扇贝多肽保护单次UVA氧化损伤HaCaT细胞

目的探讨扇贝多肽对单次长波紫外线辐射HaCaT角质形成细胞氧化损伤的保护作用机制。方法从栉孔扇贝中提取扇贝多肽(PCF,Mr=879)。UVA辐射强度为5J·cm-2。酶生化法测定胞质SOD,GSH-px活性,ROS、MDA水平;透射电镜观察细胞超微结构的改变;原位杂交技术检测细胞内p21mRNA的变化。结果在5J·cm-2UVA辐射下,在给定浓度范围内PCF能剂量依赖性降低胞质ROS、MDA水平;提高SOD,GSH-px活力;电镜下可见PCF可保护细胞超微结构;p21mRNA原位杂交发现PCF可明显抑制其表达。结论扇贝多肽对单次UVA诱导的人HaCaT细胞氧化损伤有保护作用。其机制与清除氧自由基、提高抗氧化酶活性、抑制p21mRNA表达及细胞凋亡有关。     

扇贝多肽保护单次UVA氧化损伤HaUaT细胞

目的探讨扇贝多肽对单次长波紫外线辐射HaCaT角质形成细胞氧化损伤的保护作用机制。方法从栉孔扇贝中提取扇贝多肽(PCF,Mr=879)。UVA辐射强度为5J·cm-2。酶生化法测定胞质SOD,GSH-px活性,ROS、MDA水平;透射电镜观察细胞超微结构的改变;原位杂交技术检测细胞内p21mRNA的变化。结果在5J·cm-2UVA辐射下,在给定浓度范围内PCF能剂量依赖性降低胞质ROS、MDA水平;提高SOD,GSH-px活力;电镜下可见PCF可保护细胞超微结构;p21mRNA原位杂交发现PCF可明显抑制其表达。结论扇贝多肽对单次UVA诱导的人HaCaT细胞氧化损伤有保护作用。其机制与清除氧自由基、提高抗氧化酶活性、抑制p21mRNA表达及细胞凋亡有关。     

Inhibitory effect of polypeptide from Chlamys farreri on ultraviolet A-induced oxidative damage on human skin fibroblasts in vitro.

We have previously reported that polypeptide from Chlamys farreri (PCF) inhibits the oxidative damage of ultraviolet A (UVA) on HeLa cells in vitro [Acta Pharm. Sin. 23 (2002) 961]. To further elucidate a possible role for PCF on UVA-damaged normal human cells, we established the oxidative damage models of normal human dermal fibroblasts (NHDF) exposed to UVA to study the protective effect of PCF on human dermal fibroblasts in vitro. In this study, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method was used to detect the cell viability. The intracellular superoxide dismutase (SOD), glutathione peroxidase (GSH-px), catalase (CAT), xanthine oxidase (XOD), malondialdehyde (MDA), reactive oxygen species (ROS), total antioxidative capacity (T-AOC), and anti-superoxide anion capacity (A-ASC) were measured. The effect of PCF on UVA-induced apoptosis were investigated by Annexin V-FITC assay. Intracellular calcium was determined with the calcium-sensitive fluorochrome Fluo-3, and mitochondrial transmembrane potential with rhodamine 123. Comet assay was employed to detect the UVA-induced DNA damage. The ultrastructure of cell was observed under transmission electron microscope. The results indicated that PCF could greatly enhance the viability of NHDF and markedly promote SOD, GSH-px, T-AOC, and A-ASC, while the amounts of MDA and ROS, the activity of XOD were decreased. PCF could inhibit UVA-induced apoptosis and DNA damage in NHDF. The concentration of cellular free calcium was decreased and the mitochondrial transmembrane potential was increased by PCF. In ultrastructure of NHDF, PCF could greatly decrease UVA-induced damage, especially membrane. Our results suggest that the supplementation of PCF appears to reduce the UVA-induced normal human dermal fibroblasts damage efficiently. It may be involved in the PCF's abilities of scavenging oxygen free radical, inhibiting lipid peroxidation, increasing antioxidative enzymes, decreasing intracellular calcium and protection of membrane structure in NHDF irradiated by UVA.     

扇贝多肽通过调节c-jun和COX-2抑制UVA诱导的HaCaT细胞凋亡

目的复制UVA诱导的HaCaT细胞凋亡模型,研究UVA对细胞内c-jun和环氧合酶-2(cyclooxygenase-2,COX-2)的影响,从而探究扇贝多肽(Polypeptide from Chlamys far-reri,PCF)抑制UVA引起的HaCaT细胞凋亡的分子机制。方法实验分为5组:正常对照组、UVA模型组、UVA+5.69mmol.L-1PCF组、UVA+2.84mmol.L-1PCF组、UVA+1.42mmol.L-1PCF组。应用实时荧光定量PCR和蛋白印迹法检测细胞内c-jun的表达;RT-PCR结合蛋白质印迹法检测细胞内COX-2的表达;琼脂糖凝胶电泳分析PCF和COX-2特异性抑制剂celecoxib对UVA诱导HaCaT细胞凋亡的影响。结果预先加入PCF和celecoxib均可明显抑制8J.cm-2UVA诱导的HaCaT细胞凋亡;UVA照射HaCaT细胞后COX-2mRNA及蛋白表达水平增加,与对照组相比差异有显著性(P<0.01);1.42~5.69mmol.L-1剂量范围内的PCF可剂量依赖性抑制UVA引起的细胞内COX-2mR-NA及蛋白表达(P<0.05,P<0.01);PCF也抑制了UVA引起的HaCaT细胞内c-jun表达的增加,且呈量效关系(P<0.05,P<0.01)。结论UVA诱导HaCaT细胞发生凋亡时,细胞内COX-2和c-jun的表达明显增加,PCF通过抑制细胞内COX-2和c-jun的表达而发挥其抗凋亡作用。     

肿瘤坏死因子相关凋亡诱导配体及扇贝多肽在UVA诱导HaCaT细胞凋亡中的作用

目的研究UVA对HaCaT细胞肿瘤坏死因子相关凋亡诱导配体(tumour necrosis factor related apoptosis inducing ligand,TRAIL)表达的影响;复制UVA诱导的HaCaT细胞凋亡模型,探究TRAIL在UVA诱导的HaCaT细胞凋亡中的作用及扇贝多肽(Polypeptide from Chlamys farreri,PCF)对UVA诱导的TRAIL凋亡通路的影响。方法实验设计分为5组:对照组、UVA模型组、UVA+5.69mmol·L-1PCF组、UVA+2.84mmol·L-1PCF组、UVA+1.42mmol·L-1PCF组。Real-TimePCR检测TRAIL mRNA表达;蛋白质印迹法检测TRAIL蛋白表达及caspase-8活性;琼脂糖凝胶电泳分析TRAIL中和性抗体对UVA诱导的HaCaT细胞凋亡的影响。结果8J·cm-2UVA照射HaCaT细胞后TRAIL mR-NA及蛋白表达增加,与对照组相比差异有显著性(P<0.01);TRAIL中和性抗体对UVA诱导的HaCaT细胞凋亡有抑制作用;1.42~5.69mmol·L-1剂量范围内的PCF可剂量依赖性抑制UVA引起的HaCaT细胞TRAIL mRNA及蛋白表达(P<0.05,P<0.01);PCF对UVA引起的HaCaT细胞caspase-8的活化有抑制作用,且呈量效关系。结论UVA可增强HaCaT细胞TRAIL表达;TRAIL参与了UVA诱导的HaCaT细胞凋亡;PCF对UVA诱导的HaCaT细胞TRAIL表达有抑制作用,也可减弱UVA诱导的caspase-8活化,以其抗氧化活性抑制TRAIL凋亡通路而发挥抗凋亡作用。     

扇贝多肽经由死亡受体和线粒体通路抑制UVB诱导的HaCaT细胞凋亡

目的从凋亡相关分子Fas(CD95)、半胱天冬酶-3(caspase-3)和活性氧(ROS)、细胞色素C的角度,研究扇贝多肽(polypeptide fromChlamys farreri,PCF)抑制UVB引起的人角质HaCaT细胞凋亡的分子机制。方法实验设计为5组:对照组、UVB模型组、UVB+5.69mmol.L-1PCF组、UVB+2.84mmol.L-1PCF组、UVB+1.42mmol·L-1PCF组。应用小干扰RNA(siRNA)干扰UVB辐射的HaCaT细胞的Fas表达;琼脂糖凝胶电泳分析Fas siRNA及ROS清除剂NAC对细胞凋亡的影响;以DCFH-DA为荧光探针,检测细胞内ROS的含量;免疫印迹法检测细胞色素C及用RNAi干扰降低Fas表达后caspase-3的表达。结果干扰Fas后,UVB辐射的HaCaT细胞凋亡受到明显抑制及caspase-3的表达降低;NAC对UVB诱导的HaCaT细胞凋亡有抑制作用;1.42~5.69mmol.L-1剂量范围内的PCF可剂量依赖性抑制UVB引起的ROS的生成及细胞色素C的释放。结论PCF可抑制UVB诱导的HaCaT细胞凋亡,其作用机制与抑制Fas-caspase-3及ROS-细胞色素C通路有关。     

Molecular mechanisms of polypeptide from Chlamys farreri protecting HaCaT cells from apoptosis induced by UVA plus UVB

Aim： To investigate the mechanism of polypeptide from Chlamysfarreri （PCF） protecting HaCaT cells from apoptosis induced by UVA plus UVB in vitro. Methods： An apoptotic model of UV irradiation-induced HaCaT cells was established. The 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2H-tetrazolium bromide assay, agarose gel electrophoresis, biochemical methods, and Western blotting were employed in the study. Results： PCF inhibited the UV irradiation-induced apoptosis of HaCaT cells. PCF strongly reduced the intracellular reactive oxygen species level, enhanced activities of superoxide dismutase and glutathione per- oxidase and increased the total anti-oxidative capacity in HaCaT cells following UV irradiation. Furthermore, we found that PCF could inhibit the phosphorylation of c-Jun amino-terminal kinase and the activity of caspase-3 in a concentration- dependent manner. Conclusion： PCF protected HaCaT cells from apoptosis induced by UVA plus UVB, mainly through decreasing the intracellular ROS level and increasing the activities of anti-oxidative enzymes to block the ROS-JNK-caspase-3-apoptosis signaling pathway.     

Polypeptide from Chlamys farreri attenuates murine thymocytes damage induced by ultraviolet B

Aim： Polypeptide from Chlamysfarreri （PCF, molecular mass is 879） is a new marine polypeptide compound isolated from Chlamysfarreri. This study investigates the possible protective roles and the mechanism of PCF against ultraviolet B （UVB）-induced apoptosis in murine thymocytes. Methods： The rate of apoptosis and caspase-3 activation was measured by flow cytometry. The expression of stress-response genes c-fos and c-jun was observed by RT-PCR. Western blot analysis was performed to determine the release of cytochrome c. Results： It was found that UVB induced murine thymocyte death. The cells treated with UVB showed an increase in cytochrome c release, caspase-3 activity, as well as in the expression of c-fos and c-jun. In addition, all were involved in UVB-induced cell apoptosis. Conclusion： Our present observations pointed to the ability of PCF to avert UVB-induced apoptosis in thymocytes by modulating c-fos and c-jun expression, cytochrome c release, and the consequent activation of caspase-3, which were essential components of the UV-induced cell apoptotic pathway. The results suggested that PCF is a promising protective substance against UV radiation.     

扇贝多肽在中波紫外线诱导的HaCaT细胞凋亡中对p38MAPK-HSP27通路的影响

目的从p38MAPK-HSP27通路方面研究扇贝多肽(polypeptide from Chlamys farreri,PCF)抑制中波紫外线(UVB)诱导的HaCaT细胞凋亡的作用机制。方法复制UVB诱导的HaCaT细胞凋亡模型;实验设计为6组:对照组、UVB模型组、UVB+5.69mmol.L-1维生素C阳性对照组、UVB+5.69mmol.L-1PCF组、UVB+2.84mmol.L-1PCF组、UVB+1.42mmol.L-1PCF组。PCF预处理30min,荧光染色(Hoechst33258)结合琼脂糖凝胶电泳分析PCF在p38抑制剂(SB203580)存在和不存在两种条件下对UVB诱导的HaCaT细胞凋亡的影响;蛋白质印迹法检测p38MAPK、HSP27的表达及磷酸化水平的改变,同时分析HSP27在细胞内定位的改变。结果PCF可明显抑制UVB诱导的HaCaT细胞凋亡;PCF在1.42~5.69mmol.L-1范围内,可剂量依赖性抑制p38MAPK和HSP27的磷酸化水平,并抑制HSP27由胞质向胞核中的移位。结论PCF通过阻断p38MAPK-HSP27通路来抑制UVB诱导的HaCaT细胞凋亡,其作用机制与抑制p38MAPK的活化从而阻止HSP27的磷酸化及其细胞内移位有关。     

扇贝多肽对UVB损伤HaCaT细胞的抗氧化作用

目的研究扇贝多肽(PCF)对UVB损伤HaCaT细胞的抗氧化作用。方法HaCaT细胞与PCF孵育1h后，接受UVB辐射，继续孵育18h后，采用酶化学法测定细胞超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH—Px)、过氧化氢酶(CAT)活性、以及测定其总抗氧化能力(T—AOC)、丙二醛(MDA)水平。结果PCF能提高HaCaT细胞内抗氧化酶SOD，GSH—Px，CAT活性，增强细胞总抗氧化能力并减少脂质过氧化产物MDA的产生。结论PCF能增加细胞内抗氧化酶活性，抑制脂质过氧化反应，具有抗氧化作用。     

扇贝多肽对紫外线B辐射人真皮成纤维细胞线粒体的保护作用

目的:研究扇贝多肽(PCF)对中波紫外线(UVB)辐射人真皮成纤维细胞线粒体的影响。方法:检测丙二醛(MDA)含量以及细胞内抗氧化酶(SOD、GSH-PX)的活性;流式细胞术测定线粒体膜电位;透射电镜观察细胞超微结构的变化。结果:UVB(1.176×10~(-4)J·cm~(-2))导致真皮成纤维细胞线粒体损伤,PCF(0.25％-1％)剂量依赖性地减轻UVB对线粒体的损伤;而且,PCF也可剂量依赖性地维持线粒体膜电位的相对稳定,PCF能够减少MDA的生成量,提高SOD及GSH-PX的活性,PCF各组与UVB模型组相比差异有显著性(P<0.05,P<0.01)。结论:PCF保护成纤维细胞的线粒体免受UVB的损伤。     

扇贝多肽对UVA损伤HaCaT细胞UCP2表达及线粒体功能的影响

目的探究紫外线A(ultraviolet A,UVA)损伤对HaCaT角质形成细胞线粒体解偶联蛋白2(uncoupling protein2,UCP2)表达的影响以及扇贝多肽(polypeptide from Chlamysfarreri,PCF)的调节作用,并研究PCF对UVA损伤HaCaT细胞线粒体功能的保护作用。方法复制8 J.cm-2 UVA辐射损伤HaCaT角质形成细胞模型,免疫印迹法检测UVA损伤后HaCaT细胞UCP2蛋白表达的变化及PCF的影响、PCF对UVA损伤HaCaT细胞凋亡蛋白酶激活因子(apoptot-ic protease-activating factor 1,Apaf-1)、细胞色素C(Cyto-chrome C,Cyt C)蛋白表达的影响;电子自旋共振(electronspin resonance,ESR)技术检测ROS的释放量;流式细胞术检测线粒体膜电位;紫外分光光度法测定PCF对UVA损伤HaCaT细胞线粒体呼吸链复合酶Ⅰ(NADH-辅酶Q还原酶)活性的影响。结果正常HaCaT细胞UCP2几乎不表达,UVA照射后表达升高,3 h达高峰,6 h开始逐渐下降;1.42～5.69 mmol.L-1剂量范围内的PCF对UCP2的表达有抑制作用;PCF可明显抑制UVA诱导的ROS产生、线粒体膜电位下降、Cyt C的释放及Apaf-1的表达,可有效抑制UVA损伤后HaCaT细胞呼吸链复合酶I活性的下降。结论 PCF对UVA引起的HaCaT细胞线粒体损伤具有保护作用。     

扇贝多肽保护Hela细胞免受紫外线UVA氧化损伤

目的:建立紫外线UVA(辐照强度为3650μJ·cm~(-2)对Hela细胞氧化损伤模型.探究扇贝多肽(PCF)对Hela细胞紫外线UVA氧化损伤的保护作用.方法:MTT法测定细胞活性;酶法测定抗氧化酶(GSH-Px、CAT、SOD)活性;流式细胞仪AnnexinV法测定细胞的凋亡率和死亡率;Fluo-3 AM为荧光染料,流式细胞仪测定细胞内游离Ca~(2 )的含量.结果:PCF(0.5％-2％)能明显增加 Hela细胞的增殖活性和细胞内游离Ca~(2 )的浓度.显著提高Hela细胞GSH-Px、CAT、SOD活性,且呈量效关系.同时降低Hela细胞的凋亡率和死亡率.PCF组与模型对照组比较各项指标均有统计学意义(P< 0.0 5,P<0.01).结论:扇贝多肽具有抗紫外线UVA对Hela细胞氧化损伤的作用.其机制与扇贝多肽提高抗氧化酶含量,抑制脂质过氧化有关.     

扇贝多肽对UVB辐射小鼠胸腺淋巴细胞的保护作用

目的：研究扇贝多肽(polypeptide ftom Chlamys，farreri)对中波紫外线(UVB)辐射损伤小鼠胸腺淋巴细胞的保护作用。方法：UVB辐射小鼠胸腺淋巴细胞，MTT法检测胸腺淋巴细胞的活性；酶生化法检测细胞谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性；流式细胞术检测细胞内活性氧(ROS)的产生量以及细胞凋亡率。结果：PCF能明显减轻UVB辐射对小鼠胸腺淋巴细胞的损伤；提高细胞GSH-Px、SOD和CAT活性；降低细胞中ROS的产生量和细胞凋亡率。结论：PCF对小鼠胸腺淋巴细胞具有明显的辐射保护作用。     

Saponins from the roots of Platycodon grandiflorum suppress ultraviolet A-induced matrix metalloproteinase-1 expression via MAPKs and NF-κB/AP-1-dependent signaling in HaCaT cells

Saponins from the roots of Platycodon grandiflorum (CKS) have been shown to exhibit many pharmacological activities, including anti-cancer and anti-inflammatory activities and antioxidant effects. However, anti-skin photoaging effects of CKS have not yet been reported. In this study, we investigated the protective effects of CKS against UVA damage on immortalized human keratinocytes (HaCaT). We then explored the inhibitory effects of CKS on UVA-induced MMP-1 and investigated the molecular mechanism underlying those effects. CKS increased the cell viability and inhibited reactive oxygen species (ROS) production in HaCaT cells exposed to UVA irradiation. Pre-treatment of HaCaT cells with CKS inhibited UVA-induced production of MMP-1 and MMP-9. In addition, CKS decreased UVA-induced expression of the inflammatory cytokines IL-1β and IL-6. Western blot analysis further revealed that CKS markedly suppressed the enhancement of collagen degradation in UVA-exposed HaCaT cells. CKS also suppressed UVA-induced activation of NF-κB or c-Jun and c-Fos, and the phosphorylation of MAPKs, which are upstream modulators of NF-κB and AP-1.     

扇贝多肽对UVB照射HaCaT细胞后NO释放及热休克蛋白70表达的影响

目的观察UVB诱导的HaCaT细胞一氧化氮(nitricoxide,NO)释放及诱导型一氧化氮合酶(iNOS)表达的动态变化,探究扇贝多肽(polypeptide from Chlamys farreri,PCF)对UVB照射后NO释放、热休克蛋白70(heat shock protein70,HSP70)表达的影响。方法以电子自旋共振(electronspin resonance,ESR)技术检测NO的释放量;蛋白质印迹法检测iNOS、HSP70蛋白表达。结果 20 mJ.cm-2 UVB照射HaCaT细胞后NO释放明显增多,有3、24 h两个释放高峰期;PCF对各时间点NO的释放均有抑制作用;iNOS蛋白表达在UVB照射后6 h开始逐渐升高,18～24 h达到高峰;iN-OS抑制剂可抑制UVB照射后24 h时NO的释放,对3 h时NO的释放无影响;PCF可剂量依赖性增强UVB照射后HSP70的蛋白表达。结论 UVB照射HaCaT细胞诱导iNOS高表达从而引起NO产生增加,但在照射前期也可通过非iNOS途径生成NO;PCF可能通过增强UVB照射后HSP70的表达,进而抑制iNOS蛋白表达、减少NO的生成而保护HaCaT细胞免受UVB辐射损伤。     

扇贝多肽对紫外线B所致人真皮成纤维细胞凋亡和DNA损伤的抑制作用

目的:研究扇贝多肽(PCF)对中波紫外线(UVB)所致体外培养人真皮成纤维细胞的凋亡和DNA损伤的影响。方法:用噻唑兰(MTT)法检测成纤维细胞的存活率;流式细胞术测定细胞的凋亡率和胞浆内游离[Ca~(2+)]_i;彗星电泳法检测单细胞DNA受损的程度。结果:PCF(0.25％-1％)能显著提高UVB(1.176×10~(-4) J·cm~(-2))辐射后人真皮成纤维细胞的增殖活性,降低UVB所致细胞的凋亡率和死亡率,减轻受损的程度,同时可降低胞浆内游离[Ca~(2+)]_i(和UVB模型组相比,P<0.01).结论:PCF对抗UVB所致的光老化是基于其减轻UVB对DNA的损伤和降低UVB所致细胞的凋亡,因此,PCF能够在起始阶段阻止紫外线所致光老化的发展。     

二氢石蒜碱对过氧化氢损伤的PC12细胞的保护作用

目的:探讨二氢石蒜碱(DL)对H2O2诱导的PC12细胞氧化损伤的影响及其可能机制。方法:用H2O2(200μmol.L-1)处理PC12细胞建立氧化应激模型,并加入二氢石蒜碱预处理作为保护。噻唑蓝(MTT)法和乳酸脱氢酶(LDH)检测细胞存活率和细胞损伤程度,利用荧光探针DCFH-DA和JC-1分别检测细胞内活性氧(ROS)和线粒体膜电位。结果:H2O2作用于PC12细胞后,细胞存活率下降,LDH活性和ROS含量增高,线粒体膜电位降低,与正常对照组比较具有显著性差异(P<0.01);10-7～10-5mol.L-1DL预处理后,细胞存活率提高,LDH和ROS变低,线粒体膜电位回升,且在一定范围呈剂量依赖性。结论:DL对H2O2诱导PC12细胞氧化损伤具有保护作用,其作用机制可能与减少ROS产生和稳定线粒体膜电位有关。     

海洋肽的体外抗氧化作用

海洋肽系栉了孔扇贝中提取的多肽,分子量为800～1000。为研究其抗皮肤衰老的作用机制,测定了海洋肽对由邻苯三酚自氧化产生的超氧阴离子、Fenton反应产生羟自由基的清除率,过氧化脂质的最终分解产物丙二醛(MDA),油脂过氧化值(peroxide value,POV)。结果表明,海洋肽(3％,15％)对超氧阴离子的清除率分别为11.7％和41.8％,对羟自由基的清除率分别为8.6％和38.4％,与对照组比较差异有显著性(P<0.01)。同时,海洋肽(0.05％,0.50％)能降低5d和10d的POV和MDA含量(P<0.05,0.01)。实验证明,海洋肽通过有效清除超氧阴离子、羟自由基并抑制脂质过氧化而抗皮肤老化。