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class="articleHeader__authors_author">Başak Kandemir,<sup>3</sup></span> <span class="articleHeader__authors_author">Elif Mertoğlu,<sup>4</sup></span> <span class="articleHeader__authors_author">阿里Karagoz,<sup>1</sup></span> <span class="articleHeader__authors_author">和Nazlı阿尔<sup>1</sup></span> </div> <div class="simpleShowMore"> <button class="simpleShowMore__button">显示更多</button> </div> <div class="articleHeader__academicEditor"> <strong>学术编辑器:</strong> <span>何塞·路易斯·里奥斯</span> </div> <div class="articleHeader__timeline"> <div class="articleHeader__timeline_item "> <strong>收到了</strong> <span>2012年6月14日</span> </div> <div class="articleHeader__timeline_item "> <strong>接受</strong> <span>2012年7月27日</span> </div> <div class="articleHeader__timeline_item articleHeader__timeline_item_sticky"> <strong>发表</strong> <span>2012年9月04</span> </div> </div> </div> <div class="articleBody"> <div class="xml-content"> <h4 class="header" id="abstract">文摘</h4> <p><i>槲寄生专辑</我>l是一种半寄生植物生长在树木和广泛用于治疗许多疾病在传统和补充疗法。众所周知,一些活动<我>槲寄生专辑</我>提取多种多样取决于宿主树木,如抗氧化、凋亡诱导,植物的抗癌活性。本研究的目的是检查比较methanolic提取的影响<我>诉专辑</我>生长在三个不同的主机上树(刺槐、酸橙树和对冲枫树)在H<sub>2</sub>O<sub>2</sub>全身的海拉细胞DNA损伤。在线粒体DNA氧化损伤和两个核区域被QPCR试验评估。这些细胞被使用methanolic提取(10<我>μ</我>g / mL) 48 h,治疗以750紧随其后<我>μ</我>M H<sub>2</sub>O<sub>2</sub>1小时。引起的DNA损伤明显H<sub>2</sub>O<sub>2</sub>虽然它被抑制<我>诉专辑</我>提取物。所有提取完全防止核DNA损伤。而从酸橙树中提取或白色槐树完全抑制线粒体DNA损伤,仅从对冲枫树抑制50%。这些结果表明,methanolic提取的<我>诉专辑</我>可以防止氧化DNA损伤,活动是依赖于宿主树。</p> <span class="end-abs"></span> <h4 id="introduction">1。介绍</h4> <p>活性氧(ROS),源于一系列的细胞过程,外部因素和/或各种疾病会损伤细胞组件(<一个href="#B1">1</一个>]。酶和非酶的抗氧化防御系统是自然的保护者对氧化应激引起的活性氧。然而,这些机制不能完全保护DNA免受损失(<一个href="#B2">2</一个>]。尽管氧化可以修复DNA损伤,未修理的损害可以积累在细胞中。最具戏剧性的结果累积的突变和细胞死亡<一个href="#B3">3</一个>]。因此,氧化DNA损伤是衰老过程的一个重要因素和与年龄相关的疾病,比如癌症(<一个href="#B1">1</一个>,<一个href="#B4">4</一个>,<一个href="#B5">5</一个>]。</p> <p><i>槲寄生专辑</我>l .(槲寄生)是一种半寄生多年生植物,生长在不同的主机树(<一个href="#B6">6</一个>]。不同的<我>槲寄生专辑</我>提取物已被用于传统医学用于治疗各种疾病,如中风,动脉粥样硬化,高血压和糖尿病<一个href="#B7">7</一个>]。这种植物具有许多生物活性如抗癌、抗病毒、抗氧化、凋亡诱导和免疫调节特性(<一个href="#B8">8</一个>- - - - - -<一个href="#B15">15</一个>]。尽管methanolic提取<我>槲寄生专辑</我>叶子能够降低丙二醛(MDA)和减少谷胱甘肽(GSH)含量在肾脏和心脏的糖尿病大鼠体外实验,具有较强的抗氧化活性<一个href="#B11">11</一个>- - - - - -<一个href="#B13">13</一个>),对DNA氧化损伤的影响还没有被研究在海拉细胞。本研究的主要目的是调查是否methanolic提取的<我>槲寄生专辑</我>保护nDNA和mtDNA H<sub>2</sub>O<sub>2</sub>全身的海拉细胞氧化应激。</p> <h4 id="materials-and-methods">2。材料和方法</h4> <h5 id="sec2.1">2.1。植物材料</h5> <p><i>槲寄生专辑</我>l .植物生长在三个不同的主机树木(石灰树(<我>椴树属银膜</我>Desf。前,电视),对冲枫树(<我>宏碁campestre</我>l . ssp。c<我>ampestre,</我>Av)和槐树(<我>洋槐pseudoacacia</我>l,Rv))were harvested from the Northern part of Istanbul in September 2006. The voucher specimen was identified and deposited in the Herbarium of Biology Department, Istanbul University, Istanbul, Turkey (Tv, ISTF 37486; Av, ISTF 37487; Rv, ISTF 37488). Fresh leaves of each sampling were picked and washed by tap water, followed by distilled water. After drying they were cut into small pieces, weighed, and used immediately or stored separately at −20°C until use.</p> <h5 id="sec2.2">2.2。准备的提取</h5> <p>提取准备如前所述[<一个href="#B12">12</一个>]。新鲜的叶子(20 g)浸渍在甲醇(160毫升)在一个潜伏的瓶(150转速/分钟,25°C)为24小时。删除后过滤的植物残体,每个滤液蒸发在真空下干燥,干物质重。原油提取被溶解在DMSO溶液的浓度40毫克/毫升,储存在−20°C到使用。他们指定根据树木的植物收集电视(<我>椴树属槲寄生</我>)、Av (<我>宏碁槲寄生</我>)和房车(<我>洋槐槲寄生</我>)。</p> <h5 id="sec2.3">2.3。细胞培养</h5> <p>人类希拉宫颈癌细胞培养在鹰的最低基本培养基(EMEM)补充10% (v / v) heat-inactivated胎牛血清和antibiotic-antimycotic混合物(青霉素(100 U /毫升),链霉素(100<我>μ</我>0.25 g / mL)、两性霉素B (<我>μ</我>g / mL))。细胞被播种的浓度为10<sup>5</sup>细胞/毫升和维持在37°C在大气中5%的股份有限公司<sub>2</sub>。<我>诉专辑</我>提取物添加到生长介质,在DMSO溶液溶解后最终浓度不超过0.5% (v / v)自DMSO高于此浓度能够抑制细胞生长(数据没有显示)。</p> <h5 id="sec2.4">2.4。细胞毒性测试</h5> <p>提取的细胞毒性活性测试在海拉细胞通过使用3 - (4 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium溴化(MTT)分析基于麻省理工一个有色有色甲产品的减少线粒体脱氢酶,这是活动只有在活细胞(<一个href="#B16">16</一个>]。股票的解决方案提取与EMEM稀释。细胞(10<sup>5</sup>细胞/毫升)被播种到每一个96孔板,包含200<我>μ</我>L EMEM。达到融合后(24小时后),这些细胞被处理浓度增加(1 - 500<我>μ</我>g / mL)<我>诉专辑</我>甲醇提取物稀释与EMEM 48 h。确定细胞毒性的H<sub>2</sub>O<sub>2</sub>细胞治疗与不同浓度(0.05 - 5毫米)的H<sub>2</sub>O<sub>2</sub>稀释和汉克的平衡盐溶液(HBBS) 1 h 72年底h。在下一步中,上层是丢弃。洗后的贴壁细胞磷酸缓冲盐(PBS)的干扰降到最低上层残渣,10<我>μ</我>L (MTT原液(5毫克/毫升)被添加到每个好,和板块进一步孵化4小时37°C。形成的水不溶性紫色甲瓒晶体后,他们于200年解散<我>μ</我>L (DMSO,由此产生的光密度是衡量标(<我>μ</我>定量,<我>BioTek</我>美国Winooski仪器公司,VT)在波长570 nm和690 nm(参考)。可行的细胞的细胞生存能力计算百分比在实验组与对照组治疗使用以下公式:<span class="equation" id="eq1"> <svg height="39.799999" id="M1" preserveaspectratio="xMaxYMin" style="vertical-align:-0pt;width:700.9375px;" version="1.1" viewbox="0 0 700.9375 39.799999" width="700.9375" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,39.8)"> <g transform="translate(72,-40.16)"> <text transform="matrix(1,0,0,-1,107.81,52.46)"> <tspan style="font-size: 12.50px; " x="103.34" y="7.3299999"> </tspan> <tspan style="font-size: 12.50px; " x="0" y="0"> c</tspan> <tspan style="font-size: 12.50px; " x="5.551332" y="0"> e</tspan> <tspan style="font-size: 12.50px; " x="11.102664" y="0"> l</tspan> <tspan style="font-size: 12.50px; " x="14.578498" y="0"> l</tspan> <tspan style="font-size: 12.50px; " x="22.217831" y="0"> v</tspan> <tspan style="font-size: 12.50px; " x="28.469332" y="0"> 我</tspan> <tspan style="font-size: 12.50px; " x="31.945166" y="0"> 一个</tspan> <tspan style="font-size: 12.50px; " x="37.496498" y="0"> b</tspan> <tspan style="font-size: 12.50px; " x="43.747997" y="0"> 我</tspan> <tspan style="font-size: 12.50px; " x="47.223831" y="0"> l</tspan> <tspan style="font-size: 12.50px; " x="50.699665" y="0"> 我</tspan> <tspan style="font-size: 12.50px; " x="54.175499" y="0"> t</tspan> <tspan style="font-size: 12.50px; " x="57.651333" y="0"> y</tspan> <tspan style="font-size: 12.50px; " x="70.154335" y="0"> (</tspan> <tspan style="font-size: 12.50px; " x="74.317833" y="0"> %</tspan> <tspan style="font-size: 12.50px; " x="83.65757" y="0"> )</tspan> <tspan style="font-size: 12.50px; " x="91.296906" y="0"> =</tspan> <tspan style="font-size: 12.50px; " x="118.5" y="-11.04"> O</tspan> <tspan style="font-size: 12.50px; " x="127.52717" y="-11.04"> D</tspan> </text> <text transform="matrix(1,0,0,-1,244.37,60.37)"> <tspan style="font-size: 8.75px; " x="0" y="0"> e</tspan> <tspan style="font-size: 8.75px; " x="3.8858881" y="0"> x</tspan> <tspan style="font-size: 8.75px; " x="8.2618876" y="0"> p</tspan> </text> <g> <path d="m 221.72,55.58 43.29,0" style="fill:none;stroke:#000000;stroke-width:0.82499999;stroke-linecap:butt;stroke-linejoin:miter;stroke-miterlimit:10;stroke-opacity:1;stroke-dasharray:none"></path> </g> <text transform="matrix(1,0,0,-1,221.72,43.71)"> <tspan style="font-size: 12.50px; " x="0" y="0"> O</tspan> <tspan style="font-size: 12.50px; " x="9.0271664" y="0"> D</tspan> </text> <text transform="matrix(1,0,0,-1,239.77,40.58)"> <tspan style="font-size: 8.75px; " x="0" y="0"> c</tspan> <tspan style="font-size: 8.75px; " x="3.8858881" y="0"> o</tspan> <tspan style="font-size: 8.75px; " x="8.2618876" y="0"> n</tspan> <tspan style="font-size: 8.75px; " x="12.637888" y="0"> t</tspan> <tspan style="font-size: 8.75px; " x="15.070944" y="0"> r</tspan> <tspan style="font-size: 8.75px; " x="17.941601" y="0"> o</tspan> <tspan style="font-size: 8.75px; " x="22.3176" y="0"> l</tspan> </text> <text transform="matrix(1,0,0,-1,266.21,45.13)"> <tspan style="font-size: 12.50px; " x="0" y="0"> </tspan> <tspan style="font-size: 12.50px; " x="12.16" y="-7.3299999"> ×</tspan> <tspan style="font-size: 12.50px; " x="22.925083" y="-7.3299999"> 1</tspan> <tspan style="font-size: 12.50px; " x="29.176582" y="-7.3299999"> 0</tspan> <tspan style="font-size: 12.50px; " x="35.428082" y="-7.3299999"> 0</tspan> <tspan style="font-size: 12.50px; " x="41.679585" y="-7.3299999"> 。</tspan> <tspan style="font-size: 12.50px; " x="207.91" y="-7.8200002"> (</tspan> <tspan style="font-size: 12.50px; " x="212.0735" y="-7.8200002"> 1</tspan> <tspan style="font-size: 12.50px; " x="218.325" y="-7.8200002"> )</tspan> </text> </g> </g> </svg></span>欧共体<sub>50</sub>剂量计算海拉细胞从细胞生存能力与methanolic提取的图<我>诉专辑</我>和无毒剂量的植物提取物(10<我>μ</我>g / mL)是用于进一步的实验。</p> <h6 id="sec2.4.1">2.4.1。治疗中提取</h6> <p>海拉细胞(10<sup>5</sup>细胞/毫升)与noncytotoxic剂量的孵化<我>诉专辑</我>提取(10<我>μ</我>g / mL)实验团体或只与EMEM未经处理的控制在过去的48小时内的总孵化时间72小时。</p> <h6 id="sec2.4.2">2.4.2。诱导的氧化应激</h6> <p>氧化应激诱导的H<sub>2</sub>O<sub>2</sub>在50岁<我>μ</我>米和100<我>μ</我>M和H的损耗<sub>2</sub>O<sub>2</sub>在细胞培养中测量。ROS生成增强了与200年治疗的细胞<我>μ</我>M H<sub>2</sub>O<sub>2</sub>。恶劣的应力条件诱导DNA损伤是由750年<我>μ</我>M H<sub>2</sub>O<sub>2</sub>。提取物在治疗后,这些细胞被洗PBS然后治疗H<sub>2</sub>O<sub>2</sub>1 h。汉克的平衡盐溶液(hbs)是用来稀释H<sub>2</sub>O<sub>2</sub>作为一个空白的化验。H<sub>2</sub>O<sub>2</sub>浓度是由吸光度检查在240 nm,如Aebi所述<一个href="#B17">17</一个>]。</p> <p>消耗H<sub>2</sub>O<sub>2</sub>在文化媒体测量10、30和60分钟,显示在图<一个href="//www.newsama.com/journals/ecam/2012/958740/fig2/" target="_blank">2</一个>比色法的选择和Keisari<一个href="#B18">18</一个>]。在这个分析H<sub>2</sub>O<sub>2</sub>酚红排泌量减少而被氧化合的作用。最后的紫色产品展览最大吸收612海里。简单地说,细胞(10<sup>5</sup>细胞/毫升)被播种到每一个,包含200<我>μ</我>L EMEM 96孔板。(负面)控制游离与实验小组并行运行。细胞培养基(100<我>μ</我>包含哈佛商学院+ 50 L)<我>μ</我>米和100<我>μ</我>M H<sub>2</sub>O<sub>2</sub>与200年完全混合吗<我>μ</我>L酚红/辣根过氧化物酶解决方案。的混合是在37°C的环境中5分钟,通过添加2,反应终止<我>μ</我>L 1 N氢氧化钠。H<sub>2</sub>O<sub>2</sub>使用标准曲线的浓度计算H<sub>2</sub>O<sub>2</sub>与吸光度(610海里)。吸光度的标准曲线显示一个线性关系在610 nm和H<sub>2</sub>O<sub>2</sub>在-100年12.5浓度<我>μ</我>米的范围内。</p> <h6 id="sec2.4.3">2.4.3。细胞内ROS水平</h6> <p>细胞内ROS水平估计通过使用荧光探针,2′,7′二氯荧光素二乙酸(DCFH-DA) [<一个href="#B19">19</一个>]。孵化和接触氧化应激后,培养基立即被移除,细胞用PBS孵化15分钟10紧随其后<我>μ</我>M DCFH-DA (100<我>μ</我>L) 37°C在大气中5%的股份有限公司<sub>2</sub>。水解2的荧光,7-dichlorofluorescein (DCF)测量每隔10分钟1 h与485 nm激发荧光计板机/ 530 nm发射波长(FLx800 BioTek仪器公司,Winooski, VT,美国)。活性氧产量的相对百分比计算根据以下方程:<span class="equation" id="eq2"> <svg height="38.575001" id="M2" preserveaspectratio="xMaxYMin" style="vertical-align:-0pt;width:700.9375px;" version="1.1" viewbox="0 0 700.9375 38.575001" width="700.9375" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,38.575)"> <g transform="translate(72,-41.14)"> <text transform="matrix(1,0,0,-1,138.44,53.45)"> <tspan style="font-size: 12.50px; " x="63.43" y="7.3299999"> </tspan> <tspan style="font-size: 12.50px; " x="74" y="-9.1400003"> </tspan> <tspan style="font-size: 12.50px; " x="0" y="0"> R</tspan> <tspan style="font-size: 12.50px; " x="8.026926" y="0"> O</tspan> <tspan style="font-size: 12.50px; " x="17.054092" y="0"> 年代</tspan> <tspan style="font-size: 12.50px; " x="30.244757" y="0"> (</tspan> <tspan style="font-size: 12.50px; " x="34.408257" y="0"> %</tspan> <tspan style="font-size: 12.50px; " x="43.747997" y="0"> )</tspan> <tspan style="font-size: 12.50px; " x="51.387329" y="0"> =</tspan> </text> <text transform="matrix(1,0,0,-1,220.17,59.46)"> <tspan style="font-size: 8.75px; " x="0" y="0"> 1</tspan> </text> <g> <path d="m 212.44,56.57 12.6,0" style="fill:none;stroke:#000000;stroke-width:0.82499999;stroke-linecap:butt;stroke-linejoin:miter;stroke-miterlimit:10;stroke-opacity:1;stroke-dasharray:none"></path> </g> <text transform="matrix(1,0,0,-1,212.44,44.69)"> <tspan style="font-size: 12.50px; " x="0" y="0"> </tspan> </text> <text transform="matrix(1,0,0,-1,220.17,41.57)"> <tspan style="font-size: 8.75px; " x="0" y="0"> 0</tspan> </text> <text transform="matrix(1,0,0,-1,226.24,46.12)"> <tspan style="font-size: 12.50px; " x="0" y="0"> </tspan> <tspan style="font-size: 12.50px; " x="12.15" y="-7.3299999"> ×</tspan> <tspan style="font-size: 12.50px; " x="22.927586" y="-7.3299999"> 1</tspan> <tspan style="font-size: 12.50px; " x="29.179087" y="-7.3299999"> 0</tspan> <tspan style="font-size: 12.50px; " x="35.430588" y="-7.3299999"> 0</tspan> <tspan style="font-size: 12.50px; " x="41.682087" y="-7.3299999"> %</tspan> <tspan style="font-size: 12.50px; " x="51.021828" y="-7.3299999"> ,</tspan> <tspan style="font-size: 12.50px; " x="247.88" y="-7.3299999"> (</tspan> <tspan style="font-size: 12.50px; " x="252.0435" y="-7.3299999"> 2</tspan> <tspan style="font-size: 12.50px; " x="258.29501" y="-7.3299999"> )</tspan> </text> </g> </g> </svg></span>F<sub>0</sub>是未经处理的对照组和F荧光强度<sub>1</sub>实验组的荧光强度。</p> <h5 id="sec2.5">2.5。DNA隔离</h5> <p>高分子量细胞DNA总量与GenElute孤立的哺乳动物基因组DNA隔离设备(σ,圣路易斯,密苏里州),根据制造商的协议。总细胞DNA的浓度取决于使用Quant-iT dsDNA高灵敏度分析工具在一个量子位荧光计(表达载体,佩斯利,英国)。</p> <h5 id="sec2.6">2.6。定量聚合酶链反应(QPCR)</h5> <p>QPCR根据的方法进行Erol et al。<一个href="#B20">20.</一个>]。分析了三种不同的基因组区域的氧化损失。寡核苷酸引物2082个基点片段的具体核APEX1基因(转录、基因库ID: X66133) [<一个href="#B21">21</一个>),2334个基点片段核<我>β</我>球蛋白基因簇(nontranscribed基因库ID: NG_000007),和一个2232个基点mtDNA的片段<b>(</b>基因库ID: AF347015)被用于PCR反应<一个href="#B20">20.</一个>]。所有PCR反应进行Techne tc - 3000年Thermocycler (Techne Inc .)、美国)。反应混合物的总量是50<我>μ</我>包含100 ng的DNA, L, 1 x缓冲区(Fermentas), 0.2毫米核苷酸,1 - 2.5毫米MgCl<sub>2</sub>,0.3<我>μ</我>引物,和2.5单位重组Taq DNA聚合酶(Fermentas)。定量控制使用的一半控制模板DNA的浓度是包含在每组PCR反应。小片段,线粒体DNA的161个基点和181个基点核区域,也放大了内部控制规范化的结果大片段和监控线粒体拷贝数。电泳的整除的PCR检查产品在1 - 2% (w / v)垂直琼脂糖凝胶在1 70 v 45分钟xtae缓冲区(Tris-acetate-EDTA, pH值8.0)。QPCR产品的数量是量化使用Quant-iT dsDNA高灵敏度分析工具。损伤频率平均每片段计算使用<我>泊松</我>方程(<一个href="#B22">22</一个>]。</p> <h5 id="sec2.7">2.7。统计分析</h5> <p>数据表示为三个独立实验的均值±SEM在一式三份完成。所有分析都使用GraphPad棱镜,5.00版本Windows, GraphPad软件公司,圣地亚哥,<一个href="http://www.graphpad.com/" target="_blank">http://www.graphpad.com/</一个>。统计分析使用单向方差分析Dunnett紧随其后的测试。两组之间的差异与H的消耗动力学的关系<sub>2</sub>O<sub>2</sub>评估使用双尾未配对吗<svg height="9.125" id="M3" style="vertical-align:-0.11285pt;width:5.0124998px;" version="1.1" viewbox="0 0 5.0124998 9.125" width="5.0124998" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,9.125)"> <g transform="translate(72,-64.7)"> <text transform="matrix(1,0,0,-1,-71.95,64.86)"> <tspan style="font-size: 12.50px; " x="0" y="0"> </tspan> </text> </g> </g> </svg>以及。的概率值<svg height="11.25" id="M4" style="vertical-align:-0.30096pt;width:56.150002px;" version="1.1" viewbox="0 0 56.150002 11.25" width="56.150002" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,11.25)"> <g transform="translate(72,-63)"> <text transform="matrix(1,0,0,-1,-71.95,63.35)"> <tspan style="font-size: 12.50px; " x="0" y="0"> </tspan> <tspan style="font-size: 12.50px; " x="10.890113" y="0"> <</tspan> <tspan style="font-size: 12.50px; " x="22.930502" y="0"> 0</tspan> <tspan style="font-size: 12.50px; " x="29.182001" y="0"> 。</tspan> <tspan style="font-size: 12.50px; " x="32.307751" y="0"> 0</tspan> <tspan style="font-size: 12.50px; " x="38.559254" y="0"> 5</tspan> </text> </g> </g> </svg>被认为是重要的。</p> <h4 id="results">3所示。结果</h4> <p>所有提取和H<sub>2</sub>O<sub>2</sub>海拉细胞的生存能力降低剂量依赖性的方式(图<一个href="//www.newsama.com/journals/ecam/2012/958740/fig1/" target="_blank">1</一个>)。如图<一个href="//www.newsama.com/journals/ecam/2012/958740/fig1/" target="_blank">1</一个>半最大抑制浓度(IC<sub>50</sub>)电视、Av、房车和H<sub>2</sub>O<sub>2</sub>是93<我>μ</我>g / mL, 165<我>μ</我>g / mL, 85<我>μ</我>克/毫升和810<我>μ</我>M,分别。所有提取的noncytotoxic剂量(10<我>μ</我>g / mL)被用于进一步的实验为了避免细胞死亡的细胞毒性。氧化应激引起了50<我>μ</我>米和100<我>μ</我>M H<sub>2</sub>O<sub>2</sub>60分钟内,其损耗被监控检查剩下的H<sub>2</sub>O<sub>2</sub>在文化媒体。没有明显差异的消耗动力学50<我>μ</我>米和100<我>μ</我>M H<sub>2</sub>O<sub>2</sub>细胞的处理提取(图<一个href="//www.newsama.com/journals/ecam/2012/958740/fig2/" target="_blank">2</一个>)。几乎80%的H<sub>2</sub>O<sub>2</sub>在文化媒体在1小时内被海拉细胞耗尽,而H<sub>2</sub>O<sub>2</sub>颗粒培养基保持稳定的水平。这些结果表明,H<sub>2</sub>O<sub>2</sub>不与组件的交互中,消耗H<sub>2</sub>O<sub>2</sub>通过细胞代谢发生(<一个href="#B23">23</一个>]。</p> <div class="partial-carousel-wrapper"> <div class="floats-partial-section"> <div class="floats-partial-content"> <div class="partial-carousel--full-image partial-carousel-55b132ecdc3bec65f868f94978a2f1ad"> <div class="partial-carousel--single-image"> <img loading="lazy" alt="(一)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/figures/958740.fig.001a.jpg"> <br> <strong>(一)</strong> </div> <div class="partial-carousel--single-image"> <img loading="lazy" alt="(b)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/figures/958740.fig.001b.jpg"> <br> <strong>(b)</strong> </div> </div> </div> <div class="floats-partial-middle"> <div class="floats-partial-middle--thumbs"> <div class="partial-carousel-dots-55b132ecdc3bec65f868f94978a2f1ad partial-carousel-thumbnail"> <button aria-label="Slide to (a) " class="partial-carousel-dot"><img loading="lazy" alt="(一)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/thumbnails/958740.fig.001a_th.jpg"><br><strong>(一)</strong></button> <button aria-label="Slide to (b) " class="partial-carousel-dot"><img loading="lazy" alt="(b)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/thumbnails/958740.fig.001b_th.jpg"><br><strong>(b)</strong></button> </div> </div> <div class="floats-partial-middle--nav"> <div class="partial-carousel-next-prev-55b132ecdc3bec65f868f94978a2f1ad partial-carousel-next-prev"> <button aria-label="Previous" class="carousel-prev"></button> <button aria-label="Next" class="carousel-next"></button> </div> </div> </div> <div class="floats-partial-footer"> <div class="floats-partial-caption"> <span class="caption-text">图1</span> <a aria-label="View full page" class="caption-partial-url" href="//www.newsama.com/journals/ecam/2012/958740/fig1/" title="" target="_blank"></a> </div> <div class="floats-partial-caption-text"> <table> <tbody> <tr> <td>(一)methanolic提取效果<我>槲寄生专辑</我>从不同的主机在海拉细胞生存能力,(b)的细胞毒性效应<sub>2</sub>O<sub>2</sub>。</td> </tr> </tbody> </table> </div> </div> </div> </div> <div class="partial-carousel-wrapper"> <div class="floats-partial-section"> <div class="floats-partial-content"> <div class="partial-carousel--full-image partial-carousel-68e0b4c7efdfb7be3c3e3d8df8ae2f07"> <div class="partial-carousel--single-image"> <img loading="lazy" alt="(一)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/figures/958740.fig.002a.jpg"> <br> <strong>(一)</strong> </div> <div class="partial-carousel--single-image"> <img loading="lazy" alt="(b)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/figures/958740.fig.002b.jpg"> <br> <strong>(b)</strong> </div> </div> </div> <div class="floats-partial-middle"> <div class="floats-partial-middle--thumbs"> <div class="partial-carousel-dots-68e0b4c7efdfb7be3c3e3d8df8ae2f07 partial-carousel-thumbnail"> <button aria-label="Slide to (a) " class="partial-carousel-dot"><img loading="lazy" alt="(一)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/thumbnails/958740.fig.002a_th.jpg"><br><strong>(一)</strong></button> <button aria-label="Slide to (b) " class="partial-carousel-dot"><img loading="lazy" alt="(b)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/thumbnails/958740.fig.002b_th.jpg"><br><strong>(b)</strong></button> </div> </div> <div class="floats-partial-middle--nav"> <div class="partial-carousel-next-prev-68e0b4c7efdfb7be3c3e3d8df8ae2f07 partial-carousel-next-prev"> <button aria-label="Previous" class="carousel-prev"></button> <button aria-label="Next" class="carousel-next"></button> </div> </div> </div> <div class="floats-partial-footer"> <div class="floats-partial-caption"> <span class="caption-text">图2</span> <a aria-label="View full page" class="caption-partial-url" href="//www.newsama.com/journals/ecam/2012/958740/fig2/" title="" target="_blank"></a> </div> <div class="floats-partial-caption-text"> <table> <tbody> <tr> <td>预处理的效果与<我>槲寄生专辑</我>提取的消耗动力学(a) 50<我>μ</我>100 M和(b)<我>μ</我>M H<sub>2</sub>O<sub>2</sub>在文化媒体。</td> </tr> </tbody> </table> </div> </div> </div> </div> <p>与200年的孵化海拉细胞<我>μ</我>M H<sub>2</sub>O<sub>2</sub>增加活性氧的水平。细胞内活性氧产量增加了19.2%,H<sub>2</sub>O<sub>2</sub>治疗细胞与未经处理的控制细胞相比(表<一个href="//www.newsama.com/journals/ecam/2012/958740/tab1/" target="_blank">1</一个>)。<我>诉专辑</我>methanolic提取物对细胞内ROS的稳态水平没有影响。Av ROS最高比200年抑制活动<我>μ</我>M H<sub>2</sub>O<sub>2</sub>处理细胞。另外,其他两个提取物(电视和Rv)被发现有显著的抑制ROS生成后氧化应激诱导的影响(表<一个href="//www.newsama.com/journals/ecam/2012/958740/tab1/" target="_blank">1</一个>)利用没食子酸(GA)作为对照品。</p> <div class="floats-partial-section partial-other-wrapper" data-section="tab1"> <div class="floats-partial-footer"> <div class="floats-partial-caption"> <span class="caption-text">表1</span> <a aria-label="View full page" class="caption-partial-url" href="//www.newsama.com/journals/ecam/2012/958740/tab1/" title="" target="_blank"></a> </div> <div class="floats-partial-caption-text"> 预处理的效果与<我>槲寄生专辑</我>在细胞内ROS生成不同的主机。</div> <a class="table-thumb-overlay" href="//www.newsama.com/journals/ecam/2012/958740/tab1" target="_blank" aria-label="View full page"></a> </div> </div> <p></p> <p>的gene-specific QPCR试验进行核(APEX1和区域<我>β</我>球蛋白)和线粒体DNA氧化损伤的检测造成的H<sub>2</sub>O<sub>2</sub>。分析是基于这样的事实,许多DNA损伤可以阻止聚合酶,因此,产品放大却降低了(<一个href="#B24">24</一个>]。当不同H<sub>2</sub>O<sub>2</sub>浓度(300年和750年<我>μ</我>米)在海拉细胞应用于确定损伤的频率,这是发现在APEX1和mtDNA地区同样感应到300年<我>μ</我>M H<sub>2</sub>O<sub>2</sub>,而在<我>β</我>球蛋白地区没有明显的感应被认为相对于控制(未发表的数据)。因此,为了确定植物提取物对细胞的保护作用,只有750<我>μ</我>M H<sub>2</sub>O<sub>2</sub>已经被应用。在这项研究中,每10 kb损伤数字核和线粒体DNA在海拉细胞孵化有或没有<我>槲寄生</我>在相应的条件下(图提取非常低<一个href="//www.newsama.com/journals/ecam/2012/958740/fig3/" target="_blank">3</一个>)。一个负值的电视<我>β</我>球蛋白地区显示病变频率远远低于其他的(<svg height="11.25" id="M12" style="vertical-align:-0.30096pt;width:56.150002px;" version="1.1" viewbox="0 0 56.150002 11.25" width="56.150002" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,11.25)"> <g transform="translate(72,-63)"> <text transform="matrix(1,0,0,-1,-71.95,63.35)"> <tspan style="font-size: 12.50px; " x="0" y="0"> </tspan> <tspan style="font-size: 12.50px; " x="10.890113" y="0"> <</tspan> <tspan style="font-size: 12.50px; " x="22.930502" y="0"> 0</tspan> <tspan style="font-size: 12.50px; " x="29.182001" y="0"> 。</tspan> <tspan style="font-size: 12.50px; " x="32.307751" y="0"> 0</tspan> <tspan style="font-size: 12.50px; " x="38.559254" y="0"> 5</tspan> </text> </g> </g> </svg>)。然而在所有地区检测到相当大的损害,在750年控制细胞治疗<我>μ</我>M H<sub>2</sub>O<sub>2</sub>。病变mtDNA频率比nDNA高2倍。正如所料,mtDNA是最敏感的,到750年,氧化应激诱导<我>μ</我>M H<sub>2</sub>O<sub>2</sub>。它包含每10 kb片段3.2病变,而转录(APEX1)和nontranscribed (<我>β</我>球蛋白)nDNAs少受损(1.6和1.7每10 kb病变,职责)。在实验小组methanolic提取<我>槲寄生专辑</我>生长在不同的宿主树木表现出不同程度的抗氧化DNA损伤的作用。预处理与Rv和电视提取完全防止损伤应力条件下核和线粒体DNA。然而Av显示只有~ 50%抑制对mtDNA损伤,而这完全是有效对抗nDNA损伤。</p> <div class="partial-carousel-wrapper"> <div class="floats-partial-section"> <div class="floats-partial-content"> <div class="partial-carousel--full-image partial-carousel-ce5098621942ef70351c6296e84840f6"> <div class="partial-carousel--single-image"> <img loading="lazy" alt="(一)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/figures/958740.fig.003a.jpg"> <br> <strong>(一)</strong> </div> <div class="partial-carousel--single-image"> <img loading="lazy" alt="(b)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/figures/958740.fig.003b.jpg"> <br> <strong>(b)</strong> </div> <div class="partial-carousel--single-image"> <img loading="lazy" alt="(c)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/figures/958740.fig.003c.jpg"> <br> <strong>(c)</strong> </div> </div> </div> <div class="floats-partial-middle"> <div class="floats-partial-middle--thumbs"> <div class="partial-carousel-dots-ce5098621942ef70351c6296e84840f6 partial-carousel-thumbnail"> <button aria-label="Slide to (a) " class="partial-carousel-dot"><img loading="lazy" alt="(一)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/thumbnails/958740.fig.003a_th.jpg"><br><strong>(一)</strong></button> <button aria-label="Slide to (b) " class="partial-carousel-dot"><img loading="lazy" alt="(b)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/thumbnails/958740.fig.003b_th.jpg"><br><strong>(b)</strong></button> <button aria-label="Slide to (c) " class="partial-carousel-dot"><img loading="lazy" alt="(c)" src="https://static.hindawi.com/articles/ecam/volume-2012/958740/thumbnails/958740.fig.003c_th.jpg"><br><strong>(c)</strong></button> </div> </div> <div class="floats-partial-middle--nav"> <div class="partial-carousel-next-prev-ce5098621942ef70351c6296e84840f6 partial-carousel-next-prev"> <button aria-label="Previous" class="carousel-prev"></button> <button aria-label="Next" class="carousel-next"></button> </div> </div> </div> <div class="floats-partial-footer"> <div class="floats-partial-caption"> <span class="caption-text">图3</span> <a aria-label="View full page" class="caption-partial-url" href="//www.newsama.com/journals/ecam/2012/958740/fig3/" title="" target="_blank"></a> </div> <div class="floats-partial-caption-text"> <table> <tbody> <tr> <td>的影响<我>槲寄生专辑</我>提取和/或H<sub>2</sub>O<sub>2</sub>(一)APEX1基因;(b)<我>β</我>球蛋白;(c) mtDNA海拉细胞。C:未经处理的控制细胞;电视、Av房车:细胞处理10<我>μ</我>相应的提取48 h g / ml。<svg height="11.9" id="M15" style="vertical-align:-0.30096pt" version="1.1" viewbox="0 0 68.212502 11.9" width="68.212502" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,11.9)"> <g transform="translate(72,-62.48)"> <text transform="matrix(1,0,0,-1,-71.95,67.83)"> <tspan style="font-size: 8.75px; " x="0" y="0"> ∗</tspan> <tspan style="font-size: 8.75px; " x="4.5772958" y="0"> ∗</tspan> </text> <text transform="matrix(1,0,0,-1,-62.3,62.83)"> <tspan style="font-size: 12.50px; " x="0" y="0"> </tspan> <tspan style="font-size: 12.50px; " x="10.890113" y="0"> <</tspan> <tspan style="font-size: 12.50px; " x="22.930502" y="0"> 0</tspan> <tspan style="font-size: 12.50px; " x="29.182001" y="0"> 。</tspan> <tspan style="font-size: 12.50px; " x="32.307751" y="0"> 0</tspan> <tspan style="font-size: 12.50px; " x="38.559254" y="0"> 1</tspan> </text> </g> </g> </svg>;平均值明显不同于未经处理的控制。</td> </tr> </tbody> </table> </div> </div> </div> </div> <h4 id="discussion">4所示。讨论</h4> <p>氧化应激是形成活性氧和抗氧化防御之间的不平衡,导致潜在的细胞损伤(<一个href="#B3">3</一个>]。衰老和一些疾病如癌症、糖尿病和退行性疾病与氧化应激引起的活性氧(<一个href="#B1">1</一个>,<一个href="#B25">25</一个>]。在活细胞活性氧的主要来源是氧化DNA损伤在生理和增加氧化应激条件下(<一个href="#B25">25</一个>]。真核细胞保护酶等抗氧化防御机制,激进的食腐动物,氢捐助者、电子给体,过氧化的分解者,单线态氧饮料、酶抑制剂、增效剂、金属络合剂(<一个href="#B26">26</一个>]。抗氧化剂也可能修复氧化损伤或增强抗氧化防御通过诱导二期酶或刺激线粒体生物起源(<一个href="#B27">27</一个>]。因此,DNA、蛋白质和脂质成分的细胞受到抗氧化剂对氧化损伤的保护。植物含有多种抗氧化成分,以不同的方式,在传统医学使用。根据生物活性的不同,植物提取物以及许多化合物分离是广泛应用于制药行业作为一个成分药物或化妆品;其中一些能够防止分子损伤(<一个href="#B28">28</一个>]。例如,不同的酚醛树脂化合物和抗氧化剂减少氧化DNA损伤在不同细胞类型(<一个href="#B20">20.</一个>,<一个href="#B29">29日</一个>,<一个href="#B30">30.</一个>]。</p> <p>本研究的目的是评估的保护作用<我>槲寄生专辑</我>methanolic提取物对海拉细胞的核和线粒体DNA损伤。<我>在体外</我>抗氧化活性的提取物(早些时候报道<一个href="#B12">12</一个>,<一个href="#B13">13</一个>]。在我们先前的研究的组成和活动<我>槲寄生专辑</我>methanolic提取发现依赖寄主树的时间收获(<一个href="#B12">12</一个>]。本研究首先显示<我>槲寄生专辑</我>methanolic提取物能够抑制活性氧的形成引起的H<sub>2</sub>O<sub>2</sub>在海拉细胞。在动物研究也获得了类似的调查结果报告的保护作用<我>槲寄生专辑</我>对氧化应激在糖尿病大鼠的肾脏和心脏<一个href="#B11">11</一个>]。因此,建议<我>诉专辑</我>可以保护活细胞的抑制活性氧的形成。令人惊讶的是,实验剂量的<我>诉专辑</我>(10<我>μ</我>g / mL)用于测定并没有改变基底的水平损害nDNA和mtDNA在没有感应的压力(图<一个href="//www.newsama.com/journals/ecam/2012/958740/fig3/" target="_blank">3</一个>)。这个结果可能表明,在生理条件下无法觉察的损伤发生。我们还发现,线粒体基因组显著(<svg height="11.0625" id="M14" style="vertical-align:-0.30096pt;width:63.962502px;" version="1.1" viewbox="0 0 63.962502 11.0625" width="63.962502" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,11.0625)"> <g transform="translate(72,-63.15)"> <text transform="matrix(1,0,0,-1,-71.95,63.5)"> <tspan style="font-size: 12.50px; " x="0" y="0"> </tspan> <tspan style="font-size: 12.50px; " x="10.890113" y="0"> <</tspan> <tspan style="font-size: 12.50px; " x="22.930502" y="0"> 0</tspan> <tspan style="font-size: 12.50px; " x="29.182001" y="0"> 。</tspan> <tspan style="font-size: 12.50px; " x="32.307751" y="0"> 0</tspan> <tspan style="font-size: 12.50px; " x="38.559254" y="0"> 0</tspan> <tspan style="font-size: 12.50px; " x="44.810753" y="0"> 1</tspan> </text> </g> </g> </svg>对H)更敏感<sub>2</sub>O<sub>2</sub>比这两个核位点(图全身的氧化应激<一个href="//www.newsama.com/journals/ecam/2012/958740/fig3/" target="_blank">3</一个>)。先前的研究表明,mtDNA比nDNA[更容易氧化的代理<一个href="#B23">23</一个>,<一个href="#B31">31日</一个>]。mtDNA易感性的氧化损伤是由于链增长反应或从呼吸链电子泄漏(<一个href="#B32">32</一个>]。mtDNA没有内含子的转录率高,提供一个高概率的氧化修饰表达区域(<一个href="#B33">33</一个>]。在线粒体也局部金属离子的存在可能对ROS的生成函数作为催化剂和二级ROS的刺激反应由于损坏等和/或通过脂质过氧化<一个href="#B23">23</一个>]。</p> <p>此外,我们证明<我>槲寄生专辑</我>methanolic提取物能够防止氢引起的DNA损伤<sub>2</sub>O<sub>2</sub>。据预测,几千每天每细胞DNA损伤发生在人类不同的压力因素(<一个href="#B34">34</一个>]。如果没有有效的DNA修复和/或细胞抗氧化防御机制,这在DNA损伤积累(特别是mtDNA),这可能导致与年龄相关的疾病。DNA损伤肿瘤抑制基因和致癌基因也可能是癌症的一个可能原因(<一个href="#B3">3</一个>- - - - - -<一个href="#B5">5</一个>]。因此尤其重要<我>槲寄生专辑</我>可以保护mtDNA,一个重要的角色在衰老和APEX1基因(积极转录DNA修复酶)对这种积累通过抑制氧化DNA损伤。然而,DNA损伤预防和相关性不显著抑制ROS的,暗示的抗氧化潜力<我>槲寄生专辑</我>提取是由一个复杂的协同作用和不同机制的活跃分子,不仅抑制ROS。</p> <p>植物的抗氧化活性与生物活性的化合物,主要是抗氧化剂的酚醛树脂,由于其清除自由基的能力。在<我>诉专辑</我>methanolic提取物,槲皮素含量测定为4.92<我>μ</我>3.92 g / g粗提物的电视<我>μ</我>Av g / g粗提取液,7.97<我>μ</我>g / g粗提取液分别为房车(未公开的数据)。因此,槲皮素含量高<我>诉专辑</我>提取可能是负责保护海拉细胞的DNA损伤。</p> <p>总之,综上所述这些结果表明methanolic提取的<我>槲寄生专辑</我>可以通过直接抑制ROS防止DNA损伤形成和/或间接通过其他机制,通过诱导的氧化损伤修复和二期的酶。因此,饮食摄入<我>槲寄生专辑</我>提取的风险可以降低氧化stress-mediated某些癌症等疾病,糖尿病并发症,退化和胃肠道疾病,或通过减少氧化DNA损伤和动脉粥样硬化细胞内ROS水平。还需要进一步的研究,以确定可能的有益成果的膳食使用和识别机制的行动。</p> <h4 id="abbreviations">缩写</h4> <table class="gloss-abbr"> <tbody> <tr> <td align="left">Av:</td> <td align="left">生长在宏碁campestre槲寄生专辑</td> </tr> <tr> <td align="left">遗传算法:</td> <td align="left">没食子酸</td> </tr> <tr> <td align="left">GAE:</td> <td align="left">没食子酸当量</td> </tr> <tr> <td align="left">哈佛商学院:</td> <td align="left">汉克的平衡盐溶液</td> </tr> <tr> <td align="left">mtDNA:</td> <td align="left">线粒体DNA</td> </tr> <tr> <td align="left">nDNA:</td> <td align="left">核DNA</td> </tr> <tr> <td align="left">QPCR:</td> <td align="left">定量聚合酶链反应</td> </tr> <tr> <td align="left">房车:</td> <td align="left"><i>槲寄生专辑</我>生长在<我>洋槐pseudoacacia</我></td> </tr> <tr> <td align="left">电视:</td> <td align="left"><i>槲寄生专辑</我>生长在<我>椴树属银膜</我>。</td> </tr> </tbody> </table> <h4 id="conflict-of-interests">利益冲突</h4> <p>作者宣称没有利益冲突。</p> <h4 id="acknowledgment">承认</h4> <p>这项研究得到了伊斯坦布尔大学研究基金会(项目没有。1462)。</p> </div> </div> <div class="ArticleReferences_xmlContent__p_40j"> <h4 class="ArticleReferences_references__GH2t_" id="references">引用</h4> <ol class="ArticleReferences_orderedReferences__mJr9M"> <li class="ArticleReferences_articleReference__ouEuh" 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