Tempol抗中波紫外线照射所致皮肤光老化的作用机制及影响因素的探讨
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摘要
前言
紫外线是波长为200～400 nm的电磁波,其中波长为200～290 nm的紫外线称为短波紫外线(UVC),波长为290～320nm的紫外线称为中波紫外线(UVB),波长为320～400nm的紫外线称为长波紫外线(UVA)。UVC对人类皮肤的损害作用最大,但由于太阳辐射到地球表面的UVC大部分被大气层吸收,而达不到地球表面,所以UVC对人类皮肤的作用可被忽视。紫外线引起的皮肤损伤主要与UVA及UVB有关,其中UVB是造成皮肤损伤的主要原因。近年来由于大气臭氧层的破坏,辐射到地球表面的紫外线逐渐增多,由此引起的各种皮肤病明显增多,长期紫外线照射能引起皮肤的光老化及皮肤癌等。怎样有效地预防及治疗紫外线引起的皮肤损伤,已成为国际皮肤界关注的课题,尤其是有关UVB引起皮肤光损害的研究日益受到人们的关注,并正成为当今的研究热点。
为了减轻紫外线对皮肤的多种损伤,抗氧化剂的应用已成为人们防护皮肤和治疗皮肤病的有效选择。有关于这方面的研究正在进行当中。紫外线照射人及动物的皮肤后,能在人及动物的皮肤中产生较多的氧自由基,这已被大量的研究所证实。正常情况下,体内产生的氧自由基能被细胞内抗氧化系统转化、分解掉,当紫外线照射后,产生较多的氧自由基时,体内的抗氧化系统遭到破坏,这些自由基则引起皮肤组织内DNA,脂质、蛋白质等成分的损伤,破坏细胞结构及各种生物代谢功能,从而引起光老化,诱发皮肤癌等。针对这种由氧自由基介导的紫外线引起的皮肤损伤,近年来报道了较多的有关抗紫外线氧化性损伤的物质,如:SOD,生育酚,抗坏血酸,β-胡萝卜素,过氧化氢酶,维甲酸等等。这些物质有的在实验室动物及培养的细胞系统有很好的抗紫外线损伤作用,但在人体上的效果不确切,有的需要较高的血液浓度才能起到抗紫外线损伤的作用,而长期维持这种高浓度对人体是有害的。
氮氧化物是一种常用的生物物理制剂,与SOD等相比,氮氧化物具有分子量小,稳定性强,细胞膜通透性好等诸多优点。国外的研究结果显示氮氧化物具有明显的抗氧化性损伤的作用。4-羟基-2,2,6,6-四甲基氧基哌啶(Tempol,分子式C_9H_(18)NO_2)是一种在抗氧化研究中常用的氮氧化物,我们早期的实验已经证实,Tempol对于紫外线照射引起的皮肤光老化具有防护作用。
本研究共分为两部分,分别以HaCaT细胞和活体昆明无毛小鼠为UVB照射的体外和体内实验对象,利用Tempol为光保护剂,建立了Tempol抗UVB照射所致皮肤光老化的实验模型;通过分析实验模型各种观察指标的变化,探讨Tempol抗UVB照射所致光老化的作用机制,为防治光老化性疾病提供实验依据。
第一部分Tempol对中波紫外线照射所致HaCaT细胞光损伤防护作用机制的研究
背景
紫外线分为A、B、C三种(UVA、UVB、UVC)。能穿过大气层达到地球的只有UVA和UVB两种。UVA的波长为320～400nm;UVB的波长为290～320nm。表皮位于皮肤的最外层,是机体的第一道天然保护屏障,使机体免受外界物理、化学以及微生物侵害,维持机体内环境的稳定。这些作用主要是由占表皮细胞90%以上的角质形成细胞完成。现代研究证明角质形成细胞不仅是机体的保护细胞,而且参与各种细胞生物学过程如免疫、炎症、增生以及肿瘤转化等。有资料表明:照射到皮肤的95%的紫外线是被角质形成细胞吸收的。国际上已分别建立了UVA、UVB对角质形成细胞氧化损伤的模型,并进行了研究,主要集中在免疫抑制、DNA损伤、细胞凋亡、炎症等方面。UVB被认为是太阳光中引起非黑素皮肤癌最主要的紫外线。在美国每年有100多万人被诊断患有皮肤癌,平均每小时有一个病人死于皮肤源性恶性肿瘤,预计到2060年,美国的皮肤癌患者将达4000万人。到目前为止,我国虽然还没有相关的具体统计数据,但对紫外线诱发的皮肤疾病已引起了学者们的高度重视,尤其是有关UVB引起皮肤光损害的研究日益受到人们的关注,并正成为当今的研究热点。
为了减轻紫外线对皮肤的多种损伤,抗氧化剂的应用已成为人们防护皮肤和治疗皮肤病的有效选择。有关这方面的研究正在进行当中。Stewart MS和Cameron GS等发现,在预先应用维生素C或维生素E后,可以抑制UVB照射角质形成细胞后引起的DNA损伤。多个研究发现,在应用抗坏血酸或羟基乙酸后可以抑制UVB照射角质形成细胞引起的与凋亡相关的转录因子AP-1、C-fos的表达。Valeria M等发现,抗氧化剂维生素C可以通过抑制紫外线诱导的角质形成细胞的JNK的表达,抑制AP-1的表达,从而抑制角质形成细胞的凋亡。这些物质有的在实验室动物及培养的细胞系统有很好的抗紫外线损伤作用,但在人体上的效果不确切,有的需要较高的血液浓度才能起到抗紫外线损伤的作用,而长期维持这种高浓度对人体是有害的。氮氧化物是一种常用的生物物理制剂,自1988年Samuni A.发现其有类似于SOD的歧化超氧阴离子的作用后,其抗氧化性损伤的功能受到了重视。与SOD等相比,氮氧化物具有分子量小,稳定性强,细胞膜通透性好等诸多优点。国外实验室曾先后将氮氧化物应用于细菌、培养的心肌细胞、离体心脏灌注及活体动物等研究体系中,观察其抗氧化性损伤的作用,研究结果显示氮氧化物具有明显的抗氧化性损伤的作用。4-羟基-2,2,6,6-四甲基氧基哌啶(Tempol,分子式C_9H_(18)NO_2)是一种在抗氧化研究中常用的氮氧化物,我们早期的实验已经证实,Tempol对于紫外线照射引起的皮肤光损伤具有防护作用。
HaCaT细胞是角质形成细胞系细胞,是一种被转化但非致癌的细胞,由人腹部表皮衍化而成,保留了所有的表皮分化能力,与正常角质形成细胞非常相似,它在生物学特性上与正常人角质形成细胞相似,所以我们用30mJ/cm~2的UVB照射HaCaT细胞以建立UVB对角质形成细胞光损伤的模型,目的是模仿UVB对表皮的角质形成细胞的光损伤;同时以Tempol为光防护剂,探讨Tempol对UVB照射所致HaCaT细胞光损伤防护作用的机制及其影响因素,在国内外还是首次,为Tempol和其它的抗氧化剂对UVB照射所致角质形成细胞光损伤防护作用的研究提供了一条崭新的思路,更为Tempol对UVB照射所致HaCaT细胞光损伤防护作用机制的研究奠定了理论基础。
目的
1.建立UVB照射损伤HaCaT钿胞的光损伤模型,观察Tempol对UVB照射所致HaCaT细胞光损伤防护作用的机制;
2.观察Tempol对UVB照射后HaCaT细胞增殖效应的影响及其影响因素的研究;
3.观察Tempol对UVB照射后HaCaT细胞细胞凋亡的影响及其影响因素的研究;
4.观察Tempol对UVB照射后HaCaT细胞FoxO3a mRNA表达的影响及其影响因素的研究。
方法
1.HaCaT细胞的光损伤模型的建立
1.1 HaCaT细胞培养
用含10%胎牛血清(FBS)的DMEM置37℃CO_2培养箱中进行培养。
1.2紫外线光源
UVB灯管,波长范围290～320nm,峰值297 nm,40W,4只,4只灯管制成灯箱。
2.Tempol对光损伤防护作用实验
2.1 HaCaT细胞分组
将HaCaT细胞分为七组,分别标记为A、B、C、D、E、F和G组。
A组:为正常对照组,不加入Tempol,不照射UVB;
B组:为UVB照射对照组,不加入Tempol,只照射UVB;
C组:为含0.5mMTempol的实验组;
D组:为含1mMTempol的实验组;
E组:为含2mMTempol的实验组;
F组:为含4mMTempol的实验组;
G组:为含8mMTempol的实验组。
2.2细胞培养和照射剂量
HaCaT细胞在培养板上生长到每孔汇合至80%以上时,从细胞培养箱中取出,无菌条件下加入Tempol。正常对照组和照射对照组只加角质形成细胞专用培养基以使各孔的终体积相等。细胞放入37℃、5%CO_2细胞培养箱中孵育1h后,除正常对照组外,其余六组均照射UVB,照射剂量为30mJ/cm~2,光源与细胞的垂直照射距离为15cm。
2.3 HaCaT细胞增殖效应的检测
HaCaT细胞经UVB照射损伤并孵育18小时后,加入MTT继续培养4h,再加入二甲基亚砜,使结晶物充分溶解,在酶联免疫检测仪490nm处测量各孔吸光值。
2.4 HaCaT细胞凋亡率的检测
HaCaT细胞经UVB照射损伤并孵育18小时后,经消化、离心,调节细胞浓度为1×10~6/ml,加入Annexin V-FITC和PI溶液,流式细胞仪分析,测定HaCaT细胞的凋亡率。
2.5 HaCaT细胞FoxO3a mRNA表达的检测
HaCaT细胞经UVB照射损伤并孵育18小时后,将所收集的细胞提取总RNA,经逆转录反应得到cDNA,通过RT-PCR技术检测FoxO3amRNA的表达。
结果
1.Tempol对UVB照射后HaCaT细胞增殖效应的影响
A、C、D、E和F组OD值显著升高,与B和G组比较,差异有统计学意义(P＜0.01);其中A与C组OD值升高最明显,两组之间比较差异无统计学意义(P＞0.05),D、E和F组OD值依次递减,各组之间比较差异有统计学意义(P＜0.01);而B和G组比较,差异无统计学意义(P＞0.05)。
2.Tempol对UVB照射后HaCaT细胞凋亡率的影响
B、C、D、E、F和G组HaCaT细胞凋亡率显著升高,与A比较,差异有统计学意义(P＜0.01);其中B组HaCaT细胞凋亡率升高最明显,其余各组HaCaT细胞凋亡率随Tempol药物浓度的增加而升高,各组之间比较差异有统计学意义(P＜0.01)。
3.Tempol对UVB照射后HaCaT细胞FoxO3a mRNA表达的影响
B、C、D、E、F和G组HaCaT细胞FoxO3a mRNA表达显著升高,与A组比较,差异有统计学意义(P＜0.01);其中B组HaCaT细胞FoxO3a mRNA表达升高最明显,其余各组HaCaT细胞FoxO3a mRNA表达随Tempol药物浓度的增加而升高,各组之间比较差异有统计学意义(P＜0.01)。
结论
一定浓度范围的Tempol(0.5mM,1mM,2mM,4mM,8mM)对UVB照射所致HaCaT细胞光损伤具有防护作用,其作用机制可能与提高HaCaT细胞增殖效应,抑制HaCaT细胞凋亡以及抑制HaCaT细胞FoxO3a过度表达有关;其保护作用随Tempol浓度的升高而降低,0.5mMTempol的防护作用最强。
第二部分Tempol抗中波紫外线照射所致无毛鼠皮肤光老化的作用机制及其影响因素的探讨
背景
近年来由于大气臭氧层的破坏,辐射到地球表面的紫外线逐渐增多,由此引起的各种皮肤病明显增多.长期紫外线照射能引起皮肤的光老化及皮肤癌等。怎样有效地预防及治疗紫外线引起的皮肤损伤,已成为国际皮肤界关注的课题。紫外线是波长为200-400 nm的电磁波,其中波长为200-290 nm的紫外线称为短波紫外线(UVC),波长为290-320nm的紫外线称为中波紫外线(UVB),波长为320-400nm的紫外线称为长波紫外线(UVA)。UVC对人类皮肤的损害作用最大,但由于太阳辐射到地球表面的UVC大部分被大气层吸收,而达不到地球表面,所以UVC对人类皮肤的作用可被忽视。日光中同时含有UVA、UVB,紫外线引起的皮肤损伤主要与UVA及UVB有关,其中UVB是造成皮肤损伤的主要原因。
紫外线照射人及动物的皮肤后,能在人及动物的皮肤中产生较多的氧自由基,这已被大量的研究所证实。紫外线照射皮肤后,氧自由基的产生是由内源性光敏分子吸收光子,而后变成激活的光敏物,通过电子或氢原子的传递与底物分子反应生成的。正常情况下,体内产生的氧自由基能被细胞内抗氧化系统转化、分解掉,当紫外线照射后,产生较多的氧自由基时,体内的抗氧化系统遭到破坏,这些自由基则引起皮肤组织内DNA、脂质、蛋白质等成分的损伤,破坏细胞结构及各种生物代谢功能,从而引起光老化,诱发皮肤癌等。针对这种由氧自由基介导的紫外线引起的皮肤损伤,近年来报道了较多的有关抗紫外线氧化性损伤的物质,如:生育酚,抗坏血酸,β-胡萝卜素,过氧化氢酶,SOD,维甲酸等等。这些物质有的在实验室动物及培养的细胞系统有很好的抗紫外线损伤作用,但在人体上的效果不确切,有的需要较高的血液浓度才能起到抗紫外线损伤的作用,而长期维持这种高浓度对人体是有害的。
氮氧化物是一种常用的生物物理制剂,自1988年Samuni A.发现其有类似于SOD的歧化超氧阴离子的作用后,其抗氧化性损伤的功能受到了重视。与SOD相比,氮氧化物具有分子量小,稳定性强,细胞膜通透性好等诸多优点。国外实验室曾先后将氮氧化物应用于细菌、培养的心肌细胞、离体心脏灌注及活体动物等研究体系中,观察其抗氧化性损伤的作用,研究结果显示氮氧化物具有明显的抗氧化性损伤的作用。4-羟基-2,2,6,6-四甲基氧基哌啶(Tempol,分子式C_9H_(18)NO_2)是一种在抗氧化研究中常用的氮氧化物,我们早期的实验已经证实,Tempol对于紫外线照射引起的皮肤光老化具有防护作用。
因此本研究以活体昆明无毛小鼠为UVB照射的实验对象,利用Tempol为光保护剂,建立了Tempol抗UVB照射所致皮肤光老化的动物实验模型;通过对无毛小鼠皮肤外观状态变化、皮肤胶原纤维和弹力纤维结构变化、皮肤超微结构变化、皮肤丙二醛和羟脯氨酸含量变化以及皮肤突变型p53蛋白表达的观察,对UVB引起模型动物的各种皮肤观察指标变化进行分析,探讨Tempol抗UVB照射所致光老化的作用机制,并首次探讨了时间因素对Tempol外用抗UVB照射所致光老化作用的影响,为防治光老化性疾病提供了实验依据。
目的
1.观察无毛小鼠外用Tempol后不同时段照射UVB对皮肤外观状态变化的影响;
2.观察无毛小鼠外用Tempol后不同时段照射UVB对皮肤胶原纤维和弹力纤维结构变化的影响;
3.观察无毛小鼠外用Tempol后不同时段照射UVB对皮肤超微结构变化的影响;
4.观察无毛小鼠外用Tempol后不同时段照射UVB对皮肤丙二醛和羟脯氨酸含量变化的影响;
5.观察无毛小鼠外用Tempol后不同时段照射UVB对皮肤突变型p53蛋白表达的影响。
6.探讨时间因素对Tempol抗光老化作用的影响。
方法
1.光老化动物实验模型的建立
1.1实验动物
清洁级昆明种无毛小白鼠72只,鼠龄6～8周,雌雄各半,北京大学医学部实验动物科学部提供,体重20～25g,按同窝、同性别、同条件下饲养。
1.2紫外线光源
UVB灯管,波长范围290～320nm,峰值297 nm,40W,4只。将4只灯管制成灯箱,照射高度为50cm。
2.Tempol抗光老化实验
2.1动物分组
将72只昆明种无毛小鼠随机分为六组,每组12只,分别标记为A、B、C、D、E和F组。
A组:为正常对照组,外用三蒸水,不照射UVB;
B组:为UVB照射对照组,外用三蒸水,照射UVB;
C组:为外用0.36%Tempol 1h后照射UVB实验组;
D组:为外用0.36%Tempol 2h后照射UVB实验组;
E组:为外用0.36%Tempol 4h后照射UVB实验组;
F组:为外用0.36%Tempol 8h后照射UVB实验组。
2.2照射剂量
除正常对照组外,其余五组均隔日照射UVB一次,照射剂量为0.111J/(cm~2·次),共14周,总剂量5.45J/cm~2。
2.3无毛小鼠皮肤外观状态变化的检测
选择昆明无毛小鼠背部皮肤2×3cm~2范围,外用三蒸水或0.36%Tempol后,按上述分组情况进行紫外线照射实验,隔周记录皮肤外观状态并进行评分,共14周。
2.4无毛小鼠皮肤胶原纤维和弹力纤维变化的检测
末次紫外线照射实验结束后,颈椎脱臼法将各组动物处死,用锐利刀片取其背部照射区面积0.5cm~2大小的皮肤组织,经固定、脱水、包埋、切片、HE及间苯二酚品红法染色,在显微镜下观察真皮内胶原纤维和弹力纤维含量的变化及病理形态学改变。
2.5无毛小鼠皮肤成纤维细胞和胶原纤维超微结构变化的检测
在常规组织病理取材的同时,取背部0.1cm~3的皮肤组织2～3块,经固定、脱水、包埋、定位,制成50nm超薄切片,醋酸铀、柠檬酸铅双重染色,HITACHI透射电子显微镜观察成纤维细胞和胶原纤维超微结构的变化。
2.6无毛小鼠皮肤丙二醛(MDA)和羟脯氨酸(HYP)含量变化的检测
2.6.1组织匀浆的制备和MDA含量的检测
在常规组织病理取材的同时,取各组无毛小鼠背部照射区皮肤,去除皮下脂肪称重,制成10%组织匀浆;取0.1ml 10%组织匀浆,先后加入1、2、3号MDA测定试剂并混匀,95℃水浴中40min,3500-4000r/min,离心10min,取上清液,分光光度计532nm比色,计算MDA含量。
2.6.2 HYP含量的检测
在常规组织病理取材的同时,取各组无毛小鼠背部照射区皮肤,精确称取皮肤(湿重)30-100mg放入试管,加入1ml水解液,加盖后95℃或沸水浴水解20min,调PH值至6.0-6.8左右,加蒸馏水至10ml,取3ml稀释的水解液加活性炭混匀,3500r/min,离心10min,取上清液1ml先后加入试剂1、2、3并混匀,60℃水浴中15min,冷却后3500r/min,离心10min,取上清,分光光度计550nm比色,计算HYP含量。
2.7无毛小鼠皮肤突变型p53蛋白表达的检测
在常规组织病理取材的同时,取各组无毛小鼠背部照射区皮肤,进行突变型p53蛋白表达的免疫组化检测,检测结果经美国Leica DMI4000B图像处理系统进行图像的积分吸光度(A)分析。
结果
1.无毛小鼠皮肤外观状态的变化
B、C、E和F组无毛小鼠皮肤外观评分均显著升高,与A和D组比较差异有统计学意义(P＜0.01);其中B与F组皮肤外观评分升高最明显,C与E组皮肤外观评分升高次之,而A与D组比较、B与F组比较、C与E组比较差异无统计学意义(P＞0.05)。
2.无毛小鼠皮肤胶原纤维和弹力纤维的变化
无毛小鼠皮肤切片经HE常规染色及间苯二酚品红法染色后在光学显微镜下观察,A和D组,胶原纤维及弹力纤维粗细较均匀,没有明显断裂及排列紊乱现象。B、C、E和F组表皮角化过度,乳头层变平;真皮内胶原纤维及弹力纤维均增粗、断裂、排列紊乱。
3.无毛小鼠皮肤成纤维细胞和胶原纤维超微结构的变化
电镜下可见,A组,真皮成纤维细胞及胶原纤维结构未见明显异常;D组真皮内成纤维细胞的细胞核内异染色质轻度聚集、胶原纤维略肿胀,纤维间间隙略增大,境界仍较清楚,极少数纤维出现断裂、溶解现象;B、C、E和F组真皮内成纤维细胞的细胞核固缩、凋亡,胶原纤维结构紊乱,纤维间境界模糊不清,部分纤维出现溶解现象。
4.无毛小鼠皮肤丙二醛(MDA)和羟脯氨酸(HYP)含量的变化
MDA含量:B、C、E和F组无毛小鼠皮肤的MDA含量均显著升高,与A和D组比较差异有统计学意义(P＜0.01);其中B与F组皮肤MDA含量升高最明显,C与E组皮肤MDA含量升高次之,而A与D组比较、B与F组比较差异无统计学意义(P＞0.05)。
HYP含量:B、C、E和F组无毛小鼠皮肤的HYP含量均显著下降,与A和D组比较差异有统计学意义(P＜0.01);其中B与F组皮肤HYP含量下降最明显,C与E组皮肤HYP含量下降次之,而A与D组比较、B与F组比较、C与E组比较差异无统计学意义(P＞0.05)。
5.无毛小鼠皮肤突变型p53蛋白表达的变化
经免疫组化法染色后观察,B、C、E和F组无毛小鼠表皮突变型p53蛋白的表达显著升高,与A和D组比较差异有统计学意义(P＜0.01);其中B与F组表皮突变型p53蛋白表达的升高最明显,C与E组突变型p53蛋白表达的升高次之,而A与D组比较、B与F组比较、C与E组比较差异无统计学意义(P＞0.05)。
6.时间因素对Tempol抗光老化作用的影响
经过分析上述检测数据,结果显示外用0.36%的Tempol后,1.4小时之内对UVB所致无毛小鼠皮肤光老化具有一定的防护作用,用药后2小时左右的防护作用更好,8小时之后已无防护作用。
结论
外用0.36%的Tempol对UVB照射所致无毛小鼠的皮肤光老化具有防护作用,其防护作用受时间因素影响,有效防护时段在用药后1-4小时之间,最佳防护时段在用药后2小时左右。Tempol对UVB照射所致无毛小鼠皮肤光老化的作用机制可能与抑制真皮胶原纤维及弹力纤维降解、变性,抑制真皮成纤维细胞凋亡,抑制皮肤脂质过氧化反应,提高胶原蛋白含量以及抑制表皮p53基因突变有关。
Ultraviolet is an electromagnetic wave.Its wave length is from 200 to 400 nm.It is divided into three forms according to its wave length,200nm to 290nm is called UVC,290nm to 320nm is called UVB and 320nm to 400 nm is called UVA.UVC is the most harmful to skin.but can be almost completerly absorbed by atmosphere;so its damage can be ignored.Skin damage induced by ultraviolet is related to UVB and UVA.and UVB is the main reason.In recent years,skin diseases are increasing which results from increasing ultraviolet radiation to the surface of the earth owing to the ozonosphere destruction.Exposure to ultraviolet extensively can cause skin photodamaging,cutaneous carcinoma and etc.Scientists have followed with interest in how to effectively prevent and treat skin damage caused by ultraviolet,especially in the field of skin photodamaging induced by UVB which has become a hot research field in nowadays
In order to reduce skin damage caused by ultraviolet,anti-oxidant application has akeady become utility preference to protect the skin and to treat skin diseases. These researches are carrying on.Many studies have demonstrated that more oxygen free radicals are produced in human and animal skin after radiation with ultraviolet. Endogenous light sensitive molecules absorb photons and are activated to become light substances,then the substances through the transmit of electrons and hydrogen atoms interact with substrates producing oxygen free radicals.Oxygen free radicals in vivo can be transformed and broken down by antioxidant system in cells.After radiation with abundant ultraviolet,more free radicals are produced and antioxidant system is destroyed,which causes injury of DNA,proteins and fat,destruction of cells structures and biological metabolism,resulting in skin photoaging and cutaneous carcinoma.As for the skin photodamage caused by ultraviolet,it has been reported that some anti-oxidative damage substances can prevent from skin photodamage caused by ultraviolet,such as tocophero,antiscorbic acid,β-carotene,catalase, SOD and Tretinoin etc.They have good effect on animals and cultrured cell system, but the effect on human is unclear.Some substances require a high concentration to become effective,however to maintain the concentration is harmful to the health.
Nitroxide is a common biophysics agent.Compared with SOD.it has a small molecular weight,good stability,permeability and innocuity.Many studies have shown that nitroxide has a significant effect against oxidative damage.Tempol is a common nitroxide;it has a protective effect on skin photoaging caused by ultraviolet in our recent studies.
The study was divided into two parts,HaCaT cell and KM hairless mice were used as in vitro and in vivo models respectively.Tempol was used as protectant to establish the models against skin photoaging.We investigated the mechanisms of Tempol against skin photoaging by analyzed the indexes of the models,so that to provide laboratory data for prevention and treatment of photoaging diseases.
PART ONE
The mechanisms of Tempol's protective effects on photodamaging in HaCat cells under UVB radiation
Background
Ultraviolet is divided into UVA.UVB and UVC.Only UVA and UVB can pass through atmosphere to the Earth.The wave length of UVA is 320 to 400 nm and UVB is 280 to 320 nm.Epidermis is outer layer of the skin.which is the first natural barrier to protect organism from physical,chemical as well as microorganism damage and maintain internal environment stable.This function is performed by keratinocytes, which account for 90%of epidermal cells.Keratinocytes are not only protective cells but also participate in biological processes(e.g.immunity,inflammation, proliferation,tumor transformation and etc.).Some studies showed that 95% ultraviolet are absorbed by keratinocytes.International investigations have already separately established model of keratinocytes oxidation damage caused by UVA, UVB.and conducts some researches,which mainly concentrates on immunosuppression,DNA injury,apoptosis and inflammation.It was showed that UVB is the main reason,which causes non-melanocyte skin cancer.More than one million people were diagnosed with skin cancer every year in the USA.One patient dies of the skin malignant tumor each hour.It was estimated that there will be 40,000,000 skin cancer patients in the U.S.A.at 2060.So far,although we don't have the statistical data.but skin diseases caused by ultraviolet have attracted much people high attention,particularly skin photoaging caused by UVB,which becomes a hot research field now.
In order to reduce skin damage caused by ultraviolet,anti-oxidant application has already become utility preference to protect the skin and to treat skin diseases. These researches are carrying on,Stewart MS and Cameron GS have discovered that application of the Vitamin C or E may suppress keratinocytes damage after UVB radiation.Many researchers have discovered that after application of anti-scorbutic acid or hydroxyacetic acid may suppress apoptosis related transcription factor expression about apoptosis after UVB radiation.Valeria M et al.have discovered that the anti-oxidant and Vitamin C may suppress JNK expression of keratinocytes,which suppresses AP-1 expression,and suppresses the keratinocytes apoptosis.These substances have good effect against ultraviolet on animals and cultured cell system. but the effect on human is unclear.Some substances require a high concentration to become effective,but to maintain the concentration is harmful to the health.Nitroxide is a common biophysics agent.Samuni A.found thatit is similar to SOD disproportionate superoxide anion in 1988.so its effects against oxidative damage were attracted attention.Compared with SOD,it has a smaller molecular weight. better stability,permeability and innocuity.Nitroxide was widely applied in the reaserch of bacterium,cultured myocardial cells,isolated heart perfusion and animals in vivo.The results showed nitroxide has a significant effect against oxidative damage.Tempol is a common nitroxide,which has a protective effect on skin photoaging caused by ultraviolet in our recent studies.
HaCaT cell belongs to keratinocytes,which is one kind of transformed but non-carcinogenic cell and is evolved from abdomen epidermis.HaCaT cell retains all epidermis differentiation ability,which is similar to the normal cell.It is similar to the normal cell on bionomics.Therefore we have established cell damage model with HaCaT cells radiated by 30mJ/cm~2 UVB to imitate the keratinocytes photodamage. Take Tempol as the protecting agent,we investigated the possible mechanism and the influencing factors of Tempol against UVB radiation on HaCaT cells damage.The study was unique in domestic and foreign,and it has provided a brand-new mentality for Tempol and other anti-oxidants and has laid the rationale for the possible mechanism.
Objective
To establish a phtodamage model of HaCaT cells with UVB radiation and study the mechanisms of Tempol on HaCaT cells against UVB and the influencial factors.
Materials and methods
1.Phtodamage model establishment
1.1 HaCaT cells culture
Human keratinocytes cell line HaCaT cells were cultured with DMEM supplemented with 10%fetal calf serum(FBS)at 37℃in a humidified incubator containing 5%CO_2.
1.2 UV light
The wave length of UVB ranges from 290 to 320 nm and the peak is 297 nm. Each lamp box contians four lamps and each lamp's power is 40w.Put the lamps into lamp box and radiated HaCaT cells.
2.Experiment of Tempol's protective effects on photodamaging
2.1 HaCaT cells groups
HaCaT cells were divided into seven groups,which were marked group A、B、C、D、E、FandG.
Group A was control group,which was not applied Tempol and without radiation.
Group B was UVB radiation control,which was not applied Tempol but radiated with ultraviolet.
Group C was supplied with 0.5mM Tempol.
Group D was supplied with 1mM Tempol.
Group E was supplied with 2mM Tempol.
Group F was supplied with 4mM Tempol.
Group G was supplied with 8mM Tempol.
2.2 Cell culture and Dose of radiation
When the cytomixis was to 80%,they were taken out from cell incubator and Tempol was added aseptically.Medium was added to control group and radiation control group,the volume is the same in these groups at least.Put human keratinocytes cell line HaCaT cells to be cultured in a humidified incubator containing 5%CO_2 at 37℃for one hour.Except for control group,other six groups were radiated with UVB;the dose was 30mJ/cm~2 and the vertical dimension from the light to the cells was 15cm.
2.3 Measurement of cell proliferation in HaCaT cells
HaCaT cells were incubated for 18 hours and add MTT to culture for 4 hours. Then add dimethyl sulfoxide was added to dissolve the crystal.Measure the extinction value with enzyme immunity instrumentation on 490 nm.
2.4 Measurement of apoptosis in HaCaT cells HaCaT cells
After HaCaT cells were incubated for i8 hours,digested,centrifugated and adjusted to 1×10~6/ml.Added Annexin V-FITC and PI,analyzed with FCM and measured the rate of apoptosis in HaCaT cells.
2.5 Measurement ofFoxO3a mRNA expression in HaCaT cells
HaCaT cells were incubated with UVB for 18 hours.The total RNA was extracted from HaCaT cells.The cDNA was obtained by reverse transcription, and FoxO3a mRNA expression was measured with RT-PCR.
Results
1.The effect of Tempol on the HaCaT cell proliferation The OD scores were elevated in group A、C、D、E and F significantly.The differences between group A、C、D、E、F and group B、G were significant(p＜0.01). The score in group A and C was higher than that of other groups,and the differences between group A and C was no significant(p＞0.05).The OD scores in group D、E and F decreased one by one.The differences between group B and G were no significant(p＞0.05).
2.The effect of Tempol on HaCaT cell apoptosis
The apoptosis rates were elevated in group B、C、D、E、F and G significantly.
The differences between group B、C、D、E、F、G and group A were significant (P＜0.01).The apoptosis rate in group B was the highest.The apoptosis rate was decreasing as the concentration of Tempol increased.The differences between each
group were significant(p＜0.01).
3.The effect of Tempol on FoxO3a mRNA expressions in HaCaT cell
FoxO3a mRNA expression was increased in group B、C、D、E、F and G significantly compared with group A(p＜0.01).FoxO3a mRNA expression in group B was the highest.FoxO3a mRNA expression was increasing as the concentration of Tempol increased in other groups.The differences between each group were significant(p＜0.01).
Conclusion
At a certain range of concentration(0.5mM,1mM,2mM,4mM,8mM), Tempol could have a protective effect on photodamaging caused by radiation with
UVB.The study showed that Tempol may increase HaCaT cell proliferation. inhibit HaCaT cell apoptosis and inhibit over-expression of FoxO3a in HaCaT cell. Its protective effect of Tempol was decreased as the concentration increased.Tempol at concent ration of 0.5mM had the st rongest protective effect.
PART TWO
The investigation of mechanisms and influential factors of Tempol against UVB- induced skin photoaging in hairless mice
Background
In recent years,skin diseases are increasing which result from increasing ultraviolet radiation to the surface of the earth owing to the ozonosphere destruction. Exposure to ultraviolet can cause skin photoaging,cutaneous carcinoma and etc. Scientists have followed with interest in how to effectively prevent and treat skin damage caused by ultraviolet.Ultraviolet is an electromagnetic wave,and its wave length is from 200 to 400 nm.It is divided into three forms according to its wave length.200nm to 290nm called UVC,290nm to 320nm called UVB and 320nm to 400 nm called UVA.UVC is the most harmful to skin.but can be almost completely absorbed by atmosphere;so its damage can be ignored.Skin damage induced by ultraviolet is related to UVB and UVA,of which UVB is the main reason.
Many studies have demonstrated that skin photodamage by ultraviolet related with oxygen free radicals.EPR equipment has detected more oxygen free radicals in human and animal skin after radiation with ultraviolet.Endogenous light sensitive molecules absorb photons and are activated to become light substances,then the substances through the transmit of electrons and hydrogen atoms interact with substrates producing oxygen free radicals.Oxygen free radical can be transformed and broken down by antioxidant system in cells.After radiation with abundant ultraviolet,more free radicals are produced and antioxidant system is destroyed, which causes injury of DNA,proteins and fat,cells structures destruction and disorder of biological metabolism,resulting in skin photoaging and cutaneous carcinoma.As for the skin photodamage caused by ultraviolet,it has been reported that some anti-oxidative damage substances can protect skin from photodamage caused by ultraviolet,such as SOD,tocophero,antiscorbic acid,β-carotene,catalase and Tretinoin etc.They have good effect on animals and cultured cell systems,but the effect on human is unclear.Some substances require a high concentration to come effective,however to maintain the concentration is harmful to the health.
Nitroxide is a common biophysics agent.Samuni A.found that it is similar to SOD disproportionate superoxide anion in 1988.Then its effects against oxidative damage was attracted more attention.It has a smaller molecular weight,better stability,permeability and mnocuity compared with SOD.Nitroxide was widely applied in the research of bacterium,cultured myocardial cells,isolated heart perfusion and animals.The results showed nitroxide has a significant effect against oxidative damage.Tempol is a common nitroxide;it has a protective effect on skin photoaging caused by ultraviolet in our recent studies.
KM hairless mice were selected as objects against skin photoaging to establish a model,and Tempol as protectant.We analyzed the skin surface,the changes of collagen and elastic fibers in the dermis,the malondialdehyde(MDA)and hydroxyproline(HYP)content,and mtp53 expression.These indexes were analyzed; we investigated the mechanisms of the protective effect of Tempol against skin photoaging and effects of time on the protection,in order to provide laboratory data for photoaging disease therapy.
Objective
The study was designed to investigate the changes of skin surface condition、collagen fibers and elastic fibers in the dermis、MDA and HYP content in the skin、fibroblast and collagen fiber ultrastructure,mtp53 expression in hairless mice induced by UVB radiation after application with Tempol in different time-spans.The effect of time on Tempol against photoaging was observed.
Materials and methods
1:Animal models establishment
1.1 Animals
Seventy-two KM hairless mice(6-8 weeks old;20-25 grams of body weight: half male and half female)were used in the study.They were kindly supplied by Beijing University and were fed on the same condition.
1.2 UV light
The wave length of UVB ranges was from 290 to 320 nm and the peak was 297 nm.Each light lamp power was 40w.Put the lamps into lamp box and the height of radiation was 30cm.
2.Experiment of Tempol against photoaging
2.1 Animal groups
Seventy-two hairless mice were divided into six groups randomly and each group had twelve mice.
Group A was control group.The skin was applied TDW without radiation.
Group B was UVB radiation control.The skin was applied TDW but radiated with ultraviolet.
Group C was applied 0.36%Tempol and radiated after an hour.
Group D was applied 0.36%Tempol and radiated after two hours.
Group E was applied 0.36%Tempol and radiated after four hours.
Group F was applied 0.36%Tempol and radiated after eight hours.
2.2 Dose of radiation
Except for the control group,other five groups were radiated with UVB every other day.The radiation dose was 0.111J/cm~2 every time.The total time was 14 weeks and the total dose was 5.45J/cm~2.
2.3 Measurement of surface condition changes on hairless mice skin
0.36%Tempol or TDW was applied on dorsal skin(2×3cm~2 areas)of hairless mice and the study according to above experimental design,and then the surface changes were scored every other week.The total time was 14 weeks.
2.4 Measurement of collagen and elastic fibers changes in hairless mice skin
After the last radiation,the mice were killed and shin tissue was cut off the tissue (about 0.5cm~2 areas),then the cut sheets were fixated,dehydrated,embedded and stained,the content and histological changes of collagen and elastic fibers in the dermis were observed.
2.5 Measurement of fibroblast and collagen fiber ultrastructure changes in hairless mice skin
The skin tissues were cut from the dorsal of the mice(2 or 3 pieces from each mouse and each was about 0.1cm~3 area),then were fixated,dehydrated,embedded and located.The samples were cut to extra thin sections(50nm)and stained by uranyl acetate and lead citrate.Ultrastructure changes of fibroblast and collagen fibers were observed by transmission electron microscope.
2.6 Measurement of MDA and HYP content changes in hairless mice skin
2.6.1 The homogenate preparation and measurement of MDA content
The skin was drilled down with corneal trephine,and then it was weighed without subcutaneous fat to prepare 10%homogenizer.Put 0.1ml 10%homogenizer and then added No.1、2、3 reagents misce bene.The mixture was warmed for 40 minutes in 95℃water,centrifugated 10 minutes at 3500-4000r/min.The supernatant was taken,and was shaded selection with 532nm spectrophotometer.
2.6.2 Measurement of HYP content
The skin was drilled down with corneal trephine,and then about 30 to 100 grams tissues(wet weight)was put into test tube.Added 1ml water to digest,warmed the mixture for 20 minutes.Adjusted PH to 6.0 to 6.8 and added distilled water to 10ml. Put active carbon into 3ml solution to misce bene.Centrifugated the mixture for 10 minutes at 3500r/mm and taken 1ml the clear supernatant and added No.1、2、3 agents to misce bene.The mixture was warmed for 60 minutes in 60℃water,centrifugated for 10 minutes in 3500r/min.The supernatant was taken and shaded selection with 532nm spectrophotometer.
2.7 Measurement ofmtp53 expression in hairless mice skin
The materials were cut from the dorsal skin of the mice and the expression of mtp53 was measured with immunohistochemical method.The results were analyzed by America Leica DMI4000B Image Processing System.
Results
1.The skin surface changes on hairless mice
The scores were elevated in group B、C、E and F significantly.The differences between group B、C、E、F and group A、D was significant(P＜0.01).The score in group B and F was the highest;the score in group C and E was lower than that of group B and F.The differences between group A and D,group B and F,group C and E was no significant(p＞0.05).
2.The changes of collagen and elastic fibers in hairless mice skin
The collagen and elastic fibers in the dermis arranged in bunchy regularly without marked rupture in group A and D.Epidermis in group B、C、E and F was hyperkeratinization.Papillar layer was flatted.The collagen and elastic fibers in the dermis of hairless mice became thicker,fragmented and disarranged.
3.The ultrastryctue changes of fibroblast and collagen fibers in hairless mice skin
The fibroblast and collagen fibers were normal in group A and D.We could observe karyopycnosis,the collagen and elastic fibers disarranged,the boundary among fibers unclear and some fibers was dissolved in group B、C、E and F.
4.The changes of MDA and HYP content in hairless mice skin
The MDA content
The MDA content was significant increased in group B、C、E and F.The differences between group B、C、E、F and group A,D was significant(P＜0.01). The MDA content in group B and F was the highest;the MDA content in group C and E was lower than that of group B and F.The differences between group A and D. group B and F was no significant(p＞0.05).
The HYP content
The HYP content was significant decreased in group B、C、E and F.The differences between group B、C、E、F and group A、D was significant(P＜0.01). The HYP content in group B and F was the lowest;the HYP content in group C and E was higher than that of group B and F.The differences between group A and D,group B and F,group C and E was considered no significant(p＞0.05).
5.The changes of mtp53 expression in hairless mice skin
Mtp53 expression measurement was increased significant in group B、C、E and F.The differences between group B、C、E、F and group A、D was significant (P＜0.01).The mtp53 expression in group B and F was the highest;the mtp53 expression in group C and E was lower than that of group B and F.The differences between group A and D,group B and F,group C and E was no significant(p＞0.05).
6.The affection of time on the effect of Tempol against photoaging
The results showed that 0.36%Tempol had determinate protection to skin photoaging of hairless mice caused by radiation with UVB for 1 to 4 hours after external application,and the protection effect would be the best in 2 hours after external application,no effect after 8 hours.
Conclusions
External application with 0.36%Tempol had protection to skin photoaging of hairless mice caused by radiation with UVB,and its protection were affected by time. The effective time-spans are from 1 to 4 hours after external application,and the best time-span is around 2 hours after medication.Its mechanisms may have a relationship to inhibiting degradation and degeneration of the collagen fibers and elastic fibers. inhibiting apoptosis of fibroblasts,inhibiting lipid peroxidation and inhibiting gene mutation of p53 in dermis.
紫外线是波长为200～400 nm的电磁波,其中波长为200～290 nm的紫外线称为短波紫外线(UVC),波长为290～320nm的紫外线称为中波紫外线(UVB),波长为320～400nm的紫外线称为长波紫外线(UVA)。UVC对人类皮肤的损害作用最大,但由于太阳辐射到地球表面的UVC大部分被大气层吸收,而达不到地球表面,所以UVC对人类皮肤的作用可被忽视。紫外线引起的皮肤损伤主要与UVA及UVB有关,其中UVB是造成皮肤损伤的主要原因。近年来由于大气臭氧层的破坏,辐射到地球表面的紫外线逐渐增多,由此引起的各种皮肤病明显增多,长期紫外线照射能引起皮肤的光老化及皮肤癌等。怎样有效地预防及治疗紫外线引起的皮肤损伤,已成为国际皮肤界关注的课题,尤其是有关UVB引起皮肤光损害的研究日益受到人们的关注,并正成为当今的研究热点。
为了减轻紫外线对皮肤的多种损伤,抗氧化剂的应用已成为人们防护皮肤和治疗皮肤病的有效选择。有关于这方面的研究正在进行当中。紫外线照射人及动物的皮肤后,能在人及动物的皮肤中产生较多的氧自由基,这已被大量的研究所证实。正常情况下,体内产生的氧自由基能被细胞内抗氧化系统转化、分解掉,当紫外线照射后,产生较多的氧自由基时,体内的抗氧化系统遭到破坏,这些自由基则引起皮肤组织内DNA,脂质、蛋白质等成分的损伤,破坏细胞结构及各种生物代谢功能,从而引起光老化,诱发皮肤癌等。针对这种由氧自由基介导的紫外线引起的皮肤损伤,近年来报道了较多的有关抗紫外线氧化性损伤的物质,如:SOD,生育酚,抗坏血酸,β-胡萝卜素,过氧化氢酶,维甲酸等等。这些物质有的在实验室动物及培养的细胞系统有很好的抗紫外线损伤作用,但在人体上的效果不确切,有的需要较高的血液浓度才能起到抗紫外线损伤的作用,而长期维持这种高浓度对人体是有害的。
氮氧化物是一种常用的生物物理制剂,与SOD等相比,氮氧化物具有分子量小,稳定性强,细胞膜通透性好等诸多优点。国外的研究结果显示氮氧化物具有明显的抗氧化性损伤的作用。4-羟基-2,2,6,6-四甲基氧基哌啶(Tempol,分子式C_9H_(18)NO_2)是一种在抗氧化研究中常用的氮氧化物,我们早期的实验已经证实,Tempol对于紫外线照射引起的皮肤光老化具有防护作用。
本研究共分为两部分,分别以HaCaT细胞和活体昆明无毛小鼠为UVB照射的体外和体内实验对象,利用Tempol为光保护剂,建立了Tempol抗UVB照射所致皮肤光老化的实验模型;通过分析实验模型各种观察指标的变化,探讨Tempol抗UVB照射所致光老化的作用机制,为防治光老化性疾病提供实验依据。
第一部分Tempol对中波紫外线照射所致HaCaT细胞光损伤防护作用机制的研究
背景
紫外线分为A、B、C三种(UVA、UVB、UVC)。能穿过大气层达到地球的只有UVA和UVB两种。UVA的波长为320～400nm;UVB的波长为290～320nm。表皮位于皮肤的最外层,是机体的第一道天然保护屏障,使机体免受外界物理、化学以及微生物侵害,维持机体内环境的稳定。这些作用主要是由占表皮细胞90%以上的角质形成细胞完成。现代研究证明角质形成细胞不仅是机体的保护细胞,而且参与各种细胞生物学过程如免疫、炎症、增生以及肿瘤转化等。有资料表明:照射到皮肤的95%的紫外线是被角质形成细胞吸收的。国际上已分别建立了UVA、UVB对角质形成细胞氧化损伤的模型,并进行了研究,主要集中在免疫抑制、DNA损伤、细胞凋亡、炎症等方面。UVB被认为是太阳光中引起非黑素皮肤癌最主要的紫外线。在美国每年有100多万人被诊断患有皮肤癌,平均每小时有一个病人死于皮肤源性恶性肿瘤,预计到2060年,美国的皮肤癌患者将达4000万人。到目前为止,我国虽然还没有相关的具体统计数据,但对紫外线诱发的皮肤疾病已引起了学者们的高度重视,尤其是有关UVB引起皮肤光损害的研究日益受到人们的关注,并正成为当今的研究热点。
为了减轻紫外线对皮肤的多种损伤,抗氧化剂的应用已成为人们防护皮肤和治疗皮肤病的有效选择。有关这方面的研究正在进行当中。Stewart MS和Cameron GS等发现,在预先应用维生素C或维生素E后,可以抑制UVB照射角质形成细胞后引起的DNA损伤。多个研究发现,在应用抗坏血酸或羟基乙酸后可以抑制UVB照射角质形成细胞引起的与凋亡相关的转录因子AP-1、C-fos的表达。Valeria M等发现,抗氧化剂维生素C可以通过抑制紫外线诱导的角质形成细胞的JNK的表达,抑制AP-1的表达,从而抑制角质形成细胞的凋亡。这些物质有的在实验室动物及培养的细胞系统有很好的抗紫外线损伤作用,但在人体上的效果不确切,有的需要较高的血液浓度才能起到抗紫外线损伤的作用,而长期维持这种高浓度对人体是有害的。氮氧化物是一种常用的生物物理制剂,自1988年Samuni A.发现其有类似于SOD的歧化超氧阴离子的作用后,其抗氧化性损伤的功能受到了重视。与SOD等相比,氮氧化物具有分子量小,稳定性强,细胞膜通透性好等诸多优点。国外实验室曾先后将氮氧化物应用于细菌、培养的心肌细胞、离体心脏灌注及活体动物等研究体系中,观察其抗氧化性损伤的作用,研究结果显示氮氧化物具有明显的抗氧化性损伤的作用。4-羟基-2,2,6,6-四甲基氧基哌啶(Tempol,分子式C_9H_(18)NO_2)是一种在抗氧化研究中常用的氮氧化物,我们早期的实验已经证实,Tempol对于紫外线照射引起的皮肤光损伤具有防护作用。
HaCaT细胞是角质形成细胞系细胞,是一种被转化但非致癌的细胞,由人腹部表皮衍化而成,保留了所有的表皮分化能力,与正常角质形成细胞非常相似,它在生物学特性上与正常人角质形成细胞相似,所以我们用30mJ/cm~2的UVB照射HaCaT细胞以建立UVB对角质形成细胞光损伤的模型,目的是模仿UVB对表皮的角质形成细胞的光损伤;同时以Tempol为光防护剂,探讨Tempol对UVB照射所致HaCaT细胞光损伤防护作用的机制及其影响因素,在国内外还是首次,为Tempol和其它的抗氧化剂对UVB照射所致角质形成细胞光损伤防护作用的研究提供了一条崭新的思路,更为Tempol对UVB照射所致HaCaT细胞光损伤防护作用机制的研究奠定了理论基础。
目的
1.建立UVB照射损伤HaCaT钿胞的光损伤模型,观察Tempol对UVB照射所致HaCaT细胞光损伤防护作用的机制;
2.观察Tempol对UVB照射后HaCaT细胞增殖效应的影响及其影响因素的研究;
3.观察Tempol对UVB照射后HaCaT细胞细胞凋亡的影响及其影响因素的研究;
4.观察Tempol对UVB照射后HaCaT细胞FoxO3a mRNA表达的影响及其影响因素的研究。
方法
1.HaCaT细胞的光损伤模型的建立
1.1 HaCaT细胞培养
用含10%胎牛血清(FBS)的DMEM置37℃CO_2培养箱中进行培养。
1.2紫外线光源
UVB灯管,波长范围290～320nm,峰值297 nm,40W,4只,4只灯管制成灯箱。
2.Tempol对光损伤防护作用实验
2.1 HaCaT细胞分组
将HaCaT细胞分为七组,分别标记为A、B、C、D、E、F和G组。
A组:为正常对照组,不加入Tempol,不照射UVB;
B组:为UVB照射对照组,不加入Tempol,只照射UVB;
C组:为含0.5mMTempol的实验组;
D组:为含1mMTempol的实验组;
E组:为含2mMTempol的实验组;
F组:为含4mMTempol的实验组;
G组:为含8mMTempol的实验组。
2.2细胞培养和照射剂量
HaCaT细胞在培养板上生长到每孔汇合至80%以上时,从细胞培养箱中取出,无菌条件下加入Tempol。正常对照组和照射对照组只加角质形成细胞专用培养基以使各孔的终体积相等。细胞放入37℃、5%CO_2细胞培养箱中孵育1h后,除正常对照组外,其余六组均照射UVB,照射剂量为30mJ/cm~2,光源与细胞的垂直照射距离为15cm。
2.3 HaCaT细胞增殖效应的检测
HaCaT细胞经UVB照射损伤并孵育18小时后,加入MTT继续培养4h,再加入二甲基亚砜,使结晶物充分溶解,在酶联免疫检测仪490nm处测量各孔吸光值。
2.4 HaCaT细胞凋亡率的检测
HaCaT细胞经UVB照射损伤并孵育18小时后,经消化、离心,调节细胞浓度为1×10~6/ml,加入Annexin V-FITC和PI溶液,流式细胞仪分析,测定HaCaT细胞的凋亡率。
2.5 HaCaT细胞FoxO3a mRNA表达的检测
HaCaT细胞经UVB照射损伤并孵育18小时后,将所收集的细胞提取总RNA,经逆转录反应得到cDNA,通过RT-PCR技术检测FoxO3amRNA的表达。
结果
1.Tempol对UVB照射后HaCaT细胞增殖效应的影响
A、C、D、E和F组OD值显著升高,与B和G组比较,差异有统计学意义(P＜0.01);其中A与C组OD值升高最明显,两组之间比较差异无统计学意义(P＞0.05),D、E和F组OD值依次递减,各组之间比较差异有统计学意义(P＜0.01);而B和G组比较,差异无统计学意义(P＞0.05)。
2.Tempol对UVB照射后HaCaT细胞凋亡率的影响
B、C、D、E、F和G组HaCaT细胞凋亡率显著升高,与A比较,差异有统计学意义(P＜0.01);其中B组HaCaT细胞凋亡率升高最明显,其余各组HaCaT细胞凋亡率随Tempol药物浓度的增加而升高,各组之间比较差异有统计学意义(P＜0.01)。
3.Tempol对UVB照射后HaCaT细胞FoxO3a mRNA表达的影响
B、C、D、E、F和G组HaCaT细胞FoxO3a mRNA表达显著升高,与A组比较,差异有统计学意义(P＜0.01);其中B组HaCaT细胞FoxO3a mRNA表达升高最明显,其余各组HaCaT细胞FoxO3a mRNA表达随Tempol药物浓度的增加而升高,各组之间比较差异有统计学意义(P＜0.01)。
结论
一定浓度范围的Tempol(0.5mM,1mM,2mM,4mM,8mM)对UVB照射所致HaCaT细胞光损伤具有防护作用,其作用机制可能与提高HaCaT细胞增殖效应,抑制HaCaT细胞凋亡以及抑制HaCaT细胞FoxO3a过度表达有关;其保护作用随Tempol浓度的升高而降低,0.5mMTempol的防护作用最强。
第二部分Tempol抗中波紫外线照射所致无毛鼠皮肤光老化的作用机制及其影响因素的探讨
背景
近年来由于大气臭氧层的破坏,辐射到地球表面的紫外线逐渐增多,由此引起的各种皮肤病明显增多.长期紫外线照射能引起皮肤的光老化及皮肤癌等。怎样有效地预防及治疗紫外线引起的皮肤损伤,已成为国际皮肤界关注的课题。紫外线是波长为200-400 nm的电磁波,其中波长为200-290 nm的紫外线称为短波紫外线(UVC),波长为290-320nm的紫外线称为中波紫外线(UVB),波长为320-400nm的紫外线称为长波紫外线(UVA)。UVC对人类皮肤的损害作用最大,但由于太阳辐射到地球表面的UVC大部分被大气层吸收,而达不到地球表面,所以UVC对人类皮肤的作用可被忽视。日光中同时含有UVA、UVB,紫外线引起的皮肤损伤主要与UVA及UVB有关,其中UVB是造成皮肤损伤的主要原因。
紫外线照射人及动物的皮肤后,能在人及动物的皮肤中产生较多的氧自由基,这已被大量的研究所证实。紫外线照射皮肤后,氧自由基的产生是由内源性光敏分子吸收光子,而后变成激活的光敏物,通过电子或氢原子的传递与底物分子反应生成的。正常情况下,体内产生的氧自由基能被细胞内抗氧化系统转化、分解掉,当紫外线照射后,产生较多的氧自由基时,体内的抗氧化系统遭到破坏,这些自由基则引起皮肤组织内DNA、脂质、蛋白质等成分的损伤,破坏细胞结构及各种生物代谢功能,从而引起光老化,诱发皮肤癌等。针对这种由氧自由基介导的紫外线引起的皮肤损伤,近年来报道了较多的有关抗紫外线氧化性损伤的物质,如:生育酚,抗坏血酸,β-胡萝卜素,过氧化氢酶,SOD,维甲酸等等。这些物质有的在实验室动物及培养的细胞系统有很好的抗紫外线损伤作用,但在人体上的效果不确切,有的需要较高的血液浓度才能起到抗紫外线损伤的作用,而长期维持这种高浓度对人体是有害的。
氮氧化物是一种常用的生物物理制剂,自1988年Samuni A.发现其有类似于SOD的歧化超氧阴离子的作用后,其抗氧化性损伤的功能受到了重视。与SOD相比,氮氧化物具有分子量小,稳定性强,细胞膜通透性好等诸多优点。国外实验室曾先后将氮氧化物应用于细菌、培养的心肌细胞、离体心脏灌注及活体动物等研究体系中,观察其抗氧化性损伤的作用,研究结果显示氮氧化物具有明显的抗氧化性损伤的作用。4-羟基-2,2,6,6-四甲基氧基哌啶(Tempol,分子式C_9H_(18)NO_2)是一种在抗氧化研究中常用的氮氧化物,我们早期的实验已经证实,Tempol对于紫外线照射引起的皮肤光老化具有防护作用。
因此本研究以活体昆明无毛小鼠为UVB照射的实验对象,利用Tempol为光保护剂,建立了Tempol抗UVB照射所致皮肤光老化的动物实验模型;通过对无毛小鼠皮肤外观状态变化、皮肤胶原纤维和弹力纤维结构变化、皮肤超微结构变化、皮肤丙二醛和羟脯氨酸含量变化以及皮肤突变型p53蛋白表达的观察,对UVB引起模型动物的各种皮肤观察指标变化进行分析,探讨Tempol抗UVB照射所致光老化的作用机制,并首次探讨了时间因素对Tempol外用抗UVB照射所致光老化作用的影响,为防治光老化性疾病提供了实验依据。
目的
1.观察无毛小鼠外用Tempol后不同时段照射UVB对皮肤外观状态变化的影响;
2.观察无毛小鼠外用Tempol后不同时段照射UVB对皮肤胶原纤维和弹力纤维结构变化的影响;
3.观察无毛小鼠外用Tempol后不同时段照射UVB对皮肤超微结构变化的影响;
4.观察无毛小鼠外用Tempol后不同时段照射UVB对皮肤丙二醛和羟脯氨酸含量变化的影响;
5.观察无毛小鼠外用Tempol后不同时段照射UVB对皮肤突变型p53蛋白表达的影响。
6.探讨时间因素对Tempol抗光老化作用的影响。
方法
1.光老化动物实验模型的建立
1.1实验动物
清洁级昆明种无毛小白鼠72只,鼠龄6～8周,雌雄各半,北京大学医学部实验动物科学部提供,体重20～25g,按同窝、同性别、同条件下饲养。
1.2紫外线光源
UVB灯管,波长范围290～320nm,峰值297 nm,40W,4只。将4只灯管制成灯箱,照射高度为50cm。
2.Tempol抗光老化实验
2.1动物分组
将72只昆明种无毛小鼠随机分为六组,每组12只,分别标记为A、B、C、D、E和F组。
A组:为正常对照组,外用三蒸水,不照射UVB;
B组:为UVB照射对照组,外用三蒸水,照射UVB;
C组:为外用0.36%Tempol 1h后照射UVB实验组;
D组:为外用0.36%Tempol 2h后照射UVB实验组;
E组:为外用0.36%Tempol 4h后照射UVB实验组;
F组:为外用0.36%Tempol 8h后照射UVB实验组。
2.2照射剂量
除正常对照组外,其余五组均隔日照射UVB一次,照射剂量为0.111J/(cm~2·次),共14周,总剂量5.45J/cm~2。
2.3无毛小鼠皮肤外观状态变化的检测
选择昆明无毛小鼠背部皮肤2×3cm~2范围,外用三蒸水或0.36%Tempol后,按上述分组情况进行紫外线照射实验,隔周记录皮肤外观状态并进行评分,共14周。
2.4无毛小鼠皮肤胶原纤维和弹力纤维变化的检测
末次紫外线照射实验结束后,颈椎脱臼法将各组动物处死,用锐利刀片取其背部照射区面积0.5cm~2大小的皮肤组织,经固定、脱水、包埋、切片、HE及间苯二酚品红法染色,在显微镜下观察真皮内胶原纤维和弹力纤维含量的变化及病理形态学改变。
2.5无毛小鼠皮肤成纤维细胞和胶原纤维超微结构变化的检测
在常规组织病理取材的同时,取背部0.1cm~3的皮肤组织2～3块,经固定、脱水、包埋、定位,制成50nm超薄切片,醋酸铀、柠檬酸铅双重染色,HITACHI透射电子显微镜观察成纤维细胞和胶原纤维超微结构的变化。
2.6无毛小鼠皮肤丙二醛(MDA)和羟脯氨酸(HYP)含量变化的检测
2.6.1组织匀浆的制备和MDA含量的检测
在常规组织病理取材的同时,取各组无毛小鼠背部照射区皮肤,去除皮下脂肪称重,制成10%组织匀浆;取0.1ml 10%组织匀浆,先后加入1、2、3号MDA测定试剂并混匀,95℃水浴中40min,3500-4000r/min,离心10min,取上清液,分光光度计532nm比色,计算MDA含量。
2.6.2 HYP含量的检测
在常规组织病理取材的同时,取各组无毛小鼠背部照射区皮肤,精确称取皮肤(湿重)30-100mg放入试管,加入1ml水解液,加盖后95℃或沸水浴水解20min,调PH值至6.0-6.8左右,加蒸馏水至10ml,取3ml稀释的水解液加活性炭混匀,3500r/min,离心10min,取上清液1ml先后加入试剂1、2、3并混匀,60℃水浴中15min,冷却后3500r/min,离心10min,取上清,分光光度计550nm比色,计算HYP含量。
2.7无毛小鼠皮肤突变型p53蛋白表达的检测
在常规组织病理取材的同时,取各组无毛小鼠背部照射区皮肤,进行突变型p53蛋白表达的免疫组化检测,检测结果经美国Leica DMI4000B图像处理系统进行图像的积分吸光度(A)分析。
结果
1.无毛小鼠皮肤外观状态的变化
B、C、E和F组无毛小鼠皮肤外观评分均显著升高,与A和D组比较差异有统计学意义(P＜0.01);其中B与F组皮肤外观评分升高最明显,C与E组皮肤外观评分升高次之,而A与D组比较、B与F组比较、C与E组比较差异无统计学意义(P＞0.05)。
2.无毛小鼠皮肤胶原纤维和弹力纤维的变化
无毛小鼠皮肤切片经HE常规染色及间苯二酚品红法染色后在光学显微镜下观察,A和D组,胶原纤维及弹力纤维粗细较均匀,没有明显断裂及排列紊乱现象。B、C、E和F组表皮角化过度,乳头层变平;真皮内胶原纤维及弹力纤维均增粗、断裂、排列紊乱。
3.无毛小鼠皮肤成纤维细胞和胶原纤维超微结构的变化
电镜下可见,A组,真皮成纤维细胞及胶原纤维结构未见明显异常;D组真皮内成纤维细胞的细胞核内异染色质轻度聚集、胶原纤维略肿胀,纤维间间隙略增大,境界仍较清楚,极少数纤维出现断裂、溶解现象;B、C、E和F组真皮内成纤维细胞的细胞核固缩、凋亡,胶原纤维结构紊乱,纤维间境界模糊不清,部分纤维出现溶解现象。
4.无毛小鼠皮肤丙二醛(MDA)和羟脯氨酸(HYP)含量的变化
MDA含量:B、C、E和F组无毛小鼠皮肤的MDA含量均显著升高,与A和D组比较差异有统计学意义(P＜0.01);其中B与F组皮肤MDA含量升高最明显,C与E组皮肤MDA含量升高次之,而A与D组比较、B与F组比较差异无统计学意义(P＞0.05)。
HYP含量:B、C、E和F组无毛小鼠皮肤的HYP含量均显著下降,与A和D组比较差异有统计学意义(P＜0.01);其中B与F组皮肤HYP含量下降最明显,C与E组皮肤HYP含量下降次之,而A与D组比较、B与F组比较、C与E组比较差异无统计学意义(P＞0.05)。
5.无毛小鼠皮肤突变型p53蛋白表达的变化
经免疫组化法染色后观察,B、C、E和F组无毛小鼠表皮突变型p53蛋白的表达显著升高,与A和D组比较差异有统计学意义(P＜0.01);其中B与F组表皮突变型p53蛋白表达的升高最明显,C与E组突变型p53蛋白表达的升高次之,而A与D组比较、B与F组比较、C与E组比较差异无统计学意义(P＞0.05)。
6.时间因素对Tempol抗光老化作用的影响
经过分析上述检测数据,结果显示外用0.36%的Tempol后,1.4小时之内对UVB所致无毛小鼠皮肤光老化具有一定的防护作用,用药后2小时左右的防护作用更好,8小时之后已无防护作用。
结论
外用0.36%的Tempol对UVB照射所致无毛小鼠的皮肤光老化具有防护作用,其防护作用受时间因素影响,有效防护时段在用药后1-4小时之间,最佳防护时段在用药后2小时左右。Tempol对UVB照射所致无毛小鼠皮肤光老化的作用机制可能与抑制真皮胶原纤维及弹力纤维降解、变性,抑制真皮成纤维细胞凋亡,抑制皮肤脂质过氧化反应,提高胶原蛋白含量以及抑制表皮p53基因突变有关。
Ultraviolet is an electromagnetic wave.Its wave length is from 200 to 400 nm.It is divided into three forms according to its wave length,200nm to 290nm is called UVC,290nm to 320nm is called UVB and 320nm to 400 nm is called UVA.UVC is the most harmful to skin.but can be almost completerly absorbed by atmosphere;so its damage can be ignored.Skin damage induced by ultraviolet is related to UVB and UVA.and UVB is the main reason.In recent years,skin diseases are increasing which results from increasing ultraviolet radiation to the surface of the earth owing to the ozonosphere destruction.Exposure to ultraviolet extensively can cause skin photodamaging,cutaneous carcinoma and etc.Scientists have followed with interest in how to effectively prevent and treat skin damage caused by ultraviolet,especially in the field of skin photodamaging induced by UVB which has become a hot research field in nowadays
In order to reduce skin damage caused by ultraviolet,anti-oxidant application has akeady become utility preference to protect the skin and to treat skin diseases. These researches are carrying on.Many studies have demonstrated that more oxygen free radicals are produced in human and animal skin after radiation with ultraviolet. Endogenous light sensitive molecules absorb photons and are activated to become light substances,then the substances through the transmit of electrons and hydrogen atoms interact with substrates producing oxygen free radicals.Oxygen free radicals in vivo can be transformed and broken down by antioxidant system in cells.After radiation with abundant ultraviolet,more free radicals are produced and antioxidant system is destroyed,which causes injury of DNA,proteins and fat,destruction of cells structures and biological metabolism,resulting in skin photoaging and cutaneous carcinoma.As for the skin photodamage caused by ultraviolet,it has been reported that some anti-oxidative damage substances can prevent from skin photodamage caused by ultraviolet,such as tocophero,antiscorbic acid,β-carotene,catalase, SOD and Tretinoin etc.They have good effect on animals and cultrured cell system, but the effect on human is unclear.Some substances require a high concentration to become effective,however to maintain the concentration is harmful to the health.
Nitroxide is a common biophysics agent.Compared with SOD.it has a small molecular weight,good stability,permeability and innocuity.Many studies have shown that nitroxide has a significant effect against oxidative damage.Tempol is a common nitroxide;it has a protective effect on skin photoaging caused by ultraviolet in our recent studies.
The study was divided into two parts,HaCaT cell and KM hairless mice were used as in vitro and in vivo models respectively.Tempol was used as protectant to establish the models against skin photoaging.We investigated the mechanisms of Tempol against skin photoaging by analyzed the indexes of the models,so that to provide laboratory data for prevention and treatment of photoaging diseases.
PART ONE
The mechanisms of Tempol's protective effects on photodamaging in HaCat cells under UVB radiation
Background
Ultraviolet is divided into UVA.UVB and UVC.Only UVA and UVB can pass through atmosphere to the Earth.The wave length of UVA is 320 to 400 nm and UVB is 280 to 320 nm.Epidermis is outer layer of the skin.which is the first natural barrier to protect organism from physical,chemical as well as microorganism damage and maintain internal environment stable.This function is performed by keratinocytes, which account for 90%of epidermal cells.Keratinocytes are not only protective cells but also participate in biological processes(e.g.immunity,inflammation, proliferation,tumor transformation and etc.).Some studies showed that 95% ultraviolet are absorbed by keratinocytes.International investigations have already separately established model of keratinocytes oxidation damage caused by UVA, UVB.and conducts some researches,which mainly concentrates on immunosuppression,DNA injury,apoptosis and inflammation.It was showed that UVB is the main reason,which causes non-melanocyte skin cancer.More than one million people were diagnosed with skin cancer every year in the USA.One patient dies of the skin malignant tumor each hour.It was estimated that there will be 40,000,000 skin cancer patients in the U.S.A.at 2060.So far,although we don't have the statistical data.but skin diseases caused by ultraviolet have attracted much people high attention,particularly skin photoaging caused by UVB,which becomes a hot research field now.
In order to reduce skin damage caused by ultraviolet,anti-oxidant application has already become utility preference to protect the skin and to treat skin diseases. These researches are carrying on,Stewart MS and Cameron GS have discovered that application of the Vitamin C or E may suppress keratinocytes damage after UVB radiation.Many researchers have discovered that after application of anti-scorbutic acid or hydroxyacetic acid may suppress apoptosis related transcription factor expression about apoptosis after UVB radiation.Valeria M et al.have discovered that the anti-oxidant and Vitamin C may suppress JNK expression of keratinocytes,which suppresses AP-1 expression,and suppresses the keratinocytes apoptosis.These substances have good effect against ultraviolet on animals and cultured cell system. but the effect on human is unclear.Some substances require a high concentration to become effective,but to maintain the concentration is harmful to the health.Nitroxide is a common biophysics agent.Samuni A.found thatit is similar to SOD disproportionate superoxide anion in 1988.so its effects against oxidative damage were attracted attention.Compared with SOD,it has a smaller molecular weight. better stability,permeability and innocuity.Nitroxide was widely applied in the reaserch of bacterium,cultured myocardial cells,isolated heart perfusion and animals in vivo.The results showed nitroxide has a significant effect against oxidative damage.Tempol is a common nitroxide,which has a protective effect on skin photoaging caused by ultraviolet in our recent studies.
HaCaT cell belongs to keratinocytes,which is one kind of transformed but non-carcinogenic cell and is evolved from abdomen epidermis.HaCaT cell retains all epidermis differentiation ability,which is similar to the normal cell.It is similar to the normal cell on bionomics.Therefore we have established cell damage model with HaCaT cells radiated by 30mJ/cm~2 UVB to imitate the keratinocytes photodamage. Take Tempol as the protecting agent,we investigated the possible mechanism and the influencing factors of Tempol against UVB radiation on HaCaT cells damage.The study was unique in domestic and foreign,and it has provided a brand-new mentality for Tempol and other anti-oxidants and has laid the rationale for the possible mechanism.
Objective
To establish a phtodamage model of HaCaT cells with UVB radiation and study the mechanisms of Tempol on HaCaT cells against UVB and the influencial factors.
Materials and methods
1.Phtodamage model establishment
1.1 HaCaT cells culture
Human keratinocytes cell line HaCaT cells were cultured with DMEM supplemented with 10%fetal calf serum(FBS)at 37℃in a humidified incubator containing 5%CO_2.
1.2 UV light
The wave length of UVB ranges from 290 to 320 nm and the peak is 297 nm. Each lamp box contians four lamps and each lamp's power is 40w.Put the lamps into lamp box and radiated HaCaT cells.
2.Experiment of Tempol's protective effects on photodamaging
2.1 HaCaT cells groups
HaCaT cells were divided into seven groups,which were marked group A、B、C、D、E、FandG.
Group A was control group,which was not applied Tempol and without radiation.
Group B was UVB radiation control,which was not applied Tempol but radiated with ultraviolet.
Group C was supplied with 0.5mM Tempol.
Group D was supplied with 1mM Tempol.
Group E was supplied with 2mM Tempol.
Group F was supplied with 4mM Tempol.
Group G was supplied with 8mM Tempol.
2.2 Cell culture and Dose of radiation
When the cytomixis was to 80%,they were taken out from cell incubator and Tempol was added aseptically.Medium was added to control group and radiation control group,the volume is the same in these groups at least.Put human keratinocytes cell line HaCaT cells to be cultured in a humidified incubator containing 5%CO_2 at 37℃for one hour.Except for control group,other six groups were radiated with UVB;the dose was 30mJ/cm~2 and the vertical dimension from the light to the cells was 15cm.
2.3 Measurement of cell proliferation in HaCaT cells
HaCaT cells were incubated for 18 hours and add MTT to culture for 4 hours. Then add dimethyl sulfoxide was added to dissolve the crystal.Measure the extinction value with enzyme immunity instrumentation on 490 nm.
2.4 Measurement of apoptosis in HaCaT cells HaCaT cells
After HaCaT cells were incubated for i8 hours,digested,centrifugated and adjusted to 1×10~6/ml.Added Annexin V-FITC and PI,analyzed with FCM and measured the rate of apoptosis in HaCaT cells.
2.5 Measurement ofFoxO3a mRNA expression in HaCaT cells
HaCaT cells were incubated with UVB for 18 hours.The total RNA was extracted from HaCaT cells.The cDNA was obtained by reverse transcription, and FoxO3a mRNA expression was measured with RT-PCR.
Results
1.The effect of Tempol on the HaCaT cell proliferation The OD scores were elevated in group A、C、D、E and F significantly.The differences between group A、C、D、E、F and group B、G were significant(p＜0.01). The score in group A and C was higher than that of other groups,and the differences between group A and C was no significant(p＞0.05).The OD scores in group D、E and F decreased one by one.The differences between group B and G were no significant(p＞0.05).
2.The effect of Tempol on HaCaT cell apoptosis
The apoptosis rates were elevated in group B、C、D、E、F and G significantly.
The differences between group B、C、D、E、F、G and group A were significant (P＜0.01).The apoptosis rate in group B was the highest.The apoptosis rate was decreasing as the concentration of Tempol increased.The differences between each
group were significant(p＜0.01).
3.The effect of Tempol on FoxO3a mRNA expressions in HaCaT cell
FoxO3a mRNA expression was increased in group B、C、D、E、F and G significantly compared with group A(p＜0.01).FoxO3a mRNA expression in group B was the highest.FoxO3a mRNA expression was increasing as the concentration of Tempol increased in other groups.The differences between each group were significant(p＜0.01).
Conclusion
At a certain range of concentration(0.5mM,1mM,2mM,4mM,8mM), Tempol could have a protective effect on photodamaging caused by radiation with
UVB.The study showed that Tempol may increase HaCaT cell proliferation. inhibit HaCaT cell apoptosis and inhibit over-expression of FoxO3a in HaCaT cell. Its protective effect of Tempol was decreased as the concentration increased.Tempol at concent ration of 0.5mM had the st rongest protective effect.
PART TWO
The investigation of mechanisms and influential factors of Tempol against UVB- induced skin photoaging in hairless mice
Background
In recent years,skin diseases are increasing which result from increasing ultraviolet radiation to the surface of the earth owing to the ozonosphere destruction. Exposure to ultraviolet can cause skin photoaging,cutaneous carcinoma and etc. Scientists have followed with interest in how to effectively prevent and treat skin damage caused by ultraviolet.Ultraviolet is an electromagnetic wave,and its wave length is from 200 to 400 nm.It is divided into three forms according to its wave length.200nm to 290nm called UVC,290nm to 320nm called UVB and 320nm to 400 nm called UVA.UVC is the most harmful to skin.but can be almost completely absorbed by atmosphere;so its damage can be ignored.Skin damage induced by ultraviolet is related to UVB and UVA,of which UVB is the main reason.
Many studies have demonstrated that skin photodamage by ultraviolet related with oxygen free radicals.EPR equipment has detected more oxygen free radicals in human and animal skin after radiation with ultraviolet.Endogenous light sensitive molecules absorb photons and are activated to become light substances,then the substances through the transmit of electrons and hydrogen atoms interact with substrates producing oxygen free radicals.Oxygen free radical can be transformed and broken down by antioxidant system in cells.After radiation with abundant ultraviolet,more free radicals are produced and antioxidant system is destroyed, which causes injury of DNA,proteins and fat,cells structures destruction and disorder of biological metabolism,resulting in skin photoaging and cutaneous carcinoma.As for the skin photodamage caused by ultraviolet,it has been reported that some anti-oxidative damage substances can protect skin from photodamage caused by ultraviolet,such as SOD,tocophero,antiscorbic acid,β-carotene,catalase and Tretinoin etc.They have good effect on animals and cultured cell systems,but the effect on human is unclear.Some substances require a high concentration to come effective,however to maintain the concentration is harmful to the health.
Nitroxide is a common biophysics agent.Samuni A.found that it is similar to SOD disproportionate superoxide anion in 1988.Then its effects against oxidative damage was attracted more attention.It has a smaller molecular weight,better stability,permeability and mnocuity compared with SOD.Nitroxide was widely applied in the research of bacterium,cultured myocardial cells,isolated heart perfusion and animals.The results showed nitroxide has a significant effect against oxidative damage.Tempol is a common nitroxide;it has a protective effect on skin photoaging caused by ultraviolet in our recent studies.
KM hairless mice were selected as objects against skin photoaging to establish a model,and Tempol as protectant.We analyzed the skin surface,the changes of collagen and elastic fibers in the dermis,the malondialdehyde(MDA)and hydroxyproline(HYP)content,and mtp53 expression.These indexes were analyzed; we investigated the mechanisms of the protective effect of Tempol against skin photoaging and effects of time on the protection,in order to provide laboratory data for photoaging disease therapy.
Objective
The study was designed to investigate the changes of skin surface condition、collagen fibers and elastic fibers in the dermis、MDA and HYP content in the skin、fibroblast and collagen fiber ultrastructure,mtp53 expression in hairless mice induced by UVB radiation after application with Tempol in different time-spans.The effect of time on Tempol against photoaging was observed.
Materials and methods
1:Animal models establishment
1.1 Animals
Seventy-two KM hairless mice(6-8 weeks old;20-25 grams of body weight: half male and half female)were used in the study.They were kindly supplied by Beijing University and were fed on the same condition.
1.2 UV light
The wave length of UVB ranges was from 290 to 320 nm and the peak was 297 nm.Each light lamp power was 40w.Put the lamps into lamp box and the height of radiation was 30cm.
2.Experiment of Tempol against photoaging
2.1 Animal groups
Seventy-two hairless mice were divided into six groups randomly and each group had twelve mice.
Group A was control group.The skin was applied TDW without radiation.
Group B was UVB radiation control.The skin was applied TDW but radiated with ultraviolet.
Group C was applied 0.36%Tempol and radiated after an hour.
Group D was applied 0.36%Tempol and radiated after two hours.
Group E was applied 0.36%Tempol and radiated after four hours.
Group F was applied 0.36%Tempol and radiated after eight hours.
2.2 Dose of radiation
Except for the control group,other five groups were radiated with UVB every other day.The radiation dose was 0.111J/cm~2 every time.The total time was 14 weeks and the total dose was 5.45J/cm~2.
2.3 Measurement of surface condition changes on hairless mice skin
0.36%Tempol or TDW was applied on dorsal skin(2×3cm~2 areas)of hairless mice and the study according to above experimental design,and then the surface changes were scored every other week.The total time was 14 weeks.
2.4 Measurement of collagen and elastic fibers changes in hairless mice skin
After the last radiation,the mice were killed and shin tissue was cut off the tissue (about 0.5cm~2 areas),then the cut sheets were fixated,dehydrated,embedded and stained,the content and histological changes of collagen and elastic fibers in the dermis were observed.
2.5 Measurement of fibroblast and collagen fiber ultrastructure changes in hairless mice skin
The skin tissues were cut from the dorsal of the mice(2 or 3 pieces from each mouse and each was about 0.1cm~3 area),then were fixated,dehydrated,embedded and located.The samples were cut to extra thin sections(50nm)and stained by uranyl acetate and lead citrate.Ultrastructure changes of fibroblast and collagen fibers were observed by transmission electron microscope.
2.6 Measurement of MDA and HYP content changes in hairless mice skin
2.6.1 The homogenate preparation and measurement of MDA content
The skin was drilled down with corneal trephine,and then it was weighed without subcutaneous fat to prepare 10%homogenizer.Put 0.1ml 10%homogenizer and then added No.1、2、3 reagents misce bene.The mixture was warmed for 40 minutes in 95℃water,centrifugated 10 minutes at 3500-4000r/min.The supernatant was taken,and was shaded selection with 532nm spectrophotometer.
2.6.2 Measurement of HYP content
The skin was drilled down with corneal trephine,and then about 30 to 100 grams tissues(wet weight)was put into test tube.Added 1ml water to digest,warmed the mixture for 20 minutes.Adjusted PH to 6.0 to 6.8 and added distilled water to 10ml. Put active carbon into 3ml solution to misce bene.Centrifugated the mixture for 10 minutes at 3500r/mm and taken 1ml the clear supernatant and added No.1、2、3 agents to misce bene.The mixture was warmed for 60 minutes in 60℃water,centrifugated for 10 minutes in 3500r/min.The supernatant was taken and shaded selection with 532nm spectrophotometer.
2.7 Measurement ofmtp53 expression in hairless mice skin
The materials were cut from the dorsal skin of the mice and the expression of mtp53 was measured with immunohistochemical method.The results were analyzed by America Leica DMI4000B Image Processing System.
Results
1.The skin surface changes on hairless mice
The scores were elevated in group B、C、E and F significantly.The differences between group B、C、E、F and group A、D was significant(P＜0.01).The score in group B and F was the highest;the score in group C and E was lower than that of group B and F.The differences between group A and D,group B and F,group C and E was no significant(p＞0.05).
2.The changes of collagen and elastic fibers in hairless mice skin
The collagen and elastic fibers in the dermis arranged in bunchy regularly without marked rupture in group A and D.Epidermis in group B、C、E and F was hyperkeratinization.Papillar layer was flatted.The collagen and elastic fibers in the dermis of hairless mice became thicker,fragmented and disarranged.
3.The ultrastryctue changes of fibroblast and collagen fibers in hairless mice skin
The fibroblast and collagen fibers were normal in group A and D.We could observe karyopycnosis,the collagen and elastic fibers disarranged,the boundary among fibers unclear and some fibers was dissolved in group B、C、E and F.
4.The changes of MDA and HYP content in hairless mice skin
The MDA content
The MDA content was significant increased in group B、C、E and F.The differences between group B、C、E、F and group A,D was significant(P＜0.01). The MDA content in group B and F was the highest;the MDA content in group C and E was lower than that of group B and F.The differences between group A and D. group B and F was no significant(p＞0.05).
The HYP content
The HYP content was significant decreased in group B、C、E and F.The differences between group B、C、E、F and group A、D was significant(P＜0.01). The HYP content in group B and F was the lowest;the HYP content in group C and E was higher than that of group B and F.The differences between group A and D,group B and F,group C and E was considered no significant(p＞0.05).
5.The changes of mtp53 expression in hairless mice skin
Mtp53 expression measurement was increased significant in group B、C、E and F.The differences between group B、C、E、F and group A、D was significant (P＜0.01).The mtp53 expression in group B and F was the highest;the mtp53 expression in group C and E was lower than that of group B and F.The differences between group A and D,group B and F,group C and E was no significant(p＞0.05).
6.The affection of time on the effect of Tempol against photoaging
The results showed that 0.36%Tempol had determinate protection to skin photoaging of hairless mice caused by radiation with UVB for 1 to 4 hours after external application,and the protection effect would be the best in 2 hours after external application,no effect after 8 hours.
Conclusions
External application with 0.36%Tempol had protection to skin photoaging of hairless mice caused by radiation with UVB,and its protection were affected by time. The effective time-spans are from 1 to 4 hours after external application,and the best time-span is around 2 hours after medication.Its mechanisms may have a relationship to inhibiting degradation and degeneration of the collagen fibers and elastic fibers. inhibiting apoptosis of fibroblasts,inhibiting lipid peroxidation and inhibiting gene mutation of p53 in dermis.
引文
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5.Black HS.Potential involvement of free radical reactions in ultraviolet light-mediated cutaneous damage.Photochem Photobiol.1987 Aug;46(2):213-221.
6.Norins AL.Free radical formation in the skin following exposure to ultraviolet light.J Invest Dermatol.1962;39:445-448.
7.Agarwal R.Mukhtar H.Evidence for UV light as an oxidative stressor in skin chemical carcinogenesis.In:Fuchs J,Packer L(eds).Oxidative Stress in Dermatology.New York:Marcel Dekker,1993;pp 207-241.
8.Biesalski HK,Hemmes C,Hopfenmuller W,Schmid C,Gollnick HP.Effects of controlled exposure of sunlight on plasma and skin levels of beta-carotene.Free Radic Res.1996:24:215-224.
9.Jurkiewicz BA,Bissett DL,Buettner GR:Effect of topically applied tocophero on ultraviolet radiation-mediated free radical damage in skin J.Invest.Dermatol.1995.104,484-488.
10.Saperstein H,Rapaport M,Rietschel RL.Topical vitamin E as a cause of erythema multiform-like eruption.Arch Dermatol 1984;120:906-908.
11.Willet WC.Vitamins A,E,and carotene:effects of supplementation on their plasma levels.Am.J.Clin.Nutr.1983,559-566.
12.Winkler BS,Orselli SM.Rex TS.The redox couple between glutathione and ascorbic acid:a chemical and physiological perspective.Free Radic.Biol.Med.1994;17:333-349.
13.Barnadas M.A.;Curell R.;De Moragas J.M.Effect of topical tretinoin on epidermal Langerhan's cells in photodamaged skin.International Journal of Dermatology 1995,34(8),580-582.
14.Poswig A.Wenk J,Brenneisen P,et al:Adaptive antioxidant response of manganese-superoxide dismutase following repetitive UVA irradiation.J Invest Dermatol 1999:112:13-18.
15.Mitchell JB,Samuni A,Krishna MC,et al.Biologically active metal-independent superoxide dismutase mimics.Biochemistry.1990:29:2802-2807.
16.Damiani E,Paganga G,Greci L.Inhibition of copper-mediated low density lipoprotein peroxidation by quinoline and indolinone nitroxide radical.Biochem Pharmacol.1994;48(6):1155-1161.
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18.Gelvan D.Saltman P.Powell SR,et al.Cardiac reperfusion damage prevented by a nitroxide free radical.Proc Natl Acad Sci USA.1991:88(11):4680-4684.
19.Samuni A,Winkelsberg D,Pinson A.Nitroxide stable radicals protect beating cardiomyocytes against oxidative damage.J Clin Invest.1991;87:1526-1530.
20.Samuni A,Godinger D,Aronovitch J.et al.Nitroxides block DNA scission and protect cells from oxidative damage.Biochemistry.1991;30(2):555-561.
21.Samuni A,Barenholz Y.et al.Stable nitroxide radicals protect lipid acyl chains from radiation damage.Free Radic Biol Med.1997;22(7):1165-1174.
22.Wang GY,Godinger D,Aronovitch J,et al.Opposing effects of nitroxide free radicals in Escherichia coli mutants deficient in DNA repair.Biochim Biophys Acta.1996;1305(1-2):71-78.
23.Zhang R,Shohami E,Beit-Yannai,et al.Mechanism of brain protection by nitroxide radicals in experimental model of closed-head injury.Free Radic Biol Meal.1998;24(2):332-340.
24.陈宏泉,王国英,王桂芝,等.Tempol对紫外线照射下豚鼠皮肤的防护作用.中国麻风皮肤病杂志.2006;22(9):729-730.
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7. Damiani E, Paganga G. Greci L. Inhibition of copper-mediated low density lipoprotein peroxidation by quinoline and indolinone nitroxide radical. Biochem Pharmacol. 1994; 48(6): 1155-1161.
8. Damiani E, Greci L, Parsons R. et al. Nitroxide radicals protect DNA from damage when illuminated in vitro in the presence of dibenzoylmethane and a common sunscreen ingredient. Free Radic Biol Med. 1999; 26: 809-816.
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10. Samuni A, Winkelsberg D. Pinson A. Nitroxide stable radicals protect beating cardiomyocytes against oxidative damage. J Clin Invest. 1991: 87: 1526-1530.
11. Samuni A, Godinger D. Aronovitch J, et al. Nitroxides block DNA scission and protect cells from oxidative damage. Biochemistry. 1991; 30(2): 555-561.
12. Samuni A, Barenholz Y, et al. Stable nitroxide radicals protect lipid acyl chains from radiation damage. Free Radic Biol Med . 1997; 22(7): 1165-1174.
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[1] Black HS. Potential involvement of free radical reactions in ultraviolet light-mediated cutaneous damage. Photochem Photobiol. 1987 Aug; 46(2):213-221.
[2] Norins AL. Free radical formation in the skin following exposure to ultraviolet light. J Invest Dermatol. 1962; 39: 445-448.
[3] Agarwal R, Mukhtar H. Evidence for UV light as an oxidative stressor in skin chemical carcinogenesis. ln: Fuchs J, Packer L(eds). Oxidative Stress in Dermatology. New York: Marcel Dekker, 1993; pp 207-241.
[4] Samuni A, Krishna CM. Riesz P, et al. A novel metal-free low molecular weight superoxide dismutase mimic. J Biological Chemistry. 1988: 263(34): 17921-17924.
[5] Mitchell JB, Samuni A. Krishna MC, et al. Biologically active metal-independent superoxide dismutase mimics. Biochemistry. 1990; 29: 2802-2807.
[6] Damiani E, Paganga G, Greci L. Inhibition of copper-mediated low density lipoprotein peroxidation by quinoline and indolinone nitroxide radical. Biochem Pharmacol. 1994:48(6): 1155-1161.
[7] Damiani E, Greci L, Parsons R, et al. Nitroxide radicals protect DNA from damage when illuminated in vitro in the presence of dibenzoylmethane and a common sunscreen ingredient. Free Radic Biol Med. 1999; 26: 809-816.
[8] Gelvan D, Saltman P, Powell SR, et al. Cardiac reperfusion damage prevented by a nitroxide free radical. Proc Natl Acad Sci USA. 1991; 88(11): 4680-4684.
[9] Samuni A, Winkelsberg D, Pinson A. Nitroxide stable radicals protect beating cardiomyocytes against oxidative damage. J Clin Invest. 1991; 87: 1526-1530.
[10] Samuni A, Godinger D, Aronovitch J, et al. Nitroxides block DNA scission and protect cells from oxidative damage. Biochemistry. 1991; 30(2): 555-561.
[11] Samuni A, Barenholz Y, et al. Stable nitroxide radicals protect lipid acyl chains from radiation damage. Free Radic Biol Med . 1997; 22(7): 1165-1174.
[12] Wang GY, Godinger D, Aronovitch J, et al. Opposing effects of nitroxide free radicals in Escherichia coli mutants deficient in DNA repair. Biochim Biophys Acta. 1996; 1305(1-2):71-78.
[13] Zhang R, Shohami E, Beit-Yannai, et al. Mechanism of brain protection by nitroxide radicals in experimental model of closed-head injury. Free Radic Biol Med. 1998; 24(2): 332-340.
[14] Chi Mingliang, Cao Pengli, Yu Guoying, et al. Protective effect of topically applied Polypeptide from Chlamys farreri against ultraviolet radiation-induced chronic skin damage in guinea pig. Chin J Oceanol Limnol, 2003, 21:319-323.
[15] Jurkiewicz BA, Bissett DL, Buettner GR: Effect of topically applied tocophero on ultraviolet radiation-mediated free radical damage in skin J. Invest. Dermatol. 1995, 104, 484-488.
[16] Gilchrest BA. Prior chronic sun exposure decreases the lifespan of human skin fibroblasts in vitro. J. Gerontol 1980; 35:537-541
[17] Johnston KJ, Oikarinen AI, Lowe NJ, Clark JG, Uitto J. Ultraviolet radiation-induced connective tissue changes in the skin of hairless mice. J Invest Dermatol, 1984, 82:587-590.
[18] Brenneisen P, Wenk J, Klotz LO, et al. Central role of Ferrous/ Ferric iron in the ultraviolet B irradiation-mediated signaling pathway leading to increased interstitial Collagenase [matrix-degrading metalloproteinases ( MMP ) 21] and stromelysin-1 (MMP23 ) mRNAlevels in cultured human dermal fibroblasts. J Biol Chem, 1998, 273: 5279-5287.
[19] Wenk J , Brenneisen P , Meewes C , et al. UV-induced oxidative stress and photoaging. Curr Probl Dermatol, 2001, 29: 83-94.
[1] Norins AL. Free radical formation in the skin following exposure to ultraviolet light. J Invest Dermatol. 1962: 39: 445-448.
[2] Black HS. Potential involvement of free radical reactions in ultraviolet light-mediated cutaneous damage. Photochem Photobiol. 1987 Aug; 46(2):213-221.
[3] Agarwal R. Mukhtar H. Evidence for UV light as an oxidative stressor in skin chemical carcinogenesis. In: Fuchs J, Packer L(eds). Oxidative Stress in Dermatology. New York: Marcel Dekker, 1993: pp 207-241.
[4].Samuni A. Krishna CM. Riesz P. et al. A novel metal-free low molecular weight superoxide dismutase mimic. J Biological Chemistry. 1988; 263(34): 17921-17924.
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2.Paul BS,Parrish JA.The interaction of UVA and UVB in the production of threshold erythema.J Invest Dermatol.1982;78:371-374.
3.Peter EM,Butler FRCS,Salvaldore G,et al.Quantitative and qualitative effects of chemical peeling on photo-aged skin:An experimental study.Plast Reconstr Surg.2001;107:222-228.
4.朱彦君,冯光珍,孟宇宏,等.皮肤光老化动物模型的建立.中国体视学与图像分析.2004:9(1):51-54.
5.Black HS.Potential involvement of free radical reactions in ultraviolet light-mediated cutaneous damage.Photochem Photobiol.1987 Aug;46(2):213-221.
6.Norins AL.Free radical formation in the skin following exposure to ultraviolet light.J Invest Dermatol.1962;39:445-448.
7.Agarwal R.Mukhtar H.Evidence for UV light as an oxidative stressor in skin chemical carcinogenesis.In:Fuchs J,Packer L(eds).Oxidative Stress in Dermatology.New York:Marcel Dekker,1993;pp 207-241.
8.Biesalski HK,Hemmes C,Hopfenmuller W,Schmid C,Gollnick HP.Effects of controlled exposure of sunlight on plasma and skin levels of beta-carotene.Free Radic Res.1996:24:215-224.
9.Jurkiewicz BA,Bissett DL,Buettner GR:Effect of topically applied tocophero on ultraviolet radiation-mediated free radical damage in skin J.Invest.Dermatol.1995.104,484-488.
10.Saperstein H,Rapaport M,Rietschel RL.Topical vitamin E as a cause of erythema multiform-like eruption.Arch Dermatol 1984;120:906-908.
11.Willet WC.Vitamins A,E,and carotene:effects of supplementation on their plasma levels.Am.J.Clin.Nutr.1983,559-566.
12.Winkler BS,Orselli SM.Rex TS.The redox couple between glutathione and ascorbic acid:a chemical and physiological perspective.Free Radic.Biol.Med.1994;17:333-349.
13.Barnadas M.A.;Curell R.;De Moragas J.M.Effect of topical tretinoin on epidermal Langerhan's cells in photodamaged skin.International Journal of Dermatology 1995,34(8),580-582.
14.Poswig A.Wenk J,Brenneisen P,et al:Adaptive antioxidant response of manganese-superoxide dismutase following repetitive UVA irradiation.J Invest Dermatol 1999:112:13-18.
15.Mitchell JB,Samuni A,Krishna MC,et al.Biologically active metal-independent superoxide dismutase mimics.Biochemistry.1990:29:2802-2807.
16.Damiani E,Paganga G,Greci L.Inhibition of copper-mediated low density lipoprotein peroxidation by quinoline and indolinone nitroxide radical.Biochem Pharmacol.1994;48(6):1155-1161.
17.Damiani E,Greci L,Parsons R,et al.Nitroxide radicals protect DNA from damage when illuminated in vitro in the presence of dibenzoylmethane and a common sunscreen ingredient.Free Radic Biol Med.1999;26:809-816.
18.Gelvan D.Saltman P.Powell SR,et al.Cardiac reperfusion damage prevented by a nitroxide free radical.Proc Natl Acad Sci USA.1991:88(11):4680-4684.
19.Samuni A,Winkelsberg D,Pinson A.Nitroxide stable radicals protect beating cardiomyocytes against oxidative damage.J Clin Invest.1991;87:1526-1530.
20.Samuni A,Godinger D,Aronovitch J.et al.Nitroxides block DNA scission and protect cells from oxidative damage.Biochemistry.1991;30(2):555-561.
21.Samuni A,Barenholz Y.et al.Stable nitroxide radicals protect lipid acyl chains from radiation damage.Free Radic Biol Med.1997;22(7):1165-1174.
22.Wang GY,Godinger D,Aronovitch J,et al.Opposing effects of nitroxide free radicals in Escherichia coli mutants deficient in DNA repair.Biochim Biophys Acta.1996;1305(1-2):71-78.
23.Zhang R,Shohami E,Beit-Yannai,et al.Mechanism of brain protection by nitroxide radicals in experimental model of closed-head injury.Free Radic Biol Meal.1998;24(2):332-340.
24.陈宏泉,王国英,王桂芝,等.Tempol对紫外线照射下豚鼠皮肤的防护作用.中国麻风皮肤病杂志.2006;22(9):729-730.
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7. Damiani E, Paganga G. Greci L. Inhibition of copper-mediated low density lipoprotein peroxidation by quinoline and indolinone nitroxide radical. Biochem Pharmacol. 1994; 48(6): 1155-1161.
8. Damiani E, Greci L, Parsons R. et al. Nitroxide radicals protect DNA from damage when illuminated in vitro in the presence of dibenzoylmethane and a common sunscreen ingredient. Free Radic Biol Med. 1999; 26: 809-816.
9. Gelvan D. Saltman P, Powell SR, et al. Cardiac reperfusion damage prevented by a nitroxide free radical. Proc Natl Acad Sci USA. 1991; 88(11): 4680-4684.
10. Samuni A, Winkelsberg D. Pinson A. Nitroxide stable radicals protect beating cardiomyocytes against oxidative damage. J Clin Invest. 1991: 87: 1526-1530.
11. Samuni A, Godinger D. Aronovitch J, et al. Nitroxides block DNA scission and protect cells from oxidative damage. Biochemistry. 1991; 30(2): 555-561.
12. Samuni A, Barenholz Y, et al. Stable nitroxide radicals protect lipid acyl chains from radiation damage. Free Radic Biol Med . 1997; 22(7): 1165-1174.
13. Wang GY, Godinger D. Aronovitch J, et al. Opposing effects of nitroxide free radicals in Escherichia coli mutants deficient in DNA repair. Biochim Biophys Acta. 1996; 1305(1-2):71-78.
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[1] Black HS. Potential involvement of free radical reactions in ultraviolet light-mediated cutaneous damage. Photochem Photobiol. 1987 Aug; 46(2):213-221.
[2] Norins AL. Free radical formation in the skin following exposure to ultraviolet light. J Invest Dermatol. 1962; 39: 445-448.
[3] Agarwal R, Mukhtar H. Evidence for UV light as an oxidative stressor in skin chemical carcinogenesis. ln: Fuchs J, Packer L(eds). Oxidative Stress in Dermatology. New York: Marcel Dekker, 1993; pp 207-241.
[4] Samuni A, Krishna CM. Riesz P, et al. A novel metal-free low molecular weight superoxide dismutase mimic. J Biological Chemistry. 1988: 263(34): 17921-17924.
[5] Mitchell JB, Samuni A. Krishna MC, et al. Biologically active metal-independent superoxide dismutase mimics. Biochemistry. 1990; 29: 2802-2807.
[6] Damiani E, Paganga G, Greci L. Inhibition of copper-mediated low density lipoprotein peroxidation by quinoline and indolinone nitroxide radical. Biochem Pharmacol. 1994:48(6): 1155-1161.
[7] Damiani E, Greci L, Parsons R, et al. Nitroxide radicals protect DNA from damage when illuminated in vitro in the presence of dibenzoylmethane and a common sunscreen ingredient. Free Radic Biol Med. 1999; 26: 809-816.
[8] Gelvan D, Saltman P, Powell SR, et al. Cardiac reperfusion damage prevented by a nitroxide free radical. Proc Natl Acad Sci USA. 1991; 88(11): 4680-4684.
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