夏枯草对UVA诱导的豚鼠皮肤光老化的防护作用

皮肤光老化是指紫外光长期辐射引起的慢性损容性皮肤损伤,表现为面部等曝光部位皮肤粗糙、深皱纹、色斑增多等.为了寻找安全有效的天然防光物质,我们应用夏枯草进行皮肤光老化的防护研究.     

皮肤光老化发生机制的研究进展

皮肤光老化是由于紫外线的长期慢性辐射损害而造成的皮肤损伤,在临床表现、组织病理学等方面与自然老化有着不同的表现.近年国内外关于光老化发生机制方面的研究有了很大的进展,研究已经证实,激活蛋白-1和核因子-кB活化导致基质金属蛋白酶的合成、线粒体损伤、蛋白质氧化和端粒在光老化发生中的作用.理解并认识这些发生机制有助于在防治慢性紫外线辐射造成的皮肤损伤方面提出新的预防和治疗策略.     

核因子E2相关因子2信号通路及其在紫外线致皮肤蛋白氧化应激损伤中的作用

紫外线照射产生活性氧及自由基,与皮肤光老化、皮肤癌等皮肤光损伤疾病的发生相关.活性氧及自由基不仅通过攻击氨基酸主侧链等方式氧化蛋白质,而且能够激活丝裂原活化蛋白激酶产生基质金属蛋白酶,降解皮肤结缔组织中的胶原蛋白等蛋白质,使皮肤发生光损伤.核因子E2相关因子2信号通路是细胞氧化应激反应的核心调控机构,在应激条件下,激活核因子E2相关因子2,通过调节抗氧化酶类和Ⅱ相解毒酶基因的表达,清除过多的核因子E2相关因子2和自由基,抑制基质金属蛋白酶的分泌,保护蛋白质,增强机体抗氧化应激的能力,对皮肤起到保护作用.核因子E2相关因子2信号通路及其调控机制与皮肤蛋白氧化应激损伤间关系的深入研究,将有利于寻找蛋白氧化应激损伤导致的相关皮肤光损伤疾病新的有效的治疗防御措施.     

神经肽在紫外线辐射诱导皮肤光损伤性疾病中的作用

神经肽是一类小分子肽,属于细胞外信使家族,作为维系皮肤与神经-免疫-内分泌网络系统的重要信号分子,神经肽在维持皮肤内环境稳态和多种病理生理过程中起到重要的作用。近年来研究发现,当皮肤受到紫外线辐射后,中枢神经细胞、外周感觉神经纤维和皮肤细胞可分泌多种神经肽,参与调控细胞凋亡、炎症反应、黑素生成、免疫抑制、细胞外基质降解等多种急慢性皮肤光损伤发病过程。本文综述常见神经肽在皮肤光损伤性疾病中的主要作用,以期为其防治提供新的思路和分子靶点。     

基质金属蛋白酶在曝光及非曝光部位皮肤中的表达

目的 检测基质金属蛋白酶－1、3、9（MMP-1、3、9）在曝光部位和非曝光部位皮肤中的表达,探讨它们在皮肤光老化机制中的作用。方法 应用免疫组化方法分别对23例女性健康志愿者前臂伸侧（曝光）和上臂内侧（非曝光部位）皮肤石蜡标本中的MMP-1、MMP-3、MMP-9进行检测,表达强度以免疫反应强度分布指数（IRIDI）表示,采用Wilcoxon符号秩和检验、Mann-Whitney秩和检验及Spearman秩相关分析进行统计学处理。结果 MMP-1、MMP-3、MMP-9在曝光和非曝光部位均表达,曝光部位皮肤组织中MMP-1、MMP-3、MMP-9的IRIDI均值（范围）分别为7.70（3 ～ 12）、9.22（6 ～ 12）、8.30（6 ～ 12）,非曝光部位分别为4.26（2 ～ 6）、5.39（2 ～ 9）、4.04（1 ～ 6）,曝光部位MMP-1、MMP-3、MMP-9的IRIDI均显著高于非曝光部位（均P < 0.01）。50岁以上组曝光部位皮肤组织MMP-1、MMP-3、MMP-9的IRIDI分别为9.17（6 ～ 12）、10.58（8 ～ 12）、8.92（8 ～ 12）,非曝光部位分别为4.75（2 ～ 6）、6.42（4 ～ 9）、4.33（3 ～ 6）,曝光部位均显著高于非曝光部位（P < 0.05）;50岁以下组曝光部位皮肤组织MMP-1、MMP-3、MMP-9的IRIDI分别为6.09（3 ～ 8）、7.73（6 ～ 9）、7.64（6 ～ 12）,非曝光部位分别为3.73（2 ～ 6）、4.27（2 ～ 8）、3.73（1 ～ 6）,曝光部位均显著高于非曝光部位（P < 0.05）;50岁以上组曝光部位显著高于50岁以下组,而两组非曝光部位皮肤组织MMP-1、MMP-9的IRIDI值差异无统计学意义（P > 0.05）。曝光部位皮肤组织MMP-1、MMP-3、MMP-9的IRIDI值与年龄呈正相关（r = 0.66、0.69、0.74,P < 0.01）,非曝光部位皮肤组织MMP-1、MMP-9的IRIDI值与年龄无相关性。结论 MMP-1、MMP-3、MMP-9在曝光部位表达显著高于非曝光部位,三者在光老化的发生发展中均起作用,但作用机制可能不同。     

免疫荧光法检测cathepsins在人慢性光损伤皮肤中的表达变化

目的:用免疫荧光检测cathepsins家族的几个重要亚型在人慢性光损伤皮肤中的表达变化并探讨其意义。方法:用日光模拟紫外线照射以1倍最小红斑量(MED)照射10名受试者上臀部皮肤,5天/周,共6周,人工诱导皮肤慢性光损伤。免疫荧光法检测cathepsin B、D、K、G在人慢性光损伤皮肤中的表达变化。结果:在慢性光损伤人皮肤组织中,cathepsin B和cathepsin D表达下调,表达改变主要发生于皮肤表皮层;cathepsin K表达下调,表达改变主要发生于皮肤真皮层;cathepsin G表达上调,表达改变主要发生于皮肤真皮层。结论:cathepsins家族在人皮肤慢性光损伤皮肤表达发生改变,其可能参与皮肤表皮及真皮的慢性光损伤发生机制。     

组织蛋白酶B在光老化皮肤中的表达及意义

目的 探讨组织蛋白酶B在光老化皮肤中的表达意义。方法 6例成人曝光和非曝光皮肤标本,采用免疫组化法定位及对比组织蛋白酶B的表达。体外培养原代人皮肤成纤维细胞,甲氧沙林 ＋ UVA法体外诱导培养细胞光老化。衰老相关-β-半乳糖苷酶染色证明老化诱导成功。Western印迹技术及RT-PCR对比检测光老化成纤维细胞及正常成纤维细胞组织蛋白酶B蛋白及基因表达。结果 6例成人曝光和非曝光活体皮肤均见组织蛋白酶B阳性染色,和非曝光部位相比,曝光部位皮肤阳性染色A值降低。Western印迹结果示,成纤维细胞光老化诱导组蛋白表达较UVA诱导组、甲氧沙林孵育组及空白组明显下调。光老化成纤维细胞诱导后1周,组织蛋白酶B与内参的灰度比由28.099 ± 0.054下降为25.103 ± 0.102,诱导后3周灰度值进一步下降为17.693 ± 0.099。实时定量RT-PCR结果示,光老化细胞组织蛋白酶B mRNA表达下调为正常组的64％（P < 0.05）。结论 组织蛋白酶B在光老化皮肤及老化成纤维细胞中表达降低,且有时间依赖性,与光老化皮肤自我修复能力下降有关。     

皮肤光损伤与氧化应激研究进展

长期或大量紫外线照射后皮肤发生氧化应激,不仅从分子水平上改变了蛋白质、脂质、DNA等的结构和功能,同时于转录水平上激活丝裂原活化蛋白激酶通路、核转录因子通路、信号转导和转录激活因子通路,抑制核转录因子E2相关因子(Nrf)2通路等信号转导途径,引起皮肤细胞凋亡、细胞外基质降解,最终出现红斑、脱屑、皱纹、甚至肿瘤等光损伤表现。近年来针对氧化应激在光损伤发生及发展过程中的作用机制,以及光损伤的防治亦有较多新进展,该文从氧化应激角度对近年光损伤的发病机制和研究进展进行了回顾和总结,同时对其治疗前景进行展望。     

皮肤光老化组织学改变研究进展

皮肤光老化是不断累积的日光照射所致的一个连续性过程,是指长期的日光照射(主要是紫外线)导致皮肤衰老.在组织学方面,表皮变化主要是角质形成细胞、黑素细胞、朗格汉斯细胞,真皮变化主要是弹力纤维变性和胶原减少.其机制主要涉及免疫系统、细胞因子改变、基质金属蛋白酶生成增加、活性氧簇增加及DNA损伤等.概述并探讨皮肤光老化组织学变化及其相应的发生机制.     

慢性荨麻疹患者血清组胺释放活性检测

目的 检测慢性荨麻疹患者血清组胺释放活性,探讨慢性荨麻疹的发病机制.方法 通过体外分离人皮肤肥大细胞,进行肥大细胞组胺释放试验,测定组胺释放率.结果 62例慢性荨麻疹患者中,自体血清皮肤试验阳性者24例占38.71%.混合细胞悬液中肥大细胞的组胺自发释放率<5%.血清活化皮肤肥大细胞引起的组胺释放率从3.1%～79.5%(16.44%±14.26%),明显高于正常人对照组(P<0.01),其中27例组胺释放率>15%(43.55%);自体血清皮肤试验(+)组的组胺释放率及阳性率均明显高于自体血清皮肤试验(-)组(P<0.01).结论 部分慢性荨麻疹患者血清中存在组胺释放活性,可直接活化肥大细胞,释放组胺等血管活性介质,引起荨麻疹.     

Mast cell tryptase and photoaging: possible involvement in the degradation of extra cellular matrix and basement membrane proteins

Mast cells are widely distributed in the connective tissue of the body, but are particularly prominent in tissues such as skin. An increased number of mast cells can be found in the dermis under inflammatory conditions and ultraviolet (UV) exposed skin. Previous investigations have identified matrix metalloproteinases (MMPs) as key enzymes in the degradation of extra cellular matrix (ECM). This study reports about the potential contribution of human mast cell tryptase as a new triggering enzyme in matrix degradation process. Recent studies suggest that mast cell-derived proteases can activate MMPs. We investigated both the degradation of cellular matrix components and activation of MMPs by human tryptase. Mast cells are increased in photoaged skin and the increase of mast cell tryptase in UV irradiated skin was confirmed. Human mast cell tryptase was purified from human tonsils by a series of standard chromatographic procedures. Degradation of collagen type I was achieved by incubation of human type I collagen with tryptase and the fragments were quantified by SDS-PAGE and staining with Coomassie Brilliant Blue 250-R (CBB). Treatment with tryptase resulted in the activation of proMMP-9 as revealed by gelatinolytic activity in type IV collagen zymography. When tryptase was incubated with human type IV collagen, gradual degradation of intact collagen was detected by Western blotting. Furthermore, type IV collagen degradation was observed in the basement membrane (BM) of a three-dimensional (3D) skin model. Degranulation of mast cells, which release tryptase, can activate MMPs and causes direct damage to ECM proteins. These findings strongly implicate that tryptase either alone or in conjunction with activation of MMPs, can participate in ECM damage and the possible destruction of BM leading to photoaging.     

Mast cell tryptase and photoaging: possible involvement in the degradation of extra cellular matrix and basement membrane proteins

Mast cells are widely distributed in the connective tissue of the body, but are particularly prominent in tissues such as skin. An increased number of mast cells can be found in the dermis under inflammatory conditions and ultraviolet (UV) exposed skin. Previous investigations have identified matrix metalloproteinases (MMPs) as key enzymes in the degradation of extra cellular matrix (ECM). This study reports about the potential contribution of human mast cell tryptase as a new triggering enzyme in matrix degradation process. Recent studies suggest that mast cell-derived proteases can activate MMPs. We investigated both the degradation of cellular matrix components and activation of MMPs by human tryptase. Mast cells are increased in photoaged skin and the increase of mast cell tryptase in UV irradiated skin was confirmed. Human mast cell tryptase was purified from human tonsils by a series of standard chromatographic procedures. Degradation of collagen type I was achieved by incubation of human type I collagen with tryptase and the fragments were quantified by SDS-PAGE and staining with Coomassie Brilliant Blue 250-R (CBB). Treatment with tryptase resulted in the activation of proMMP-9 as revealed by gelatinolytic activity in type IV collagen zymography. When tryptase was incubated with human type IV collagen, gradual degradation of intact collagen was detected by Western blotting. Furthermore, type IV collagen degradation was observed in the basement membrane (BM) of a three-dimensional (3D) skin model. Degranulation of mast cells, which release tryptase, can activate MMPs and causes direct damage to ECM proteins. These findings strongly implicate that tryptase either alone or in conjunction with activation of MMPs, can participate in ECM damage and the possible destruction of BM leading to photoaging.     

Histopathologic differences in the photoaging process in facial versus arm skin.

We used clinical criteria to study skin biopsy specimens with mild to moderate photoaging taken from the face and dorsal forearms of 74 Caucasian volunteers between the ages of 30 and 50. Facial skin had a greater number of granular cell layers, a higher degree of keratinocytic atypia, and more often showed a compact stratum corneum than arm skin. Furthermore, the dermis of facial skin had a more extensive perivascular and perifollicular lymphocytic infiltrate, more perifollicular fibrosis, a greater number of mast cells and melanophages, and thinner vascular walls than forearm skin. This study demonstrated that the photoaging process is different for face and arm skin. Appreciation of these differences should permit more refined studies of photoaging and the development of more efficient therapies.     

Human skin mast cells rapidly release preformed and newly generated TNF-alpha and IL-8 following stimulation with anti-IgE and other secretagogues

Several groups have previously reported that rodent or human leukemic mast cells produce inflammatory cytokines such as TNF-alpha and IL-8 as well as the pro-allergic cytokines IL-4, IL-5 and IL-13. Comparatively little is known, however, regarding the ability of normal human skin mast cells to secrete these factors following either IgE-dependent or IgE-independent modes of activation. We therefore investigated whether normal human skin mast cells produce these cytokines following stimulation by a variety of secretagogues. Enriched isolated skin mast cells released both TNF-alpha and IL-8 following activation with either anti-IgE, SCF, substance P, compound 48/80 or A23187. This release was dose- and time-dependent, with maximal levels being reached within 4 h of stimulation involving, in part, the secretion of preformed stores of both cytokines. In accordance with this, using lysates of highly purified (>90%) skin mast cells, we could demonstrate that both TNF-alpha and IL-8 mRNA and protein were present in both unstimulated as well as stimulated mast cells. In stark contrast to these results, no significant levels of either IL-4, IL-5 or IL-13 were detected, regardless of the secretagogue used or the period of stimulation. These results show that human skin mast cells are capable of rapidly secreting pro-inflammatory cytokines like TNF-alpha and IL-8 following IgE-dependent activation and stimulation by the neuropeptide substance P, SCF and the basic polypeptide analogue compound 48/80. In contrast to other types of human mast cells however, human skin mast cells were incapable of secreting IL-4, IL-5 or IL-13 in these settings.     

Low-energy green light alleviates senescence-like phenotypes in a cell model of photoaging

Background

Ultraviolet B (UVB) affects diverse pathways in skin cells, resulting in skin photoaging. Skin fibroblasts internalize and degrade elastin and collagen, playing prominent roles in photoaging. Green light is used in many fields of dermatology, but few studies have examined its role in photoaging. The present work aimed to assess low-energy green light for its effects in a previously proposed cell model of photoaging and to explore the possible anti-photoaging mechanism.

Methods

The stress-induced premature senescence (SIPS) model was constructed via repeated treatment of MDFs with UVB. Senescence-like phenotypes were compared among normal, low-energy green light pretreatment and UVB groups, for example, cell morphological properties, senescence-associated β-galactosidase (SA-β-gal) amounts, extracellular matrix (ECM) biosynthesis and degradation, and autophagy.

Results

In comparison with the UVB group, the green light pretreatment group showed significantly decreased number of senescent mast cells and markedly declined signal intensity and amounts of SA-β-gal-positive cells. Furthermore, green light pretreatment directly affected ECM by increasing type I and type III collagen production and decreasing MMP-1 amounts. Moreover, changes in autophagy levels induced by green light pretreatment provided a potential mechanism underlying its anti-aging property.

Conclusions

Low-energy green light pretreatment improves senescence-like phenotypes in vitro, indicating a possible application for anti-aging in clinic after future research has uncovered the potential mechanism.     

Numbers of murine dermal mast cells remain unchanged during chronic ultraviolet B irradiation

Dermal mast cell numbers reportedly increase in response to chronic ultraviolet irradiation in both humans and in the HRS/Skh-1 mouse model of human photoaging. It has been hypothesized that these increased numbers of mast cells are responsible, at least in part, for the damage in this chronically irradiated or photoaged skin. However, few actual quantitative data have been reported to support this claim of increased dermal mast cell numbers caused by chronic ultraviolet irradiation. We sought to quantify the numbers of dermal mast cells in the skin of chronic ultraviolet-irradiated and control HRS/Skh-1 hairless mice. Dermal mast cells from irradiated and age-matched control mice were quantified by digital image analysis during a 20-week period of exposure to ultraviolet B (UVB) radiation. During the entire course of irradiation, there was no difference in the numbers of dermal mast cells between the irradiated and nonirradiated age-matched control mice. Visible physical evidence of the effects of chronic UVB irradiation, i.e., skin wrinkling, was evident after 6 weeks of treatment. The numbers of dermal mast cells in unirradiated age-matched NSA (CF-1) haired mice were three- to four-fold lower than those in either ultraviolet-exposed or unexposed HRS/Skh-1 mice. These findings indicate that dermal mast cell numbers in HRS/Skh-1 mice are not increased by chronic exposure to UVB radiation.     

Extracellular localization of human connective tissue mast cell granule contents.

In early phases of cutaneous inflammation, connective tissue mast cell degranulation is associated with apparent secretion and externalization of immunoreactive chymotryptic serine proteinase. To determine whether this event is associated with structural evidence of granule externalization, we studied the sequential evolution of IgE-mediated hypersensitivity in vivo, as well as mast cell degranulation provoked by a variety of stimuli in cultured explants of human skin. By 1 min after intradermal antigen challenge with ragweed extract, mast cell degranulation was associated with apparent extrusion of intragranule constituents into the pericellular connective tissue. Similar features typified cultured skin explants exposed for 45 min to anti-IgE and other mast cell secretagogues (morphine sulfate, calcium ionophore A23187, compound 48/80, and substance P). Once externalized, granule constituents could be identified within the dermal matrix by their rounded contour and structural similarity to solubilized granule matrices remaining within actively secreting cells. These data indicate that externalization of connective tissue mast cell granule contents occurs early after secretagogue exposure, potentially accounting for infrequent documentation of this event in naturally occurring dermatoses. The ability to recognize externalized granule products at a morphologic level should facilitate the understanding of interactions between mast cell-derived mediators and target structures of the dermal microvasculature.     

The effect of reactive oxygen species on the biosynthesis of collagen and glycosaminoglycans in cultured human dermal fibroblasts

The purpose of this study was to evaluate the possibility that the biological changes observed in connective tissue matrix components of photoaging skin may be induced by an alteration of biosynthesis in fibroblasts damaged by reactive oxygen species (ROS). We investigated the effect of ROS induced by xanthine and the xanthine oxidase system on the biosynthesis of connective tissue matrix components, collagen and glucosaminoglycans (GAGs) in cultured human dermal fibroblasts. ROS decreased collagen production and increased GAGs synthesis. Interestingly, these changes were consistent with the biological alterations of connective tissue matrix components observed in photoaging skin. Moreover, catalase and alpha-tocopherol completely prevented the ROS-induced alterations of collagen and GAGs biosynthesis, whereas superoxide dismutase had no effect on the ROS-induced changes. These results suggest that ROS may be one of the factors which cause the biological changes of connective tissue matrix components observed in photoaging skin.     

Melasma: Updates and perspectives

Management of melasma is highly challenging due to inconsistent treatment results and frequent relapses. However, recent studies revealed that melasma may not only be a disease of melanocytes, but also a photoaging skin disorder. Herein, we attempt to validate that melasma is indeed a photoaging disorder by presenting the histopathologic findings of melasma: solar elastosis, altered basement membrane, increased vascularization and increased mast cell count. We also provide some therapeutic implications based on these findings and a discussion on the latest updates and perspectives regarding treatment.