海洋生物面膜对小鼠皮肤的抗辐射作用研究

目的 本文探讨中波紫外线（UVB）照射下海洋生物面膜对小鼠皮肤氧化损伤的保护作用和抗氧化酶的关系及其Bcl-2、NOS的表达。方法 建立UVB（辐照强度为5.15×10-2J·cm-2×30d）对昆明种无毛小鼠氧化损伤模型。昆明种无毛小鼠,随机分为双蒸水未照射组和UVB模型组（双蒸水对照组、10％MFP组、10％维生素C组）。电镜观察皮肤组织超微结构。并应用免疫组织化学的方法测定小鼠表皮细胞的Bcl-2蛋白表达、NOS的活性。酶法测定皮肤匀浆上清液中抗氧化酶（GSH-Px、SOD）活性和MDA的含量及总抗氧化能力（T-AOC）。结果 电镜下UVB损伤的对照组小鼠皮肤的表皮细胞胞质内可见空泡形成,真皮的成纤维细胞内可见囊泡状扩张的滑面内质网,粗面内质网等细胞器减少。①MFP组表皮细胞结构正常,成纤维细胞的粗面内质网增多,②MFP可以增强小鼠表皮细胞内Bcl-2蛋白的表达,并抑制NOS的免疫活性;③MFP可提高小鼠皮肤组织的总抗氧化能力、SOD活性,降低MDA含量,与维生素C的抗氧化作用一致。结论 海洋生物面膜与维生素C一样具有抗UVB氧化损伤的作用。其作用机制可能与海洋生物面膜增强Bcl-2的蛋白表达,降低NOS的活性,提高抗氧化酶含量、清除自由基有关。     

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.     

扇贝多肽对长期紫外线A辐射无毛小鼠皮肤所致突变型p53,EGFR和SP表达的抑制作用

探讨局部应用扇贝多肽(PCF)对长期长波紫外线(UVA)辐射无毛小鼠皮肤所致突变型p53,表皮生长因子受体(EGFR)和P物质(SP)表达的影响。建立长期长波紫外线辐射无毛小鼠皮肤模型,免疫组化法测定皮肤组织突变型p53,表皮生长因子受体和P物质的表达。无毛小鼠背部皮肤每天一次应用5％和20％扇贝多肽可显著降低长期长波紫外线辐射(剂量为4556.4 J·cm-2)所致突变型p53,表皮生长因子受体和P物质的过表达。和模型组相比,5％扇贝多肽可分别降低突变型p53,表皮生长因子受体和P物质的表达至86.7％,81.7％和85.2％。20％扇贝多肽几乎可完全对抗长期长波紫外线辐射所致的过表达。扇贝多肽可通过抑制无毛小鼠皮肤中突变型p53,表皮生长因子受体和P物质的过表达,从而可保护皮肤防止光致癌和光老化。     

扇贝多肽对抗UVB辐照所致小鼠胸腺淋巴细胞凋亡的研究

目的研究扇贝多肽(PCF)对中波紫外线(UVB)辐照损伤小鼠胸腺淋巴细胞的保护作用。方法以UVB辐照制备损伤模型,预先给予PCF,应用透视电镜观察细胞超微结构的变化;免疫细胞化学检测细胞p53,Bcl-2,Bax基因蛋白的表达;原位杂交检测细胞P38mRNA的基因表达。结果UVB辐照引起小鼠胸腺淋巴细胞凋亡,PCF能明显减轻UVB辐射损伤引起的细胞超微结构改变。PCF还能抑制突变型p53蛋白和促凋亡蛋白Bax的表达,增强凋亡抑制蛋白Bcl-2的表达以及抑制P38 mRNA基因的表达。结论PCF对UVB辐照的体外小鼠胸腺淋巴细胞具有明显的保护作用。     

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。     

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。     

扇贝多肽经由EGFR-CDK-4通路抑制UVA诱导的HaCaT细胞凋亡

目的建立8J.cm-2紫外线A(UVA)辐射损伤永生化的人角质形成细胞株(HaCaT)细胞的病理模型,经由信号通路的表皮生长因子受体(EGFR),磷脂酰肌醇-3激酶(AKT),细胞周期蛋白(CyclinD1)至周期蛋白依赖激酶4(CDK-4)角度研究扇贝多肽(Polypeptide from Chlamys farreri,PCF)抑制UVA诱导HaCaT细胞凋亡的分子机制。方法采用琼脂糖凝胶电泳检测细胞凋亡;RT-PCR和DNA测序法检测胞内表皮生长因子受体EGFR的mRNA表达及基因变化;蛋白印迹法检测AKT,p-AKT,CyclinD1及CDK-4的蛋白表达水平。结果EGFR抑制剂AG1478和AKT抑制剂PHZ1023均可阻断UVA引起的细胞凋亡;1.42～5.68mmol.L-1范围内的PCF可抑制UVA辐射后细胞内EGFR的表达量。预先加入AG1478和PHZ1023则分别抑制UVA引起的AKT及CyclinD1,CDK-4蛋白水平的表达。结论PCF可以通过阻断EGFR-CDK-4通路来抑制UVA诱导的HaCaT细胞凋亡。     

扇贝多肽对大鼠神经元的保护作用研究

目的:探究扇贝多肽对大鼠大脑中动脉缺血再灌注模型(MCAO)损伤后神经元的保护作用,并探讨其作用机理。方法:Wistar大鼠 64只随机分为 4组:假手术组、扇贝多肽治疗组、蒸馏水组和模型组。利用焦油紫染色观察大鼠脑缺血区神经元;酶法测定脑组织匀浆抗氧化酶(SOD、GSH-Px)的活性和MDA的含量。结果:焦油紫染色观察发现模型组顶叶缺血神经细胞呈重度缺血样变性,而扇贝多肽组则表现为神经元轻度缺血样变性,说明扇贝多肽能减轻顶叶缺血区神经细胞的损伤程度。模型组脑组织匀浆中 SOD的活性(x±s)为 74.65±17.18 Nu·mL~(-1)、GSH-Px活性为 21.29±3.07U,MDA含量为 79.65±15.62;扇贝多肽组脑组织匀浆中SOD的活性(x±s)为120.37±20.35 Nu·mL~(-1)、GSH-Px活性为29.38±2.15U、MDA含量为 39.56±13.19,与模型组比较,扇贝多肽组脑组织匀浆中 SOD、GSH-Px活性显著提高,MDA含量明显降低(P<0.01)。结论:扇贝多肽对大鼠大脑中动脉缺血再灌注损伤后的神经元具有保护作用。其机制与扇贝多肽能提高抗氧化酶含量,抑制脂质过氧化有关。     

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。     

扇贝多肽对紫外线辐照小鼠胸腺淋巴细胞的保护作用

建立体外培养的小鼠胸腺淋巴细胞紫外线（UV）辐射损伤的病理模型，采用MTT法检测细胞活性；酶生化法测定胞浆超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、活性氧（ROS）、丙二醛（MDA）含量以及细胞抗超氧阴离子能力（A-SAC）和总抗氧化能力（T-AOC）；流式细胞仪Rhodamine 123荧光染色测定线粒体膜电位（△ψM）等方法，探扇讨贝多肽（PCF）对小鼠胸腺淋巴细胞辐射损伤的保护作用及机制。结果显示PCF能显著提高小鼠胸腺淋巴细胞的活性；抑制紫外线辐射所致淋巴细胞的损伤，显著提高细胞荣中SOD、GSH-Px的活性，降低ROS，MDA含量，提高A-SAC及T-AOC；稳定线拉体的膜电位；表明扇贝多肽对紫外线辐射损伤的小鼠胸腺淋巴细胞具有保护作用。其作用机制与PCF能清除氧自由基、提高抗氧化酶活性，保护细胞膜组织结构有关。     

Inhibitory effect of polypeptide from Chlamys farreri on UVA-induced apoptosis in human keratinocytes

Polypeptide from Chlamys farreri (PCF, Mr = 879) is a novel marine active product isolated from gonochoric Chinese scallop Chlamys farreri which has been served as sea food for several thousand years. As an octapeptide, PCF consists of 8 amino acids, namely, Pro, Asn, Ser, Thr, Arg, Hyl, Cys, and Gly. PCF had been identified as a marine chemopreventive drug that protected hairless mice's epidermis against UV-induced damage in our previous study. However, the molecular mechanisms that underlie the effect of PCF on ultraviolet A-induced apoptosis in ketatinocytes are not well understood yet. In the present study, PCF was investigated as a potential inhibitory agent for UVA-induced apoptosis in a human keratinocyte cell line, HaCaT. The effects of PCF on UVA-induced generation of ROS and MDA, DNA damage, apoptosis rate were examined. We also investigated whether PCF could inhibit UVA-induced decreasing of mitochondrial membrane potential and the changing of morphology of the cells. We found that, compared with UVA only group, PCF attenuated UVA-induced generation of ROS and MDA, increased the mitochondrial membrane potential, and decreased the apoptosis rate. These results indicate that PCF may protect HaCaT keratinocytes against UVA-induced apoptosis.     

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。     

Effects of ginseng saponins isolated from red ginseng on ultraviolet B-induced skin aging in hairless mice

It is well-known that chronic ultraviolet B (UVB) exposure at low-dose causes skin photoaging including increases in skin thickness and wrinkle formation and reduction in skin elasticity. This study examined the effects of total saponins and ginsenoside Rb₁ isolated from Red Ginseng roots on skin thickness, elasticity, and wrinkle formation caused by long-term, low-dose UVB irradiation in hairless mice. The topical application of total ginseng saponins (10 pg or 100 ng/mouse) and ginsenoside Rb₁ (100 fg, 10 pg, or 1 ng/mouse) significantly inhibited increases in skin thickness and wrinkle formation and the reduction in skin elasticity induced by long-term UVB irradiation. Furthermore, we examined the histological effects of total saponins and ginsenoside Rb₁ in the skin of UVB-irradiated hairless mice. The increases in apoptotic, Ki-67-, and 8-hydroxy-2′-deoxyguanosine-positive cells induced by UVB exposure were prevented by the topical application of total saponins and ginsenoside Rb₁. Furthermore, total saponins and ginsenoside Rb₁ prevented the disruption of collagen fibers induced by the long-term UVB irradiation. Ginsenoside Rb₁ (100 fg, 10 pg, and 1 ng/ml) increased the Bcl-2 expression level in UVB-treated human keratinocytes. The protective effect of ginsenoside Rb₁ on UVB-mediated apoptosis may be due to the up-regulation of Bcl-2 expression. These results suggest that the protective effect of ginsenoside Rb₁ on skin photoaging induced by chronic UVB exposure may be due to the increase in collagen synthesis and/or the inhibition of matrix metalloproteinase expression in dermal fibroblasts.     

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.     

Prevention of the photodamage in the hairless mouse dorsal skin by kojic acid as an iron chelator

Kojic acid, a fungal metabolic product, has been used as a skin-depigmenting agent in skin care products marketed in Japan. Iron in the skin is known to be involved in wrinkling as a result of chronic photodamage. Kojic acid was expected to have anti-wrinkling activity, since it possesses iron-chelating activity. We now evaluated the anti-wrinkling activity of kojic acid by using hairless mice exposed to chronic solar-simulating ultraviolet (UV) irradiation as model animal. At the end of a 20-week irradiation period, topical application of kojic acid before UV irradiation was observed to dramatically prevent: (1) the wrinkling, (2) hyperplasia of the epidermis, (3) fibrosis of the lower dermis, and (4) the increase of extracellular matrix components in the upper dermis. These findings indicate that kojic acid is a typical agent preventing wrinkling of the skin due to chronic photodamage.