 protein. Process to compare with the quantity,
(D) A step of evaluating or selecting a test substance that increases the expression level of the TIMP-1 gene or TIMP-1 protein as a preventive or healing agent for skin photodamage based on the result of (C) above,
A method for evaluating or selecting a preventive or healing agent for skin photodamage, comprising:

  According to the present invention, it is possible to more accurately and simply evaluate the prevention or healing effect of various substances on skin photodamage represented by surface shape change, and more accurately determine substances having the prevention or improvement effect. It becomes possible to select.

FIG. 1 is a graph showing the effect of neutralizing antibody on photodamage using as an index the surface shape change of human transplanted skin to which TIMP-1 neutralizing antibody was administered intradermally. Upper row: non-specific IgG administration, UV non-irradiated group, middle row: non-specific IgG administration, UV irradiation group, lower row: TIMP-1 neutralizing antibody administration, UV irradiation group. FIG. 2 is a diagram showing mRNA expression of TIMPs. A) Relative ratio of the expression level of TIMP in the inner skin of the upper arm not irradiated with ultraviolet light, B) Change in the expression level of each TIMP after ultraviolet irradiation in human transplanted skin. FIG. 3 is a diagram showing an outline of a screening method for a preventive or healing agent for skin photodamage.

  Examples of cells capable of expressing the TIMP-1 gene or TIMP-1 protein used in the present invention include mammalian skin fibroblasts, lung fibroblasts, fibroblasts such as gingival fibroblasts, cartilage synovial cells, and the like. Is mentioned. Examples of mammals include humans, mice, rats, and rabbits. These cells may be primary culture cells or in vitro aged cells after repeated passages, and may be fetal or aged. In addition, these cells may be collected from a tissue by a known method and used, or commercially available products may be purchased. Among these, human-derived fibroblasts are preferable, and human-derived skin fibroblasts are more preferable.

For the contact between the cell and the test substance, for example, after adding the test substance to the culture solution in advance to a predetermined concentration, the cell is placed in the culture solution, or the cell is placed. It can be performed by adding the test substance to the culture solution so as to have a predetermined concentration.
Here, the cell concentration at the time of seeding of cells capable of expressing the TIMP-1 gene or TIMP-1 protein is preferably 0.5 × 10 ^{3 to} 1.0 × 10 ⁵ cells / well when a 24-well plate is used. In particular, it is preferably 1.0 × 10 ^{3 to} 3.5 × 10 ⁴ cells / well. Further, the concentration of the substance to be added is preferably 0.00001 to 10% by mass (solid residue), and particularly preferably 0.0001 to 3% by mass (solid residue).

  The test substance is not particularly limited as long as it is expected to directly or indirectly affect the photodamage of the skin. For example, animal and plant extracts, compounds, chemical substances and the like can be used.

  As a medium for culturing cells capable of expressing the TIMP-1 gene or TIMP-1 protein, a conventional medium capable of culturing the cells can be used, and examples thereof include 10% FBS-containing Dulbecco's Modified Eagle's Medium. It is preferable to add growth additives such as growth factors, antibacterial agents, insulin, and hydrocortisone to these media during cell passage and growth. Next, the culture supernatant is collected and the expression level of the TIMP-1 gene or TIMP-1 protein is measured.

  When measuring the expression level of TIMP-1 gene at the mRNA level, for example, extract total RNA from cells and use real-time RT-PCR, RNase protection assay, Northern blot analysis, etc. Then, the mRNA transcribed from the TIMP-1 gene may be detected and quantified.

  Further, the expression level of TIMP-1 protein can be measured by an ordinary immunoassay, for example, RIA, EIA, ELISA, bioassay, Western blot, etc. Inexpensive, simple and desirable.

  Evaluation of prevention or healing agent for skin photodamage is performed by measuring the expression level of TIMP-1 gene or TIMP-1 protein in cells capable of expressing TIMP-1 gene or TIMP-1 protein in contact with the test substance. When the expression level increases compared to the expression level in the control group (control cells) not contacted with the substance, the test substance can be evaluated as having a skin photodamage prevention or healing effect, and the substance is selected. be able to. Substances evaluated or selected in this way include, for example, pharmaceuticals for preventing or curing skin photodamage represented by surface shape changes such as loss of skin elasticity, skin relaxation, and skin surface roughness. Can be.

Test Example 1 Examination of surface shape change of transplanted skin by intradermal administration of TIMP-1 neutralizing antibody (1) Abdominal skin transplantation to severe combined immunodeficient (SCID) mice
A white (Caucasian, 41 year old) female abdominal skin from an abdominoplasty operation at Cincinnati University Hospital (Cincinnati, OH) is trimmed to an appropriate size (approx. 2.0 cm x 2.0 cm to approx. 3.0 cm x 3.0 cm) and Phosphate Buffered After washing with Saline (PBS), it was immersed in Dulbecco's Modified Eagle's Medium (DMEM) containing L-glutamine and antibiotic / antimycotic (Invitrogen) and stored until transplantation. After shaving the back of a 4-6 week old female SCID mouse (Taconic, NY) acclimatized for about 1 week at the Cincinnati Children's Hospital Animal Facility (Children's Hospital Research Foundation, Cincinnati, OH), isofluorane / oxygen (3 % / 0.8 liter). Thereafter, the mouse back skin was excised (diameter: about 2 to 3 cm) while aspirating isofluorane / oxygen (2% / 0.7 liter), and the human abdominal skin was sutured to the mouse skin. Sensorcaine was added to the boundary between the transplanted abdominal skin and mouse skin to treat sensory loss. After transplantation, the mice were maintained at 37 ° C. for 1 hour or until they woke up from anesthesia.

(2) Induction of photodamage to human transplanted abdominal skin by chronic UVB irradiation and intradermal administration of TIMP-1 neutralizing antibody When 10 weeks have passed since transplanted abdominal skin was completely healed, Photodamage was induced in human transplanted skin by the method shown (Hachiya A, et al., Am J Pathol, 174, 401 (2009)). That is, irradiation was started using a UVB lamp (34-0044-01 lamps, UVB, Upland, CA) with a filter irradiated with UVB having a peak at 302 nm. The amount of UVB energy was measured using a UV LIGHT METER, UV-340 MSR7000 (Lutron Electronic Enterprise Co. ltd, Taiwan). The distance from the filter to the surface of the transplanted skin was about 30 cm, and 40 mJ / cm ² of UVB corresponding to 1 minimal erythema dose (MED) was irradiated for the first week. The dose was increased by 10 mJ / cm ² until the third week, and then maintained at 60 mJ / cm ² until the sixth week. Since it was irradiated for 5 days a week, it means that a total of 1.65 J / cm ² of UVB was irradiated. During UVB irradiation, the mouse was allowed to move freely in a transparent container having a bottom area of about 100 cm ² . Moreover, the TIMP-1 neutralizing antibody was intradermally administered immediately after UVB irradiation every other day. The administration concentration was set to 20 μg / mL corresponding to 4 times the concentration used in Reed MJ, et al., Microvasc Res, 65, 9 (2003).

(3) Results
The shape change induced on the transplanted abdominal skin by UVB irradiation was visually observed. In the group that received nonspecific IgG intracutaneously without UVB irradiation (the upper part of Figure 1), there was no significant change in appearance, whereas nonspecific IgG was peeled while continuously irradiating UVB. In the internally administered group, a rough structure began to be observed on the surface of the skin after 4 weeks, and it was clearly formed after 6 weeks of continuous irradiation (middle of FIG. 1). On the other hand, in the group in which TIMP-1 neutralizing antibody was intradermally administered while continuously irradiating UVB (lower part of Figure 1), it was continuous for 4 weeks earlier than the group in which nonspecific IgG was intradermally administered while continuously irradiating UVB At the time of irradiation, it was confirmed that the surface of the skin was clearly rough. Moreover, remarkable desquamation as well as the surface roughness was observed.

Test Example 2 Examination of mRNA expression level of TIMPs in skin (1) Expression of each TIMPs in non-ultraviolet irradiated skin
We asked the Dallas contract laboratory, Stephens & Associates, Inc. (Carrollton, TX), to collect the inner skin of the upper arm of Caucasian women in their 20s and 50s with a punch biopsy, and always use a portion of the skin. Total RNA was extracted according to the method. After preparing cDNA, quantitative gene expression analysis of TIMP-1, TIMP-2 and TIMP-3 was performed using specific probe and 7300 Real Time PCR System (Applied Biosystems). RPLP0 expression was used as an internal standard for gene expression analysis.

(2) Expression of each TIMPs after ultraviolet irradiation The human skin collected in (1) above was subjected to continuous irradiation (40-60 mJ / cm ² ) for 6 weeks. RNA was extracted, cDNA was prepared, and quantitative gene expression analysis of TIMP-1, TIMP-2 and TIMP-3 was performed using a specific probe and 7300 Real Time PCR System (Applied Biosystems). As an internal standard, the expression of RPLP0 was used as described above.

(3) Results When the relative expression levels of TIMP-1, TIMP-2 and TIMP-3 were analyzed using total RNA derived from the inner skin of the upper arm, the expression ratio was 5: 1: 5 (1: 0.2: 1) (Figure 2A)
In addition, the expression levels of TIMP-1, TIMP-2, and TIMP-3 in photodamaged model skin and UVB-unimplanted skin were compared. TIMP-1 expression was prominent in chronic UVB-irradiated photodamage. On the other hand, the expression of TIMP-2 and TIMP-3 did not change even during photodamage (Figure 2B).
From the above results, it was considered that among TIMPs, a marked decrease in the expression of TIMP-1 particularly contributes to the enhancement of photodamage.

Test Example 3 Screening for Preventing or Curing Skin Photodamage (1) Method The outline of this screening method is shown in FIG. That is, normal human skin fibroblasts were seeded on a 24-well plate at a cell density of 2 × 10 ⁴ cells / well, and culture was started using 0.5 mL of 10% FBS-containing DMEM (Sigma). After 24 hours, the medium was replaced with 0.5 mL of serum-free medium, and at the same time, various plant extracts (50% EtOH extract) were added as test substances. The extract concentration was 0.001% (solid residue concentration), and an equivalent amount of 50% EtOH was used as a control. Forty-eight hours after the addition of the extract, the medium and cells were collected as measurement samples. The medium was collected as it was from each well, centrifuged at 12,000 rpm for 2 minutes, and the amount of TIMP-1 protein in the supernatant was measured using a human TIMP-1 assay system (GE Healthcare Biosciences). .
Further, after recovering the medium from each well, the cells were washed twice with 0.5 mL of PBS, and 125 μL of 0.1 N NaOH was added and the cells were lysed at room temperature for about 5 minutes, and recovered as a cell lysate. The amount of protein in this cell lysate was measured using BCA Protein Assay Kit (Pierce) to evaluate the toxicity of various plant extracts added.

(2) Results When the effects of various plant extracts (extraction solvent 50% EtOH) on TIMP-1 production were evaluated, the amount of TIMP-1 protein was 2.0 relative to the control (control group) in perilla extract and mint. More than doubled (Table 1). In addition, proteins with a protein amount ratio of 0.5 or less with respect to the control (50% EtOH addition) were excluded. In addition, no cytotoxicity was observed in any plant extract.
Perilla extract is already known as a photoaging protection agent (JP 2002-104924 A), and mint extract (peppermint) is known as a collagenase (MMP-1) inhibitor (JP 2000-2000 A). -159631).
From these results, it was confirmed that a substance having a preventive or healing effect against photodamage of the skin can be evaluated or selected using the TIMP-1 production increasing action as an index.

Claims (3)

The following steps (A) to (D):
(A) a step of bringing a test substance into contact with a cell capable of expressing a TIMP-1 gene or TIMP-1 protein;
(B) a step of measuring the expression level of TIMP-1 gene or TIMP-1 protein in the cell,
(C) Expression of TIMP-1 gene or TIMP-1 protein in a control group in which the expression level calculated in (B) above is not brought into contact with a cell capable of expressing TIMP-1 gene or TIMP-1 protein. Process to compare with the quantity,
(D) A step of evaluating or selecting a test substance that increases the expression level of TIMP-1 gene or TIMP-1 protein as a preventive or curative agent for skin photodamage based on the result of (C) above,
A method for evaluating or selecting a prophylactic or healing agent for skin photodamage. The method for evaluating or selecting a preventive or curative agent for skin photodamage according to claim 1, wherein the cells capable of expressing the TIMP-1 gene or TIMP-1 protein are human-derived skin fibroblasts. 3. The method for evaluating or selecting a skin photodamage prevention or healing agent according to claim 1 or 2, wherein the skin photodamage is skin photodamage induced by prolonged UVB irradiation.