WO2012105706A1 - Agent thérapeutique contre les chéloïdes - Google Patents

Agent thérapeutique contre les chéloïdes Download PDF

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WO2012105706A1
WO2012105706A1 PCT/JP2012/052559 JP2012052559W WO2012105706A1 WO 2012105706 A1 WO2012105706 A1 WO 2012105706A1 JP 2012052559 W JP2012052559 W JP 2012052559W WO 2012105706 A1 WO2012105706 A1 WO 2012105706A1
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keloid
resveratrol
bond
fibroblasts
therapeutic agent
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PCT/JP2012/052559
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English (en)
Japanese (ja)
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佳奈枝 池田
俊彦 鳥越
佳隆 松本
龍哉 藤田
高敏 四ツ柳
昇志 佐藤
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北海道公立大学法人札幌医科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders

Definitions

  • the present invention relates to a novel keloid therapeutic agent and a treatment method using the same.
  • Keloid is a fibrotic skin disease characterized by the proliferation of fibroblasts that occur during the wound healing process and excessive accumulation in the dermis of extracellular matrix mainly composed of type I collagen [1]. Keloids occur after trauma, inflammation, surgery, burns, and sometimes occur spontaneously. Keloids are red, hard scars that are itchy and painful. Hypertrophic scars do not exceed the original wound, whereas keloids grow beyond the boundaries of the original wound, do not regress spontaneously, and tend to recur after resection. The ears, deltoid region, precordial region, and presternal area are the prevalent parts of keloids in the body.
  • keloids intralesional corticosteroid injections alone or in combination with other methods, or endokeloid resection in combination with radiation therapy.
  • endokeloid resection in combination with radiation therapy.
  • the basic mechanisms involved in keloid development remain unknown, most treatments fail, and the rate of recurrence after treatment is high.
  • Keloids are likely to occur in races with dark skin tones, suggesting the genetic involvement of keloids. [2, 3]. Other factors that promote keloid development are trauma morphology, wound tension, infection, foreign body response, and endocrine factors [4]. Keloids show biological differences between marginal and central keloids, including proliferation and apoptosis, and cultured fibroblasts derived from keloid lesions lose keloid-specific features during cell culture It has also been suggested to do [5, 6]. Although the characteristics of keloids are generally known, the mechanism of keloid formation has not yet been clarified. Since keloids occur only in humans and not in animals, there is no ideal animal experimental model, hindering the elucidation of the pathogenesis of keloids and the development of treatments [7].
  • resveratrol is strong in liver fibroblast cell proliferation, collagen production, ⁇ -smooth muscle actin ( ⁇ -SMA) expression, and matrix metalloproteinase 2 (MMP-2) secretion It has been demonstrated to show a suppressive effect [11]. Moreover, in the rat cirrhosis CCl4 model, resveratrol has been shown to have a strong antifibrotic effect by suppressing NF-kb activity and by inhibiting production of transforming growth factor- ⁇ (TGF- ⁇ ). [12]. However, there have been no reports that show that resveratrol has a similar effect on keloids.
  • the present inventors diligently studied the effect of resveratrol on keloids and found that resveratrol suppresses the proliferation of keloid fibroblasts and induces apoptosis. Our data suggest that resveratrol is applicable to keloid therapy.
  • an object of the present invention is to provide a novel keloid therapeutic agent capable of remarkably suppressing true keloid fibroblasts and a therapeutic method using the same.
  • a keloid therapeutic agent comprising the polyphenol of formula (I): (Where R 1 is CH or an oxygen atom, R 2 is a hydrogen atom, CH or C (OH), R 3 is a hydrogen atom, C ( ⁇ O), C ( ⁇ NH), CH 2 or CH (OH), R 4 is a hydrogen atom or a hydroxyl group, where When R 1 is a CH group, the bond between R 1 and the carbon represented by * is a double bond, the bond between R 2 and the carbon of * is a single bond, and R 2 and There is no bond between R 3 and R 2 and R 3 are hydrogen atoms; When R 1 is an oxygen atom, the bond between R 1 and the carbon of * is a single bond, the bond between R 2 and the carbon of * is a double bond, R 2 and R 3 , The bond between is a single bond, and R 2 is a CH group or a C (OH) group, and R 3 is C ( ⁇ O), C ( ⁇ NH), CH 2 or CH (I):
  • a method for treating keloid comprising administering an effective amount of the polyphenol described in formula (I) to a keloid lesion site.
  • the proliferation of intrinsic keloid fibroblasts can be significantly suppressed.
  • Keloid fibroblasts treated with resveratrol for 48 hours were evaluated for expression of type I collagen and HSP47 by RT-PCR. G3PDH was used as an internal control. It is a figure which shows the effect of the resveratrol with respect to a keloid fibroblast.
  • B Levels of TGF- ⁇ 1 in keloid fibroblast culture supernatant after addition of resveratrol for 48 hours. Data are the mean ⁇ SEM of triplicate experiments for each group. D. ( * P ⁇ 0.05).
  • Keloid fibroblasts treated with resveratrol for 48 hours are evaluated for expression of type I collagen, ⁇ -SMA and HSP47 by RT-PCR. It is a figure which shows the effect of the resveratrol with respect to cell proliferation.
  • the WST-1 assay was performed up to 48 hours. Each point is the mean ⁇ SEM of data collected from 5 wells. D. ( * P ⁇ 0.05). It is a photograph of keloid fibroblasts treated with resveratrol.
  • Keloid central fibroblasts (a, b) and keloid marginal fibroblasts (c, d) were cultured for 48 hours in DMEM supplemented with 10% FCS.
  • FIG. 5 shows apoptosis of keloid fibroblasts induced by resveratrol.
  • Fig. 5 shows apoptosis of keloid fibroblasts induced by resveratrol. FACS analysis of apoptotic cells after staining with annexin and propidium iodide (PI). The number of apoptotic cells in the cell cycle is increased by resveratrol treatment. Cell cycle analysis by flow cytometry.
  • Keloid central fibroblasts (a, b) and keloid marginal fibroblasts (c, d) treated or not treated with resveratrol (100 ⁇ M) for 48 hours.
  • A, c Control;
  • Figure 5 shows that resveratrol activated caspase-3 in keloid fibroblasts. After treatment with resveratrol for 8 hours, the cells were lysed and analyzed for caspase-3 activity. Data are the mean ⁇ SEM of triplicate experiments for each group. D. ( * P ⁇ 0.05). It is a figure which shows the scheme of the in vivo study regarding the antifibrotic effect of a resveratrol. Collagen gel (5 ⁇ 10 5 cells / ml) embedded with keloid fibroblasts was transplanted under the skin of nude mice. Resveratrol (100 mM, 100 ⁇ l) was then injected into the transplanted gel.
  • the result of having investigated the inhibitory effect with respect to the proliferation of the keloid marginal fibroblast by a quercetin by a WST-1 assay is shown. Data are the mean ⁇ SEM of triplicate experiments for each group. D. ( * P ⁇ 0.05). The inhibitory effect with respect to the proliferation of the keloid central fibroblast by nicotinamide is shown by the result examined by WST-1 assay. Data are the mean ⁇ SEM of triplicate experiments for each group. D. Indicates. The result of having investigated the inhibitory effect with respect to the proliferation of the keloid marginal fibroblast by a nicotinamide by a WST-1 assay is shown. Data are the mean ⁇ SEM of triplicate experiments for each group. D. Indicates.
  • keloid is a lesion in which scar tissue is excessively proliferated, is classified as a benign skin fibroproliferative lesion, and is also referred to as a true keloid. Therefore, the “keloid” of the present invention is distinguished from hypertrophic scars, which are related lesions.
  • the keloid therapeutic agent of the present invention is characterized by comprising the polyphenol of formula (I) as an active ingredient. It is surprising that such polyphenols of formula (I) have significant growth inhibitory activity against genuine keloid fibroblasts.
  • R 2 is a hydrogen atom or C (OH), and R 3 is a hydrogen atom or C ( ⁇ O).
  • R 1 is a CH group
  • the bond between R 1 and the carbon of * is a double bond
  • the bond between R 2 and the carbon of * is a single bond
  • the bond between R 2 and R 3 is absent
  • R 2 and R 3 are hydrogen atoms.
  • R 4 is a hydrogen atom and the polyphenol of formula (I) is resveratrol.
  • the polyphenol of formula (I) is resveratrol, it is particularly advantageous in inducing apoptosis of keloid fibroblasts.
  • R 1 is an oxygen atom
  • the bond between R 1 and the carbon of * is a single bond
  • the bond between R 2 and the carbon of * is two.
  • a double bond, a bond between R 2 and R 3 is a single bond
  • R 2 is a CH group or a C (OH) group
  • R 3 is C ( ⁇ O), C ( ⁇ NH) , CH 2 or CH (OH).
  • R 2 is a C (OH) group
  • R 3 is C ( ⁇ O).
  • R 4 is a hydroxyl group and the polyphenol of formula (I) is quercetin.
  • the polyphenol of the present invention is not particularly limited as long as it is included in the formula (I), and stilbenoids (Kai Xiao et al. Stilbenoids: Chemistry and bioactivities. Studies in Natural Products Chemistry Volume 34, 646, etc.).
  • the keloid therapeutic activity of stilbenoids and polyphenols encompassed by the present invention can be confirmed, for example, using the methods disclosed in the present application (Examples 1 to 7).
  • the polyphenol of the present invention may be a natural product, a synthetic product or a commercial product.
  • the polyphenol of the present invention is a known polyphenol such as resveratrol or quercetin, those skilled in the art can easily obtain it by isolating and purifying from known citrus fruits and plant components.
  • the polyphenol of formula (I) may be used as it is as a therapeutic agent, or may be used as a composition together with other components described later.
  • the content of polyphenol in the keloid therapeutic agent of the present invention is not particularly limited, and is, for example, 0.0002 to 20% by weight, preferably 0.02 to 2% by weight.
  • the concentration of polyphenol in the preparation is not particularly limited, and can usually be in the range of 1 mM to 1M. However, in the case of an injection, for example, the concentration range may be 200 ⁇ M to 500 mM, preferably 1 mM to 100 mM.
  • the form of the keloid therapeutic agent of the present invention is not particularly limited, and in the case of transdermal administration, forms of pharmaceutical preparations such as ointments, gel preparations, creams, liquids, sprays, patches, and lotions Can be suitably used.
  • These formulations include polyphenols of formula (I), pharmaceutically acceptable salts, esters, amides, prodrugs, analogs thereof (US Pat. No. 6,790,869, US patent application Ser. No. 10/071). , 124, and others) and combinations thereof can be used in conjunction with other pharmaceutically acceptable ingredients to prepare by conventional methods.
  • Such other components include, but are not limited to, for example, known pharmaceutically acceptable carriers or diluents such as hydrocarbons (eg, petrolatum, squalane, liquid paraffin, etc.), higher alcohols (eg, stearyl alcohol).
  • hydrocarbons eg, petrolatum, squalane, liquid paraffin, etc.
  • alcohols eg, stearyl alcohol
  • the keloid therapeutic agent can be provided for oral or parenteral administration.
  • other components as shown above may be included, and pharmaceutically acceptable carriers, vehicles, additives and the like particularly suitable for oral or parenteral drug administration may be selected and contained.
  • Such formulations can be provided, for example, as polyphenol-containing microemulsions.
  • the keloid treatment can be effectively performed by administering the polyphenol of the formula (I) having the suppression of keloid fibroblast proliferation to the keloid lesion site of the patient.
  • the polyphenol of the formula (I) is used for inhibiting the growth of keloid fibroblasts.
  • the polyphenol of the present invention can be suitably used for inducing apoptosis of keloid fibroblasts.
  • the polyphenol of the present invention is preferably resveratrol.
  • polyphenol of the present invention can effectively suppress the expression of type I collagen, HSP47 or TGF- ⁇ 1 in true keloid fibroblasts. Therefore, according to another aspect of the present invention, there is provided an expression inhibitor of type I collagen, HSP47 or TGF- ⁇ 1 in authentic keloid fibroblasts, comprising a polyphenol of formula (I).
  • the effective amount, dose and usage of the keloid therapeutic agent of the present invention is the level of keloid fibroblast growth inhibitory or apoptosis-inducing activity of polyphenols of formula (I), polyphenol content, keloid severity and condition, keloid continuation Those skilled in the art can appropriately determine the number of years and the age and weight of the patient.
  • the keloid therapeutic agent of the present invention can be administered by topical application.
  • the keloid therapeutic agent is not particularly limited, but is preferably administered at about 0.1 to about 50 mg / cm 2 (skin area) per application to the keloid region or its peripheral region.
  • the keloid therapeutic agent of the present invention is preferably administered by intralesional injection into a keloid lesion site. It is well known in the art that injection requires a lower effective concentration than topical application.
  • the keloid therapeutic agent is not particularly limited, and is administered, for example, at about 0.004 to about 10 mg / cm 2 , preferably about 0.02 to about 2 mg / cm 2 (skin area).
  • the keloid therapeutic agent of the present invention is not particularly limited, and can be administered once to several times per day, for example.
  • the patient to which the keloid therapeutic agent of the present invention is applied is usually a human, but may be a mammal, and the present invention includes such an embodiment.
  • the keloid lesion site in the present invention is not particularly limited, and preferred examples include the ear, shoulder, chest, or public region, but preferably the shoulder, chest, or pubic region.
  • a method for treating keloid fibroblasts which is effective in suppressing the proliferation of keloid fibroblasts and / or inducing apoptosis of keloid fibroblasts.
  • a method comprising providing an amount of the composition to keloid fibroblasts, wherein the composition consists essentially of resveratrol.
  • resveratrol is provided to the keloid fibroblasts by intralesional injection.
  • resveratrol is preferably provided in an amount of about 0.02 to about 2 mg per cm 2 of skin.
  • resveratrol in the method for treating keloid fibroblasts, is provided to keloid fibroblasts by topical application.
  • resveratrol is preferably provided in an amount of about 0.1 to about 50 mg per cm 2 of skin.
  • DMEM Medium and Chemical Dulbecco's Modified Eagle Medium
  • FCS Fetal Bovine Serum
  • EDTA Ethylenediaminetetraacetic acid
  • Resveratrol was purchased from Cayman Chemical Company, Ann Arbor, Michigan, USA.
  • keloid lesions were excised, full-thickness biopsy materials were obtained from the raised red areas (marginal areas) and retracted soft areas (center areas) of each keloid lesion.
  • a cell culture of fibroblasts by explant method was prepared. A small amount of tissue was cut to remove the fat and cut into small pieces less than 1 mm in diameter. Tissue pieces were cultured in DMEM supplemented with 10% FCS in a humidified atmosphere of 5% CO 2 at 37 ° C. The medium was changed every 2-3 days. Seven to 14 days after the primary culture, the cells grown from the marginal part of the cultured tissue were passaged. Keloid fibroblasts from passage 2 to 5 were used in all experiments. Fibroblasts collected from the central keloid region were referred to as keloid central fibroblasts, and those derived from the peripheral region were referred to as keloid peripheral fibroblasts.
  • More specific culture methods include, for example, inoculating keloid fibroblasts (2 ⁇ 10 3 cell / cm 2 ) in a 100 mm dish or 75 cm 2 flask, or keloid fibroblasts (1 ⁇ 10 3 to 10 4 cell / cm 2 ). cm 2 ) was seeded in a 96-well plate and then cultured in DMEM supplemented with 10% FCS for 24 hours. Unless otherwise specified, a serum-free medium was used in the subsequent treatment with resveratrol.
  • Stimulant When the stimulating substance was resveratrol, cells were seeded in a tissue culture plate at a concentration of 1 ⁇ 10 4 / ml. After incubation in DMEM with 10% FCS for 24 hours, cells were treated with resveratrol (0, 25, 50, or 100 ⁇ M) for the indicated period in serum-free medium.
  • the cells were tested in the same manner as for resveratrol except that quercetin or nicotinamide was treated at various concentrations (0, 10, 100 ⁇ M) in serum-free medium for the indicated period. went.
  • RNA of pretreated cells was extracted using the RNeasy mini kit (QIAGEN, Tokyo, Japan) according to the manufacturer's instructions.
  • Reverse transcription (RT) was performed according to the manufacturer's instructions using Superscript III reverse transcriptase (Invitrogen, Tokyo, Japan) using 2 ⁇ g of total RNA as a template.
  • Polymerase chain reaction (PCR) was performed using taq DNA polymerase (QIAGEN, Tokyo, Japan) and specific primers (Table 2-1).
  • the sample was incubated in a PCR thermal cycler (Gene Amp, Life Technologies Japan, Tokyo, Japan).
  • the temperature profile consisted of 30 cycles of denaturation at 94 ° C. for 15 seconds, annealing at 60 ° C. for 30 seconds, and extension at 72 ° C. for 30 seconds.
  • Amplified product (15 ⁇ l) was loaded onto a 2% agarose gel in TAE buffer and the separation bands were visualized by ethidium bromide staining. The relative density of the bands was quantified using Dolphin-View (Kurashiki Boseki, Osaka, Japan).
  • the treatment concentration of quercetin or nicotinamide is 0, 10, or 100 ⁇ M
  • the specific primers shown in Table 2-1 are used, and in addition to type I collagen and HSP47 mRNA, Reverse transcription polymerase chain reaction was performed in the same manner as for resveratrol except for - ⁇ 1 mRNA.
  • WST-1 assay When the stimulant was resveratrol, the cell suspension was added to each well of a 96 well flat bottom microtiter plate and incubated for 24 hours before treatment. Thereafter, resveratrol solution was added to each well at a concentration of 0, 25, 50, or 100 ⁇ M. 12, 24, 36, and 48 hours after exposure to resveratrol, 10 ⁇ l of WST-1 solution (manufactured by Dojindo Laboratories, Kumamoto, Japan) was added to each well and incubated at 37 ° C. for 1 hour. The optical density at 450 nm was measured with a microtiter plate reader (ARVO SX, PerkinElmer, Massachusetts, USA).
  • Enzyme-linked antibody immunosorbent assay Keloid fibroblasts were treated with resveratrol at concentrations of 0, 25, 50, or 100 ⁇ M for 48 hours. The treated cell culture supernatant was collected in a sterile test tube. TGF- ⁇ 1 concentration was determined with enzyme-linked antibody immunosorbent assay (ELISA) technology (R & D Systems, Minnesota, USA). Total TGF- ⁇ 1 levels were assayed using a commercial ELISA according to manufacturer's instructions (R & D Systems, Minnesota, USA).
  • ELISA enzyme-linked antibody immunosorbent assay
  • Caspase-3 activity assay Treated with resveratrol (100 ⁇ M) for 48 hours in serum-free medium.
  • the level of caspase activity in the treated keloid fibroblasts was determined using a commercially available fluorescence assay kit (caspase colorimetric protease assay kit; MBL, Nagoya, Japan) according to the manufacturer's instructions [13. ].
  • caspase-3 activity was measured by cleavage of the fluorescent substrate DEVD-pNA. Cells, including isolated cells, were collected and lysed. The lysate was then centrifuged and the clarified supernatant was collected. The supernatant was incubated with DAVE-pNA at 37 ° C. for 1 hour. The optical density of each sample was determined at 405 nm using a plate reader (ARVO SX, PerkinElmer, Massachusetts, USA).
  • Annexin-V assay Keloid fibroblasts were treated with resveratrol (100 ⁇ M) for 48 hours in serum-free medium. Incubation was terminated and cells were harvested using 0.02% EDTA solution and washed with phosphate buffered saline (PBS). Apoptotic cells were then determined by using Annexin-V-Fluos and staining (Annexin-V-FLUOS staining kit, Roche Diagnostics, Tokyo, Japan) according to the manufacturer's instructions. The labeled cells were then counted with a FACSCalibur flow cytometer (Becton Dickinson, NJ, USA).
  • Keloid fibroblasts were treated with resveratrol (100 ⁇ M) for 48 hours in serum-free medium. Cells were then harvested with 0.02% EDTA solution, washed and collected. Fibroblasts were fixed with 75% (volume / volume) cold ethanol for at least 1 hour at ⁇ 20 ° C. Fixed cells were collected by centrifugation, resuspended in RNase staining buffer (Phoenix Flow Systems, CA, USA) to stain the DNA, and finally FACSCalibur flow cytometer (Becton Dickinson, New Jersey). State, USA).
  • the cell cycle distribution of keloid-derived fibroblasts was analyzed with a FACSCalibur flow cytometer. Cell cycle distribution was calculated using the Cell Quest software program (Becton Dickinson, NJ, USA). The percentage of cells in the G0 / G1, G2 / M, S, and sub-G1 phases of the cell cycle were automatically analyzed using the accompanying ModFit LT software program (Verity Software House, Maine, USA). Data are expressed as the percentage of cells in G0 / G1, G2 / M, S, and sub-G1 phases.
  • Keloid fibroblast culture The human keloid fibroblast used in this study was obtained by culturing keloid tissue excised from a patient during surgery using the explant method. Cells were maintained in DMEM supplemented with 10% fetal calf serum (FCS) and grown at 37 ° C. in 5% CO 2 atmosphere.
  • FCS fetal calf serum
  • collagen gel A collagen solution was prepared by mixing with a type I atelo-clagen solution, 10-fold concentrated minimal essential medium (10 ⁇ MEM), and FCS, 8: 1: 1 (volume / volume). / Volume). Fibroblasts were dispersed with 0.05% trypsin and 0.02% EDTA and suspended in a collagen gel solution at a density of 5 ⁇ 10 5 cells / ml. The cell suspension was dispersed in 6-well tissue culture plates at 2 ml / dish and incubated at 37 ° C. in a 5% CO 2 atmosphere. Then, 24 hours later, 2 ml of DMEM supplemented with 10% FCS was poured onto the gel and cultured for one week.
  • Example 1 Inhibition of keloid fibroblast type I collagen and HSP47 mRNA expression by resveratrol
  • the effect of resveratrol on type I collagen and ⁇ -SMA mRNA expression of keloid fibroblasts was first studied. Keloid fibroblasts were treated with resveratrol for 48 hours. Total RNA of pretreated cells was extracted and analyzed by RT-PCR. Type I collagen and ⁇ -SMA mRNA expression decreased in keloid fibroblasts in a dose-dependent manner (FIGS. 1A and C).
  • HSP47 is a collagen-specific molecular chaperone that interacts with procollagen during the folding, assembly, and transport processes from the endoplasmic reticulum [14]. HSP47 expression is closely related to collagen expression in fibrotic diseases including keloids [15]. Resveratrol treatment showed the same inhibitory effect on HSP47 mRNA expression as type I collagen (FIG. 1A). Inhibition was evident on the second day after resveratrol treatment. This result suggested that resveratrol suppresses collagen production of keloid fibroblasts.
  • TGF- ⁇ 1 Reduction of keloid fibroblast TGF- ⁇ 1 production by resveratrol TGF- ⁇ 1 is one of the important mediators of fibrosis.
  • TGF- ⁇ 1 is overexpressed in keloid tissues and is known to stimulate fibroblast proliferation.
  • TGF- ⁇ 1 also promotes type I collagen synthesis and suppresses collagenase transcription [16]. Therefore, we next performed an assay using the TGF- ⁇ 1 ELISA kit to quantify the amount of TGF- ⁇ 1 protein present in the culture supernatant. Keloid fibroblasts were treated with resveratrol for 48 hours. Cell culture supernatant was collected and the TGF- ⁇ 1 concentration was determined. Resveratrol (100 ⁇ M) significantly suppressed keloid marginal fibroblast TGF- ⁇ 1 production (P ⁇ 0.05), consistent with suppression of fibrosis (FIG. 1B).
  • Example 3 Inhibition of keloid fibroblast proliferation by resveratrol
  • the present inventors studied the effect of resveratrol on keloid fibroblast proliferation. Cells were treated with resveratrol at concentrations of 0, 25, 50, or 100 ⁇ M and cultured for up to 48 hours (FIG. 2). It was shown that resveratrol treatment inhibited the proliferation of keloid fibroblasts in a concentration-dependent manner.
  • Example 4 Induction of Keloid Fibroblast Apoptosis by Resveratrol We have determined that apoptotic cells that were treated with resveratrol were rounded, regressed, and floated, which was not seen in the untreated control. Were observed (FIG. 3).
  • two-color staining with Annexin-V and PI was used to detect the proportion of cells undergoing early apoptosis and subsequent cell death.
  • the present inventors evaluated cell apoptosis only in 48 hours based on the inventors' initial observation on the time course of the inhibition of keloid fibroblast proliferation by resveratrol.
  • resveratrol 100 ⁇ M induced cell apoptosis in keloid fibroblasts.
  • central keloid fibroblasts 9.58% of all cells had early apoptosis, late apoptosis was 24.51%, and necrosis was 6.57%.
  • necrosis 6.57%.
  • keloid marginal fibroblasts 3.14% of all cells had early apoptosis, late apoptosis was 18.49%, and necrosis was 0.98%.
  • keloid fibroblasts were treated with resveratrol (100 ⁇ M), and the cell cycle was analyzed by flow cytometry. Data are expressed as the percentage of cells in G0 / G1, G2 / M, S, and sub-G1 (apoptosis). Both central and marginal keloid fibroblasts showed a marked increase in the number of apoptotic cells after 48 hours of treatment with resveratrol (FIG. 5). The degree of this effect was greater in keloid marginal fibroblasts than in the central keloid fibroblasts. Thus, keloid marginal fibroblasts may be more susceptible to apoptosis than keloid central fibroblasts.
  • caspase-3 is known to be an important effector molecule that stimulates apoptosis [17, 18], we have found that resveratrol activates caspase-3 in keloid fibroblasts [17, 18], we have found that resveratrol activates caspase-3 in keloid fibroblasts [17, 18], we have found that resveratrol activates caspase-3 in keloid fibroblasts [17, 18], we have found that resveratrol activates caspase-3 in keloid fibroblasts We investigated whether or not. To measure caspase-3 activity, lysates from keloid fibroblasts treated with resveratrol were analyzed for caspase-3 activity using DEVD-pNA as a substrate. We observed that caspase-3 was activated in keloid fibroblasts treated with resveratrol 8 hours after treatment (FIG. 6). These results suggested that caspase-3 may be activated by resveratrol in the early stages of
  • Example 5 In vivo study on the anti-fibrotic effect of resveratrol
  • One collagen gel (5 ⁇ 10 5 cells / ml) embedded with keloid fibroblasts was implanted under the skin of nude mice.
  • resveratrol 100 mM, 100 ⁇ l
  • control medium PBS, 100 ⁇ l
  • the result was as shown in FIG.
  • resveratrol was injected, embedded keloid fibroblasts were significantly reduced.
  • keloid a benign collagenous tumor characterized by massive accumulation of extracellular matrix and occurring during abnormal wound healing.
  • Type I collagen is the most important element in keloids. Indeed, in our preliminary experiments, it was found that the expression of type I collagen mRNA is overexpressed in keloid fibroblasts compared to normal skin fibroblasts derived from the same patient. (Data not shown). Because dark-skinned races, including African Americans, Latin Americans, and East Asians, are susceptible to keloid formation [19], genetic factors may play an important role in it. It has been.
  • Keloid fibroblasts have a higher proliferative capacity than normal skin fibroblasts, which are resistant to apoptosis induced by several stimuli such as anti-Fas antibody, staurosporine, and C2 ceramide. It has been reported [21, 22]. Apoptosis may mediate the transition from granulation stage to normal scar in the wound healing process.
  • keloids are the result of abnormal wound healing processes that prolong cell proliferation and are resistant to apoptosis, resulting in collagen accumulation.
  • Resveratrol has been reported to exhibit antifibrotic effects by various mechanisms [23, 24], but its effect on keloids is not known at all.
  • the inventors first studied the effect of resveratrol on the expression of type I collagen, which is the main component of the extracellular matrix. Its overexpression is one of the pathophysiological features of keloid formation. The results by the present inventors have shown that resveratrol suppresses the expression of type I collagen mRNA in a dose-dependent manner. ⁇ -SMA is known to be a marker for myofibroblasts. The results by the inventors showed that resveratrol also suppressed ⁇ -SMA mRNA expression in a dose-dependent manner. Next, the inventors investigated keloid fibrogenic properties, including the effects of HSP47 and TGF- ⁇ 1.
  • HSP47 is a collagen-specific molecular chaperone that increases with collagen expression in fibrotic diseases [14, 25].
  • keloid tissue was stained more strongly with anti-HSP47 antibody than with normal skin tissue in immunohistochemical analysis, which was consistent with the literature [15] report.
  • the present inventors also found that HSP47 mRNA expression was significantly reduced in keloid fibroblasts treated with resveratrol, suggesting that it may inhibit collagen accumulation.
  • TGF- ⁇ 1 exhibits the most prominent fibrogenic properties [28], and the main role of TGF- ⁇ 1 in keloid fibroblasts is thought to be the positive regulation of type I collagen accumulation [16]. It was shown by keloid fibroblast ELISA that the total TGF- ⁇ 1 production of keloid marginal fibroblasts was significantly suppressed after treatment with resveratrol 100 ⁇ M. On the other hand, with RT-PCR, TGF- ⁇ 1 mRNA expression in keloid fibroblasts is not suppressed by resveratrol treatment (data not shown), and resveratrol affects TGF- ⁇ 1 expression in fibroblasts at the transcriptional level. It was suggested that there is a case not to affect.
  • resveratrol suppresses TGF- ⁇ 1 mRNA expression and proliferation of hepatic stellate cells, thereby explaining its anti-fibrotic mechanism [12, 23].
  • TGF- ⁇ 1 activation effect on type I collagen appears to be complex.
  • the present inventors have found that resveratrol can directly act on keloid fibroblasts and suppress endogenous synthesis of TGF- ⁇ 1, resulting in suppression of type I collagen expression. I guess it will be brought.
  • TGF- ⁇ 1 activates HSF1 and thus stimulates transcription of HSP47 mRNA, resulting in increased expression of HSP47 protein in human fibroblasts.
  • resveratrol reduced keloid fibroblast proliferation in a dose-dependent manner.
  • the mechanism of its inhibitory effect on cell proliferation remains unknown.
  • growth inhibition by resveratrol could be a common finding because resveratrol inhibits the growth of many types of cells, including cancer cells [30-33].
  • Another effect of resveratrol is the ability to induce apoptosis.
  • Apoptosis is a major factor in the regression of keloid tissues, and resveratrol-induced increased apoptosis of keloid fibroblasts may be involved in the molecular mechanism of resveratrol's anti-fibrotic activity.
  • cell cycle analysis and Annexin-V assay results showed resveratrol-induced apoptosis of keloid fibroblasts.
  • resveratrol stimulated caspase-3 activation.
  • Resveratrol has a variety of mechanisms, including suppression of the PI3K / Akt pathway, promotion of p53 phosphorylation, and mitochondrial apoptosis pathway, resulting in leukemia cell lines, breast cell lines, myeloma cell lines, skin cell lines, fetuses It has been shown to cause apoptosis in multiple rhabdomyosarcoma and glioblastoma cell lines [9, 34-37]. In keloid fibroblasts, it has been suggested that resveratrol induces apoptosis through a caspase-3-dependent mechanism, although multiple apoptotic triggers functioning by various signal pathways have been suggested.
  • TGF- ⁇ 1 exhibits anti-apoptotic effects in keloid fibroblasts [21], or because it may induce apoptosis by suppressing NFkb activity [38], apoptosis by resveratrol in keloid fibroblasts Another mechanism may be by suppression of TGF- ⁇ 1.
  • the in vivo effect needs to be further investigated. Lu et al. [39] reported that there was a significant difference in cell cycle distribution between keloid central fibroblasts and keloid marginal fibroblasts. Most keloid marginal fibroblasts are in the proliferative phase (G2 / M and S) and their high proliferative capacity can explain the hyperplastic growth characteristics of the marginal region of keloids.
  • the present inventors have found that resveratrol is more effective as an inducer of apoptosis in keloid marginal fibroblasts than in keloid central fibroblasts.
  • the present inventors studied cultured keloid fibroblasts derived from patient keloids to determine whether resveratrol exhibits an antifibrotic effect on keloids.
  • the inventors have demonstrated that resveratrol is effective in suppressing type I collagen accumulation in keloid fibroblasts.
  • resveratrol suppressed keloid fibroblast proliferation and induced apoptotic cell death of keloid fibroblasts.
  • resveratrol's anti-fibrotic effect may be due to suppression of keloid fibroblast activation and a proapoptptic effect.
  • Our studies suggest the therapeutic potential of patients with resveratrol and thus provide new insights into the therapeutic strategies for keloid treatment.
  • Example 6 Inhibition of Keloid Fibroblast Type I Collagen, HSP47 and TGF- ⁇ mRNA Expression by Quercetin Inhibition of keloid fibroblast type I collagen, HSP47 and TGF- ⁇ mRNA expression by using quercetin
  • quercetin suppressed the expression of type I collagen, HSP47 and TGF- ⁇ in a dose-dependent manner in keloid marginal fibroblasts (FIGS. 9B, Q10 and Q100).
  • nicotinamide known as a Sirt1 inhibitor as a comparative example, expression of type I collagen, HSP47 and TGF- ⁇ was not affected (FIGS. 9A and B, N10 and N100).
  • Example 7 Inhibition of Keloid Fibroblast Growth by Quercetin
  • the inhibitory effect on the proliferation of keloid central and keloid marginal fibroblasts was examined using quercetin according to the procedure of the WST-1 assay.
  • the result in the keloid central part fibroblast is shown in FIG. 11, and the result in the keloid marginal fibroblast is shown in FIG.
  • Quercetin showed a significant cell growth inhibitory effect in both the central keloid and the marginal fibroblasts.
  • the same experiment was conducted using nicotinamide as a comparative example.
  • the results for keloid central fibroblasts are shown in FIG. 13, and the results for keloid marginal fibroblasts are shown in FIG. Nicotinamide did not show a significant cytostatic effect in both the central keloid and the peripheral keloid fibroblasts.
  • Resveratrol inhibits dimethylnitrosamine-induced hepatic fibrosis in rats. Arch Pharm Res 2010; 33: 925-932. [24] Li J, Qu X, Ricardo SD, et al. Resveratrol inhibits renal fibrosis in the obstructed kidney: potential role in deacetylation of Smad 3. Am J Pathol 2010; 177: 1065-1071. [25] Masuda H, Fukumoto M, Hirayoshi K, et al. Coexpression of the collagen-binding stress protein HSP47 gene and the alpha 1 (I) and alpha 1 (III) collagen genes in carbon tetrachloride-induced rat liver fibrosis. Clin Invest 1994; 94: 2481-2488.
  • Resveratrol-caused apoptosis of human prostate carcinoma LNCaP cells is mediated via modulation of phosphtidylinositol 3'-kinase / Akt pathway and Bcl-2 family proteins. Mol Cancer Ther 2006; 5: 1335-1341. [36] She QB, Bode AM, Ma WY, et al. Resveratrol-induced activation of p53 and apoptosis is mediated by extracellular-signal regulated protein kinases and p38 kinase. Cancer Res 2001; 61: 1604-1610. [37] Delmas D, Rebe C, Micheau O, et al.

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Abstract

La présente invention concerne une composition destinée à inhiber la prolifération d'un fibroblaste chéloïdien, qui contient un polyphénol représenté par la formule (I).
PCT/JP2012/052559 2011-02-04 2012-02-03 Agent thérapeutique contre les chéloïdes WO2012105706A1 (fr)

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WO2015156328A1 (fr) * 2014-04-09 2015-10-15 江崎グリコ株式会社 Inhibiteur de vieillissement de la peau et concentré de resvératrol 3-o-α-glucoside
CN108795873A (zh) * 2018-06-25 2018-11-13 宁波美奈生物科技有限公司 一种成纤维细胞的制备方法及其试剂盒

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JP2007084446A (ja) * 2005-09-20 2007-04-05 Pola Chem Ind Inc 皮膚外用剤

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WO2001043705A2 (fr) * 1999-12-16 2001-06-21 Johnson & Johnson Consumer Companies, Inc. Compositions a base de retinoides et de stilbenes pour le soin de la peau
JP2007084446A (ja) * 2005-09-20 2007-04-05 Pola Chem Ind Inc 皮膚外用剤

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015156328A1 (fr) * 2014-04-09 2015-10-15 江崎グリコ株式会社 Inhibiteur de vieillissement de la peau et concentré de resvératrol 3-o-α-glucoside
JPWO2015156328A1 (ja) * 2014-04-09 2017-04-13 江崎グリコ株式会社 皮膚老化防止剤、及びレスベラトロール３−Ｏ−α−グルコシドの濃縮液
US10149809B2 (en) 2014-04-09 2018-12-11 Ezaki Glico Co., Ltd. Skin aging inhibitor and concentrate of resveratrol 3-O-α-glucoside
CN108795873A (zh) * 2018-06-25 2018-11-13 宁波美奈生物科技有限公司 一种成纤维细胞的制备方法及其试剂盒

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