| IntroductionCutaneous exposure to solar ultraviolet(UV) irradiation has a variety of adverse effects including erythema,edema,sunburn,photoaging,photo immunosuppression and photocarcinogenesis.Except for sunscreens,antioxidants have been suggested as a useful supplement to prevent UV-mediated cutaneous damages.Many studies have shown that antioxidants,including ascorbic acid,vitamins C and E,N-acetylcysteine (NAC) and genistein,reduce UV-induced photoaging or carcinogenesis in human and hairless mouse skin.However,vitamins C and E are highly unstable and easy to be oxidized.In addition,other well-known compounds,such as NAC,are only effective at unreasonably high working concentrations.Thus,there is a need for the identification and development of new antioxidants.Sandalwood oil is an extract from the dark rough bark and scented nature wood of Santalum album L,a medium-sized evergreen tree with drooping branches,sandalwood oil is a potent antiseptic,astringent,anti-inflammatory agent.It stimulates cellular regeneration and assists in healing scars,wounds,wrinkles,acne,and other skin conditions.In animals,sandalwood oil and its active ingredient a-santalol significantly inhibite chemical and UV induced skin carcinogenesis.Although there is substantial evidence indicating that sandalwood oil is a potential agent for photoprotection in cultured cells and animal studies,it is still unknown whether sandalwood oil really provides efficient photoprotection in humans.Langerhans cells(LCs) are the major antigen presenting cells in the epidermis.UV radiation induces depletion of LCs causing immune suppression.Interleukin- 10(IL-10) plays a critical role in UVB-induced immune suppression by inhibiting cell-mediated immune reactions.Exposure of human or mouse skin to UV irradiation results in the induction of a series of matrix metalloproteinases(MMPs),degrading the collagen framework and other components of skin connective tissue which have been implicated in photoaging,p53 is a transcriptional factor upregulated in epidermal cells responding to UVB-induced DNA damage.DNA mutation and photoimmunosuppression eventually lead to carcinogenesis.This study aimed to investigate the protective effects of sandalwood oil on UVB-induced human skin damage by evaluating erythema index,sunburn cell formation,LCs,IL-10,p53 and photoaging related endpoints(MMP-1 and MMP-9).Materials and methods1.SubjectsThirty women aged from 42 to 50 years with Fitzpatrick skin typeⅢ-Ⅳwere recruited.All were in good health with no evidence of acute or chronic diseases, without known photosensitivity.Subjects were instructed not to take any other medicines until the study was ended.Written consent was obtained from all participants prior to the study.2.Sandalwood oil1%and 5%sandalwood oil was prepared by diluting sandalwood oil in vehicle (ethanol:propylene glycol=2:3)just before application.3.UVB irradiationEach subject's minimal erythema dose(MED) was determined by exposing the back skin to graded doses of UVB radiation.The lowest dose of inducing uniform erythema 24 hours after exposure was considered to be the MED.Seven areas of back skin were irradiated with 1.5-MED UVB.50μL 1%and 5%sandalwood oil solution was applied to 2×2 cm area on the back 60 min before and 5 min after UVB irradiation. Controls included irradiated skin with vehicle,irradiated skin without either vehicle or sandalwood oil,and nonirradiated skin without sandalwood oil.4.Assessement of erythema and discomfortThe intensity of erythema was evaluated visually and discomfort was recorded by two investigators 24 hours after irradiation.EI was determined by using Mexameter(?) before(EI0),24 hours after(EI24h) and 7 days after(EI7d)UVB irradiation.The increase in EI(△EI) at irradiated sites was determined by subtracting the EI0 from the value at each site.5.BiopsiesSkin biopsies were obtained 24 hours after the UVB irradiation.The biopsies were divided into 3～4 parts and processed for respective assessments.6.Hematoxylin-eosin(HE) stainingNumber of sunburn cells was counted in five 400×magnification fields on paraffin-embedded sections stained with HE.The results were expressed as mean number of cells±SD.7.IHC techniques(1) MMP-1 and MMP-9IHC assessments of MMP-1 and MMP-9 protein expressions were carded out on frozen skin blocks with application of primary antibodies(mouse against human monoclonal antidody,Santa Cruz,USA) and secondary antibody labeled with biotin-streptavidin amplification system.The results were evaluated by two investigators under a light-field microscope at 400×magnification,counting the positive staining of five fields in the dermis.The results were expressed as mean number of positive staining±SD.(2) LCsMonoclonal mouse anti human CDla antibody was used for staining LCs in fresh epidermal sheet.The number of CD1a positive LCs was quantified under 400 magnification.The results were expressed as mean number of cells±SD/mm2. (3) p53IHC was carried out on formaldehyde fixated paraffin sections with application of primary antibodies and secondary antibody labeled with biotin-streptavidin amplification system.The results were evaluated by two investigators under a light-field microscope at 400×magnification,counting the positive keratinocytes of five fields.The results were expressed as mean number of cells±SD.8.RT-PCR(1) RNA isolation and eDNA synthesisTotal cellular RNA was isolated from cryopreserved skin samples using RNAiso kit following the manufacturer's protocol,eDNA was synthesized by reverse transcription.(2) Real-time quantitative RT-PCR detection of MMP-1 and MMP-9 mRNAThe expressions of MMP-1 and MMP-9 mRNA in human skin tissues were analyzed by quantitative analysis of real time RT-PCR.13 -actin mRNA served as an internal control to ensure that similar amounts of RNA and cDNA were assessed.9.RT-PCR detection of IL-10 mRNAThe expression of IL-10 mRNA in human skin tissues was analyzed by RT-PCR. The effect of sandalwood oil on IL-10 mRNA expression was quantified by densitometry analysis using Labworks 4.0 software.The changes in the intensity of the IL-10 gene relative to GAPDH(internal control) were calculated.10.Statistical analysisThe data were analyzed by using the statistical package SPSS 13.0 for windows. The statistical significance of difference between groups was determined by one-way analysis of variance(ANOVA).P value＜0.05 was considered statistically significant.Results1.Effects of topical sandalwood oil on UVB-indueed erythema and discomfort All subjects developed erythema in the UVB irradiation sites.Either pre-UVB or post-UVB application of sandalwood oil showed some effects in inhibiting UVB-induced erythema and discomfort.In all subjects,5%sandalwood oil was found to be more efficient than 1%sandalwood oil.2.sandalwood oil inhibited UVB-induced sunburn cellsTopical application of sandalwood oil either pre-UVB or post-UVB irradiation significantly reduced UVB-induced sunburn cells(P＜0.05).3.sandalwood oil inhibited UVB-induced expressions of MMP-1 and MMP-9 mRNA and protein in human skinThe expressions of MMP-1 and MMP-9 mRNA and protein were upregulated by UVB irradiation at similar levels in the areas of UVB exposure,pre-UVB and post-UVB application of vehicle.The upregulation was completely inhibited by application of 5%sandalwood oil either pre-UVB or post-UVB.4.Effects of topical sandalwood oil on UVB-reduced LCsThe UVB-induced LCs depletion was partially inhibited by application of 1% sandalwood oil pre-UVB or post-UVB(P＜0.05) and almost completely inhibited by 5%sandalwood oil.5.sandalwood oil inhibited UVB-induced IL-10 mRNA expression in human skinIL-10 mRNA expression was upregulated in UVB-irradiated and UVB-irradiated vehicle-treated sites compared with unirradiated normal skin.Application of 1%and 5%sandalwood oil resulted in suppression of IL-10 mRNA expression(P＜0.05).6.Effects of topical sandalwood oil on UVB-induced p53Immunohistochemical analysis showed sparse nuclear p53 staining keratinocytes in the skin of unirradiated control.A significant higher expression of p53 was observed in the epidermis in the UVB irradiated and UVB plus vehicle treated skin.Either pre- or post-UVB application of 5%sandalwood oil exterted a significant reduction(P＜0.05) of p53-positive cells.Conclusions1.Topical application of sandalwood oil could attenuate UVB-induced erythema, discomfort and sunburn cell formation.No irritation or allergy was induced showing the safety of application of sandalwood oil in human.2.Topical application of 5%sandalwood oil could inhibit UVB-induced upregulation of MMP-1 and MMP-9 mRNA and protein,thus preventing photoaging.3.Topical application of sandalwood oil could inhibit UVB-induced LCs reduction in the epidermal sheet and UVB-induced increase of IL-10 mRNA, protecting the immune function of the skin.4.Topical application of sandalwood oil could reduce p53 expression in the UVB-irradiated skin,which might play a role in inhibiting UVB-induced photocarcinogenesis.5.Application of sandalwood oil either pre- or post-UVB irradiation exerted similar effects indicating the photoprotective effects of sandalwood oil in human skin do not result from the sunscreen effect.
|
PDF Full Text Request