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Cited by 206Skin Protection and AgingProtease and Inhibitor MechanismsDermatologic Treatments and Research

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Biological Mechanisms Underlying the Ultraviolet Radiation-Induced Formation of Skin Wrinkling and Sagging I: Reduced Skin Elasticity, Highly Associated with Enhanced Dermal Elastase Activity, Triggers Wrinkling and Sagging

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Imokawa, Genji, Ishida, Koichi·2015·International Journal of Molecular Sciences
DOI 10.3390/ijms16047753PMC PMC4425048
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Abstract

The repetitive exposure of skin to ultraviolet B (UVB) preferentially elicits wrinkling while ultraviolet A (UVA) predominantly elicits sagging. In chronically UVB or UVA-exposed rat skin there is a similar tortuous deformation of elastic fibers together with decreased skin elasticity, whose magnitudes are greater in UVB-exposed skin than in UVA-exposed skin. Comparison of skin elasticity with the activity of matrix metalloproteinases (MMPs) in the dermis of ovariectomized rats after UVB or UVA irradiation demonstrates that skin elasticity is more significantly decreased in ovariectomized rats than in sham-operated rats, which is accompanied by a reciprocal increase in elastase activity but not in the activities of collagenases I or IV. Clinical studies using animal skin and human facial skin demonstrated that topical treatment with a specific inhibitor or an inhibitory extract of skin fibroblast-derived elastase distinctly attenuates UVB and sunlight-induced formation of wrinkling. Our results strongly indicated that the upregulated activity of skin fibroblast-derived elastase plays a pivotal role in wrinkling and/or sagging of the skin via the impairment of elastic fiber configuration and the subsequent loss of skin elasticity.

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Figure 1 Curing of elastin fibers in a three-dimensional configuration following repetitive UVB exposure. (a) Elastic fibers in non-exposed skin; (b) Elastic fibers in repeatedly UVB exposed skin. Rats were placed in cages individually and irradiated by a bank of five Toshiba SE lamps (UVB) without any filtering. The UVB lights have no detectable emission below 340 nm and a peak of emission near 312 nm with the irradiance between 290 and 320 nm corresponding to 55% of the total amount of UVB. The distance from the UVB lamps to the animals’ hind limbs was 42 cm (irradiance approximately 0.72 mW/cm2). UVB radiation at a dose of 130 mJ/cm2 (rat 1 minimal erytyemal dose (MED) = 170 mJ/cm2) was given three times weekly from three to eight weeks of age.
Figure 1 Curing of elastin fibers in a three-dimensional configuration following repetitive UVB exposure. (a) Elastic fibers in non-exposed skin; (b) Elastic fibers in repeatedly UVB exposed skin. Rats were placed in cages individually and irradiated by a bank of five Toshiba SE lamps (UVB) without any filtering. The UVB lights have no detectable emission below 340 nm and a peak of emission near 312 nm with the irradiance between 290 and 320 nm corresponding to 55% of the total amount of UVB. The distance from the UVB lamps to the animals’ hind limbs was 42 cm (irradiance approximately 0.72 mW/cm2). UVB radiation at a dose of 130 mJ/cm2 (rat 1 minimal erytyemal dose (MED) = 170 mJ/cm2) was given three times weekly from three to eight weeks of age.CC BY · PubMed Central
Figure 2 Elastase in the dorsal skin of mice at different ages. Elastase activity was measured per unit area of each skin biopsy. Values represent means ± S.D. from five independent experiments. ** p < 0.01; * p < 0.05 vs. 2 months (ANOVA, Dunnett’s test).
Figure 2 Elastase in the dorsal skin of mice at different ages. Elastase activity was measured per unit area of each skin biopsy. Values represent means ± S.D. from five independent experiments. ** p < 0.01; * p < 0.05 vs. 2 months (ANOVA, Dunnett’s test).CC BY · PubMed Central
Figure 3 (A) Effects of ovariectomy with or without UVA irradiation on skin sagging. (a) Photographs after UVA irradiation (five times weekly for 12 weeks) of hairless mouse skin. White arrow shows distinctly increased sagging around the neck; (b) Visual scoring (scale = 0–3) of sagging after UVA irradiation. One week after the operations (i.e., when the mice were seven weeks old), half of the 96 mice were divided into six groups of eight mice each, as follows: (i) group 1, nonoperation/UVA nonirradiated (UVA−) group; (ii) group 2, sham operation/UVA− group; (iii) group 3, ovariectomy/UVA− group; (iv) group 4, nonoperation/UVA irradiated (UVA+) group; (v) group 5, sham operation/UVA+ group; and (vi) group 6, ovariectomy/UVA+ group. The mice were irradiated using a bank of 12 Toshiba BL lamps with a glass filter (0.5 cm thick) for UVA (peak of emission near 351 nm, no emission below 320 nm, the irradiance between 320 and 380 nm corresponding to 93% of the total amount of UVA). The distance from the lamps to the animals’ backs was 35 cm. The animals were exposed to a UVA dose of 22.3 J/cm2/day, five times weekly, for 14 weeks; (B) Evaluation of sagging by pinch testing. (a) Photographs of pinch testing. Left: Mouse dorsal skin on the midline was picked up with the fingers as much as possible; Right: Photographs 1 s after the skin was released; (b) Recovery time in the pinch test using UVA exposed mice (five times weekly for 12 weeks). Data represent means ± SD; (C) Changes in skin elastic parameters after ovariectomy with or without UVA irradiation. Data represent means ± SD, n = 8. **, * p < 0.01, 0.05, a (vs. sham/UVA−); b (vs. ovariectomy/UVA−); c (vs. sham/UVA+). Ue/Ue*: immediate distension, Uv/Uv*: delayed distension, Ur/Ur*: immediate retraction, Ue/Ue*: final distension, All these variables are a function of skin thickness and thus cannot be compared simply between subjects and regions. Each individual measurement of the skin thickness was used to calculate mechanical variable values for a given animal for a skin thickness of 1 mm. These normalized values are marked with an asterisk (Ue*, Uf*, Ur*, Uv*), as described by Agache et al. [38].
Figure 3 (A) Effects of ovariectomy with or without UVA irradiation on skin sagging. (a) Photographs after UVA irradiation (five times weekly for 12 weeks) of hairless mouse skin. White arrow shows distinctly increased sagging around the neck; (b) Visual scoring (scale = 0–3) of sagging after UVA irradiation. One week after the operations (i.e., when the mice were seven weeks old), half of the 96 mice were divided into six groups of eight mice each, as follows: (i) group 1, nonoperation/UVA nonirradiated (UVA−) group; (ii) group 2, sham operation/UVA− group; (iii) group 3, ovariectomy/UVA− group; (iv) group 4, nonoperation/UVA irradiated (UVA+) group; (v) group 5, sham operation/UVA+ group; and (vi) group 6, ovariectomy/UVA+ group. The mice were irradiated using a bank of 12 Toshiba BL lamps with a glass filter (0.5 cm thick) for UVA (peak of emission near 351 nm, no emission below 320 nm, the irradiance between 320 and 380 nm corresponding to 93% of the total amount of UVA). The distance from the lamps to the animals’ backs was 35 cm. The animals were exposed to a UVA dose of 22.3 J/cm2/day, five times weekly, for 14 weeks; (B) Evaluation of sagging by pinch testing. (a) Photographs of pinch testing. Left: Mouse dorsal skin on the midline was picked up with the fingers as much as possible; Right: Photographs 1 s after the skin was released; (b) Recovery time in the pinch test using UVA exposed mice (five times weekly for 12 weeks). Data represent means ± SD; (C) Changes in skin elastic parameters after ovariectomy with or without UVA irradiation. Data represent means ± SD, n = 8. **, * p < 0.01, 0.05, a (vs. sham/UVA−); b (vs. ovariectomy/UVA−); c (vs. sham/UVA+). Ue/Ue*: immediate distension, Uv/Uv*: delayed distension, Ur/Ur*: immediate retraction, Ue/Ue*: final distension, All these variables are a function of skin thickness and thus cannot be compared simply between subjects and regions. Each individual measurement of the skin thickness was used to calculate mechanical variable values for a given animal for a skin thickness of 1 mm. These normalized values are marked with an asterisk (Ue*, Uf*, Ur*, Uv*), as described by Agache et al. [38].CC BY · PubMed Central
Figure 4 Changes in skin elastic parameters after ovariectomy, with or without UVB irradiation. Data represent means ± SD, n = 8. **, * p < 0.01, 0.05, a (vs. sham/UVB−); b (vs. ovariectomy/UVB−); c (vs. sham/UVB+). At seven weeks old, 48 mice were divided into six groups of eight mice each, as follows: (i) group 1, nonoperation/UVB− group; (ii) group 2, sham operation/UVB− group; (iii) group 3, ovariectomy/UVB− group; (iv) group 4, nonoperation/UVB+ group; (v) group 5, sham operation/UVB+ group; and (vi) group 6, ovariectomy/UVB+ group. A bank of six Toshiba SE lamps was used without any filtering for UVB (peak of emission near 312 nm, the irradiance between 290 and 320 nm corresponding to 55% of the total amount of UVB). The distance from the lamps to the animals’ backs was 35 cm. A progressive UVB exposure regimen was used starting at approximately 50 mJ/cm2 (non-operated mice: 1MED ¼ 70 mJ/cm2/day at week 1, which was then increased by 5 mJ/cm2/week until week 4. The exposure dose of 65 mJ/cm2 was then kept constant for the remaining period of exposure, the mice being irradiated five times weekly for 14 weeks. The total UVB dose was approximately 4.4 J/cm2. Ue/Ue*: immediate distension, Uv/Uv*: delayed distension, Ur/Ur*: immediate retraction, Ue/Ue*: final distension.
Figure 4 Changes in skin elastic parameters after ovariectomy, with or without UVB irradiation. Data represent means ± SD, n = 8. **, * p < 0.01, 0.05, a (vs. sham/UVB−); b (vs. ovariectomy/UVB−); c (vs. sham/UVB+). At seven weeks old, 48 mice were divided into six groups of eight mice each, as follows: (i) group 1, nonoperation/UVB− group; (ii) group 2, sham operation/UVB− group; (iii) group 3, ovariectomy/UVB− group; (iv) group 4, nonoperation/UVB+ group; (v) group 5, sham operation/UVB+ group; and (vi) group 6, ovariectomy/UVB+ group. A bank of six Toshiba SE lamps was used without any filtering for UVB (peak of emission near 312 nm, the irradiance between 290 and 320 nm corresponding to 55% of the total amount of UVB). The distance from the lamps to the animals’ backs was 35 cm. A progressive UVB exposure regimen was used starting at approximately 50 mJ/cm2 (non-operated mice: 1MED ¼ 70 mJ/cm2/day at week 1, which was then increased by 5 mJ/cm2/week until week 4. The exposure dose of 65 mJ/cm2 was then kept constant for the remaining period of exposure, the mice being irradiated five times weekly for 14 weeks. The total UVB dose was approximately 4.4 J/cm2. Ue/Ue*: immediate distension, Uv/Uv*: delayed distension, Ur/Ur*: immediate retraction, Ue/Ue*: final distension.CC BY · PubMed Central
Figure 5 Effects of ovariectomy with or without UVA irradiation on the activities of elastase, collagenase I, and collagenase IV. (A) elastase activity; (B) type I collagenase activity; (C) type IV collagenase activity. Data represent means ± SD. ∆□○◊: each measured value.
Figure 5 Effects of ovariectomy with or without UVA irradiation on the activities of elastase, collagenase I, and collagenase IV. (A) elastase activity; (B) type I collagenase activity; (C) type IV collagenase activity. Data represent means ± SD. ∆□○◊: each measured value.CC BY · PubMed Central
Figure 6 Elastase activity measured using N-succinyl-tri-alanyl-p-nitroaniline (STANA) as a substrate in UVB-exposed skin. Skin homogenates excluding subcutaneous tissue were used as the enzyme source for elastase activity. Mouse skin was biopsied 1 day after the indicated time of UVB irradiation, homogenized and processed for elastase activity. * p < 0.05 (vs. 0 week); n = 5. Data represent means ± SD. The mice were irradiated using a bank of Toshiba SE20 lamps (Toshiba Lighting & Technology Corp., Tokyo, Japan) as the source, being rich in UVB. The distance from the lamp to the animals’ backs was 35 cm. A progressive UV exposure regimen was used starting at approximately 65 mJ/cm2 (just below the minimum erythemal dose [MED]) at week 1, which was then increased by 10 mJ/cm2/week until week 4. An exposure dose of 95 mJ/cm2 was then kept constant for the remaining period of exposure. The mice were irradiated five times a week for 18 weeks. It should be noted that there was no induction of erythema or desquamation at any time during this study.
Figure 6 Elastase activity measured using N-succinyl-tri-alanyl-p-nitroaniline (STANA) as a substrate in UVB-exposed skin. Skin homogenates excluding subcutaneous tissue were used as the enzyme source for elastase activity. Mouse skin was biopsied 1 day after the indicated time of UVB irradiation, homogenized and processed for elastase activity. * p < 0.05 (vs. 0 week); n = 5. Data represent means ± SD. The mice were irradiated using a bank of Toshiba SE20 lamps (Toshiba Lighting & Technology Corp., Tokyo, Japan) as the source, being rich in UVB. The distance from the lamp to the animals’ backs was 35 cm. A progressive UV exposure regimen was used starting at approximately 65 mJ/cm2 (just below the minimum erythemal dose [MED]) at week 1, which was then increased by 10 mJ/cm2/week until week 4. An exposure dose of 95 mJ/cm2 was then kept constant for the remaining period of exposure. The mice were irradiated five times a week for 18 weeks. It should be noted that there was no induction of erythema or desquamation at any time during this study.CC BY · PubMed Central
Figure 7 Inhibitory profiles of fibroblast elastase (A) and neutrophil elastase (B). To measure fibroblast elastase, an enzyme solution obtained from a lysate of human fibroblasts was used at a concentration of 100 μg/mL. Neutrophil elastase was used at a concentration of 20 U/mL. Elastase activity was measured using STANA as a substrate in the presence of various inhibitors. Data represent means ± SD.
Figure 7 Inhibitory profiles of fibroblast elastase (A) and neutrophil elastase (B). To measure fibroblast elastase, an enzyme solution obtained from a lysate of human fibroblasts was used at a concentration of 100 μg/mL. Neutrophil elastase was used at a concentration of 20 U/mL. Elastase activity was measured using STANA as a substrate in the presence of various inhibitors. Data represent means ± SD.CC BY · PubMed Central
Figure 8 Inhibitory effect of NPLT on hairless mouse skin elastase stimulated by UVB irradiation. Hairless mouse skin was biopsied 24 h after 6 weeks of UVB irradiation or age-matched control, homogenized, and processed for elastase activity. NPLT was used at the indicated concentrations. The UVB radiation was carried out in the same conditions described in the legend of Figure 6. * p < 0.05; ** p < 0.01 (vs. control); # p < 0.05; ## p < 0.01 (vs. 0 mM NPLT). Data represent mean ± SD.
Figure 8 Inhibitory effect of NPLT on hairless mouse skin elastase stimulated by UVB irradiation. Hairless mouse skin was biopsied 24 h after 6 weeks of UVB irradiation or age-matched control, homogenized, and processed for elastase activity. NPLT was used at the indicated concentrations. The UVB radiation was carried out in the same conditions described in the legend of Figure 6. * p < 0.05; ** p < 0.01 (vs. control); # p < 0.05; ## p < 0.01 (vs. 0 mM NPLT). Data represent mean ± SD.CC BY · PubMed Central
Figure 9 Photographs of wrinkle formation on the dorsal skin of hairless mice at week 18. (A) UVB-exposed and 80% ethanol–treated skin; (B) UVB-exposed and NPLT-treated skin; (C) UVB-exposed and Parsol-MCX–treated skin. The UVB radiation was carried out in the same conditions described in the legend of Figure 6.
Figure 9 Photographs of wrinkle formation on the dorsal skin of hairless mice at week 18. (A) UVB-exposed and 80% ethanol–treated skin; (B) UVB-exposed and NPLT-treated skin; (C) UVB-exposed and Parsol-MCX–treated skin. The UVB radiation was carried out in the same conditions described in the legend of Figure 6.CC BY · PubMed Central
Figure 10 Effects of NPLT on UVB-induced wrinkling. (A) Experimental protocol. Rats were placed in cages individually and were irradiated by a bank of five Toshiba SE lamps (UVB) without any filtering, three times a week for a total of six weeks. The distance from the lamp to the animal’s hind limbs was 42 cm (irradiance was approximately 0.72 mW/cm2), and a dose of 130 mJ per cm2 (rat 1 suberythemal dose = 170 mJ/cm2) was given three times weekly. The relative spectral contributions of Toshiba SE lamps used in this study were 63%, 36%, and 0.5% in the UVB (290–320 nm), UVA (320–400 nm), and UVC (<290 nm) regions, respectively; (B) Effect of topical application of NPLT on elasticity of UVB-irradiated rat hind limb skin. The skin elasticity, expressed as physical parameters Ue, Uv, Ur, and Uf, was measured with a Cutometer. These measurements were carried out at an age of nine weeks. ***, **, * p < 0.005, 0.01, 0.05 (vs. irradiated/EtOH treatment); ###, ## p < 0.005, 0.01 (vs. unirradiated/untreated); Ue, immediate distension measured at 0.1 s; Uv, delayed distention; Ur, immediate retraction; Uf, final distention. n = 10, values represent mean ± SD; (C) SEM images after topical treatment followed by intravascular injection of Mercox resin and selective digestion at nine weeks; (D) Interrelationship between wrinkle formation, skin elasticity, and elastic fiber linearity.
Figure 10 Effects of NPLT on UVB-induced wrinkling. (A) Experimental protocol. Rats were placed in cages individually and were irradiated by a bank of five Toshiba SE lamps (UVB) without any filtering, three times a week for a total of six weeks. The distance from the lamp to the animal’s hind limbs was 42 cm (irradiance was approximately 0.72 mW/cm2), and a dose of 130 mJ per cm2 (rat 1 suberythemal dose = 170 mJ/cm2) was given three times weekly. The relative spectral contributions of Toshiba SE lamps used in this study were 63%, 36%, and 0.5% in the UVB (290–320 nm), UVA (320–400 nm), and UVC (<290 nm) regions, respectively; (B) Effect of topical application of NPLT on elasticity of UVB-irradiated rat hind limb skin. The skin elasticity, expressed as physical parameters Ue, Uv, Ur, and Uf, was measured with a Cutometer. These measurements were carried out at an age of nine weeks. ***, **, * p < 0.005, 0.01, 0.05 (vs. irradiated/EtOH treatment); ###, ## p < 0.005, 0.01 (vs. unirradiated/untreated); Ue, immediate distension measured at 0.1 s; Uv, delayed distention; Ur, immediate retraction; Uf, final distention. n = 10, values represent mean ± SD; (C) SEM images after topical treatment followed by intravascular injection of Mercox resin and selective digestion at nine weeks; (D) Interrelationship between wrinkle formation, skin elasticity, and elastic fiber linearity.CC BY · PubMed Central
Figure 11 Concentration-dependent inhibition of skin fibroblast elastase by Zingiber officinale (L.) Rose (A) and chemical structures of active substances (B). An enzyme solution obtained from a cell lysate of human fibroblasts was used at a concentration of 200–500 μg/mL. Fibroblast-derived elastase (filled circles) and neutrophil-derived elastase (open circles) activities were measured using STANA as a substrate in the presence of Zingiber officinale (L.) Rose at the indicated concentrations. Data represent means ± SD.
Figure 11 Concentration-dependent inhibition of skin fibroblast elastase by Zingiber officinale (L.) Rose (A) and chemical structures of active substances (B). An enzyme solution obtained from a cell lysate of human fibroblasts was used at a concentration of 200–500 μg/mL. Fibroblast-derived elastase (filled circles) and neutrophil-derived elastase (open circles) activities were measured using STANA as a substrate in the presence of Zingiber officinale (L.) Rose at the indicated concentrations. Data represent means ± SD.CC BY · PubMed Central
Figure 12 Clinical study using a skin fibroblast inhibitory extract on wrinkling of human facial skin. (A) Representative photos of the corners of the eyes before and 1 year after topical treatment with the Zingiber officinale (L.) Rose extract; (B) Replicas; (C) Wrinkle score; (D) Fine Wrinkle percent evaluated by image analysis; (E) Skin elasticity; (F) Water Content.
Figure 12 Clinical study using a skin fibroblast inhibitory extract on wrinkling of human facial skin. (A) Representative photos of the corners of the eyes before and 1 year after topical treatment with the Zingiber officinale (L.) Rose extract; (B) Replicas; (C) Wrinkle score; (D) Fine Wrinkle percent evaluated by image analysis; (E) Skin elasticity; (F) Water Content.CC BY · PubMed Central

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  1. Tsukahara, 2004, A clinical effect of Zingiber officinale (L.) Rose on facial wrinkles and skin elasticity, Jichi Med. School J., 27, 19

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Metadata

Authors
Imokawa, G., & Ishida, K.
Affiliations
  • Chubu University (JP)
  • Kao Corporation (Japan) (JP)
Corresponding author
Imokawa, Genji
ORCID
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2015-04-08
Volume
16
Issue
4
Pages
7753-7775
ISSN
1422-0067 (ISSN-L)
DOI
10.3390/ijms16047753
PMC
PMC4425048
OpenAlex
W2089161677
Cited by
206
References
59
Citations by year
2026: 19 · 2025: 19 · 2024: 35 · 2023: 17 · 2022: 40 · 2021: 17 · 2020: 21 · 2019: 14 · 2018: 8 · 2017: 6 · 2016: 9 · 2015: 1
FWCI
1.28
Type
journal article
Language
en
Open access
Licence
Locations
  • International Journal of Molecular Sciences (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Topics
  • Health Sciences › Medicine › Dermatology › Skin Protection and Aging
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Protease and Inhibitor Mechanisms
  • Health Sciences › Medicine › Dermatology › Dermatologic Treatments and Research
Keywords
ultraviolet radiation, skin aging, skin wrinkling, skin sagging, skin elasticity, elastase, matrix metalloproteinases, elastic fibers, UV-B radiation, UVA, ovariectomy, collagenase, dermal fibroblasts, elastase inhibitors, facial skin
MeSH terms
Animals, Elasticity, Fibroblasts, Humans, Pancreatic Elastase, Ultraviolet Rays, Skin Aging, Dermis

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