EGCG对急性UVB辐射后表皮角质形成细胞的保护机制探讨及对慢性UVA辐射后小鼠皮肤光老化的防护作用
摘要
上篇EGCG对急性UVB照射HaCaT细胞保护机制的探讨
研究背景:
目光中的长波紫外线(UVA)和中波紫外线(UVB)辐射可引起急性损伤如日晒伤和慢性累积性损伤如光老化及皮肤癌,对人类健康构成潜在危害。波长短能量较高的UVB(290-320nm)主要作用于表皮,角质形成细胞是其靶细胞,可诱导皮肤不良反应如红斑、水肿、水疱、皮肤色素沉着、异常增生、光老化和非黑色素皮肤癌,而机体通过多种保护性措施如凋亡和炎症来保护皮肤监视及对抗潜在致癌细胞的增殖。
近几年紫外线诱导的炎症反应和细胞恶性转化过程中信号通路的作用受到广泛关注,其中一个研究热点即是丝裂原活化蛋白激酶(MAPK)信号转导通路,该途径可被紫外线激活,故在调节紫外线诱导的多重细胞反应中至关重要。MAPK主要由以下四个家族组成:ERK,JNK,p38和ERK5/BMK1,其中p38介导的炎症反应和凋亡在维护表皮完整性和对抗紫外线诱导的肿瘤效应方面具有重要作用。
绿茶是全球广泛受欢迎的饮料,绿茶中的酚类是儿茶素,其中的没食子儿茶素没食子酸酯(EGCG)是有效的自由基清除剂和抗氧化剂,可以预防化学致癌作用和紫外线诱导的氧化应激效应。我们以前的研究证实单次UVB辐射可引起急性光损伤包括细胞活性减低及增殖减慢、细胞凋亡增加、光加合物形成增多。累积性UVA/UVB照射甚至可以导致DNA突变及皮肤癌发生,当加入EGCG干预后,均可以减少UVB照射所致上述各种损伤性改变,但关于p38丝裂原活化蛋白激酶(p38MAPK)信号途径在UVB所致凋亡及炎症中的作用,及其EGCG的保护机制是否也与此途径有关在国内尚未见报告,因此我们以人皮肤角质形成细胞株HaCaT为观察对象,研究p38MAPK途径在UVB照射诱导的细胞凋亡及炎症介质分泌活动中的作用,并以p38抑制剂为阳性参照对比观察EGCG的干预保护作用是否也与p38MAPK通路有关。
目的:第一部分研究UVB辐射引起HaCaT细胞发生凋亡的概率、细胞因子TNF-α、IL-1β及IL-6的分泌及p38MAPK和磷酸化p38丝裂原活化蛋白激酶(p-p38MAPK)的表达,探讨凋亡、细胞因子分泌与p38MAPK之间的联系,确定p38MAPK信号转导途径是否参与了UVB诱导的细胞凋亡及细胞因子的分泌。第二部分研究EGCG对UVB辐射后细胞凋亡、细胞因子分泌及p38MAPK、p-p38MAPK表达的影响,探讨EGCG的光保护机制是否也与p38MAPK通路有关。
方法:使用0,20,30,40,60,70及90mJ/cm~2UVB照射HaCaT细胞,照射后Oh,1h,2h,3h,4h,6h,24h及48h收集细胞和上清。流式细胞仪检测细胞周期和凋亡;ELISA检测上清液中TNF-α、IL-1β、IL-6水平;WesternBlot方法检测p38MAPK和p-p38MAPK的表达。
结果:①与空白对照组细胞凋亡率(0)相比,30mJ/cm~2UVB照射后24h,HaCaT细胞凋亡率为21.25±0.33%,应用p38抑制剂后测细胞凋亡率降为10.14±0.27%,较UVB组降低近1/2。②30mJ/cm~2UVB照射HaCaT细胞,照射后HaCaT细胞分泌TNF-α渐增加,24h达高峰,后渐下降,p38抑制剂SB203580完全抑制TNF-α的分泌。UVB辐射后IL-1β,IL-6则随孵育时间而大幅度增加,SB203580明显抑制了IL-1β和IL-6的分泌水平。③不同剂量UVB照射后可激活HaCaT细胞p38MAPK途径,磷酸化p-p38水平增高,照光组与非照光组间相比具有统计学意义。以30mJ/cm~2UVB照射时p-p38表达量最高,但非磷酸化p38表达量没有类似明显变化。以上数据说明急性UVB照射后p38MAPK途径的激活、细胞因子的分泌及凋亡细胞的增加之间存在时间先后上的调控关系,即p38MAPK信号通路中p38的快速磷酸化参与调控了后续的细胞因子的分泌活动及其由炎症介质介导的细胞凋亡作用。另外本实验结果显示p38抑制剂不能抑制UVB对p38磷酸化的诱导作用。④应用EGCG后细胞凋亡率降为6.63±7.5%,同时加入EGCG能部分抑制p-p38MAPK的表达以及TNF-α、IL-1β与IL-6的分泌。
结论:UVB辐射对HacaT细胞具有明显的损伤作用;UVB可以通过激活p38MAPK途径和p38磷酸化,以促进炎症因子分泌增加而诱导HaCaT细胞凋亡。加用EGCG干预处理可抑制p38MAPK途径活化和炎症因子分泌活动,从而减少了UVB诱导的细胞凋亡,发挥了EGCG抵抗UVB急性损伤的功效。
下篇EGCG对慢性UVA照射后小鼠皮肤光老化的防护作用
研究背景:
高紫外线暴露人群皮肤老化危险性要比低暴露人群高,老化发生提前。皮肤光老化不仅有损于人的美容,而且与皮肤病的发生有着病因学的联系。在光老化的发病因素中,UVA由于其波长长可穿透表皮到达真皮后发挥作用,而使其在光老化进程中具有重要意义。皮肤光老化临床上特征性改变包括:皱纹增多,纹理加深,干燥粗糙,皮肤松弛,弹性降低,斑点状色素沉着以及癌变。组织学上主要表现为:真皮结缔组织的变化,即胶原成分的减少和异常弹性纤维沉积。胶原使皮肤具有强度和弹性,是皮肤蛋白质的主要成分,在人体真皮中约占蛋白质的90%,主要由Ⅰ型胶原(80%)和少量Ⅲ型胶原(10%)组成。当皮肤被紫外线辐射损伤后,结缔组织重建:细胞外基质降解和清除,新成分合成和沉积。胶原作为细胞外基质中最丰富的成分,在此过程中发挥重要作用。
本实验小组已对EGCG乳膏保护UVB所致BALB/c小鼠皮肤的急慢性光损伤作了研究,结果示:EGCG可干预慢性UVB辐射诱导的皮肤组织病理改变、p53蛋白表达及炎症因子的变化,还可促进受损细胞的凋亡。但是在国内外目前对慢性UVA辐射所导致动物皮肤的光老化情况以及使用EGCG乳膏有无干预作用目前研究甚少,故设计本实验,每次UVA照射前外用EGCG乳膏,通过形态学方法观察EGCG对小鼠皮肤的保护作用。
目的:研究EGCG对慢性UVA辐射BALB/c小鼠皮肤光老化的防护作用。
方法:将实验小鼠分为6组:对照组、EGCG组、基质组、UVA照射组、EGCG+UVA组以及基质+UVA组,EGCG乳膏和基质在UVA照射前30分钟外涂,UVA照射剂量第1周为0.5J/min×20min,以后每周逐渐递增20min,直至辐射时间为2h时停止增加,共持续12周后处理小鼠,取皮肤进行普通组织病理检查及扫描电镜观察。
结果:小鼠暴露于紫外线辐射12周后,组织病理学出现显著的皮肤变化,真皮厚度增加。外涂EGCG乳膏组可减少紫外线诱导的皮肤光老化效应。扫描电镜见UVA组皮肤真皮层增厚,胶原之间排列疏松,扭曲而不规则,而空白对照组真皮胶原纤维致密。EGCG+UVA组和UVA组相比,真皮厚度及胶原纤维的排列方向均明显改善,而基质+UVA组在镜下见改善不明显。
结论:局部应用EGCG乳膏可预防紫外线辐射后的组织损伤,延迟光老化进程。
Chapter One
Background
UV radiation is an important environmental factor with inducible health hazards for mankind, particularly UVB has been demonstrated to be the pivotal casual factor in human. UVB exposure has been established to induce adverse effects, such as erythema, edema, blistering, skin pigmentation, sunburn cell formation, hyperplasia, photoaging and nonmelanoma skin cancer. Substantial UVB exposure may lead to detrimental acute effects such as tissue damage owing in part to the organism's surveillance mechanism(s) attempting to eliminate damaged cells through apoptosis and inflammation. Scientific interest in UV-induced information highways from the cell surface to the nucleus has explored over the past years, one of the best-studied signaling routes is the mitogen activated protein kinase (MAPK) signal transduction pathway, which plays a crucial role in the regulation of the multitude of UV-induced cellular responses. MAPK is composed of at least four families that include extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38, and ERK5/BMK1. P38 mediated apoptosis and inflammation are critical in protecting the integrity of the epidermis against the tumorigenic effects of UV-induced damage. Green tea is one of the most widely consumed beverages worldwide. The polyphenols in green tea are catechins, a family of compounds which includes (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin, (-)-epicatechin-3-gallate, (-)-epicatechin . EGCG , which are known to be effective free radical scavengers and potent antioxidants, protect against chemical carcinogenesis and UV-induced oxidative stress. Here We used HaCaT cells to assess the effects of EGCG on UVB-induced damage, such as apoptosis and cytokine secretion. We also attempted to ascertain the regulation mechanism of p38MAPK pathways, by which UVB irradiation and EGCG treatment could function in HaCaT cell culture system.
Objectives: Part I to investigate the incidence of apoptosis, secretion of cytokine, expression of p38MAPK and pp38MAPK induced by UVB irradiation in HaCaT cells; Part IIto investigate the EGCG effects on the apoptosis, secretion of cytokine and expression of p38MAPK.
Methods: Flow cytometry was used to detect the apoptosis of the cells. ELISA was used to detect the TNF-αconcentration. Western blot analysis was performed to determine the expression of p38 mitogen-activated protein kinase (MAPK).
Results: PartⅠ: After UVB irradiation P38MAPK phosphorylation was detectable immediately and peaked at one hour. Again TNF-α, IL-1β, IL-6 secretion and cell apoptosis were significantly increased. P38 inhibitor, SB203580 inhibited the activity of p38MAPK, reduced cytokine secretion of TNF-α, IL-1β, IL-6 and decreased cell apoptosis. These data suggest that p38 MAPK activation is one aspect of the signaling cascade which culminates in the secretion of TNF-α, IL-1β, IL-6 and contributes to cell apoptosis after UVB irradiation. PartⅡ:We found that EGCG reduced levels of the phosphorylation of UVB-induced p38 MAPK. coupled with a marked down-regulation of UVB-induced cytokine secretion and apoptosis.
Conclusions: These data suggest p38 MAPK activation is one aspect of the signaling cascade that culminates in the secretion of cytokine and contributes to cell apoptosis after UVB irradiation and demonstrate that EGCG exerts photoprotective effects against UVB irradiation. Pretreatment with EGCG protects against UVB irradiation via the suppression p38 MAPK activation. Our results suggest that EGCG may be useful in the prevention of UVB-induced human skin damage.
Chapter Two
Background
Protection against UVA exposure is also important since UVA radiation (320-400 nm), which makes up 95% of natural sunlight, is not filtered by standard glass. UVA is a potent inducer of reactive oxygen species (ROS), which can induce various biological processes. Chronic UVA irradiation has been reported to induce photoaging and photocarcinogenesis, because of its capacity to reach dermal layers, unlike UVB (290-320 run) which is absorbed in the epidermis. Clinically and histopathologically, photoaging and photocarcinogenesis behaved themselves many characteristics. Our researches have proved that EGCG cream showed photoprotections in BALB/c mice skin induced by acute and chronic UVB irradiation, such as reduced apoptosis,etc.Here we evaluated the photoprotective effects of EGCG cream against chronic UVA-induced damages in BALB/c mice.
Objective: To investigate the potency of EGCG cream in reducing the degradative changes photoaging induced in BALB/c mouse skin upon exposure to chronic UVA radiation for 12 weeks.
Methods: EGCG was prepared in hydrophilic ointment as the vehicle. Mice were divided into 6 groups and each group has 5 mice. The first received no treatment and was the control group. The second and third groups received EGCG and vehicle treatment ,respectively. The fourth , fifth and sixth group received UVA treatment only, UVA+EGCG treatment and UVA+vehicle, respectively. After 12 weeks' treatment as above, all mice were sacrificed and the skin specimens were examined by scanning electron microscope and common light microscope.
Results: Upon exposure of mice to UVA radiation, the results of common light microscope and scanning electron microscope indicated that among groups one ,four, and five, the thickness of dermal layers have significant difference. There are elastosis with accumulation of elastotic fibers. Comparison of Groups one, two and three shows similar results.
Conclusions: It can be concluded that topical EGCG cream is capable of preventing the structural damage caused by chronic UVA irradiation and EGCG is also found very useful in decelerating the photoaging process.
研究背景:
目光中的长波紫外线(UVA)和中波紫外线(UVB)辐射可引起急性损伤如日晒伤和慢性累积性损伤如光老化及皮肤癌,对人类健康构成潜在危害。波长短能量较高的UVB(290-320nm)主要作用于表皮,角质形成细胞是其靶细胞,可诱导皮肤不良反应如红斑、水肿、水疱、皮肤色素沉着、异常增生、光老化和非黑色素皮肤癌,而机体通过多种保护性措施如凋亡和炎症来保护皮肤监视及对抗潜在致癌细胞的增殖。
近几年紫外线诱导的炎症反应和细胞恶性转化过程中信号通路的作用受到广泛关注,其中一个研究热点即是丝裂原活化蛋白激酶(MAPK)信号转导通路,该途径可被紫外线激活,故在调节紫外线诱导的多重细胞反应中至关重要。MAPK主要由以下四个家族组成:ERK,JNK,p38和ERK5/BMK1,其中p38介导的炎症反应和凋亡在维护表皮完整性和对抗紫外线诱导的肿瘤效应方面具有重要作用。
绿茶是全球广泛受欢迎的饮料,绿茶中的酚类是儿茶素,其中的没食子儿茶素没食子酸酯(EGCG)是有效的自由基清除剂和抗氧化剂,可以预防化学致癌作用和紫外线诱导的氧化应激效应。我们以前的研究证实单次UVB辐射可引起急性光损伤包括细胞活性减低及增殖减慢、细胞凋亡增加、光加合物形成增多。累积性UVA/UVB照射甚至可以导致DNA突变及皮肤癌发生,当加入EGCG干预后,均可以减少UVB照射所致上述各种损伤性改变,但关于p38丝裂原活化蛋白激酶(p38MAPK)信号途径在UVB所致凋亡及炎症中的作用,及其EGCG的保护机制是否也与此途径有关在国内尚未见报告,因此我们以人皮肤角质形成细胞株HaCaT为观察对象,研究p38MAPK途径在UVB照射诱导的细胞凋亡及炎症介质分泌活动中的作用,并以p38抑制剂为阳性参照对比观察EGCG的干预保护作用是否也与p38MAPK通路有关。
目的:第一部分研究UVB辐射引起HaCaT细胞发生凋亡的概率、细胞因子TNF-α、IL-1β及IL-6的分泌及p38MAPK和磷酸化p38丝裂原活化蛋白激酶(p-p38MAPK)的表达,探讨凋亡、细胞因子分泌与p38MAPK之间的联系,确定p38MAPK信号转导途径是否参与了UVB诱导的细胞凋亡及细胞因子的分泌。第二部分研究EGCG对UVB辐射后细胞凋亡、细胞因子分泌及p38MAPK、p-p38MAPK表达的影响,探讨EGCG的光保护机制是否也与p38MAPK通路有关。
方法:使用0,20,30,40,60,70及90mJ/cm~2UVB照射HaCaT细胞,照射后Oh,1h,2h,3h,4h,6h,24h及48h收集细胞和上清。流式细胞仪检测细胞周期和凋亡;ELISA检测上清液中TNF-α、IL-1β、IL-6水平;WesternBlot方法检测p38MAPK和p-p38MAPK的表达。
结果:①与空白对照组细胞凋亡率(0)相比,30mJ/cm~2UVB照射后24h,HaCaT细胞凋亡率为21.25±0.33%,应用p38抑制剂后测细胞凋亡率降为10.14±0.27%,较UVB组降低近1/2。②30mJ/cm~2UVB照射HaCaT细胞,照射后HaCaT细胞分泌TNF-α渐增加,24h达高峰,后渐下降,p38抑制剂SB203580完全抑制TNF-α的分泌。UVB辐射后IL-1β,IL-6则随孵育时间而大幅度增加,SB203580明显抑制了IL-1β和IL-6的分泌水平。③不同剂量UVB照射后可激活HaCaT细胞p38MAPK途径,磷酸化p-p38水平增高,照光组与非照光组间相比具有统计学意义。以30mJ/cm~2UVB照射时p-p38表达量最高,但非磷酸化p38表达量没有类似明显变化。以上数据说明急性UVB照射后p38MAPK途径的激活、细胞因子的分泌及凋亡细胞的增加之间存在时间先后上的调控关系,即p38MAPK信号通路中p38的快速磷酸化参与调控了后续的细胞因子的分泌活动及其由炎症介质介导的细胞凋亡作用。另外本实验结果显示p38抑制剂不能抑制UVB对p38磷酸化的诱导作用。④应用EGCG后细胞凋亡率降为6.63±7.5%,同时加入EGCG能部分抑制p-p38MAPK的表达以及TNF-α、IL-1β与IL-6的分泌。
结论:UVB辐射对HacaT细胞具有明显的损伤作用;UVB可以通过激活p38MAPK途径和p38磷酸化,以促进炎症因子分泌增加而诱导HaCaT细胞凋亡。加用EGCG干预处理可抑制p38MAPK途径活化和炎症因子分泌活动,从而减少了UVB诱导的细胞凋亡,发挥了EGCG抵抗UVB急性损伤的功效。
下篇EGCG对慢性UVA照射后小鼠皮肤光老化的防护作用
研究背景:
高紫外线暴露人群皮肤老化危险性要比低暴露人群高,老化发生提前。皮肤光老化不仅有损于人的美容,而且与皮肤病的发生有着病因学的联系。在光老化的发病因素中,UVA由于其波长长可穿透表皮到达真皮后发挥作用,而使其在光老化进程中具有重要意义。皮肤光老化临床上特征性改变包括:皱纹增多,纹理加深,干燥粗糙,皮肤松弛,弹性降低,斑点状色素沉着以及癌变。组织学上主要表现为:真皮结缔组织的变化,即胶原成分的减少和异常弹性纤维沉积。胶原使皮肤具有强度和弹性,是皮肤蛋白质的主要成分,在人体真皮中约占蛋白质的90%,主要由Ⅰ型胶原(80%)和少量Ⅲ型胶原(10%)组成。当皮肤被紫外线辐射损伤后,结缔组织重建:细胞外基质降解和清除,新成分合成和沉积。胶原作为细胞外基质中最丰富的成分,在此过程中发挥重要作用。
本实验小组已对EGCG乳膏保护UVB所致BALB/c小鼠皮肤的急慢性光损伤作了研究,结果示:EGCG可干预慢性UVB辐射诱导的皮肤组织病理改变、p53蛋白表达及炎症因子的变化,还可促进受损细胞的凋亡。但是在国内外目前对慢性UVA辐射所导致动物皮肤的光老化情况以及使用EGCG乳膏有无干预作用目前研究甚少,故设计本实验,每次UVA照射前外用EGCG乳膏,通过形态学方法观察EGCG对小鼠皮肤的保护作用。
目的:研究EGCG对慢性UVA辐射BALB/c小鼠皮肤光老化的防护作用。
方法:将实验小鼠分为6组:对照组、EGCG组、基质组、UVA照射组、EGCG+UVA组以及基质+UVA组,EGCG乳膏和基质在UVA照射前30分钟外涂,UVA照射剂量第1周为0.5J/min×20min,以后每周逐渐递增20min,直至辐射时间为2h时停止增加,共持续12周后处理小鼠,取皮肤进行普通组织病理检查及扫描电镜观察。
结果:小鼠暴露于紫外线辐射12周后,组织病理学出现显著的皮肤变化,真皮厚度增加。外涂EGCG乳膏组可减少紫外线诱导的皮肤光老化效应。扫描电镜见UVA组皮肤真皮层增厚,胶原之间排列疏松,扭曲而不规则,而空白对照组真皮胶原纤维致密。EGCG+UVA组和UVA组相比,真皮厚度及胶原纤维的排列方向均明显改善,而基质+UVA组在镜下见改善不明显。
结论:局部应用EGCG乳膏可预防紫外线辐射后的组织损伤,延迟光老化进程。
Chapter One
Background
UV radiation is an important environmental factor with inducible health hazards for mankind, particularly UVB has been demonstrated to be the pivotal casual factor in human. UVB exposure has been established to induce adverse effects, such as erythema, edema, blistering, skin pigmentation, sunburn cell formation, hyperplasia, photoaging and nonmelanoma skin cancer. Substantial UVB exposure may lead to detrimental acute effects such as tissue damage owing in part to the organism's surveillance mechanism(s) attempting to eliminate damaged cells through apoptosis and inflammation. Scientific interest in UV-induced information highways from the cell surface to the nucleus has explored over the past years, one of the best-studied signaling routes is the mitogen activated protein kinase (MAPK) signal transduction pathway, which plays a crucial role in the regulation of the multitude of UV-induced cellular responses. MAPK is composed of at least four families that include extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38, and ERK5/BMK1. P38 mediated apoptosis and inflammation are critical in protecting the integrity of the epidermis against the tumorigenic effects of UV-induced damage. Green tea is one of the most widely consumed beverages worldwide. The polyphenols in green tea are catechins, a family of compounds which includes (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin, (-)-epicatechin-3-gallate, (-)-epicatechin . EGCG , which are known to be effective free radical scavengers and potent antioxidants, protect against chemical carcinogenesis and UV-induced oxidative stress. Here We used HaCaT cells to assess the effects of EGCG on UVB-induced damage, such as apoptosis and cytokine secretion. We also attempted to ascertain the regulation mechanism of p38MAPK pathways, by which UVB irradiation and EGCG treatment could function in HaCaT cell culture system.
Objectives: Part I to investigate the incidence of apoptosis, secretion of cytokine, expression of p38MAPK and pp38MAPK induced by UVB irradiation in HaCaT cells; Part IIto investigate the EGCG effects on the apoptosis, secretion of cytokine and expression of p38MAPK.
Methods: Flow cytometry was used to detect the apoptosis of the cells. ELISA was used to detect the TNF-αconcentration. Western blot analysis was performed to determine the expression of p38 mitogen-activated protein kinase (MAPK).
Results: PartⅠ: After UVB irradiation P38MAPK phosphorylation was detectable immediately and peaked at one hour. Again TNF-α, IL-1β, IL-6 secretion and cell apoptosis were significantly increased. P38 inhibitor, SB203580 inhibited the activity of p38MAPK, reduced cytokine secretion of TNF-α, IL-1β, IL-6 and decreased cell apoptosis. These data suggest that p38 MAPK activation is one aspect of the signaling cascade which culminates in the secretion of TNF-α, IL-1β, IL-6 and contributes to cell apoptosis after UVB irradiation. PartⅡ:We found that EGCG reduced levels of the phosphorylation of UVB-induced p38 MAPK. coupled with a marked down-regulation of UVB-induced cytokine secretion and apoptosis.
Conclusions: These data suggest p38 MAPK activation is one aspect of the signaling cascade that culminates in the secretion of cytokine and contributes to cell apoptosis after UVB irradiation and demonstrate that EGCG exerts photoprotective effects against UVB irradiation. Pretreatment with EGCG protects against UVB irradiation via the suppression p38 MAPK activation. Our results suggest that EGCG may be useful in the prevention of UVB-induced human skin damage.
Chapter Two
Background
Protection against UVA exposure is also important since UVA radiation (320-400 nm), which makes up 95% of natural sunlight, is not filtered by standard glass. UVA is a potent inducer of reactive oxygen species (ROS), which can induce various biological processes. Chronic UVA irradiation has been reported to induce photoaging and photocarcinogenesis, because of its capacity to reach dermal layers, unlike UVB (290-320 run) which is absorbed in the epidermis. Clinically and histopathologically, photoaging and photocarcinogenesis behaved themselves many characteristics. Our researches have proved that EGCG cream showed photoprotections in BALB/c mice skin induced by acute and chronic UVB irradiation, such as reduced apoptosis,etc.Here we evaluated the photoprotective effects of EGCG cream against chronic UVA-induced damages in BALB/c mice.
Objective: To investigate the potency of EGCG cream in reducing the degradative changes photoaging induced in BALB/c mouse skin upon exposure to chronic UVA radiation for 12 weeks.
Methods: EGCG was prepared in hydrophilic ointment as the vehicle. Mice were divided into 6 groups and each group has 5 mice. The first received no treatment and was the control group. The second and third groups received EGCG and vehicle treatment ,respectively. The fourth , fifth and sixth group received UVA treatment only, UVA+EGCG treatment and UVA+vehicle, respectively. After 12 weeks' treatment as above, all mice were sacrificed and the skin specimens were examined by scanning electron microscope and common light microscope.
Results: Upon exposure of mice to UVA radiation, the results of common light microscope and scanning electron microscope indicated that among groups one ,four, and five, the thickness of dermal layers have significant difference. There are elastosis with accumulation of elastotic fibers. Comparison of Groups one, two and three shows similar results.
Conclusions: It can be concluded that topical EGCG cream is capable of preventing the structural damage caused by chronic UVA irradiation and EGCG is also found very useful in decelerating the photoaging process.
引文
1.Assefa Z,Vantieghem A,Garmyn M,et al.P38 Mitogen-activated Protein Kinase Regulates a Novel,Caspase-independent Pathwagy for the Mitochondrial Cytochrome C Release in Ultraviolet B Radiation-inducen Apoptosis. Biol Chem, 2000,275 (28) : 21416-21
2. Shimizu H, Banno Y, Sumi N, et al. Activation of P38 Mitogen-Activated Protein Kinase and Caspases in UVB-Induced Apoptosis of Human Keratinocyte HaCaT cells. Invest Dermatol, 1999,112 (5) :769-74
3. Hildesheim J, Awwad RT, Fornace AJ Jr. P38 Mitogen-Activated Protein Kinase Inhibitor Protects the Epidermis Against the Acute Damaging Effects of Ultraviolet Irradiation by Blocking Apoptosis and Inflammatory Response. J Invest Dermatol 2004, 122 (2) : 497-502
4. Ballard-Croft C , White DJ, Maass DL , et al. Role of P38 mitogen-activated protein kinase in cardiac myocyte secretion of the inflammatory cytokine TNF-a. Am J Physiol Heart Circ Physiol , 2001,280 (5) :H1970-81
5. Verma A, Deb DK, Sassano A, et al. Activation of the p38 mitogen-activated protein kinase mediates the suppressive effects of type I interferons and transforming growth factor-beta on normal hematopoiesis. Biol Chem ,2002, 277: 7726-35
6. Valladares A, Alvarez AM, Ventura JJ, et al.P38MAPK Activated Protein Kinase Mediates Tumor Necrosis Factor-a-Induced Apoptosis in Rat Fetal Brown Adipocytes. Endocrinology , 2000,141 (12) : 4383-95
7. Wehrli P, Viard I, Bullani R, Tschopp J, French LE. Death receptors in cutaneous biology and disease. Invest Dermatol ,2000,115:141-148
8. Van Laethem A, Van Kelst S, Lippens S, et al.Activation of P38MAPK is required for Bax translocation to mitochondria, cytochrome c release and apoptosis induced by UVB irradiation in human keratinocytes. FASEB, 2004,18 (15) : 1946-8
1.Shimizu H,Banno Y,Sumi N,et al.Activation of p38mitogen-activated protein kinase and caspases in UVB-induced apoptosis of human keratinocyte HaCaT cells.J Invest Dermatol.1999May;112(5):769-74.
2.Assefa Z,Vantieghem A,Garmyn M,et al.p38 mitogen-activated protein kinase regulates a novel,caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis.J Biol Chem.2000 Jul 14;275(28):21416-21.
3.Hildesheim J,Awwad RT,Fornace AJ Jr.p38 Mitogen-activated protein kinase inhibitor protects the epidermis against the acute damaging effects of ultraviolet irradiation by blocking apoptosis and inflammatory responses.J Invest Dennatol. 2004 Feb;122(2):497-502.
4. Ballard-Croft C, White DJ, Maass DL,et al. Role of p38 mitogen-activated protein kinase in cardiac myocyte secretion of the inflammatory cytokine TNF-alpha. Am J Physiol Heart Circ Physiol. 2001 May;280(5):H1970-81.
5. Kim SY, Kim DS, Kwon SB,et al. Protective effects of EGCG on UVB-induced damage in living skin equivalents. Arch Pharm Res. 2005 Jul;28(7):784-90.
1.Nishigori C,Hattori Y,Arima Y,Miyachi Y.Photoaging and oxidative stress.Exp Dermatol.2003;12(Suppl.2):18-21.
2.Sultana Y,Kohli K,Athar M,et al.Effect of pre-treatment of almond oil on ultraviolet B-induced cutaneous photoaging in mice.J Cosmet Dermatol.2007,6(1):14-9.
3.徐丽贤,骆丹,吉玺等.UVB诱导皮肤免疫抑制和表没食子儿茶素没食子酸酯光保护作用冲华皮肤科杂志.2006,39(9):500-502.
4.徐晶,骆丹,周炳荣等.不同剂型表没食子儿茶素没食子酸酯对中波紫外线辐射后BALB/c小鼠表皮细胞凋亡的影响.中国药学杂志.2007,42(10):1540-1542.
5.闵玮,骆丹,林向飞.中波紫外线辐射对原代及永生化角质形成细胞损伤能力的比较研究.中国麻风皮肤病杂志,2004,20(6):521-524.
6.闵玮,骆丹,林向飞.羟氯喹及没食子酸酯对HaCaT细胞光照射的影响.中华皮肤科杂志,2005,38(9):575-577.
7.Kativar SK.Skin photoprotection by green tea:antioxidant and immunomodulatory effects.Curr Drug Targets Immune Endocr Metabol Disord.2003,3(3):234-42.
8.Sevin A,Oztas P,Senen D,et al.Effects of polyphenols on skin damage due to ultraviolet A rays:an experimental study on rats.J Eur Acad Dermatol Venereol.2007,21(5):650-6.
1.Reed JC Dysregulation of apoptosis in cancer.J Clin Oncol,1999,17:2941-2953
2.Benassi L,Ottani D,Fantini F,et al.1,25- dihydroxyvitamin D3,transforming growth factor betal,calcium,and ultraviolet B radiation induce apoptosis in cultured human keratinocytes.J Invest Dermatol,1997,109(3):276-82
3.Brash DE Sunlight and the onset of skin cancer.Trends Genet, 1997, 13 (10) :410-4
4. Bang B, Gniadecki R, Larsen JK, et al. In vivo UVB irradiation induces clustering of Fas (CD95) on human epidermal cells. Exp Dermatol , 2003, 12 (6) :791-8
5. Berton TR, Mitchell DL, Guo R, et al. Regulation of epidermal apoptosis and DNA repair by E2F1 in response to ultraviolet B radiation. Oncogene, 2005 , 24 (15) :2449-60
6. Van Laethem A, Claerhout S, Garmyn M, et al .The sunburn cell: regulation of death and survival of the keratinocyte. Int J Biochem Cell Biol, 2005, 37 (8) :1547-53
7. French LE, Trent JT, Kerdel FA. Use of intravenous immunoglobulin in toxic epidermal necrolysis and Stevens-Johnson syndrome: our current understanding. Int Immunopharmacol, 2006, 6(4):543-9.
8. Nassif A, Moslehi H, Le Gouvello S, et al. Evaluation of the potential role of cytokines in toxic epidermal necrolysis. J Invest Dermatol , 2004, 123 (5) :850-5
9. Tsukada N, Kobata T, Aizawa Y, et al .Graft-versus-leukemia effect and graft-versus-host disease can be differentiated by cytotoxic mechanisms in a murine model of allogeneic bone marrow transplantation. Blood , 1999, 93 (8) :2738-47
10. Gaipl US, Kuhn A, Sheriff A, et al. Clearance of apoptotic cells in human SLE. Curr Dir Autoimmun, 2006, 9:173-87
11.Kruger-Krasagakis S,Galanopoulos VK,Giannikaki L,et al.Programmed cell death of keratinocytes in infliximab-treated plaque-type psoriasis.Br J Dermatol,2006,154(3):460-6
12.Fukuya Y,Higaki M,Higaki Y,et al Effect of vitamin D3 on the increased expression of Bcl-xL in psoriasis.Arch Dermatol Res,2002,293:620-625
13.Bianchi L,Farrace MC,Nini G,et al.Abnormal bcl-2and"Tissue"transglutaminasa expression in psoriatic skin.Invest Dermatol 1994;103:829-833.
14.Wrone-Smith T,Johnson T,Nelson B,et al.Discordant expression of bcl-x and bcl-2 by keratinocytes in vitro and psoriatsic keratinocytes in vivo.Am J Pathol 1995;146:1079-1088.
15.Stander S,Schwarz T Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)is expressed in normal skin and cutaneous inflammatory diseases,but not in chronically UV-exposed skin and non-melanoma skin cancer.Am J Dermatopathol,2005,27(2):116-21
16.Nakayama H,Ikebe T,Beppu M,et al.High expression levels of nuclear factor kappaB,IkappaB kinase alpha and Akt kinase in squamous cell carcinoma of the oral cavity.Cancer,2001,92:3037-3044
2. Shimizu H, Banno Y, Sumi N, et al. Activation of P38 Mitogen-Activated Protein Kinase and Caspases in UVB-Induced Apoptosis of Human Keratinocyte HaCaT cells. Invest Dermatol, 1999,112 (5) :769-74
3. Hildesheim J, Awwad RT, Fornace AJ Jr. P38 Mitogen-Activated Protein Kinase Inhibitor Protects the Epidermis Against the Acute Damaging Effects of Ultraviolet Irradiation by Blocking Apoptosis and Inflammatory Response. J Invest Dermatol 2004, 122 (2) : 497-502
4. Ballard-Croft C , White DJ, Maass DL , et al. Role of P38 mitogen-activated protein kinase in cardiac myocyte secretion of the inflammatory cytokine TNF-a. Am J Physiol Heart Circ Physiol , 2001,280 (5) :H1970-81
5. Verma A, Deb DK, Sassano A, et al. Activation of the p38 mitogen-activated protein kinase mediates the suppressive effects of type I interferons and transforming growth factor-beta on normal hematopoiesis. Biol Chem ,2002, 277: 7726-35
6. Valladares A, Alvarez AM, Ventura JJ, et al.P38MAPK Activated Protein Kinase Mediates Tumor Necrosis Factor-a-Induced Apoptosis in Rat Fetal Brown Adipocytes. Endocrinology , 2000,141 (12) : 4383-95
7. Wehrli P, Viard I, Bullani R, Tschopp J, French LE. Death receptors in cutaneous biology and disease. Invest Dermatol ,2000,115:141-148
8. Van Laethem A, Van Kelst S, Lippens S, et al.Activation of P38MAPK is required for Bax translocation to mitochondria, cytochrome c release and apoptosis induced by UVB irradiation in human keratinocytes. FASEB, 2004,18 (15) : 1946-8
1.Shimizu H,Banno Y,Sumi N,et al.Activation of p38mitogen-activated protein kinase and caspases in UVB-induced apoptosis of human keratinocyte HaCaT cells.J Invest Dermatol.1999May;112(5):769-74.
2.Assefa Z,Vantieghem A,Garmyn M,et al.p38 mitogen-activated protein kinase regulates a novel,caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis.J Biol Chem.2000 Jul 14;275(28):21416-21.
3.Hildesheim J,Awwad RT,Fornace AJ Jr.p38 Mitogen-activated protein kinase inhibitor protects the epidermis against the acute damaging effects of ultraviolet irradiation by blocking apoptosis and inflammatory responses.J Invest Dennatol. 2004 Feb;122(2):497-502.
4. Ballard-Croft C, White DJ, Maass DL,et al. Role of p38 mitogen-activated protein kinase in cardiac myocyte secretion of the inflammatory cytokine TNF-alpha. Am J Physiol Heart Circ Physiol. 2001 May;280(5):H1970-81.
5. Kim SY, Kim DS, Kwon SB,et al. Protective effects of EGCG on UVB-induced damage in living skin equivalents. Arch Pharm Res. 2005 Jul;28(7):784-90.
1.Nishigori C,Hattori Y,Arima Y,Miyachi Y.Photoaging and oxidative stress.Exp Dermatol.2003;12(Suppl.2):18-21.
2.Sultana Y,Kohli K,Athar M,et al.Effect of pre-treatment of almond oil on ultraviolet B-induced cutaneous photoaging in mice.J Cosmet Dermatol.2007,6(1):14-9.
3.徐丽贤,骆丹,吉玺等.UVB诱导皮肤免疫抑制和表没食子儿茶素没食子酸酯光保护作用冲华皮肤科杂志.2006,39(9):500-502.
4.徐晶,骆丹,周炳荣等.不同剂型表没食子儿茶素没食子酸酯对中波紫外线辐射后BALB/c小鼠表皮细胞凋亡的影响.中国药学杂志.2007,42(10):1540-1542.
5.闵玮,骆丹,林向飞.中波紫外线辐射对原代及永生化角质形成细胞损伤能力的比较研究.中国麻风皮肤病杂志,2004,20(6):521-524.
6.闵玮,骆丹,林向飞.羟氯喹及没食子酸酯对HaCaT细胞光照射的影响.中华皮肤科杂志,2005,38(9):575-577.
7.Kativar SK.Skin photoprotection by green tea:antioxidant and immunomodulatory effects.Curr Drug Targets Immune Endocr Metabol Disord.2003,3(3):234-42.
8.Sevin A,Oztas P,Senen D,et al.Effects of polyphenols on skin damage due to ultraviolet A rays:an experimental study on rats.J Eur Acad Dermatol Venereol.2007,21(5):650-6.
1.Reed JC Dysregulation of apoptosis in cancer.J Clin Oncol,1999,17:2941-2953
2.Benassi L,Ottani D,Fantini F,et al.1,25- dihydroxyvitamin D3,transforming growth factor betal,calcium,and ultraviolet B radiation induce apoptosis in cultured human keratinocytes.J Invest Dermatol,1997,109(3):276-82
3.Brash DE Sunlight and the onset of skin cancer.Trends Genet, 1997, 13 (10) :410-4
4. Bang B, Gniadecki R, Larsen JK, et al. In vivo UVB irradiation induces clustering of Fas (CD95) on human epidermal cells. Exp Dermatol , 2003, 12 (6) :791-8
5. Berton TR, Mitchell DL, Guo R, et al. Regulation of epidermal apoptosis and DNA repair by E2F1 in response to ultraviolet B radiation. Oncogene, 2005 , 24 (15) :2449-60
6. Van Laethem A, Claerhout S, Garmyn M, et al .The sunburn cell: regulation of death and survival of the keratinocyte. Int J Biochem Cell Biol, 2005, 37 (8) :1547-53
7. French LE, Trent JT, Kerdel FA. Use of intravenous immunoglobulin in toxic epidermal necrolysis and Stevens-Johnson syndrome: our current understanding. Int Immunopharmacol, 2006, 6(4):543-9.
8. Nassif A, Moslehi H, Le Gouvello S, et al. Evaluation of the potential role of cytokines in toxic epidermal necrolysis. J Invest Dermatol , 2004, 123 (5) :850-5
9. Tsukada N, Kobata T, Aizawa Y, et al .Graft-versus-leukemia effect and graft-versus-host disease can be differentiated by cytotoxic mechanisms in a murine model of allogeneic bone marrow transplantation. Blood , 1999, 93 (8) :2738-47
10. Gaipl US, Kuhn A, Sheriff A, et al. Clearance of apoptotic cells in human SLE. Curr Dir Autoimmun, 2006, 9:173-87
11.Kruger-Krasagakis S,Galanopoulos VK,Giannikaki L,et al.Programmed cell death of keratinocytes in infliximab-treated plaque-type psoriasis.Br J Dermatol,2006,154(3):460-6
12.Fukuya Y,Higaki M,Higaki Y,et al Effect of vitamin D3 on the increased expression of Bcl-xL in psoriasis.Arch Dermatol Res,2002,293:620-625
13.Bianchi L,Farrace MC,Nini G,et al.Abnormal bcl-2and"Tissue"transglutaminasa expression in psoriatic skin.Invest Dermatol 1994;103:829-833.
14.Wrone-Smith T,Johnson T,Nelson B,et al.Discordant expression of bcl-x and bcl-2 by keratinocytes in vitro and psoriatsic keratinocytes in vivo.Am J Pathol 1995;146:1079-1088.
15.Stander S,Schwarz T Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)is expressed in normal skin and cutaneous inflammatory diseases,but not in chronically UV-exposed skin and non-melanoma skin cancer.Am J Dermatopathol,2005,27(2):116-21
16.Nakayama H,Ikebe T,Beppu M,et al.High expression levels of nuclear factor kappaB,IkappaB kinase alpha and Akt kinase in squamous cell carcinoma of the oral cavity.Cancer,2001,92:3037-3044
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