CN113260421A

CN113260421A - Aging inhibitor

Info

Publication number: CN113260421A
Application number: CN201980085018.7A
Authority: CN
Inventors: 加治屋健太朗; 高垣知辉; 渡部彻郎; 吉松康裕
Original assignee: Shiseido Co Ltd; Tokyo Medical and Dental University NUC
Current assignee: Shiseido Co Ltd; Tokyo Medical and Dental University NUC
Priority date: 2018-12-21
Filing date: 2019-12-20
Publication date: 2021-08-13
Also published as: JPWO2020130148A1; WO2020130148A1

Abstract

The present invention provides an endothelial mesenchymal transition inhibitor and a lymphatic vessel aging inhibitor that inhibits aging of a lymphatic vessel by inhibiting endothelial mesenchymal transition of the lymphatic vessel. An endothelial mesenchymal transition inhibitor containing cortex Mori and/or folium Psidii Guajavae as effective components is provided. By applying the endothelial mesenchymal transition inhibitor of the invention, the endothelial mesenchymal transition of lymphatic vessels can be inhibited. By inhibiting endothelial mesenchymal transition of lymphatic vessels, lymphatic aging can be inhibited.

Description

Aging inhibitor

Technical Field

The present invention relates to a senescence inhibitor, in particular, an endothelial mesenchymal transition inhibitor containing cortex mori and/or guava leaves as an active ingredient, and a lymphatic vessel senescence inhibitor for inhibiting lymphatic vessel senescence by inhibiting lymphatic vessel endothelial mesenchymal transition.

Background

The lymphatic vessels play a role as drains for recovering metabolites such as inflammatory cells, proteins, and water from the interstitial spaces of the cells. On the other hand, it is known that if the function of lymphatic vessels is impaired due to aging, various problems such as the inhibition of excretion of metabolites and edema occur. Therefore, a substance for preventing/improving lymphatic aging is desired.

In recent years, it has been reported that endothelial cells of lymphatic-like schlemm's canal are differentiated and transformed into mesenchymal cells in the eyeball to impair the aqueous humor excretory function, resulting in aging-associated glaucoma (non-patent document 1). In addition, it has been reported that endothelial mesenchymal transition of vascular endothelial cells is involved in the progression of tumors and the like.

However, it is not clear whether or not endothelial mesenchymal transition of endothelial cells is involved in lymphatic aging in organs such as the skin. In addition, it is necessary to search for a substance that inhibits the differentiation and transformation of lymphatic vessels into mesenchymal cells.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open No. 2014-218479

Patent document 2: japanese patent laid-open No. 2007-22957

Patent document 3: international publication No. 2007/007732

Non-patent document

Non-patent document 1: the Journal of Clinical Investigation, 2017; 127(10): 3877-3896

Disclosure of Invention

Problems to be solved by the invention

The present invention addresses the problem of providing an inhibitor of lymphatic endothelial mesenchymal transition, and an inhibitor of lymphatic aging that inhibits lymphatic aging by inhibiting lymphatic endothelial mesenchymal transition.

Means for solving the problems

The present inventors have confirmed that the differentiation and transformation of lymphatic endothelial cells into mesenchymal cells in organs such as skin are involved in the aging of lymphatic vessels. Therefore, if differentiation and transformation of lymphatic vessels into mesenchymal cells can be suppressed, aging of lymphatic vessels in various organs of the body can be suppressed, and further, it is expected that this can contribute to aging suppression of the organs. The present inventors have made intensive studies on the effect of various components as an endothelial mesenchymal transition inhibitor for lymphatic vessels, and as a result, have found that cortex mori and guava leaves have particularly high effects, and have completed the following inventions:

(1) an endothelial mesenchymal transition inhibitor for lymphatic vessels contains cortex Mori and/or folium Psidii Guajavae as effective components.

(2) The inhibitor of endothelial mesenchymal transition according to (1), which inhibits endothelial mesenchymal transition of lymphatic vessels by inhibiting TGF- β (transforming growth factor- β).

(3) A lymphatic vessel aging inhibitor contains cortex Mori and/or folium Psidii Guajavae as effective components, and can inhibit lymphatic vessel aging by inhibiting lymphatic endothelial mesenchymal transition.

(4) The lymphatic aging inhibitor according to (3), which inhibits endothelial mesenchymal transition of lymphatic vessels by inhibiting TGF-. beta.s.

(5) TGF-beta inhibitor comprising guava leaves as an active ingredient.

(6) A composition comprising the endothelial mesenchymal transition inhibitor of (1) or (2), the lymphatic aging inhibitor of (3) or (4), and/or the TGF- β inhibitor of (5).

(7) A cosmetic method for preventing lymphatic aging in a subject, comprising administering the composition of (6) to the subject.

(8) A cosmetic counseling method for supporting cosmetic behavior in a subject, comprising recommending to the subject the composition of (6).

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, an endothelial mesenchymal transition inhibitor containing cortex mori and/or guava leaves as an active ingredient can be provided. By administering the endothelial mesenchymal transition inhibitor of the present invention, endothelial mesenchymal transition can be inhibited. By inhibiting endothelial mesenchymal transition of lymphatic vessels, it is also possible to inhibit aging of lymphatic vessels.

Drawings

FIG. 1 shows the expression of LYVE1 and SM22 α in young and advanced age groups. The upper panel is a representative diagram showing the expression of LYVE1 and SM22 α in endothelial cells of cutaneous lymphatic vessels of young and old age groups. The lower graph is a graph showing the ratio (EndMT ratio) of expression amounts of LYVE1 and SM22 α in young and old groups (n.8) (student's t-test, p < 0.05).

FIG. 2 shows the expression levels of Prox1 (upper panel) and VEGFR3 (lower panel) in human skin-derived lymphatic endothelial cells (HDLEC) for each culture passage. The vertical axis shows the ratio (%) of the expression level in each culture passage, assuming that the expression level in the fifth passage cells (kk2 p5) is 100. The horizontal axis shows the number of cultured passages from the fifth passage cell.

FIG. 3 shows the expression levels of LYVE1 and SM22 α in HDLEC. In each figure, the results of addition of SB431542(SB), 0.1% BSA/4mM HCl (Ctrl, control), and TGF-. beta. (TGF-. beta.) as controls are shown from the left (T test, p < 0.001).

FIG. 4 shows the inhibitory effect of TGF-. beta.in various samples. The results of addition of PBS alone (cont, control), TGF-beta alone (TGFb }, TGF-beta + SB431542(TGFb + SB), and TGF-beta + various samples (1 to 18) are shown.13 for the guava leaf extract and 15 for the mulberry bark extract.

FIG. 5 shows the results of determination of the expression amounts of LYVE1 and Prox1 in HDLEC by RT-PCR. The left panel shows the expression level of LYVE1, and the right panel shows the expression level of Prox 1. In each figure, the cases where PBS alone, TGF- β alone, SB431542+ TGF- β, cortex Mori extract alone, and TGF- β + cortex Mori extract were added are shown in order from the left.

FIG. 6 shows the results of determination of the expression amount of SM22 α in HDLEC by RT-PCR. The cases of addition of PBS only, TGF-beta only, SB431542+ TGF-beta, cortex Mori extract only, and TGF-beta + cortex Mori extract are shown in order from the left.

FIG. 7 shows the morphological changes of HDLEC caused by the addition of cortex Mori and TGF- β. The upper panel shows the case where only SB (SB), only BSA/4mM HCI (blank), and TGF- β (TGF- β) are added, and the lower panel shows the case where only SB431542+ TGF- β (SB + TGF- β), only the cortex Mori extract (extract), and the cortex Mori extract + TGF- β (extract + TGF- β) are added, in order from the left.

Fig. 8 shows the results of the measurement for determining the permeability of HDLECs. From the left, the results of no additive (control) and addition of TGF-beta alone (TGF-beta 2), TGF-beta + SB431542 (TGF-beta 2+ SB), and TGF-beta + cortex Mori extract (TGF-beta 2+15) are shown (Danete's multiple test, mp < 0.01). The vertical axis represents the absorbance of FITC-dextran leaked from the insert (insert) of each group, with the relative value of 1 in the case of no addition.

FIG. 9 shows the results of measuring the expression level of SM22alpha by RT-PCR. The results of addition of PBS only (control), TGF-beta only (TGF-beta), TGF-beta + SB431542 (TGF-beta + SB), and TGF-beta + guava leaf extract (TGF-beta + guava leaf) are shown from the left with the relative value of 100 in the case of addition of the control (control) (Danete multiplex test, p < 0.001).

Detailed Description

By applying the endothelial mesenchymal transition inhibitor of the invention, the endothelial mesenchymal transition of lymphatic vessels can be inhibited. By inhibiting endothelial mesenchymal transition of lymphatic vessels, the aging of lymphatic vessels can be inhibited. The endothelial mesenchymal transition inhibitor and the senescence inhibitor of the present invention are useful for excretion of metabolites, prevention or improvement of edema, promotion of metabolism, and prevention or treatment of lymphatic dysfunction. For example, by maintaining the function of the lymphatic vessels of the skin well, the removal of metabolites, the circulation of lymph fluid, and the like are normally performed, and as a result, the prevention of skin aging such as spots, wrinkles, and sagging can be expected.

The term "lymphatic endothelial mesenchymal transition" refers to the differentiation and transformation of lymphatic endothelial cells into mesenchymal cells (hereinafter, referred to as "intrinsic-mesenchymal transition"; hereinafter, sometimes abbreviated as "EndMT"). Endothelial mesenchymal transition of lymphatic vessels can be measured by decrease in the expression amount of an endothelial cell marker in lymphatic endothelial cells, increase in the expression amount of a mesenchymal cell marker, and increase in the ratio of the expression amount of a mesenchymal cell marker to that of an endothelial cell marker (EndMT ratio: calculated by the method of formula 1 below), and the like. Examples of endothelial cell markers include LYVE1, Prox1, VEGFR 3; examples of the mesenchymal cell marker include SM22 α, but the present invention is not limited thereto, and any endothelial cell marker and mesenchymal cell marker can be used, and the EndMT ratio as the ratio of these markers is also any. Further, since morphological changes such as fibrosis are observed when endothelial cells are differentiated and transformed into mesenchymal cells (fig. 7), the cells can be also measured by morphological observation. Furthermore, when endothelial cells are differentiated into mesenchymal cells, the function of the lymphatic vessels is impaired due to a decrease in the number of lymphatic endothelial cells or failure to maintain the properties of the lymphatic endothelial cells, and the fluid flowing through the lymphatic vessels leaks out. Such deterioration of lymphatic vessel function can be confirmed by measuring the permeability of an insert in which lymphatic endothelial cells are formed, for example, by the permeability measurement described in the examples. That is, endothelial mesenchymal transition of lymphatic vessels can be confirmed by hyperfiltration obtained by a permeability measurement.

[ formula 1]

The inhibition of lymphatic endothelial mesenchymal transition refers to inhibition of lymphatic endothelial cell differentiation into mesenchymal cells.

The term "inhibition of lymphatic endothelial mesenchymal transition" may mean that, if a lymphatic endothelial mesenchymal transition inhibitor is added, the amount of expression of a mesenchymal cell marker and/or the amount of expression of an EndMT ratio and the amount of expression of an endothelial cell marker in lymphatic endothelial cells are inhibited from decreasing compared to the case where the lymphatic endothelial mesenchymal transition inhibitor is not added. Inhibition may be, for example, a reduction in statistically significant differences (e.g., student's t-test) with a significance level of 5%, and/or may be, for example, inhibition of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100%.

Here, TGF-. beta.was reported to have an effect of inducing endothelial mesenchymal transition. Therefore, when the inhibitory effect of various agents on the endothelial mesenchymal transition of lymphatic vessels is measured, for example, the endothelial mesenchymal transition of lymphatic vessels can be promoted by adding a substance that promotes the endothelial mesenchymal transition, such as TGF- β. For example, the expression level of the mesenchymal cell marker and/or the increase in EndMTratio and the decrease in the expression level of the endothelial cell marker may be promoted by TGF-. beta.s.

TGF-. beta.is a multifunctional cytokine identified as a homodimer (molecular weight of about 25kDa) of a factor that promotes fibroblast transformation, and three subtypes (TGF-. beta.1 to 3) having similar structures exist in mammals such as humans. TGF- β exerts various actions of controlling cell proliferation, differentiation, and occurrence of individuals by binding to a receptor on a cell membrane. TGF- β is also known to inhibit proliferation of various cells such as epithelial cells, vascular endothelial cells, and lymphocytes, and is considered to be involved in various conditions such as cancer and renal fibrosis.

The present inventors confirmed that the expression level of endothelial cell markers in lymphatic endothelial cells decreased with age, the expression level of mesenchymal cell markers and/or the EndMT ratio increased, the number of lymphatic endothelial cells decreased, and the morphology of endothelial cells changed. Therefore, aging of lymphatic vessels may mean decrease in expression level of endothelial cell markers such as LYVE1, Prox1 and VEGFR3 in lymphatic endothelial cells with age, increase in expression level of mesenchymal cell markers such as SM22 α and/or endomt ratio, decrease or sclerosis of lymphatic endothelial cells, morphological change (e.g., fibroblast-like change) of lymphatic endothelial cells, or impairment of lymphatic function due to decrease and/or sclerosis of lymphatic endothelial cells. The endothelial cell marker, the mesenchymal cell marker, and the EndMT ratio are not limited to the above.

Lymphatic senescence inhibition refers to inhibition of lymphatic senescence as described above, and may be achieved via inhibition of lymphatic endothelial mesenchymal transition.

The lymphatic senescence inhibition may be, for example, a method in which, if a lymphatic senescence inhibitor is added, the expression level of a mesenchymal cell marker in lymphatic endothelial cells and/or the increase in EndMT ratio and the decrease in the expression level of an endothelial cell marker associated with senescence are inhibited, as compared to the case where the lymphatic senescence inhibitor is not added. The inhibition may be, for example, a decrease in the expression level of the marker having a statistically significant difference (e.g., student's t-test) with a significance level of 5%, and/or may be, for example, an inhibition of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

Here, when the senescence inhibitory effect of various agents on lymphatic vessels is measured, for example, it is possible to promote the endothelial mesenchymal transition of lymphatic vessels by adding TGF- β or the like, thereby promoting senescence. For example, the expression level of the mesenchymal cell marker and/or the increase in EndMT ratio and the decrease in the expression level of the endothelial cell marker due to aging can be promoted by using TGF-. beta.s.

Cortex Mori refers to root bark of a plant belonging to the genus Morus of the family Moraceae, such as white mulberry (Morus aIba Linne). Cortex mori has been used as a chinese medicine since ancient times, and its diuretic, analgesic, anti-inflammatory and antitussive effects have been reported (patent documents 1 and 2). Moreover, it has been reported that cortex mori has TGF- β inhibitory effect and hair growth promoting effect (patent document 2). Guava leaves refer to leaves of plants belonging to genus guava of family Myrtaceae, such as guava (Psidium guajava). Guava leaves have been reported to have effects of diabetes, hypertension, obesity, diarrhea, and the like (patent document 3). However, it is not known that cortex mori radicis and/or guava leaves have an endothelial mesenchymal transition inhibitory effect and/or a lymphatic aging inhibitory effect on lymphatic vessels in organs such as the skin.

The lymphatic mesenchymal transition inhibitor or lymphatic aging inhibitor of the present invention may contain, for example, 10 mass% or more, 20 mass% or more, 30 mass% or more, 40 mass% or more, 50 mass% or more, 60 mass% or more, 70 mass% or more, 80 mass% or more, 90 mass% or more, 95 mass% or more, or 99 mass% or more of cortex mori and/or guava leaf as an active ingredient on a dry weight basis. In one embodiment, the lymphatic endothelial mesenchymal transition inhibitor or the lymphatic aging inhibitor of the present invention is also sometimes composed of cortex mori radicis and/or guava leaf.

The cortex Mori and/or folium Psidii Guajavae can be used directly, or dried powder or extract.

As a method for obtaining the dried powder, there is a method of cutting or pulverizing cortex mori and/or guava leaves, and thereafter drying; a method for preparing dried powder by cutting or pulverizing plant after drying. Further, a method of cutting or pulverizing the plant, fermenting and/or treating with an enzyme, drying the cut or pulverized plant, and further pulverizing the whole plant to a predetermined particle size as necessary can be appropriately adopted.

In the case of using the extract of cortex mori and/or guava leaves, the extraction method may be performed by solvent extraction. In the case of solvent extraction, the mulberry bark and/or guava leaf is dried as necessary, and further cut or pulverized as necessary, and then extracted at normal temperature or under heating using an aqueous extractant such as cold water, warm water, or hot water having a boiling point or a temperature lower than the boiling point, or a hydrated organic solvent such as methanol, ethanol, 1, 3-butanediol, diethyl ether, ethyl acetate, or the like. However, the extraction method is not limited to solvent extraction, and extraction can be performed by a common method known in the art. The form of the extract may be not only the extract itself but also a liquid obtained by diluting or concentrating the extract appropriately by a usual method, and further a powdery or massive solid obtained by drying the extract.

The present invention also provides a method for preventing lymphatic aging by inhibiting lymphatic endothelial mesenchymal transition, by administering an endothelial mesenchymal transition inhibitor or a lymphatic aging inhibitor containing cortex mori and/or guava leaf as an active ingredient, or a composition containing the same. The method of the present invention is a method for cosmetic purposes, and there are cases where treatment is not performed by a doctor and/or a medical practitioner. As one embodiment, the method of the present invention may be a cosmetic method for preventing skin aging caused by aging of lymphatic vessels in the skin, such as edema, spots, wrinkles, and sagging, by administering cortex mori and/or guava leaves or a composition containing the same to prevent aging of lymphatic vessels in the skin through inhibition of endothelial mesenchymal transition of lymphatic vessels in the skin. The present invention also provides a method of cosmetic counseling comprising administering to a subject a composition comprising mulberry bark and/or guava leaf or a composition comprising the same, and supporting cosmetic behavior in the subject.

The route of administration of the endothelial mesenchymal transition inhibitor, lymphatic aging inhibitor or composition of the present invention may be arbitrarily selected, and examples thereof include oral administration, transdermal administration, subcutaneous administration, transmucosal administration, intramuscular administration, and the like.

The amount of the cortex mori radicis and/or the guava leaf in the lymphatic vessel endothelial mesenchymal transition inhibitor, the lymphatic aging inhibitor, or the composition of the present invention may be determined as appropriate depending on the type, purpose, form, method of use, and the like of the inhibitor, the lymphatic aging inhibitor, or the composition. For example, in the case of transdermal administration, the preparation can be arbitrarily prepared so as to be about 0.0001 to about 10 mass%, about 0.001 to about 1 mass%, or about 0.01 to about 0.1 mass% in terms of dry mass relative to the total mass of the external preparation. In any case, it is preferable that the active ingredient of the present invention is contained in such an amount as to sufficiently exhibit the inhibitory effect on the endothelial mesenchymal transition of lymphatic vessels.

In addition, the composition of the present invention may be a food composition, a cosmetic composition, or a pharmaceutical composition. The food composition may be in the form of powder, beverage, or tablet, and may be in the form of powder, liquid, solid, granule, paste, or gel. The cosmetic composition can be in the form of emulsion, cream, beauty lotion, cosmetic water, facial mask, facial cleanser, soap, bath lotion, shampoo, etc., and can be in the form of liquid, emulsion, paste, solid, sheet, plate, gel, foam, powder, etc. The pharmaceutical composition can be tablet, capsule, powder, granule, ointment, paste, patch, etc.

Further, components used in quasi drugs, cosmetics, medicines, and the like may be appropriately blended as necessary. For example, known components such as excipients, colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, pH adjusters, oils, surfactants, powders, colorants, water, alcohols, thickeners, chelating agents, silicones, antioxidants, ultraviolet absorbers, humectants, perfumes, various medicinal components, preservatives, pH adjusters, and neutralizers can be appropriately selected and used.

In addition, the present invention also provides cortex mori and/or guava leaves for inhibiting lymphatic aging via inhibiting endothelial mesenchymal transition of lymphatic vessels; use of cortex Mori and/or folium Psidii Guajavae in the manufacture of an inhibitor of endothelial mesenchymal transition or an inhibitor of lymphatic aging; and a method for producing an endothelial mesenchymal transition inhibitor or a lymphatic aging inhibitor or a composition containing the same, using cortex mori and/or guava leaves as an active ingredient. The mulberry bark and/or guava used in the production method may be in any form such as dried form, powder form, or extract.

Examples

Next, the present invention will be described in further detail by way of examples. In addition, the present invention is not limited by these examples.

Experiment 1: in vivo (vivo) changes due to aging of cutaneous lymphatic endothelial cells

The excess skin was obtained from healthy male and female subjects over 22 years old. These subjects were divided into 8 young groups consisting of young people aged 22 to 40 years and 8 advanced age groups consisting of elderly people over 40 years and under 73 years. LYVE1 was used as an endothelial cell marker and SM22 α was used as a mesenchymal cell marker.

Samples of endothelial cells in each group of cutaneous lymphatic vessels were prepared by staining with fluorescent immunohistochemistry using anti-LYVE-1 (Reliatech), anti-SM 22alpha antibody (Abcam), and a corresponding fluorescently labeled secondary antibody, and the expression of LYVE1 and SM22 α in the endothelial cells was visualized by confocal laser microscopy (force ルツアイス). In addition, the ratio of expression amounts of LYVE1 and SM22 α (EndMT ratio) was calculated according to the following formula 2. The student's t-test was used in the statistical significance difference test.

[ formula 2]

The results are shown in FIG. 1. The lower graph shows the results of experiment 1, showing the ratio of the expression amounts of LYVE1 and SM22 α (EndMT ratio) for each group. As seen from this figure, the EndMT ratio in the senior group was significantly increased (p < 0.05) in comparison with the young group, i.e., the expression level of SM22 α in LYVE1 was increased in the elderly. In addition, although expression of LYVE1 was observed in a large amount in young groups in fluorescence micrographs obtained by photographing lymphatic vessels, expression of LYVE1 (red) was reduced in older groups and expression of SM22 α (green) was observed in a large amount (upper panel of fig. 1). From these results, it was confirmed that the aged people have a transition from the endothelial cells of the lymphatic vessels of the skin to the mesenchymal cells.

Experiment 2: in vitro (in vitro) changes due to aging of human skin lymphatic endothelial cells (HDLEC)

2-1: culture of human skin lymphatic endothelial cells

Samples were used according to Kajiya et al, EMBO j.2005 Aug 17; 24(16): 2885 methods described in 2895, Human skin Lymphatic Endothelial Cells (HDLEC Cells: Human Dermal Lymphatic Endothelial Cells) isolated from foreskin. EGM was used for the HDLEC cells^TM-2MV Microvascular Endothelial Cell Growth Medium-2 BulletKit^TM(Lonza C-3202), and Single Quots added to the basic Medium (CC-3156) attached to the kit^TMCulture was carried out in a medium of supports (CC-4147) (hereinafter abbreviated as EBM2(+) medium). The status of the cells was confirmed by using Prox1 and VEGFR3 as endothelial cell markers, and passaging was repeated.

The Expression levels of Prox1 and VEGFR3 were measured by quantitative RT-PCR as described in detail below using TaqMan Gene Expression Assays available from applied biosystems.

2-2: recovery of cells and preparation of RNA

After washing the wells with PBS, RLT Buffer from QIAGEN RNeasy Mini Kit was added, and the cells were scraped with a cell scraper (cell scraper) and transferred to a 1.5ml volumetric centrifuge tube. RNA was extracted according to the protocol of QIAGEN RNeasy Mini Kit. The resulting solution was dissolved in 30. mu.l of RNase-free water. RNA concentration was measured using a Nano Drop2000 ultramicro spectrophotometer, and a portion was diluted to a concentration of 50 nM.

2-3: quantification of mRNA by RT-PCR

RNA expression was determined by real-time PCR using probe detection. TaqMan RNA-to-C1 Step Kit Applied Biosystems was used, and Prox1 and VEGFR3 were used as probes. The measuring instrument used Light Cycler 480II (Roche). The procedure and composition of the PCR are shown in the following table.

TABLE 1

[ procedure ]

TABLE 2

[ composition ]

RT-PCR mixture (2X)	10μl
		Primer and method for producing the same	1μl
Enzyme mixture	0.5μl
		RNA(50nM)	2μl
RNase-free water	6.5μl
		Total of	20μl

FIG. 2 shows the results of experiment 2, showing the expression levels of Prox1 (upper panel) and VEGFR3 (lower panel) for each culture generation. The more the number of passages increases, the less the expression of these endothelial cell markers. Therefore, it was confirmed that endothelial cells in lymphatic vessels of the skin did not differentiate into normal lymphatic endothelial cells with aging, or did not retain the properties as lymphatic endothelial cells, and the number thereof decreased.

Experiments

1 and 2 suggest that the endothelial mesenchymal transition of the cutaneous lymphatic vessels is associated with aging, and if the endothelial mesenchymal transition can be inhibited, the aging of the lymphatic vessels can be inhibited. Thus, substances inhibiting the endothelial mesenchymal transition were searched for.

Experiment 3: preparation of the samples

The "white mulberry root-bark" as the root bark of white mulberry (Morus alba Linne) was extracted with 70 vol% ethanol, and the resulting extract was further extracted with ethyl acetate, and then ethyl acetate was distilled off, and the residue was dissolved with 70 vol% ethanol, thereby preparing a white mulberry root-bark extract.

The guava leaf extract was prepared by extracting the leaves of "guava" as the leaves of guava (Psidium guajava) with 70 vol% ethanol, and purifying the resulting extract by 70 vol% ethanol.

In addition to the above samples, a total of 34 samples were prepared using 32 plants such as aloe vera juice.

Experiment 4: effect of TGF-beta signalling on expression of endothelial cell markers and mesenchymal cell markers in human cutaneous lymphatic endothelial cells (HDLEC)

4-1: culture of human skin lymphatic endothelial cells and addition to a sample of HDLEC cells

HDLEC cells (Lonza, cc-2810) were used at 8X 10⁴The individual cells/well were seeded in collagen-coated 6-well plates and cultured at 37 ℃ in the same EBM2(+) medium as in experiment 2. On the next day, SB431542(Wako corporation, 198-16543) (hereinafter abbreviated as SB) as a TGF-. beta.receptor kinase inhibitor was added at a final concentration of 5. mu.M, TGF-. beta.2 (hereinafter abbreviated as TGF-. beta.) was added at a final concentration of 1ng/ml, and 1. mu.l of 0.1% BSA (control) dissolved in 4mM HCl as a control was further cultured for 72 hours.

4-2: recovery of cells and preparation of RNA

After washing the wells with PBS, RNA was prepared with NucleoSpin RNA, and cDNA synthesis was performed by primescript ii (takarabio) with random hexanucleotide primers.

4-3: mRNA quantification by RT-PCR

Quantitative RT-PCR was performed using the cDNA synthesized in experiment 4-2 as a template, and the PCR primers shown in the following table and FastStart Universal SYBR Green Master (ROX) (Roche). Beta-actin was used as an endogenous control. The procedure and composition of the PCR are shown in the following table.

TABLE 3

[ primers used in quantitative RT-PCR ]

TABLE 4

[ procedure ]

TABLE 5

[ composition ]

RT-PCR mixture (2X)	7.5μl
		Forward primer (10. mu.M)	0.45μl
Reverse primer (10. mu.M)	0.45μl
		Form panel	1μl
RNase-free water	5.6μl
		Total of	15μl

4-4: results

The results of measuring the expression levels of LYVE1 and SM22 α by the quantitative RT-PCR described above are shown in FIG. 3. Expression of LYVE1 is significantly reduced compared to the control if TGF- β is added, and significantly increased compared to the control if an inhibitor of TGF- β is added. In addition, the expression of SM22 α significantly increased compared to the control when TGF- β was added, and significantly decreased compared to the control when an inhibitor of TGF- β was added.

From the results of experiment 4, it was found that endothelial mesenchymal transition of the cutaneous lymphatic vessels is promoted by TGF- β signaling, and thus it was suggested that aging of the cutaneous lymphatic vessels is also promoted by TGF- β signaling. Therefore, it is suggested that if endothelial mesenchymal transition by TGF- β signaling can be inhibited, lymphatic aging can be inhibited. Further, it was found that the endothelial mesenchymal transition inhibitor and the senescence inhibitor of lymphatic vessels can be searched by using an endothelial mesenchymal transition evaluation system that utilizes the expression of the endothelial cell marker and the mesenchymal cell marker in HDLEC, and TGF- β signals can be utilized in such an evaluation system.

Experiment 5: inhibitory Effect of TGF-. beta.s in various samples

As a pre-sieve used in the endothelial mesenchymal transition evaluation system of experiment 4, HEK-Blue TGF-. beta.cells (InvivoGen) were used to examine the effect of inhibiting TGF-. beta.of each of the samples prepared in experiment 3 by measuring absorbance (640 nm). Specifically, samples No. 1 to 18 among 34 samples prepared in experiment 3 were added together with TGF-. beta.s. PBS only, TGF- β + SB were used as controls. At 12X 10⁴Individual cells/well HEK-Blue TGF-. beta.cells were seeded in 24-well plates. The following day was stimulated separately as in the following table. In the case of SB addition, stimulation was performed with TGF-. beta.and each ligand after 2 hours from the addition of SB. After 24 hours from the stimulation, 2.5. mu.L of the supernatant was taken out, mixed with 100. mu.L of QUANTI-Blue as a substrate, incubated at 37 ℃ for 30 minutes, and the absorbance at 640nm was measured.

TABLE 6

The results (1 to 18) of adding the sample numbers 1 to 18 and TGF-beta, and the results of adding PBS alone (control), TGF-beta alone (TGFb), and TGF-beta + SB (TGFb + SB) are shown in FIG. 4. In the figure, sample No. 15 represents a cortex mori extract (final concentration 0.01 mass%), and sample No. 13 represents a guava leaf extract (final concentration 0.1 mass%). Except for the control to which no TGF-. beta.was added, it was shown that the lower the absorbance, the higher the inhibitory effect of TGF-. beta.was.

As can be seen from fig. 4, TGF- β inhibitory effects were observed for the cortex mori extract (15) and the guava leaf extract (13).

Experiment 6: analysis of expression level of endothelial cell marker by adding cortex Mori extract

The cortex mori extracts pre-screened in experiment 5 were used to analyze the expression level of the endothelial cell markers LYVE1 and Prox1 by the endothelial mesenchymal transition evaluation system of experiment 4. Specifically, the reaction was carried out at 8X 10 in the same manner as in experiment 4⁴Individual cells/well HDLEC cells were seeded in collagen-coated 6-well plates and cultured at 37 ℃ in the same EBM2(+) medium as in experiment 2. On the next day, cell adhesion was confirmed, and each sample was added in the test area set as shown in the following table. That is, 2. mu.l of PBS, 1ng/mL of TGF-. beta.and 5. mu.M of SB were added to control PBS and TGF-. beta.respectively, and 2. mu.l of the cortex Mori extract prepared in experiment 3 (final concentration: 0.1% by mass) was cultured for 72 hours, followed by quantitative RT-PCR as in experiment 4. In addition to the endothelial cell marker LYVE1, PCR primers for Prox1 were used, and a β -actin probe was used as an endogenous control. PCR primers for LYVE1 and β -actin were identical to those in experiment 4. PCR primers for Prox1 are shown below.

TABLE 7

[ primers used in quantitative RT-PCR ]

TABLE 8

The results are shown in FIG. 5. When TGF- β alone was added, the expression of LYVE1 and Prox1 decreased, but when SB was added, the expression increased. Furthermore, the addition of the extract of cortex Mori inhibited the decrease in expression of LYVE1 and Prox1 by TGF- β, and was significantly increased over that obtained without any addition. Therefore, it is suggested that cortex mori has an effect of not only inhibiting the endothelial mesenchymal transition promoted by TGF- β but also promoting the differentiation into lymphatic endothelial cells.

Experiment 7: analyzing expression amount of mesenchymal cell marker by adding cortex Mori extract

The same sample/method as in experiment 6 was performed except that the expression level of the mesenchymal cell marker SM22 α was measured instead of the endothelial cell marker.

The results are shown in FIG. 6. The increase in expression of the mesenchymal cell marker SM22 α by TGF- β was suppressed by the cortex mori extract. The inhibitory effect is stronger than that brought about by SB.

Experiment 8: changes in HDLEC morphology due to addition of cortex Mori and TGF-beta

HDLEC cells identical to those of experiment 4 were cultured at 8X 10⁴Individual cells/well were seeded in collagen-coated 6-well plates and cultured in the same EBM2(+) medium as in experiment 2 at 37 ℃. The following day, cell adhesion was confirmed, and each sample was added in the test area set as shown in the following table. That is, after SB was set to a final concentration of 5. mu.M, 0.1% BSA/4mM HCl was set to a final concentration of 1. mu.l, and TGF-. beta.was set to a final concentration of 1ng/mL, 2. mu.l of the cortex Mori extract prepared in experiment 3 was added (to a final concentration of 0.1 mass%), and after 72 hours of culture, the morphology of the cells was observed by a fluorescence microscope BZ-X710 (Keyence).

TABLE 9

The results are shown in FIG. 7. As is clear from FIG. 7, when TGF-. beta.was added, the number of cells was significantly reduced, and the morphology was also changed. Thus, it was suggested that endothelial cells in skin lymphatic vessels are affected by TGF-. beta.s and that the function as lymphatic vessels is impaired. However, it was found that even when TGF-. beta.is added, if SB and/or the extract of cortex Mori are added at the same time, the decrease in the number of cells and the change in morphology are suppressed.

Experiment 9: permeability (leakage) test of human lymphatic endothelial cells (HDLEC)

Mu.l of Fibronectin (Corning Co.) diluted to 5. mu.g/ml with PBS was added to each of the inserts (insert) of HTS transwell-24well0.4um (Corning Co.), incubated at 37 ℃ for 15 minutes, and then aspirated (Fibronectin coating). HDLEC cultured in EBM2(+) medium in the same manner as in experiment 2 was changed to 4X 10⁴Individual cells/well were seeded into fibronectin coated inserts and cultured at 37 degrees.

After 24 hours, the cells were divided into 4 groups (control, TGF-. beta. + SB, TGF-. beta. + cortex Mori extract (15)). The culture medium was changed to a culture medium prepared by adding SB to EBM2(+) medium to a final concentration of 1. mu.M, or to a culture medium prepared by adding cortex Mori extract to EBM2(+) medium to a final concentration of 0.1%, and the culture medium was changed to a culture medium without addition of EBM2(+) medium for the other groups, and the culture was carried out at 37 ℃.

After 3 hours, the medium was exchanged for EBM2(+) no-addition medium for all wells and cultured at 37 ℃. After 24 hours, the medium was changed to EBM2(+) medium prepared by adding TGF-. beta.to the group to which TGF-. beta.was added to a final concentration of 1ng/ml, and the other group was changed to EBM2(+) medium without addition, and cultured at 37 ℃. After 24 hours, 50. mu.l of FITC-dextran adjusted to 10mg/ml was added, and after incubation at 37 ℃ for 15 minutes, the FITC-dextran leakage from the insert was measured with a microplate reader.

The results are shown in FIG. 8, and the absorbance of FITC-dextran leaked from the inserts of each group is shown as a relative value in the case of the control as 1. The permeability of HDLEC was increased by addition of TGF-beta 2 alone, while the permeability was inhibited by treatment with SB and cortex Mori extract, which are TGF-beta inhibitors.

Experiment 10: analysis of expression level of mesenchymal cell marker by adding guava leaf extract

10-1: culture of lymphatic endothelial cells

The guava leaf extract pre-screened in experiment 5 is used for analyzing the expression quantity of the mesenchymal cell marker SM22alpha through the endothelial mesenchymal transition evaluation system of experiment 4. Specifically, human skin microlymphatic endothelial cells (HDLEC) obtained from LONZA were added to EBM2(+) same as in experiment 2 at a ratio of 6X 10⁴Individual cells/well were plated in 6-well plates and incubated overnight at 37 ℃. Cell adhesion was confirmed the next day, and the cells were cultured for 72 hours after stimulation with SB of 5. mu. M, TGF-. beta.of 1ng/mL, respectively.

10-2: addition to samples of HDLEC cells

HDLEC cells cultured as described above were cultured at 4X 10⁵Individual cells/well were seeded in collagen coated 6-well dishes. Addition of SingleQuots to the basic Medium (CC-3156) attached to the same kit as in experiment 2^TMCulture medium (EBM2(+) medium) obtained from supports (CC-4147), and the cell suspension was adjusted to 2ml per well. After culturing at 37 ℃ for 24 hours, the Basal Medium (CC-3156) (EBM2(-) Medium) of the kit was replaced with a Medium obtained by adding FBS at a concentration of 0.5%. After incubation at 37 ℃ for 4 hours, the samples were added to the test zones as set forth in the following table. Each sample was diluted with PBS and added at the concentrations reported in the table below. After addition, HDLEC cells were cultured at 37 ℃ for 20 hours. After the culture, the expression level of SM22 α was analyzed by quantitative RT-PCR in the same manner as in experiment 2.

Watch 10

10-3: results

The results are shown in FIG. 9. The expression of SM22a increased when only TGF-. beta.was added, while the expression decreased when SB was added. Further, it is also known that the expression of SM22 α by TGF- β is significantly reduced in the guava leaf extract. Therefore, guava leaf extract also has an effect of inhibiting endothelial mesenchymal transition of lymphatic vessels promoted by TGF- β.

Experiment 11: analysis of other samples

With respect to 32 kinds of samples other than the white mulberry root-bark and the guava leaf prepared in experiment 3, TGF- β inhibitory effects were observed with respect to sample 18. However, when SM22 α expression was confirmed by using sample 18 in the same manner as in experiment 4, the expression of SM22 α was higher in TGF- β + sample 18 than in the case of adding TGF- β alone, and endothelial mesenchymal transition was promoted instead. Furthermore, although sample 17 also observed TGF- β inhibitory effect, cytotoxicity was observed (data not shown).

From the above results, it was confirmed that cortex Mori and guava leaf have TGF- β inhibitory effects, endothelial mesenchymal transition inhibitory effect, and lymphatic aging inhibitory effect.

The present invention can inhibit the transformation of the endothelial mesenchyme by administering the endothelial mesenchyme transformation inhibitor containing the white mulberry root-bark and/or the guava leaf as an active ingredient. By inhibiting endothelial mesenchymal transition of lymphatic vessels, the aging of lymphatic vessels can be inhibited. In addition, the endothelial mesenchymal transition inhibitor of the present invention inhibits not only lymphatic vessels but also vascular endothelial mesenchymal transition, and is expected to prevent vascular aging.

Claims

1. An endothelial mesenchymal transition inhibitor for lymphatic vessels contains cortex Mori and/or folium Psidii Guajavae as effective components.

2. The inhibitor of endothelial mesenchymal transition according to claim 1, which inhibits endothelial mesenchymal transition of lymphatic vessels by inhibiting TGF- β (transforming growth factor- β).

3. A lymphatic vessel aging inhibitor contains cortex Mori and/or folium Psidii Guajavae as effective components, and can inhibit lymphatic vessel aging by inhibiting lymphatic endothelial mesenchymal transition.

4. The lymphatic senescence inhibitor of claim 3, inhibiting endothelial mesenchymal transition of lymphatic vessels by inhibiting TGF- β.

A TGF- β inhibitor comprising guava leaves as an active ingredient.

6. A composition comprising the endothelial mesenchymal transition inhibitor of claim 1 or 2, the lymphatic senescence inhibitor of claim 3 or 4, and/or the TGF- β inhibitor of claim 5.

7. A cosmetic method for preventing lymphatic aging in a subject, comprising administering to the subject the composition of claim 6.

8. A cosmetic counseling method for supporting cosmetic behavior in a subject comprising recommending to the subject the composition of claim 6.