黄芩甙对中波紫外线照射HaCaT细胞后光产物的影响

目的 探讨黄芩甙对UVB照射后HaCaT细胞光产物环丁烷嘧啶二聚体(CPD)的产生和清除的影响.方法 以一定剂量UVB照射HaCaT细胞,在照光后不同时间点采用免疫组织化学法检测CPD的产生和清除;以黄芩甙预先孵育HaCaT细胞,再观察黄芩甙对CPD的影响.结果 HaCaT细胞损伤程度随UVB照射剂量增大而加重.30mJ/cm²UVB照射后HaCaT光产物CPD的产生在0.5h左右达到高峰,同时细胞开始清除CPD,照射后4h内清除速率较快,至24h时基本清除CPD.黄芩甙预处理组UVB照射后细胞的CPD产生少于非加药组(U=2.324,P<0.05).结论 UVB照射对HaCaT细胞光损伤程度呈剂量递增性,HaCaT细胞对CPD的清除存在快速清除期及慢速清除期;黄芩甙可减少光产物生成.     

黄芩苷对中波紫外线诱导后BALB/c小鼠表皮光产物形成的干预作用

目的 观察中波紫外线（UVB）诱导BALB/c小鼠皮肤表皮细胞光产物的形成和清除情况,以及黄芩苷的干预作用。方法 将黄芩苷（1 mg/cm2）连续3 d外用于BALB/c小鼠表皮24 h后,采用免疫组织化学法及免疫印迹法检测180 mJ/cm2 UVB辐射后1 h、24 h和48 h小鼠表皮内环丁烷嘧啶二聚体（CPD）的产生和清除情况。结果 CPD仅在接受UVB辐射的小鼠皮肤内出现。相对于UVB辐射后1 h光产物的平均值,UVB辐射组1 h、24 h和48 h,CPD的信号强度分别为（100 ± 5.22）％、（75.34 ± 8.22）％和（42.11 ± 3.24）％;经过黄芩苷处理后的小鼠接受UVB辐射后1 h、24 h和48 h,CPD的信号强度分别为（81.45 ± 5.22）%、（32.14 ± 6.33）%和（5.21 ± 3.15）%;而丙酮处理后的小鼠接受UVB辐射后1 h、24 h和48 h,CPD的信号强度分别为（106 ± 8.21）%、（70.23 ± 4.13）%和（41.22 ± 4.21）%。结论 外用黄芩苷能抑制UVB辐射诱导光产物的形成,并在48 h内加速光产物的清除。黄芩苷是一种有效的紫外线防护剂。     

表没食子儿茶素没食子酸酯对中波紫外线诱导人原代表皮黑素细胞光损伤干预作用的研究

目的:观察中波紫外线(UVB)诱导人原代表皮黑素细胞光产物的形成和清除情况,以及表没食子儿茶素没食子酸酯(EGCG)的干预作用.方法:选择10、20、40、80、100 mg/L 5种浓度EGCG作用于体外培养的人表皮黑素细胞72 h,测定细胞增殖活性.采用免疫斑点印迹技术在30 mJ/cm2UVB照射及EGCG干预下.分别取照射后0.5 h和24 h检测环丁烷嘧啶二聚体(CPDs)的产生和清除量.结果:低浓度(＜20 mg/L)EGCG能促进黑素细胞增殖.UVB照射后黑素细胞内光产物形成较快,但清除缓慢.EGCG对UVB诱导光产物的形成无明显影响,但能加速光产物的清除(P＜0.05).结论:EGCG能加速UVB照射后黑素细胞内光产物的清除.     

黄芩苷对中波紫外线所致HaCaT细胞凋亡、细胞周期变化及p16、原癌基因c-myc蛋白表达的影响

目的:观察中波紫外线(UVB)辐射引起的人表皮角质形成细胞株HacaT细胞凋亡、细胞周期及p16、c-myc蛋白水平的影响及传统中药黄芩苷(baicalin)对它的干预作用.方法:以200 mg/L黄芩苷预处理HacaT细胞,以流式细胞仪检测经0、30、60和90 mJ/cm2的UVB照射后24 h细胞周期和凋亡率变化;以Western blot方法检测90 mJ/cm2 UVB照射后p16、c-myc 蛋白的表达水平.结果:UVB照射可诱导HaCaT细胞产生凋亡,凋亡率呈剂量依赖性增加;30 mJ/cm2 剂量下细胞可出现明显S期阻滞,随UVB剂量增大,S期细胞数量相对下降;照光后p16、c-myc蛋白水平均有所增高.加入黄芩苷处理可抑制UVB引起的上述变化.结论:黄芩苷可明显抑制UVB引起的凋亡与细胞周期阻滞以及p16、c-myc蛋白表达,证实该药具有有效的光保护作用.     

表没食子儿茶素没食子酸酯对中波紫外浅诱导的人皮肤成纤维细胞基质金属蛋白酶-1表达的影响

目的:研究中波紫外线(UVB)诱导人皮肤成纤维细胞(FB)表达基质金属蛋白酶(MMP)-1和表没食子儿茶素没食子酸酯(EGCG)及c-Jun氨基端激酶(JNK)抑制剂SP600125对该诱导的保护作用.方法:以30 mJ/cm2 UVB及12.5μg/mL EGCG处理FB.同时以SP600125为对照,照光和(或)加药后于相应时间点提取上清及总RNA,以ELISA法检测MMP-1表达,反转录(RT)-PCR法检测细胞中MMP-1 mRNA含量.结果:30 mJ/cm2 UVB照射FB后24h MMP-1表达为对照组的3.1倍,12h及24h时MMP-1 mRNA表达分别增加至对照组的2.60倍及2.66倍(P均<0.05).UVB照射前、后加EGCG及SP600125组较单纯UVB照射组显著抑制MMP-1的表达,MMP-1 mRNA含量分别为单纯照射组的71.9%及40.4%,MMP-1蛋白表达量分别为单纯照射组的61.8%及48.9%.结论:UVB照射FB后MMP-1 mRNA及MMP-1表达水平均显著增加,EGCG可抑制UVB所致的MMP-1 mRNA及MMP-1高表达.     

黄芪甲甙对中波紫外线损伤皮肤角质形成细胞的保护作用

目的 研究传统中药活性成分黄芪甲甙对中波紫外线(UVB)损伤皮肤角质形成细胞的保护作用.方法 采用30、60、90 mJ/cm2的UVB照射培养的人皮肤永生角质形成细胞系HaCaT细胞,加入黄芪甲甙进行干预处理,以MTT法检测细胞活性;ELISA法检测细胞因子肿瘤坏死因子(TNF-α)和白介素(IL)-6的分泌量;流式细胞仪检测细胞凋亡率.结果 UVB照射可引起皮肤角质形成细胞损伤,单纯照光组细胞增殖活性可下降23%～43%,而黄芪甲甙预处理组照光后活性仅下降7%～20%;UVB照射后炎性细胞因子TNF-α、IL-6分泌显著增加,A值分别为16.32～91.59及98.6～403.53,而黄芪甲甙预处理后其分泌量显著降低(P<0.05);UVB辐射后在G1期前出现明显的亚二倍体峰(凋亡峰),而经黄芪甲甙处理的UVB组细胞凋亡率则明显下降(P<0.05).结论　黄芪甲甙具有光保护性能,可减轻UVB对皮肤角质形成细胞的损伤作用.     

金属硫蛋白基因对小鼠UVB照射的保护作用

目的:研究金属硫蛋白基因对UVB照射反应的影响.方法:敲除小鼠金属硫蛋白基因,背部皮肤剃毛后24 h照射不同剂量的UVB(0.05 J/cm2,0.70 J/cm2,1.40 J/cm2),照光前、后24 h测量小鼠背部皮肤厚度,皮肤组织病理观察照光后24 h日晒伤细胞形成情况.正常野鼠作对照.结果:小剂量UVB照射时两种小鼠水肿程度无显著性差异,大剂量UVB照射时金属硫蛋白基因敲除小鼠背部水肿显著高于野鼠.病理显示金属硫蛋白基因敲除小鼠较野鼠有更多的日晒伤细胞产生.结论:金属硫蛋白基因对急性UVB照射具有保护作用.     

表没食子儿茶素没食子酸酯对紫外线辐射后小鼠表皮细胞凋亡的影响

目的：观察表没食子儿茶素没食子酸酯[（-）-epigallocatechin-3-gallate,EGCG]脂质体对急、慢性中波紫外线（UVB）辐射后BALB／c小鼠表皮细胞凋亡的影响。方法：EGCG脂质体局部外用于BALB／c小鼠背部皮肤,将小鼠分为5组,分别给予中波紫外线180mJ／cm^2照射1次为急性损伤组：30mJ／cm^2每天照射1次,持续30d,为慢性损伤组。采用末端转移酶介导的缺口末端标记法（TUNEL）检测小鼠表皮中的凋亡细胞。结果：急性损伤组中接受UVB照射的小鼠表皮中大部分细胞发生凋亡,EGCG脂质体未表现出对表皮细胞凋亡的影响;慢性损伤组中照光加药组的凋亡细胞多于其他组,EGCG脂质体表现为促凋亡作用（P〈0．05）。结论：EGCG脂质体不影响急性大剂量UVB辐射所致的表皮细胞凋亡;对慢性低剂量UVB辐射有促凋亡作用。     

黄芩苷对中波紫外线辐射后小鼠皮肤氧化应激及DNA损伤的影响

目的观察黄芩苷对中波紫外线(UVB)辐射后小鼠皮肤氧化应激及DNA损伤的影响。方法将黄芩苷外搽于Balb/c小鼠皮肤,检测180mJ/cm2UVB辐射后24h小鼠皮肤厚度及过氧化氢和CPDs产量。结果无论是UVB辐射前还是辐射后外用黄芩苷均明显减轻紫外线辐射造成的小鼠皮肤增厚。外用黄芩苷还可减少因UVB辐射诱导的小鼠皮肤组织中过氧化氢和CPDs产量。结论黄芩苷可抵抗UVB诱导的小鼠皮肤增厚,减少光产物表达,并通过减少活性氧簇的产生而发挥抗氧化作用。     

表没食子儿茶素没食子酸酯减轻UVB诱导的朗格汉斯细胞凋亡作用

目的 研究中波紫外线(UVB)对人表皮朗格汉斯细胞(LC)的光损伤作用以及绿茶活性成分表没食子儿茶素没食子酸酯(EGCG)的光保护作用。方法 采用密度梯度离心和免疫磁珠的方法分离纯化人表皮LC,将分离纯化的LC随机分为对照组、30 mJ/cm² UVB辐射组以及辐射后加用200μg/mL EGCG处理组,4h后以PI染色并经流式细胞仪检测细胞凋亡率。结果 30 mJ/cm² UVB辐射组的凋亡率明显高于对照组,EGCG干预组的凋亡率低于单纯UVB辐射组,但仍高于非照射对照组;且辐射后S期细胞数明显增加。几乎没有G₂/M期的细胞,EGCG处理辐射细胞后,S期细胞数减少。结论 UVB可诱导人表皮LC凋亡,而EGCG具有一定的保护作用。     

Aged human skin removes UVB-induced pyrimidine dimers from the epidermis more slowly than younger adult skin in vivo

Although many studies have been reported on the repair of ultraviolet light (UV)-induced cyclobutane-type pyrimidine dimers (CPDs) in DNA, the effects of aging on the removal of UV-induced CPDs from the human skin epidermis in vivo remains uncertain. Therefore, we employed immunoblotting and immunohistochemical methods using monoclonal antibodies (TDM-2) to CPDs to study age-related differences in the time required for the in vivo removal of UVB-induced CPDs. The flexure surfaces of the upper arms of five young men were exposed to UVB light at a fluence of 35 and 700 mJ/cm², and four older men were also irradiated with the same doses of UVB mentioned above. Each area of skin was biopsied before and immediately after irradiation, and at 4, 24 h, 2 and 4 days after irradiation in the younger group; and before and immediately after irradiation, and at 24 h, 4, 7, and 14 days after irradiation in the older group. A total of 108 DNA samples were taken from the epidermis of 108 biopsied specimens. These samples were immunoblotted using TDM-2 and the intensities of the immunoprecipitates were measured by photodensitometer. Our results show that the CPDs had been removed from the epidermis at 4 days after irradiation at either dose in the younger group, and between 7–14 days after irradiation in the aged group. The results of our immunohistochemical studies were consistent with those of our immunoblotting studies, and indicated that basal cells repair CPDs more quickly than prickle cells in the epidermis except the amounts at 24 h after UVB irradiation, and that the CPDs were removed by epidermal turnover after the nucleotide excision repair (NER). Our results showed age-associated decline in the NER in vivo, indicating high risk of UV-associated skin cancer.     

紫外线对皮肤角质形成细胞和成纤维细胞产生MMP-1和MMP-3的影响

目的:研究中波紫外线辐射对体外培养的表皮角质形成细胞和真皮成纤维细胞产生基质金属蛋白酶-1(MMP-1)和MMP-3的直接和间接影响,研究绿茶中的主要活性成分表没食子儿茶酚没食子酸酯(EGCG)对此影响的保护作用。方法:体外培养角质形成细胞株HaCaT和真皮成纤维细胞,中波紫外线辐射、不同浓度IL-6刺激及EGCG处理后,ELISA方法测定上清液中Pro-MMP-1和MMP-3蛋白含量,半定量RT-PCR方法测细胞中mRNA含量。结果:UVB30mJ/cm2辐射后角质形成细胞分泌的pro-MMP-1和MMP-3并未增加(P>0.05),真皮成纤维细胞合成和分泌MMP-1和MMP-3mRNA含量和蛋白水平均显著增加(P<0.05),IL-6(8、16、24pg/mL)可显著增加成纤维细胞产生MMP-1和MMP-3(P<0.05)。EGCG(0.15、0.3mM)能够显著抑制紫外线诱导成纤维细胞产生MMP含量的增加(P<0.05),但IL-6刺激成纤维细胞所产生的MMP-1和MMP-3的增加不受EGCG的影响(P>0.05)。结论:中波紫外线辐射并不能直接导致角质形成细胞分泌MMP-1和MMP-3增加,但紫外线辐射后角质形成细胞分泌的IL-6可促进成纤维细胞产生MMP。EGCG对IL-6刺激成纤维细胞产生MMP增加没有影响,但它可以显著抑制紫外线辐射直接导致的成纤维细胞产生MMP-1和MMP-3的增加,对防治皮肤光老化可能有一定作用。     

Baicalin protects human fibroblasts against ultraviolet B-induced cyclobutane pyrimidine dimers formation

Exposure to ultraviolet B (UVB) irradiation is a major risk factor for the development of skin cancer. Therefore, it is important to identify agents that can offer protection against UVB-caused DNA damage. Photocarcinogenesis is caused largely by mutations at the sites of incorrectly repaired DNA photoproducts, of which the most common are the cyclobutane pyrimidine dimers (CPDs). In this study, a DNA damage model of UVB irradiation-induced fibroblasts was established. The immunocytochemical staining, immuno dot blotting and Western blotting were employed in the study. We demonstrated that pre-treatment of fibroblasts with Baicalin dose-dependently reduced the amount of UVB-generated CPDs. Compared with UVB irradiated cells, UVB-induced p53 accumulation was less pronounced in Baicalin-treated cells. Taken together, these results suggest that Baicalin prevent CPDs formation induced by UVB. Baicalin is therefore a promising protective substance against UVB radiation.     

Photoprotective potential of Cordyceps polysaccharides against ultraviolet B radiation-induced DNA damage to human skin cells

Summary Background Ultraviolet (UV) radiation causes DNA damage resulting in photoageing and skin cancer. UVB (290–320 nm) interacts directly with DNA, inducing two major photoproducts: cyclobutane‐pyrimidine dimers (CPDs) and (6‐4) pyrimidine‐pyrimidone photoproducts. Cordyceps sinensis (Berk.) Sacc. is a medicinal fungus with reported anticancer and cytoprotective effects. Objectives To investigate genoprotective effects of polysaccharide‐rich Cordyceps mycelial components against UVB‐induced damage in normal human fibroblast cells. Methods Cultured human fibroblasts (BJ cells) were treated for 30 min and, separately, for 24 h with hot water extract of Cordyceps fungal mycelia or exopolysaccharides. Cells were washed, irradiated with UVB (302 nm), and immediately lysed, after which DNA damage, as strand breaks, was measured using an enzyme‐assisted comet assay that detects CPDs. Results DNA damage in UVB‐irradiated cells was significantly lowered (P < 0·01) with Cordyceps pretreatment. Similar results were seen with 30 min and 24 h pretreatment. Specifically, and in comparison with irradiated cells with no Cordyceps pretreatment, there was a 27% reduction in CPDs in irradiated cells with 24 h pretreatment with 200 μg mL^?1 of the hot water Cordyceps extract, and a 34% reduction with 24 h pretreatment with 200 μg mL^?1 of the exopolysaccharide extract. Conclusions Clear evidence of protection against UVB‐induced CPDs was seen with Cordyceps mycelial extracts. Results indicate that Cordyceps may offer photoprotection and lower the risk of basal cell carcinoma, the main skin cancer caused by CPDs. Further study is needed to identify protective mechanisms.     

紫外线诱导成纤维细胞凋亡及防护机制的初步探讨

目的 探讨紫外线诱导真皮成纤维细胞凋亡的发生机理以及绿茶多酚的主要成分表没食子儿茶素没食子酸酯(EGCG)对凋亡的保护作用。方法 用流式细胞仪及RT-PCR方法分别检测了成纤维细胞凋亡率变化以及凋亡相关基因Fas和Bcl-2 mRNA表达变化。结果 经中波紫外线(UVB)和长波紫外线(UVA)辐射的成纤维细胞,均在G1期前出现明显的亚二倍体(凋亡峰),凋亡率分别为34.79±2.24和29.69±3.05;而应用EGCG后凋亡率均明显下降为4.23±0.03和5.23±0.01。经统计学分析,差异有显著性(P<0.001)。Fas的mRNA表达在UVB及UVA辐射组均有较明显增加,分别为0.72±0.05和0.68±0.02;应用EGCG后,两组表达均明显减弱为0.35±0.02和0.47±0.03。经统计学分析,差异有显著性(P<0.001)。Bcl-2的mRNA经UVB辐射后,没有明显变化,仍有较强表达;而经UVA辐射以后,其mRNA表达明显减弱。为0.27±0.03,应用EGCG后,表达则又明显增强为0.51±0.04。结论 EGCG对UVB及UVA诱导的凋亡均有保护作用,但凋亡的发生机理以及保护机理则有所差异。     

Mitigation of acute ultraviolet B radiation-mediated damages by baicalin in mouse skin

Background: Solar ultraviolet (UV) irradiation, in particular UVB with a wavelength range between 290 and 320 nm, induces different hazardous effects on the skin, including sunburn, photoaging and cancer. Protection against sun-induced damage is therefore a highly desirable goal. Chemoprevention is being investigated as a potential approach for the management of UV damages including skin cancer.
Aim: In this study, to determine the relevance of our in vitro findings to in vivo situations, we assessed the effects of baicalin on UVB-mediated damages in mice skin.
Methods: Balb/C hairless mice were topically pretreated (24 h before UVB) or post-treated (5 min after UVB) with baicalin (1 mg/cm² skin area/mouse/100 μl acetone) and were exposed to UVB 24 h later (180 mJ/cm²). The animals were sacrificed 1 and 24 h after the UVB exposure. Skin edema, histopathology changes, hydrogen peroxide (H₂O₂) and cyclobutane pyrimidine dimers (CPDs)-positive cells were assessed to determine the UVB-induced photodamage.
Results: Our data demonstrated that a topical application of baicalin, either as a pretreatment or as a post-treatment, resulted in a significant decrease in UVB mediated increases in skin edema, skin hyperplasia and infiltration of leukocytes. Further, baicalin treatments (pre and post) also resulted in a significant decrease in UVB mediated (1) generation of H₂O₂ and (2) formation of DNA photolesions: CPDs.
Conclusion: Based on these data, we suggest that baicalin could be developed as an agent for the management of conditions elicited by UV exposure including skin cancer.     

茶多酚单体对中波紫外线辐射培养的成纤维细胞氧化损伤的保护机制研究

目的：探讨茶多酚的活性单体表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate，EGCG)对紫外线辐射氧化损伤的保护机制。方法：用722分光光度计测定培养的人皮肤成纤维细胞上清液中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH—Px)的活性，以及脂质过氧化产物丙二醛(MDA)的含量；采用逆转录—聚合酶链反应(RT—PCR)方法测定成纤维细胞合成基质金属蛋白酶1(MMP1)以及其组织抑制因子—1(TIMP—11的mRNA表达水平。结果：EGCG可以减少中波紫外线(UVB）辐射引起的丙二醛沉积，增加抗氧化酶的活性；并且可以抑制UVB诱导的MMP1 mRNA表达，减少胶原蛋白的降解。对TIMP—1的表达则没有观察到显著变化。结论：EGCG可以对UVB辐射损伤的体外培养成纤维细胞起到保护作用。