Dose-effect relationship between Q-switch ruby laser and skin pigmentation in Guinea pigs

Antioxidants such as vitamins C and E have been reported to inhibit the progression of ultraviolet (UV) radiation-induced pigmentation in the skin of hairless mice. However, little is known of the lightening effect of proanthocyanidin, a powerful polyphenolic antioxidant, on UV-induced pigmentation of the skin. We investigated the lightening effect of oral administration of a proanthocyanidin-rich grape seed extract (GSE) using guinea pigs with UV-induced pigmentation. These pigmented guinea pigs were fed diets containing 1% GSE or 1% vitamin C (w/w) for 8 weeks. GSE-feeding had an apparent lightening effect on the guinea pigs' pigmented skin. Histologic evaluation demonstrated a decrease in the number of 3,4-dihydroxyphenylalanine (DOPA)-positive melanocytes as well as 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive, Ki-67-positive, proliferating cell nuclear antigen (PCNA)-positive melanin-containing cells in the basal epidermal layer of the UV-irradiated skin in GSE-fed guinea pigs. In contrast, these parameters did not change in the skin of vitamin C-fed or control guinea pigs. GSE inhibited the activity of mushroom tyrosinase and also inhibited melanogenesis without inhibiting the growth of cultured B16 mouse melanoma cells. In conclusion, we demonstrated that oral administration of GSE is effective in lightening the UV-induced pigmentation of guinea pig skin. This effect may be related to the inhibition of melanin synthesis by tyrosinase in melanocytes and the reactive oxygen species (ROS)-related proliferation of melanocytes.

Objective To compare the accelerating effect of topical recombinant human epidermal growth factor （rhEGF） and recombinant bovine basic fibroblast growth factor （rb-bFGF） on wound healing after fractional CO2 laser therapy. Methods Twenty male guinea pigs were included in this study. After hair removal and irradiation with fractional CO2 laser, the back of each guinea pig was divided into 4 regions to be topically treated with rhEGF of 10 μg/cm： （rhEGF group）, rb-bFGF of 262.51 IU/cm2 （rb-bFGF group）, the combination of rhEGF and rb-bFGF （combination group）, or normal saline （control group）, twice daily until the healing of wound. Skin physiology parameters including elasticity index and melanin index were detected before the irradiation, 7, 14 and 28 days after the irradiation, and compared between the 4 groups by analysis of variance. Tissue specimens were obtained from 4 mice at the above time points and subjected to pathological examination for the observation of collagen fibers and quantification of fibroblasts. Results After fractional CO2 laser therapy, the crusts fall off completely in growth factor-treated regions, while partly in the control regions, within 3 to 7 days; the wounds healed completely in 14 to 28 days in all the groups, with the regenerating tissue being more tender and redder compared with the surrounding unirradiated tissue. The wound surface was smaller in area and redder in color in the 3 growth factor-treated groups than in the control group. At 28 days after the irradiation, the elasticity index was 262.29 ± 62.40 in the combination group, 202.00 ± 65.62 in the rhEGF group, 188.86 ± 35.02 in the rb-bFGF group, 167.14 ± 42.49 in the control group. Statistical difference was observed in elasticity index, but not in skin melanin index among the 4 groups. Pathological examination showed a dense and organized arrangement of collagen fibem in the combination group but a sparse and disorganized arrangement of collagen fibers in the control group. Conclusion The combined application of rhEGF and rbbFGF can accelerate the healing of wound and increase the elasticity of regenerating tissue after fractional CO2 laser therapy. Key words: Administration, cutaneous; Fibroblast growth factor; Epidermal growth factor; Lasers; Wound healing

Melanocytes stimulated by ultraviolet radiation (UVR) produce melanin and melanosomes, which causes skin pigmentation and acts as an important physiological defence process for photoprotection. Neutral luminal pH of melanosomes is critical for providing optimal conditions for the rate-limiting, pH-sensitive melanin synthesizing enzyme tyrosinase (TYR). As a major component of extraocular phototransduction pathway, transient receptor potential ankyrin1 (TRPA1) can be activated by ultraviolet B (UVB) and reported to be expressed in melanocytes. However, whether TRPA1 is involved in the regulation of melanogenesis remains unclear. Melanogenic activity of TRPA1 was evaluated in primary normal human epidermal melanocytes (HEMs) and murine B16-F10 cell cultures, and the effects of topical applications of TRPA1 specific agonist and antagonist on UVB-induced skin pigmentation were confirmed on in vivo guinea pig models. Calcium (Ca2+ ) imaging and pH imaging were performed to analyse the effects of TRPA1 on intracellular Ca2+ concentration ([Ca2+ ]ic ) and melanosome luminal pH. TRPA1 regulated melanin synthesis, UVB-induced Ca2+ influx and melanosome luminal pH in HEMs and B16-F10 cells. Topical treatment of TRPA1 specific agonist JT010 increased UVB-induced skin pigmentation in guinea pigs, while topical using of TRPA1 selective antagonist HC-030031 mitigated such pigmentation. Our results indicated that TRPA1 activated by UVB enhanced the skin pigmentation, most likely by regulating the [Ca2+ ]ic and the melanosomal pH, consequently influencing the enzymatic activity of TYR. Therefore, the results suggest TRPA1 as a potential therapeutic target in the treatment of skin pigmented disorders that are at high risk under UVB irradiation.

Resveratrol is a polyphenolic compound found in various natural products such as grapes and berries and possesses anti-cancer, anti-hyperlipidemia, and anti-aging properties. Recently, it has been reported that resveratrol inhibits α-melanocyte-stimulating hormone signaling, viability, and migration in melanoma cells. However, these effects have not been confirmed in vivo, specifically brownish guinea pigs. To evaluate the potential of resveratrol as a regulator of melanin for hyperpigmentation therapy, the influence of resveratrol on pigmentation was investigated by ultraviolet B-induced hyperpigmentation in brownish guinea pig skin. We found that resveratrol reduced the expression of melanogenesis-related proteins tyrosinase, tyrosinase-related proteins 1 and 2, and microphthalmia-associated transcription factor in melanoma cells. Furthermore, topical application of resveratrol was demonstrated to significantly decrease hyperpigmentation on ultraviolet B-stimulated guinea pig skin in vivo. Based on our histological data, resveratrol inhibits melanin synthesis via a reduction in tyrosinase-related protein 2 among the melanogenic enzymes. This study is the first to provide evidence supporting resveratrol as a depigmentation agent, along with further clinical investigation of resveratrol in ultraviolet B-induced skin disorders such as hyperpigmentation and skin photoaging.

Objective To study the inductive effect of targeted UVB phototherapy on skin hyperpigmentation and its mechanism.Methods Ten brownish guinea pigs were used to develop experimental models.After depilation,four adjacent areas were selected on the back of each guinea pigs and served as the control,low-dose,moderate-dose and high-dose group to receive targeted UVB irradiation with a cumulative dose of O,2500,3500,4500 mJ/cm2,respectively.After 6-week irradiation,the guinea pigs were sacrificed and skin sampies were obtained.The hyperpigmentation induced by UVB irradiation was estimated by naked eyes,staining for melanocytes(Imokawa method)and melanin granules(Masson-Fontana staining),respectively.Immunohistochemistry was carried out to determine the level of nitric oxide synthase and HE staining to observe epidermal histological changes.Results A statistical difference was observed in the pigmentation score,quantity of melanin granules and dopa-positive melanocyte number among the four groups (P<0.05),and the moderatedose group was higher than the high-dose group in terms of these parameters.The expression of inducible nitric oxide synthase increased in a radiation dose-dependent manner,and the median value for inducible nitric oxide synthase expression level was 0.50,1.25,1.75,2.00 in the control,low-dose,moderate-dose and highdose group,respectively(P<0.05).Conclusions Targeted UVB phototherapy can induce hyperpigmentation of the skin in brownish guinea pigs in a dose-dependent manner,but higher dose may not work better.To irradiate with an initial dose close to or slightly higher than the minimum erythema dose may result in a satisfactory effect with reduced cumulative dose and potential risk for cancer. Key words: Ultraviolet therapy; Skin pigmentation; Radiation injuries,experimental; Nitric-oxide synthase

Objective To evaluate the effect of large-spot and low-energy Q switched Nd:YAG laser on melasma,and to observe the changes of melasma lesions with confocal laser scanning microscopy (CLSM) before and after the laser treatment.Methods Totally,45 patients aged from 24 to 48 years and diagnosed with facial melasma were included in this study,and treated with large-spot and low-energy Q switched Nd:YAG once a week for 10 or more sessions.CLSM was used to estimate the melanin content in melasma lesions before each irradiation and after the last irradiation.Results Among the 45 patients,8 ( 17.78％ ) were nearly cured,25 (55.56％) markedly improved,11 (24.44％) improved,and only 1 (2.22％) unimproved after the laser irradiation.The total response rate was 73.33％.As CLSM showed,there was an increment in melanin granules in melasma lesions compared with the normal skin surrounding melasma lesions,but a reduction in melanin granules was induced by the laser treatment in melasma lesions.Conclusions Large-spot and lowenergy Q switched Nd∶YAG laser is substantially effective and highly safe for the treatment of melasma,and CLSM can be used to evaluate the therapeutic effect of laser on melasma. Key words: Confocal laser scanning microscopy; Melasma; Laser

Inhibitory effects of salidroside and paeonol on tyrosinase activity and melanin synthesis in mouse B16F10 melanoma cells and ultraviolet B-induced pigmentation in guinea pig skin

BackgroundPigment disorder dermatoses are common diseases with complex mechanisms. There are various methods for the clinical treatment of pigmentation diseases, but these have a poor curative effect and many adverse reactions. Currently, looking for safe and effective whitening agents is a popular research topic. Stromal vascular fractions (SVFs) are a compound cell component of adipose-derived stem cells (ADSCs) that can promote tissue regeneration, healing, and vascularization. The purpose of this experiment was to investigate the inhibitory effect of SVFs on pigmentation in guinea pigs.MethodsAfter guinea pig subcutaneous fat was digested and centrifuged, SVFs were isolated and quantified. SVF was injected into the pigmentation area of the prepared guinea pig pigmentation model. The amount of inducible nitric oxide synthase (iNOS) was determined using immunohistochemical analysis, histopathological staining, and the Fontana-Masson (F-M) method for measuring melanin formation.ResultsThe skin of the guinea pigs obtained stable and homogenous coloration following three treatments with narrow-band ultraviolet B (NB-UVB). Hematoxylin-eosin (HE) staining revealed that compared to the control group, the cuticle, granular layer, and spinous layer were thicker and the number of epidermal melanocytes and melanin granules increased. While the quantity of pigment granules in the treated group dramatically decreased, it did not significantly change in the blank control group. F-M staining revealed that melanin granules greatly expanded following ultraviolet irradiation and were continuously distributed in basal cells and spinous layers. The entire epidermis was evenly covered in melanin granules. The level of melanin dramatically decreased following therapy. According to immunohistochemical labeling, epidermal cells’ cytoplasm and membranes are where iNOS is primarily found. In the epidermis of the irradiated group, iNOS expression was much higher than in the control group, and following treatment, it decreased in the experimental group.ConclusionsSVFs have a reliable treatment effect on ultraviolet B (UVB)-induced pigmentation in guinea pig skin. SVFs can significantly inhibit pigmentation, effectively shorten the fading time of pigmentation, and play a role in skin whitening, providing a new breakthrough for the treatment of pigmentation diseases.

Skin, our first interface to the external environment, is subjected to oxidative stress caused by a variety of factors such as solar ultraviolet, infrared and visible light, environmental pollution, including ozone and particulate matters, and psychological stress. Excessive reactive species, including reactive oxygen species and reactive nitrogen species, exacerbate skin pigmentation and aging, which further lead to skin tone unevenness, pigmentary disorder, skin roughness and wrinkles. Besides these, skin microbiota are also a very important factor ensuring the proper functions of skin. While environmental factors such as UV and pollutants impact skin microbiota compositions, skin dysbiosis results in various skin conditions. In this review, we summarize the generation of oxidative stress from exogenous and endogenous sources. We further introduce current knowledge on the possible roles of oxidative stress in skin pigmentation and aging, specifically with emphasis on oxidative stress and skin pigmentation. Meanwhile, we summarize the science and rationale of using three well-known antioxidants, namely vitamin C, resveratrol and ferulic acid, in the treatment of hyperpigmentation. Finally, we discuss the strategy for preventing oxidative stress-induced skin pigmentation and aging.

Laser treatment has become a popular method for resolving peri-implantitis, but the full range of its effects on implant surfaces is unknown. The purpose of the present investigation was to analyze the influence of different clinically applicable erbium:yttrium-aluminum-garnet (Er:YAG), carbon dioxide (CO2), and diode laser parameters on titanium surfaces that were either polished or sandblasted, large-grit, acid-etched (SLA). Six polished and six SLA titanium disks were irradiated at nine different power settings (n = 54 polished, 54 SLA) with Er:YAG, CO2, or diode lasers. The CO2 and diode lasers were used in continuous wave mode, and the Er:YAG laser was used in a pulsed manner. The surface of each disk was analyzed by scanning electron microscopy and confocal white light microscopy. Each disk was irradiated on six circular areas of 5 mm in diameter with the same specific laser setting for 10 seconds. Within the chosen parameters, the CO2 and diode laser did not cause any visible surface alterations on either the polished or SLA disks. In contrast, both polished and SLA disks showed surface alterations when irradiated with the pulsed Er:YAG laser. The SLA surfaces showed alteration after 10 seconds of irradiation with Er:YAG laser at 300 mJ/10 Hz. The surfaces of the polished disks did not show alteration with the Er:YAG laser until they were irradiated at the higher energy of 500 mJ/10 Hz for 10 seconds. The results of confocal white light microscopy were in agreement with scanning electron micrographs. In contrast to continuous-wave diode and CO2 laser irradiation, pulsed Er:YAG laser irradiation caused distinct alterations with power settings beyond 300 mJ/10 Hz on the SLA surface and 500 mJ/10 Hz on the polished surface. Thus, it is only safe to use the Er:YAG laser for implant surface irradiation with settings no higher than 300 or 500 mJ/10 Hz.

Topical trans-4-aminomethylcyclohexanecarboxylic acid prevents ultraviolet radiation-induced pigmentation

Ultraviolet (UV) radiation under sunlight stimulates skin pigmentation through immediately affecting the oxidative modification of existing melanin pigments and the spatial redistribution of pigmented melanosomes followed by the up-regulation of melanogenic genes in delayed kinetics. However, abnormal accumulation and synthesis of melanin biopolymers are responsible for skin disorders with more pigmented patches. Chemical-based regulation of the hyperpigmented disorders has been a long-standing goal for cosmetic and pharmaceutical applications. A large number of the chemicals with antimelanogenic activity have met with limited or no success in the treatment of skin patients, since they may not overcome the challenge of penetrating the skin barrier. Guinea pig skin displays similar kinetic parameters to human skin in the transdermal absorption of numerous chemicals, thus can serve as the surrogate for human skin. Here, we provide a concise review of our current understanding of the chemical-based therapy against skin hyperpigmentation in UV-irradiated guinea pig models, suggest molecular mechanisms of the action and emphasize the translation from preclinical outcomes to skin patients.

Correction for ‘Inhibitory effect of a genistein derivative on pigmentation of Guinea pig skin’ by Quancheng Zhou et al., RSC Adv., 2017, 7, 7914–7919.

Ultraviolet (UV) B irradiation evokes erythema and delayed pigmentation in skin, where a variety of toxic and modulating events are known to be involved. Nitric oxide (NO) is generated from L-arginine by NO synthases (NOS). Production of NO is enhanced in response to UVB-stimulation and has an important role in the development of erythema. NO has recently been demonstrated as a melanogen which stimulates melanocytes in vitro, however, no known in vivo data has been reported to support this finding. In this study, we investigated the contribution of NO with UV-induced pigmentation in an animal model using an NOS inhibitor. UVB-induced erythema in guinea pig skin was reduced when an NOS inhibitor, L-NAME (N-nitro-L-arginine methylester hydrochloride), was topically applied to the skin daily, beginning 3 days before UVB-irradiation. Delayed pigmentation and an increased number of DOPA-positive melanocytes in the skin were markedly suppressed by sequential daily treatment with L-NAME. Furthermore, melanin content 13 days after UVB-irradiation was significantly lower in skin treated with L-NAME than in the controls. In contrast, D-NAME (N-nitro-D-arginine methylester hydrochloride), an ineffective isomer of L-NAME, demonstrated no effect on these UV-induced skin responses. These results suggest that NO production may contribute to the regulation of UVB-induced pigmentation.

A study was designed to evaluate the effectiveness of single q-switched ruby laser exposure to erase actinic lentigo. A peeling fluid containing glycolic acid designed to treat lentigines was evaluated comparatively. Ten female patients presenting with actinic lentigines on the forearms and dorsal aspects of their hands were treated with the q-switched ruby laser on the right side. Single ruby laser irradiation of actinic lentigines on the dorsal aspects of forearms and hands caused transient crusting due to exfoliation of the epidermal surface, which generally lasted for 2 weeks. Four weeks after treatment total fading of the lesions was evident. Topical application of peeling fluid applied on the left forearms caused burning sensations, local irritation and superficial scaling but could not clear the lentigines. A single course of q-switched ruby laser exposure is safe and efficient for the management of actinic lentigines, as it completely clears these obvious signs of aging. Topical treatment of lentigines using a commercial peeling solution leads to moderate or severe irritation and is ineffective.