摘要
前言
皮肤是被覆于人体表面的器官,与外界环境直接接触。皮肤具有复杂的抗氧化功能,决定氧化应激造成的损伤程度。皮肤的自然老化及外源性刺激可以使自身的抗氧化能力下降。紫外线(ultraviolet,UV)照射是引起皮肤抗氧化能力下降的一个因素。过度的UV照射造成细胞内活性氧基团增加,损伤皮肤内的脂质、蛋白和核酸,皮肤会出现红斑、水肿、增生、光老化、色素沉着、DNA损伤及免疫抑制,这些生物反应直接或间接地导致皮肤肿瘤的发生。近年来,由于人们生活方式的改变、户外活动增加以及大气臭氧层的破坏,由UV引起的疾病不断增多。
动物和人体实验表明天然抗氧化剂如维生素C和E、大豆异黄酮、绿茶多酚、白藜芦醇及N-乙酰半胱氨酸(N-acetylcystein,NAC)等能调节这些事件的启动和进展,减轻UV引起的皮肤损伤。这些提示在护肽品中或饮食中加入抗氧化剂可能阻止UV引起的皮肤炎症、老化,抑制肿瘤发生。但是,有些抗氧化剂如维生素C和E很不稳定,而有些如NAC要很高浓度才能起作用,而且,每种抗氧化剂都不能完全消除UV引起的光损伤。所以,有必要探索新的抗氧化剂,以增强抗光损伤的功效。
檀香油(sandalwood oil)又名东印度檀香油,由檀香科植物白檀的木片或根、枝经水蒸气蒸馏所得。檀香油用于治疗炎症性皮肤病及皮肤护理已有数千年的历史。外用檀香油具有很强的杀菌效果,能抑制痤疮丙酸杆菌、皮肤癣菌的生长,抑制单纯疱疹病毒1型复制,并且对宿主细胞无细胞毒作用。它能刺激细胞的更新,有助于瘢痕、伤口、皱纹、痤疮以及其他皮肤病的愈合。因其疗效卓著,被称作“液体黄金”。檀香油能清除活性氧基团,有很强的抗氧化作用。
Dwivedi等报道檀香油及其主要成分α-檀香醇涂抹小鼠皮肤后,能显著抑制化学致癌剂致皮肤肿瘤的启动和促进,减少肿瘤的数目。α-檀香醇亦能诱导人类表皮样细胞癌A431细胞凋亡。我们预期人体皮肤局部涂抹檀香油可以减轻UV照射引起的皮肤光损伤。但目前人体应用檀香油的有效性及安全性尚无报道。
本研究的目的是通过在人体皮肤UVB照射前、后涂抹檀香油,比较出现红斑和不适反应程度、表皮内日晒伤细胞数目,分析基质金属蛋白酶-1(matrixmetalloproteinase,MMP-1)和MMP-9、白介素-10(interleukin-10,IL-10)和表皮内朗格汉斯细胞(Langerhans cells,LCs)数目及p53的表达等指标,来探讨局部涂抹檀香油对UVB引起的人体皮肤晒伤、光老化、光免疫抑制及光致癌性有无防护作用。
方法
1、志愿者:
30名健康女性(年龄42～50岁,Fitzpatrick皮肤类型Ⅲ～Ⅳ型),未患与光线有关的皮肤病,没有系统疾病;试验部位没有皮肤病;1个月内及实验过程中没有用药(包括非处方药)。签署知情同意书。其中15名取皮肤活检。
2、最小红斑量(minimal erythema dose,MED)测定
实验前测定每个志愿者的MED。在背部或臀部选择8个部位,分别以不同剂量UVB照射,以出现红斑的最小UVB剂量作为MED。
3、UVB照射及檀香油涂抹:
选择背部作为照射区域。UVB照射剂量为1.5-MED。分别于照射UVB前1小时或照射后5分钟涂抹1%檀香油、5%檀香油(溶于基质3:2的丙二醇:乙醇中)或基质;仅照射UVB、仅涂抹5%檀香油及不涂抹檀香油也不照射UVB作为对照。每个部位涂抹50μl檀香油溶液。照射UVB前将溶液轻轻擦去。
4、红斑程度:
红斑程度分级:0:无红斑;1:淡红斑;2:明显红斑;3:显著红斑或有浸润。照射后24小时由2名医生独立评估、照相。
5、测定红斑指数(erythema index,EI):
分别于照射前、照射后24小时及7天后测定各部位的红斑指数,每个部位测5个点,取平均值作为该点的E_I0、EI_(24h)、EI_(7d)。
6、不适指数:
采用5分法进行评分:正常皮肤感觉—1;轻度瘙痒—2;明显瘙痒—3;轻度疼痛—4;明显疼痛—5。
7、皮肤活检:
15例志愿者照射后24小时实验区域取皮肤活检,每块分成3～4份:分别福尔马林固定后石蜡包埋、OCT包埋-70℃冻存、立即浸于液氮中数秒后-70℃冻存、EDTA中浸泡4℃过夜后分离表皮。
8、石蜡切片苏木素-伊红(hematoxylin-eosin,HE)染色:
4μm厚石蜡切片贴于挂有多聚赖氨酸的载玻片上,HE染色。显微镜下观察表皮内日晒伤细胞,照相,计数400倍镜下5个视野内日晒伤细胞数目。计算15例志愿者的平均值±标准差。
9、免疫组化:所有抗体都是小鼠抗人单克隆抗体
(1)冰冻切片中MMP-1、MMP-9:抗体1:50稀释。
(2)石蜡切片中p53表达:抗体1:100稀释。
(3)新鲜表皮片中CD1a表达:解剖显微镜下分离表皮,置于Eppendorf管中与抗体反应。
10、荧光Real-time定量RT-PCR检测皮肤活检中MMP-1、MMP-9mRNA水平
-70℃冻存的组织块切片后应用RNAiso试剂盒提取总RNA,应用紫外分光光度计测定浓度和纯度,反转录,得到cDNA,荧光real-time quantitative PCR扩增。根据RNA浓度,反转录体系中加入的RNA模板量调至相同,以使得到的cDNA量一致。根据各样本内参照β-actin,对mRNA进行半定量分析。
11、RT-PCR检测皮肤活检中IL-10 mRNA水平
-70℃冻存的组织块切片后应用RNAiso试剂盒提取总RNA,测定浓度和纯度。根据RNA浓度,反转录体系中加入的RNA模板量调至相同,以使得到的cDNA量一致。PCR扩增后进行琼脂糖凝胶电泳。用Labworks 4.0软件分析电泳条带,以IL-10与内参照GAPDH条带的密度比值表示IL-10 mRNA相对量。
12、统计学分析:
应用SPSS 13.0统计软件包进行ANOVA方差分析,以P＜0.05有统计学意义。
结果
1、红斑程度:
30名受试者UVB照射的部位均出现了不同程度的红斑,涂抹檀香油照射UVB的部位红斑程度都减轻,5%檀香油防护效果强于1%檀香油。空白对照及仅涂抹檀香油的部位均无红斑出现。
2、EI:
UVB照射后24小时各部位照射EI值与肉眼观察的红斑程度一致。涂抹檀香油的部位EI值明显低于UVB照射及UVB/基质的部位。7天后,照射部位遗留色素沉着斑,红斑几乎消退,红斑指数下降,但仍显著高于照射前(P＞0.05)。
3、不适指数:
除空白对照及仅涂抹檀香油的部位无任何不适外,其余各处均有不同程度的瘙痒,2名志愿者仅UVB照射的部位及UVB/基质部位出现轻度疼痛。涂抹檀香油的部位不适指数为1者居多,5%檀香油的作用略强于1%檀香油。
4、日晒伤细胞:
UVB照射后表皮内出现日晒伤细胞,表现为胞核浓缩,胞浆嗜伊红染色。涂抹檀香油的部位表皮内日晒伤细胞数显著少于UVB/基质及仪照射UVB的部位(P＜0.05),5%檀香油的作用强于1%檀香油(P＜0.05),UVB照射前、后涂抹檀香油的作用没有显著性差异(P＞0.05)。
5、MMP-1、MMP-9蛋白的表达:
UVB照射后MMP-1、MMP-9的表达增加,檀香油涂抹对UVB引起MMP-1、MMP-9蛋白表达增高有抑制作用。涂抹檀香油的部位真皮内MMP-1、MMP-9表达量显著少于仅照射UVB的部位(P＜0.05),与空白对照间差异很小(P＞0.05)。照射前、后涂抹檀香油的作用没有显著性差异。
6、MMP-1、MMP-9 mRNA水平:
UVB照射及UVB/基质的部位照射24小时后皮肤活检组织中MMP-1、MMP-9mRNA水平较空白对照部位显著升高(P＜0.05),UVB照射前、后涂抹檀香油可以抑制MMP-1、MMP-9 mRNA水平(P＜0.05),涂抹5%檀香油/UVB的部位mRNA水平与空白对照皮肤中接近(P＞0.05)。
7、表皮片中CD1a~+LCs数目:
UVB照射24小时后,UVB及UVB/基质部位表皮片中CD1a~+LCs较空白处显著减少(P＜0.05)。UVB照射前、后涂抹檀香油可以抑制UVB减少表皮内LCs的作用(P＜0.05)。涂抹5%檀香油/UVB的部位表皮片中LCs数量与空白对照表皮中相当(P＞0.05)。
8、IL-10 mRNA水平:
人体皮肤照射1.5-MED的UVB 24小时后,UVB与UVB/基质部位皮肤活检中IL-10 mRNA水平较正常皮肤中显著升高(P＜0.05)。檀香油抑制UVB诱导的IL-10mRNA的升高,5%檀香油作用强于1%檀香油(P＜0.05)。
9、p53的表达:
空白对照部位个别角质形成细胞核p53染色阳性,与UVB照射部位有显著性差异。涂抹基质前、后照射UVB部位p53阳性细胞数与仅UVB照射部位无明显差异。涂抹5%檀香油/UVB部位表皮内p53阳性细胞数显著少于仅UVB照射及UVB/基质部位(P＜0.05)。
结论
1、人体皮肤涂抹檀香油可以减轻UVB照射引起的皮肤红斑与不适,减少表皮内日晒伤细胞的形成。人体皮肤涂抹檀香油没有刺激性及过敏反应,安全性好。
2、人体皮肤涂抹5%檀香油能抑制UVB照射引起的MMP-1和MMP-9mRNA及蛋白表达的升高,从而抑制光老化。
3、人体皮肤照射UVB引起表皮内LCs数目减少及IL-10 mRNA水平升高,局部涂抹檀香油能保护表皮内的LCs,抑制IL-10水平升高,从而保护皮肤正常的免疫功能。
4、人体皮肤局部涂抹檀香油能够抑制UVB引起的p53表达,在人体皮肤中具有抗光致癌的作用。
5、UVB照射前、后涂抹檀香油有相同的功效,提示檀香油的光防护作用不是通过遮光起作用的。
Introduction
Cutaneous 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 methods
1.Subjects
Thirty 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 oil
1%and 5%sandalwood oil was prepared by diluting sandalwood oil in vehicle (ethanol:propylene glycol=2:3)just before application.
3.UVB irradiation
Each 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 discomfort
The intensity of erythema was evaluated visually and discomfort was recorded by two investigators 24 hours after irradiation.EI was determined by using Mexameter(?) before(EI_0),24 hours after(EI_(24h)) and 7 days after(EI_(7d))UVB irradiation.The increase in EI(△EI) at irradiated sites was determined by subtracting the EI_0 from the value at each site.
5.Biopsies
Skin 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) staining
Number 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-9
IHC 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) LCs
Monoclonal 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/mm~2.
(3) p53
IHC 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 synthesis
Total 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 mRNA
The 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 mRNA
The 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 analysis
The 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.
Results
1.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 cells
Topical 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 skin
The 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 LCs
The 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 skin
IL-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 p53
Immunohistochemical 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.
Conclusions
1.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.
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