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。     

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.     

UVB致成纤维细胞损伤及两种中药的保护作用研究

目的观察中波紫外线(UVB)辐射后成纤维细胞(FB)DNA光产物环丁烷嘧啶二聚体(CPD s)产生和清除情况及表没食子儿茶素没食子酸酯(EGCG)和黄芩甙的干预作用。方法以30,60,90 m J/cm2UVB照射FB并予EGCG及黄芩甙干预处理,采用免疫细胞化学法在照光后不同时间检测CPD s的产生和清除情况。结果细胞损伤程度随照光剂量加大而加重;30 m J/cm2UVB照射后细胞CPD s生成量在辐射后1 h左右达到高峰,同时细胞也开始清除CP-D s,辐射后4 h内清除速率较快,4 h后清除速率逐渐降低,至24 h基本清除CPD s;EGCG和黄芩甙处理UVB辐射的细胞CPD s少于单纯照光组(P<0.05)。结论UVB辐射可以导致FB的DNA损伤而产生光产物CPD s;细胞损伤程度显示剂量依赖性;细胞自身有修复能力;EGCG和黄芩甙均可降低UVB辐射所致的光产物水平。     

黄芩苷对培养人原代成纤维细胞长波紫外线光损伤端粒的影响

目的 探讨黄芩苷对于长波紫外线（UVA）照射导致人皮肤成纤维细胞衰老的保护作用及对端粒途径的影响。方法 分离培养人原代成纤维细胞,将亚融合状态的培养细胞分为空白组、黄芩苷组、UVA组和UVA + 黄芩苷组,照光组中以10 J/cm2 UVA进行照射,药物干预组中加入50 μg/ml黄芩苷干预处理。以流式细胞仪测定细胞周期变化;以端粒重复序列扩增-酶联免疫吸附法（TRAP-ELISA）检测细胞端粒酶水平;以实时定量PCR法检测细胞端粒长度及衰老相关基因p53、p16和c-myc mRNA水平;以蛋白印迹法检测p16和c-myc蛋白水平。结果 UVA引起显著G1期阻滞,G1期细胞比值由正常对照组59.94%升高至81.04%,而黄芩苷处理后G1期细胞比值下降为65.55%。UVA照射后细胞端粒长度缩短为正常组的31.2%,黄芩苷干预组的端粒长度可恢复至正常细胞的63.9%。此外与正常组相比,UVA诱导p53和p16 mRNA表达水平升高为2.93 ± 0.21和2.14 ± 0.09,而c-myc mRNA水平下降为0.53 ± 0.03;黄芩苷干预可抑制上述改变。照射后与正常组相比,p16蛋白水平增高为5.84 ± 0.16,c-myc蛋白表达降低为0.35 ± 0.04;黄芩苷处理后p16蛋白表达量下降为4.09 ± 0.13（P < 0.05）;c-myc蛋白表达水平无明显变化（P > 0.05）。正常对照和UVA组的端粒酶活性为阴性,加入黄芩苷对端粒酶活性无影响。结论 黄芩苷可延缓人成纤维细胞光老化进程,其机制可能与调控p53等老化相关基因表达有关,与端粒酶活性无关。     

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。     

咖啡因可保护UVB致人皮肤成纤维细胞的损伤

目的：确定咖啡因对中波紫外线（UVB）照射致体外培养人皮肤成纤维细胞氧化损伤的防护作用.方法：分离培养人成纤维细胞,分为空白对照组、咖啡因组、UVB照射组、UVB照射＋咖啡因组,UVB照射剂量为30 mJ/cm2.用MTT法检测细胞增殖活性,酶生化比色法检测细胞丙二醛（MDA）含量、超氧化物歧化酶（SOD）及谷胱甘肽过氧化物酶（GSH-Px）活性.结果：UVB照射前后用咖啡因处理能使成纤维细胞存活率提高、MDA产生减少,酶的活性增强.结论：咖啡因对UVB照射损伤成纤维细胞具有一定保护性作用,其机制可能与抑制氧化损伤和增强抗氧化能力有关.     

黄芩苷对UVB照射后小鼠表皮细胞Ki-67及PCNA表达的影响

目的:评价外用黄芩苷对UVB照射后小鼠表皮厚度及细胞增殖标志物Ki-67、PCNA表达的影响.方法:将6周龄大的雌性C57BL/6小鼠随机分为空白对照、单纯UVB照射和涂抹黄芩苷+UVB照射3组.末次照射24 h后取背部皮肤切片HE染色,免疫组化法、Western blot法检测皮肤组织中Ki-67、PCNA蛋白的表达.结果:黄芩苷预处理可抑制反复UVB照射引起小鼠皮肤表皮层增厚,真皮层单一核细胞浸润.Ki-67及PCNA在对照组表达分别(27.78±13.85)%和(20.68±10.64)%,UVB组表达分别(57.83±9.71)%、(62.48±8.73)%,两组有显著性差异(P<0.05).涂抹黄芩苷+UVB照射组Ki-67及PCNA表达为38.69±11.76、42.83±10.37,虽高于对照组,但明显低于UVB照射组(Ps<0.05).结论:外用黄芩苷可通过降低照射小鼠表皮中Ki-67及PCNA的表达从而发挥其抗UVB损伤作用.     

Caspase-3在UVB诱导人皮肤成纤维细胞凋亡中的作用

目的：评价caspase-3在UVB诱导皮肤成纤维细胞凋亡中的作用。方法：皮肤成纤维细胞经150mJ／cm2 UVB照射后,用MTF法检测细胞活性,用Hoechst33258染色法检测细胞凋亡,用抑制剂Z-DEVD-FMK抑制caspase-3活性后,检测凋亡细胞数量。结果：UVB明显抑制成纤维细胞的活性,并导致细胞凋亡,并呈时间-效应关系;加入抑制剂Z-DEVD-FMK抑制了UVB导致的细胞凋亡。结论：Caspase-3在UVB照射诱导皮肤成纤维细胞凋亡中发挥重要作用。     

p53相关的细胞周期调控在UVB诱导的皮肤成纤维细胞早衰中的作用

目的探讨中波紫外线(UVB)诱导皮肤成纤维细胞(HSF)应激诱导的早衰(SIPS)后细胞周期调控的变化。探讨p53相关的生长停滞、细胞凋亡相关基因表达在UVB诱导的SIPS中的变化情况。方法对UVB诱导发生SIPS的HSF,流式细胞仪检测细胞周期;Western blot法检测衰老信号通路中p53,p21,p16的表达;实时荧光定量PCR芯片检测与p53相关的生长停滞(p53,p21,p16,p19)、凋亡(bax,bcl-2)基因的mRNA表达水平。结果流式细胞仪对UVB诱导的SIPS模型进行细胞周期检测发现,大部分细胞发生了G1期阻滞;衰老通路中p53,p21和p16的表达都明显增加;实时荧光定量PCR芯片检测发现,与生长停滞相关的抑癌基因p53,p21,p16,p19的表达均上调;p53下游的凋亡相关基因中的抑凋亡基因bcl-2表达稍上调,而凋亡基因bax表达下调。结论G1期阻滞和衰老信号通路蛋白表达增加进一步印证了UVB诱导HSF发生的SIPS。p53信号通路相关基因的表达变化提示UVB诱导的SIPS可能具有与p53相关的抗凋亡作用,但其中p53信号通路所起的作用还需更进一步的实验探索。     

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.     

The 0.8% ultraviolet B content of an ultraviolet A sunlamp induces 75% of cyclobutane pyrimidine dimers in human keratinocytes in vitro

Tanning lamps, emitting predominantly ultraviolet (UV) A, are used widely throughout the U.K. and other countries, but little is known about the long-term risks associated with their use, especially with respect to skin cancer. We have exposed normal human epidermal keratinocytes to a commercial tanning lamp and used the comet assay in association with DNA repair enzymes T4 endonuclease V and endonuclease III to investigate the relative yields of directly formed cyclobutane pyrimidine dimers (CPDs) and indirectly formed types of oxidative DNA damage. To put the risk of using tanning lamps into perspective, the sunbed used in this study (five Philips Performance 80W-R UVA tubes at a distance of 35 cm) was found to be approximately 0.7 times as potent at inducing CPDs as U.K. natural sunlight around noon on a fine summer day. This compares with a relative risk for CPD induction and erythema of 0.8 and 0.7 times, respectively, calculated from the relevant action spectra of tanning lamps and British noontime sunlight. To determine the relative contribution of UVB and UVA to the induction of CPDs and oxidative DNA damage, we modified the spectral output of the tanning lamps with a series of Schott WG UVB filters. The induction of CPDs was more dependent on the UVB component of the sunbed than oxidative types of damage. Schott WG UVB filters with 50% transmission at 305 nm reduced the yield of T4 endonuclease V sites by 42% while there was only a 17% decrease in the yield of endonuclease III sites. CPD induction was not completely abolished after irradiation through WG335 and WG345 nm filters despite there being no detectable UVB. From these data, it was estimated that, although the tanning lamps emitted only 0.8% of their total output in the UVB range, these wavelengths were responsible for the induction of over 75% of CPDs and 50% of the oxidative damage to DNA.     

A clinical trial and molecular study of photoadaptation in vitiligo

Background Photoadaptation to ultraviolet (UV) B phototherapy is due to both pigmentary and nonpigmentary influences. Objectives To measure photoadaptation in vitiliginous skin and to compare it with normal pigmented skin. Methods Seventeen patients with Fitzpatrick skin phototypes III–VI with vitiligo received six to nine UVB treatments, two to three times weekly. Minimal erythema dose (MED) testing was done at baseline and after all treatments; the percentage change in MED was analysed as a measure of photoadaptation. The percentage decrease in cyclobutane pyrimidine dimers (CPDs) over 24 h after a single exposure of 1 MED was analysed on vitiliginous and normal skin. Results The mean ± SD percentage change in MED from before to after treatments was: treated vitiliginous skin 28·5 ± 39·9% (P = 0·015), treated normal skin 35·9 ± 49·9% (P = 0·015), untreated vitiliginous skin 11·9 ± 22·6% (P =0·070), untreated normal skin 25·1 ± 41·3% (P = 0·041). Of these patients, two‐thirds had a positive percentage change in MED (photoadaptation). The mean amount of CPDs induced per megabase of DNA immediately after exposure was significantly higher in vitiliginous skin. The mean ± SD percentage decrease in CPDs (rate of repair) in 24 h was 35·7 ± 26·8% in vitiliginous skin (P = 0·027) and 46·2 ± 19·5% in normally pigmented skin (P = 0·001); no difference was noted in the repair in vitiliginous skin compared with normal skin (P = 0·4). Conclusions Photoadaptation in vitiliginous and normal skin was observed in two‐thirds of patients. Vitiliginous skin had significantly more CPDs following UVB exposure; the rate of repair of UVB‐induced DNA damage was equivalent to that in normal skin.     

紫外线对人皮肤成纤维细胞的影响

紫外线辐射可造成皮肤损伤,引起皮肤出现红斑、光老化等。紫外线作用于成纤维细胞,引起细胞因子分泌及基因表达的改变,不仅能造成真皮层的损伤,还可被用来治疗某些疾病,如局限性硬皮病。从紫外线对皮肤成纤维细胞的损伤、成纤维细胞对紫外线的防御反应及紫外线的应用等方面阐述了紫外线对成纤维细胞的影响。     

紫外线对人皮肤成纤维细胞中Hrd1及Nrf2表达的影响

目的探讨紫外线(UV)照射对人皮肤成纤维细胞中羟甲基戊二酰辅酶A还原酶降解蛋白(Hrd)1及核因子E2相关因子(Nrf)2表达的影响及可能机制。方法共收集12份人皮肤组织标本,曝光及非曝光部位标本各6份,免疫组化检测2组Hrd1及Nrf2的表达。将体外培养人皮肤成纤维细胞分为对照组、UVA组、UVB组,照光后用Western blot法检测各组成纤维细胞中Hrd1及Nrf2的表达。应用Hrd1-siRNA转染体外培养人皮肤成纤维细胞,下调细胞中Hrd1的表达后,Western blot检测成纤维细胞Nrf2的表达。应用免疫荧光分析检测细胞中Hrd1与Nrf2在细胞中的共定位情况,应用免疫共沉淀检测体外培养人成纤维细胞中Hrd1与Nrf2是否存在内源性结合。结果临床标本实验中,曝光部位皮肤组织中Hrd1表达(0.4756±0.0785)明显高于避光部位(0.1270±0.0253,t=7.317,P<0.05),曝光部位皮肤组织中Nrf2表达(0.1190±0.0217)明显低于避光部位(0.2629±0.0263,t=7.306,P<0.05)。体外培养成纤维细胞实验中,经紫外线照射后,UVA组与对照组相比,Hrd1蛋白表达水平明显增高(t=7.227,P<0.05),Nrf2蛋白表达水平明显下降(t=6.456,P<0.05);同样,UVB组与对照组相比,Hrd1蛋白表达水平明显增高(t=2.980,P<0.05),Nrf2蛋白表达水平明显下降(t=13.52,P<0.05)。应用Hrd1-SiRNA下调体外培养人纤维细胞中Hrd1的表达后,无论是经UVA照射还是UVB照射,被下调Hrd1组细胞内Nrf2的表达较未下调组明显升高。免疫荧光分析结果显示Hrd1与Nrf2在体外培养人皮肤成纤维细胞中的均有表达,并且在细胞中存在Hrd1与Nrf2共定位。免疫共沉淀验证了Hrd1与Nrf2在体外培养人皮肤成纤维细胞中存在内源性结合。结论Hrd1与Nrf2在人皮肤成纤维细胞中存在结合,紫外线照射可通过增加细胞中Hrd1的表达从而抑制Nrf2的表达。     

窄谱中波紫外线对寻常型白癜风患者皮肤组织液内皮素-1的影响

目的 探讨窄谱中波紫外线（Narrow Band Ultraviolet B，NB-UVB）对寻常型稳定期白癜风患者白斑区皮肤组织液内皮素-1（Endothelin-1，ET-1）的影响，探讨NB-UVB治疗白癜风的作用机制。方法采用放射免疫方法测定27例白癜风患者在NB-UVB治疗前后的白斑部位和非白斑的皮肤组织液中ET-1含量。结果白斑区皮肤组织液ET-1含量为（54．87±6．89）pg／mL，高于非白斑区的（40．87±6．59）pg／mL。NB-UVB照射后的白斑区皮肤组织液的ET-1与处理前相比明显升高，分别为（62．22±4．96）pg／mL与（54．87±6．89）pg／mL。正常皮肤组织液内的ET-1含量在紫外线照射前后无明显变化。结论NB—UVB照射后皮肤组织液内ET-1含量升高，NB—UVB能促使角质形成细胞分泌ET-1，这可能是NB—UVB引起白斑着色的机制之一。