【作者】 姚晖；

【导师】 邓虹珠；

【作者基本信息】 南方医科大学 ， 中药学， 2010， 博士

【摘要】 衰老是从生殖成熟后才开始或加速的,它是具有累积性、普遍性、渐进性和内生性的生命过程，是所有生物的共同特征。机体要经过生长发育期、生殖期、衰老期的生命过程,不同物种在寿限决定上有相似机制。进入21世纪后,人类社会面临着人口老龄化的严峻挑战。2007年联合国经济和社会事务部发表了《2007年世界经济和社会调查报告》。报告显示,由于人口出生率下降和寿命增加,全球大多数国家正迅速进入老龄化社会,2005年-2050年,世界人口增加的一半为60岁以上的老年人,80岁以上人口将从9千万增加到4亿,各种老年病也随之而来,因此,如何延缓衰老已成为世界各国学者研究的热点。可见,对衰老和抗衰老领域的研究以及抗衰老药物的开发对于提高人类的生命质量是至关重要的。哈蟆油(Oviductus Ranae, OR)为蛙科动物中国林蛙(Rana temporaria chensinesis David)雌蛙的输卵管,经采制干燥而得。具有补肾益精、养阴润肺的功能。用于阴虚体弱、神疲乏力、心悸失眠、盗汗不止、痨嗽咳血。主要成分为蛋白质及氨基酸、脂肪酸(饱和和不饱和)、甾体激素、无机元素、维生素类等多种。目前已发现OR能够显著延长果蝇的平均寿命和最高寿命,其机制为升高超氧化物歧化酶(SOD)的活性,降低过氧化脂质的生成。除抗衰老作用外,OR还具有抗疲劳、改善内分泌失调、调节神经免疫、脂肪代谢等方面的作用。近年来课题组采用D-半乳糖致衰老小鼠模型,发现OR具有升高衰老模型小鼠生殖器官SOD活力,抑制自由基在生殖器官的堆积,防止细胞老化及组织器官的退行性改变的作用,提高雌性衰老模型小鼠体内雌激素水平,延长小鼠动情期,增加其卵巢和子宫指数,改善卵巢和子宫的病理改变,从而改善生殖器官的衰萎状况。已开发出以OR为君药,治疗妇女更年期综合征(Climacteric Syndrome,CS)的中药制剂益妇灵胶囊(YFL),动物实验表明YFL能显著提高体内雌激素水平,显著升高子宫指数、肾上腺指数,改善子宫内膜层萎缩性病变,增加腺体数目和分泌活动,子宫壁增厚。其作为医院制剂(许可证号：XZ20010003)在临床应用多年,取得良好的治疗效果。同时也研发了组方以OR为主,人参、当归为辅的中药复方坤丽片(国食健字G20090379,东方药林牌坤丽片),已通过技术审评即将获得生产批文,通过人体试验证明有明显的祛黄褐斑作用。并已取得国家发明专利：“治疗更年期综合症的药物及其制造方法(专利号：ZL01107471.X)”。在众多衰老学说中,以自由基学说最受重视,支持这一理论的实验证据也最为丰富。其他衰老学说虽有各自的立论依据及长处,但只有自由基学说可以将许多学说联系起来,成为诸学说的中心。近50年的理论和实验证实,自由基学说是最有说服力的衰老学说之一。而近年来研究表明衰老作为一种正常而复杂的生命过程,涉及机体的不同组织、细胞的一系列特定程序性变化。众多研究表明,细胞衰老作为衰老的基本过程是借助于信号传导途径实现的,许多细胞因子亦参与到这些信号途径中。其中最经典的细胞衰老途径为P16INK4a-Rb和p53-p21WAF1/CIP1-Rb途径,当这两条途径所涉及的关键调控因子,如p16,p21,细胞周期蛋白(cyclin),细胞周期蛋白激酶(CDK),视网膜母细胞瘤蛋白Rb等发生改变,细胞将延缓衰老或绕过衰老程序继续增殖。目前已发现这两条衰老途径中p16或p21过高表达均可使衰老细胞中活性氧自由基(reactive oxygen species, ROS)水平上升和积聚而诱导衰老,其中Rb蛋白表达(即Rb蛋白磷酸化)以及其它调控因子的变化,如Rb的调控因子cyclin和CDK,起着关键作用。前期研究和对OR近20年文献分析发现,哈蟆油延缓衰老作用机制主要与抗ROS作用相关,因此OR抑制ROS积累延缓衰老的同时,是否是也通过调控这两条细胞衰老途径过程中的关键因子的表达变化,使细胞增殖而达到延缓衰老的作用以及OR提高机体抗氧化能力与上述调控因子表达的是否具有相关性还未见报道。对此利用分子生物学手段探讨OR对上述细胞衰老途径中关键因子的影响,可进一步揭示OR延缓衰老机制。本研究采用D-半乳糖致亚急性衰老的大鼠模型,以细胞衰老信号转导途径p21-Rb和p16-Rb中涉及的关键调节因子p21、p16、cyclinD1、cyclinD3、CDK6及pRb(磷酸化Rb)等为切入点,通过检测血清、肝组织SOD及其分型铜锌-SOD(CuZn-SOD)、过氧化物酶(POD),总抗氧化能力(T-AOC)活性水平以及肝组织衰老病理变化,并且从分了生物学角度探讨哈蟆油延缓机体衰老的作用机制；在已发现的OR具有抑制自由基堆积基础上,研究OR对衰老动物上述细胞增殖关键调控因子表达的影响,揭示该药抗氧化作用与调节上述调节因子的相关性；同时研究OR对衰老雌性动物动情周期的影响,对子宫、卵巢组织衰老病理变化和对上述组织中细胞增殖关键调控因子的表达变化,探讨OR延缓生殖器官衰老的作用机制,为研究OR延缓生殖衰老机制提供新的理论和实验依据。目的：①观察哈蟆油对衰老大鼠体重、动情周期的影响,并且从肝、子宫和卵巢组织来观察OR对机体和生殖器官衰老的组织学变化情况,以及OR对血清、肝组.织SOD及其分型CuZn-SOD、POD, T-AOC等活性的影响；②应用qRT-PCR法检测OR对衰老大鼠肝组织、子宫、卵巢组织cyclinD1和p21基因的表达,免疫组化方法检测衰老大鼠肝组织、子宫、卵巢组织p16和p21蛋白的表达,Western blotting方法检测OR对衰老大鼠肝、子宫组织cyclinD1、cyclinD3、CDK6和pRb表达的影响,进一步探讨OR延缓大鼠机体和生殖器官的衰老机制；③探讨OR抗氧化作用与细胞增殖调控因子的相关性。方法：1、实验动物、分组及模型建立：48只SD雌性青年大鼠随机分为正常(Normal)、D-半乳糖(D-gal,350mg/kg)、阳性对照、给药组。阳性对照为维生素E(VE,30mg/kg)组,给药组又分哈蟆油高(OR-H,1.8g/kg)、巾(OR-M,0.9g/kg)、低剂量(OR-L,0.45g/kg)组,每组动物8只。32只SD雄性青年大鼠随机分为正常(Normal)、D-半乳糖(D-gal,350mg/kg)、维生素E(VE,30mg/kg)、哈蟆油(OR, 0.9g/kg)组,每组8只。用14%的D-半乳糖溶液颈背部注射方法连续用药42d,建立亚急性衰老模型。阳性药组和给药组在颈背部注射D-gal溶液同时灌服给予VE、OR。2、标本的采集及研究方法：①用药过程中观察衰老大鼠体重变化；采集标本前2周采用瑞氏吉姆萨染色方法检查雌性衰老大鼠阴道上皮角化情况；②连续用OR30d后,取各组大鼠的血清、肝组织,采用紫外分光光度法检测血清、肝组织SOD及其分型CuZn-SOD、POD, T-AOC等活性；③取各组大鼠肝、子宫、卵巢组织用甲醛溶液固定,制作石蜡组织切片,进行HE染色光镜下观察肝、子宫和卵巢组织病理结构的变化；④应用qRT-PCR法检测肝、子宫和卵巢组织cyclinD1、p21的基因水平；⑤应用免疫组化法检测p16和p21蛋白的表达,Western blotting方法检测cyclinD1、cyclinD3、CDK6和pRb蛋白的表达；⑥探讨肝组织SOD、POD及T-AOC等活性与细胞增殖调控因子p16、p21、cyclinD1、CDK6和pRb等蛋白表达的相关性。结果：1、OR对衰老大鼠衰老体征、组织形态学和抗氧化指标的影响1.1动物实验结果：一般体征方面,与Normal组比较,D-gal组大鼠神态萎靡,活动较少,毛发脱落较明显,色泽暗发黄；OR各剂量组大鼠神态活动度较D-gal组活泼,毛发色泽等接近Normal组。体重方面,Normal组体重均数低于D-gal组,但无统计学意义(P=0.152)；OR-H和M组雌性大鼠体重低于D-gal组,差异有显著性(P值分别为0.000,0.001),而雄性大鼠各组体重均未见差异(P=0.648)。动情周期方面,衰老组雌性大鼠与正常组比较,动情周期延长,动情期缩短。OR组能使雌性衰老大鼠动情周期较衰老模型组缩短,动情期时间延长,差异有显著性(P值均为0.000)。1.2组织形态和病理学结果：大体解剖学改变,正常组大鼠肝脏表面光滑,颜色棕红,质软而脆,外观未见异常。D-gal组大鼠肝脏表面可见白色坏死点,肝脏颜色偏白,OR各剂量组较D-gal组肝脏颜色较红,外观基本正常；与正常组比较,D-gal组子宫萎缩变细,OR各剂量组较D-gal组子宫较肥厚,外观基本正常；与正常组比较,D-gal组卵巢缩小,表面明显苍白,点状突起较少,OR各剂量组较D-gal组卵巢色泽较红,表面可见多个白色点状突起。光镜观察,正常组肝细胞镜下结构正常,肝索排列规律,肝窦正常,中央静脉无异常。D-gal组肝细胞体积缩小,核亦缩小,胞浆嗜酸性增强,部分肝细胞连接松散,肝窦明显扩张,肝索狭窄。OR-H组肝细胞及细胞核结构,大小、肝窦、肝索结构未见明显异常。OR-M和L组肝细胞增大,核大,部分肝细胞可见双核,肝索及肝窦未见明显异常。正常组子宫组织镜下内膜、肌层、外膜各层结构清晰,内膜上皮细胞排列规整,腺体丰富。D-gal组子宫内膜上皮增生但变薄,分泌减轻或不明显,间质间可见大量嗜酸性粒细胞浸润。OR各剂量组子宫具有与D-gal组类似的萎缩性病理改变特点,但内膜层较D-gal组病变有所改善,OR-H和M组子宫腺体增生明显,分泌现象明显,间质细胞肥大增生。OR-L组子宫内膜腺体及上皮增生,但较前两个剂量组轻。正常组卵巢组织镜下可见各个发育阶段的卵泡和多个黄体,卵泡内呈多层颗粒细胞,成熟卵泡各层细胞分布均匀,排列规整。D-gal组卵泡发育受阻,初级卵泡数目增多,成熟卵泡数目较少,黄体数目减少。OR各剂量组镜下可见多个各级卵泡及成熟卵泡,黄体发育体积较大,卵泡内呈多层颗粒细胞。1.3抗氧化指标结果：D-gal组大鼠血清和肝组织SOD、CuZn-SOD、POD和T-AOC活性明显降低,与Normal组比较有显著性差异(P=0.000),OR各剂量组均能提高血清和肝组织中上述指标数值(P值均为0.000),尤以OR-H组较为明显。2、衰老大鼠肝、子宫和卵巢组织cyclinDl基因表达降低,p21基因高表达,OR可提高cyclinDl基因,降低p21基因表达水平,促进细胞增殖。2.1 OR对衰老大鼠肝、子宫和卵巢组织cyclinDl基因表达的影响。2.1.1雌性衰老大鼠肝组织qRT-PCR结果显示,D-gal组cyclinD1基因表达与Normal比较下降,差异有显著性(P=0.028),给予不同剂量OR后,OR各剂量组cyclinD1基因表达与D-gal组比较升高,差异有显著性(P值分别为0.016,0.007,0.000)。OR-M组作用较明显。2.1.2雄性衰老大鼠肝组织qRT-PCR结果显示,D-gal组cyclinDl基因表达与Normal组比较下降,差异有显著性(P=0.040),OR组cyclinDl基因表达与D-gal组比较升高,差异有显著性(P=0.000)。2.1.3衰老大鼠子宫组织qRT-PCR结果显示,D-gal组cyclinD1基因表达与Normal组比较下降,差异有显著性(P=0.000)；OR各剂量组与D-gal组比较升高,差异有显著性(P值分别为0.000,0.002,0.004)。cyclinD1基因随着OR剂量增加而升高。2.1.4衰老大鼠卵巢组织qRT-PCR结果显示,D-gal组cyclinD1基因表达与Normal组比较下降,差异有显著性(P=0.002)；OR各剂量组与D-gal组比较升高,差异有显著性(P值均为0.000)。2.2 OR对衰老大鼠肝、子宫和卵巢组织p21基因表达的影响。2.2.1雌性衰老大鼠肝组织qRT-PCR结果显示,D-gal组p21基因表达与Normal比较升高,差异有显著性(P=0.000)。OR各剂量组与D-gal组比较,p21基因表达下降,差异有显著性(P值均为0.000)。OR-M组降低p21基因表达较为明显。2.2.2雄性衰老大鼠肝组织qRT-PCR结果显示,D-gal组p21基因表达与Normal组比较升高,差异有显著性(P=0.018)；OR组p21基因表达与D-gal组比较下降,差异有显著性(P=01001)。2.2.3衰老大鼠子宫组织qRT-PCR结果显示,D-gal组p21基因表达与Normal组比较升高,差异有显著性(P=0.000)。OR各剂量组p21基因表达与D-gal组比较下降,差异有显著性(P值均为0.000)。2.2.4衰老大鼠卵巢组织qRT-PCR结果显示,D-gal组p21基因表达与Normal组比较升高,差异有显著性(P=0.000)。OR各剂量组p21基因表达与D-gal组比较下降,差异有显著性(P值均为0.000)。3、免疫组化显示衰老大鼠细胞增殖调控相关蛋白p16和p21高表达,OR可降低这些蛋白的表达,促进细胞增殖。3.1雌性衰老大鼠肝组织免疫组化结果表明,p16和p21多为胞浆表达,弥漫性、灶性分布均有。D-gal组p16和p21阳性细胞积分与Normal组比较升高,差异有显著性(P值均为0.000)。OR-M和OR-L组与D-gal组相比,p16阳性细胞积分降低,差异有显著性(P值分别为0.001,0.000)。OR各剂量组与D-gal组相比,p21阳性细胞积分降低,差异有显著性(P值均为0.000)。3.2雄性衰老大鼠肝组织免疫组化结果表明,p16和p21多为胞浆表达,弥漫性、灶性分布均有。D-gal组p16和p21阳性细胞积分与Normal比较升高,差异有显著性(P值均为0.002)。OR各剂量组与D-gal组相比,p16、p21阳性细胞积分降低,差异有显著性(P值分别为0.002,0.008)。3.3衰老大鼠子宫组织免疫组化结果表明,子宫组织p16和p21阳性染色多为胞浆着色,见于子宫内膜、上皮细胞胞浆、子宫间质腺体腺上皮细胞胞浆,外膜上皮也有表达,弥漫性、灶性分布均有。D-gal组p16阳性细胞积分与Normal组比较升高,差异有显著性(P=0.009)。D-gal组p21阳性细胞积分与Normal组比较升高,差异有显著性(P=0.000)。OR各剂量组与D-gal组相比,p16、p21阳性细胞积分降低,差异有显著性(P值分别为0.000,0.001,0.002；0.001,0.000,0.000)。3.4衰老大鼠卵巢组织免疫组化结果表明,卵巢组织p16和p21多为弥漫性染色,阳性染色于各级卵泡、卵细胞、黄体,黄体表达强于卵泡,部分间质细胞胞浆染色亦呈阳性。D-gal组p16和p2l阳性细胞积分与Normal组比较升高,差异有显著性(P分别为0.019,0.000)。OR-M、OR-L组p16阳性细胞积分与D-gal比较下降,差异有显著性(P分别为0.004,0.002)。OR各剂量组与D-gal组p21阳性细胞积分比较下降,差异有显著性(P值分别为0.000,0.001,0.000)。4、Westernblotting法显示衰老大鼠肝、子宫和卵巢组织细胞衰老相关周期蛋白cyclinD1、cyclinD3,细胞周期激酶CDK6以及pRb的表达下降,OR可提高这些蛋白的表达,促进细胞增殖。4.1雌性衰老大鼠肝组织Westernblotting结果表明,D-gal组肝组织cyclinD1蛋白表达与Normal比较降低,差异有显著性(P=0.000)。OR各剂量组cyclin D1蛋白表达与D-gal组比较,表达均升高(P值均为0.000),OR-H组尤为明显。D-gal组cyclinD3蛋白表达与Normal比较降低,差异有显著性(P=0.000)。OR各剂量组cyclin D3蛋白表达与D-gal组比较均升高,差异有显著性(P值均为0.000),OR-H组尤为明显。D-gal组CDK6蛋白表达与Normal比较降低,差异有显著性(P=0.009)；OR-H组CDK6蛋白表达与D-gal组比较升高,差异有显著性(P=0.014), OR-M、L组CDK6蛋白表达与D-gal组比较降低,差异有显著性(P值均为0.000)。D-gal组pRb蛋白表达与Normal比较降低,差异有显著性(P=0.000)。OR各剂量组pRb蛋白表达与D-gal组比较显著升高(P值均为0.000),OR-H组尤为明显。4.2雄性衰老大鼠肝组织Westernblotting结果表明,D-gal组cyclinD1、cyclinD3、CDK6、pRb表达与Normal比较降低,差异有显著性(P值均为0.000)。OR组cyclin D1、cyclin D3、CDK6和pRb蛋白表达与D-gal组比较升高,差异有显著性(P值均为0.000)。4.3衰老大鼠子宫组织Westernblotting结果表明,D-gal组子宫组织cyclinD1表达与Normal相比均数低于后者,差异无显著性(P=0.257)。OR各剂量组cyclinD1蛋白表达与D-gal组比较,OR-H和OR-L表达高于D-gal组,差异有显著性(P值分别为0.000,0.001),OR-M组均数高于D-gal组,但差异无显著性(P=0.999),OR-H组表达最高。D-gal组cyclinD3表达与Normal比较降低,差异有显著性(P=0.000)。OR各剂量组子宫组织cyclin D3蛋白表达与D-gal组比较均升高,差异有显著性(P值均为0.000),OR-L组尤为明显。D-gal组CDK6表达与Normal比较降低,差异有显著性(P=0.000)。OR各剂量组子宫组织CDK6蛋白表达与D-gal组比较均升高,差异有显著性(P值均为0.000),低剂量组尤为明显。D-gal组pRb表达与Normal比较降低,差异有显著性(P=0.000)。OR各剂量组子宫组织pRb蛋白表达与D-gal组比较,OR各剂量组表达高于D-gal组,差异有显著性(P值分别为0.001,0.002,0.000)。5、肝组织SOD、POD及T-AOC等活性与细胞增殖调控因子p16、p21、cyclinD1、CDK6和pRb等蛋白表达的相关性。5.1雌性衰老大鼠肝组织SOD活性与p16或p21蛋白表达之间相关系数分别为Spearman r=-0.487；Pearson r=-0.849,SOD与p16蛋白表达或p21蛋白表达均存在显著负相关关系(P值均为0.000)。SOD活性与cyclinD1或CDK6或pRb蛋白表达之间相关系数分别为Pearson r=0.752,Spearman r=0.484,Spearman r=0.740,SOD活性与cyclinD1或CDK6或pRb蛋白表达均存在显著正相关关系(P值均为0.000)。雄性衰老大鼠肝组织SOD活性与p16或p21蛋白表达之间相关系数分别为Pearson r=-0.719: Pearson r=-0.680,SOD与p16蛋白表达或p21蛋白表达均存在显著负相关关系(P值均为0.000)。SOD活性与cyclinD1或CDK6或pRb蛋白表达之间相关系数分别为Pearson r=0.755,Spearman r=0.687,Pearson r=0.863,SOD活性与cyclinD1或CDK6或pRb蛋白表达均存在显著正相关关系(P值均为0.000)。5.2雌性衰老大鼠肝组织POD活性与p16或p21蛋白表达之间相关系数分别为Spearman r=-0.403；Pearson r=-0.712,SOD与p16蛋白表达或p21蛋白表达均存在显著负相关关系(P值均为0.000)。POD活性与cyclinD1或CDK6或pRb蛋白表达之间相关系数分别为Pearson r=0.760,Spearman r=0.396,Spearman r=0.806,POD活性与cyclinD1或CDK6或pRb蛋白表达均存在显著正相关关系(P值均为0.000)。雄性衰老大鼠肝组织POD活性与p16或p21蛋白表达之间相关系数分别为Person r=-0.765; Person r=-0.774,POD与p16蛋白表达或p21蛋白表达均存在显著负相关关系(P值均为0.000)。POD活性与cyclinD1或CDK6或pRb蛋白表达之间相关系数分别为Pearson r=0.869,Spearman r=0.716,Pearson r=0.918,POD活性与cyclinD1或CDK6或pRb蛋白表达均存在显著正相关关系(P值均为0.000)。5.3雌性衰老大鼠肝组织T-AOC活性与p16或p21蛋白表达之间相关系数分别为Spearman r=-0.552；Pearson r=-0.764,T-AOC与p16蛋白表达或p21蛋白表达均存在显著负相关关系(P值均为0.000。T-AOC活性与cyclinD1或CDK6或pRb蛋白表达之间相关系数分别为Pearson r=0.577,Spearman r=0.357,Spearman r=0.724,T-AOC活性与cyclinDl或CDK6或pRb蛋白表达均存在显著正相关关系(P值均为0.000)。雄性衰老大鼠肝组织T-AOC活性与p16或p21蛋白表达之间相关系数分别为Pearson r=-0.702; Pearson r=-0.703, T-AOC与p16蛋白表达或p21蛋白表达均存在显著负相关关系(P值均为0.000)。T-AOC活性与cyclinD1或CDK6或pRb蛋白表达之间相关系数分别为Pearson r= 0.782, Spearman r=0.615, Pearson r=0.880, T-AOC活性与cyclinD1或CDK6或pRb蛋白表达均存在显著正相关关系(P值均为0.000)。结论：1.衰老雌性大鼠动情周期延长,动情期缩短；肝、子宫、卵巢组织结构发生损伤,细胞形态发生改变；血清和肝组织SOD、CuZn-SOD、POD和T-AOC活性明显降低。应用OR可以减缓衰老组织结构的损伤,维持细胞的正常形态,改善衰老子宫和卵巢萎缩和衰老程度,能使雌鼠动情周期缩短,动情期时间延长,能提高血清和肝组织中抗氧化指标,从而延缓机体和生殖器官的衰老。2. qRT-PCR实验结果显示衰老大鼠肝组织、子宫、卵巢组织细胞增殖正性调控因子cyclinD1基因表达降低,负性调控因子p21基因高表达,应用OR可提高cyclinD1基因表达,降低p21基因表达。3.免疫组化实验结果表明衰老大鼠肝、子宫、卵巢组织p16、p21蛋白高表达,应用OR可降低上述蛋白的表达。4. Westernblotting实验显示衰老大鼠肝、子宫组织细胞增殖正性调控因子cyclinD1、cyclinD3、CDK6蛋白和关键因子pRb低表达,应用OR可显著提高上述蛋白的表达。5.OR抗氧化作用与细胞增殖调控因子相关性分析表明两者在不同性别上均有相关性,但性别之间有差异。在雄性衰老大鼠方面,SOD、POD和T-AOC活性均与p16或p21蛋白表达具有负相关性；与cyclinD1、CDK6和pRb蛋白表达具有正相关。在雌性衰老大鼠方面,SOD、POD和T-AOC活性均与p16或p21蛋白表达具有负相关性,根据相关系数比较,在雌性中,SOD、POD和T-AOC活性与p16相关性不如与p21的相关性,这与雄性有差别；SOD、POD和T-AOC活性与cyclinD1、CDK6和pRb蛋白表达具有正相关,根据相关系数比较,上述抗氧化指标与CDK6相关性不如与其它两种蛋白明显,这与雄性有差别。6.结合文献研究和实验结果,推测OR延缓衰老机制为↓ROS积聚,↑机体、抗氧化能力→同时↓衰老组织中p16和p21过表达,↑细胞增殖正性调控因子cyclinD1、cyclinD3、CDK6水平→使Rb蛋白磷酸化水平↑→释放出转录因子蛋白进入核内→与相应靶基因启动子上的对应序列结合→促进细胞的分化增殖,延缓细胞衰老,从而改善组织器官衰老。此推测还需进一步证明。更多还原

【Abstract】 Aging is begun and accelerated after reproduction maturity. It is characterized by cumulative, universal, progressive and endogenous vital process, which is the common in nature of all creatures. Organism has three vital process phases, growth and development phase, reproductive period and senescence phase. Different species have similar mechanism of duration of life decision. In 21st centuries, human society is faced with a severe challenge of aging of the population. In 2007, United Nations Economic and Social Affairs published the《2007 World Economic and Social Survey》. The report is showed that because of fertility decline and increased life expectancy, most countries in the world are rapidly entering the aging society. From 2005 to 2050 years, half of the world’s population increasing is the old over 60.Population of the senior over 80 will increase from 90 to 400 million and many kind of senile diseases will be followed as well. Therefore, how to delay senility has been one of the hot spot of international scholars to research. It is thus clear that the research of aging and anti-aging field and exploration of anti-aging drugs is of vital importance to improve the quality of human life.Oviductus Ranae (OR) is the Chinese forest female frog’s((Rana temporaria chensinesis David)) fallopian tubes, obtained by the collect and process drying. It has the ability of tonify the kidney and replenish essence, nourishing yin and moistening lung and is used in the symptom including yin asthenia and physically weak, lethargy, heart palpitations and insomnia, night sweat and coughing up blood. The ingredients mainly contain protein and amino acids, fatty acids (saturated and unsaturated), hormones, steroids, inorganic elements, vitamins and so on. Now, it had reported that OR was able to significant prolongation mean life span and maximum lifes pan of Drosophila. Its anti-aging mechanism was to heighten activity of SOD and to decrease level of LPO. OR had also many effects incuding anti-fatigue, to adjust nerve immunization and lipometbolism.In our previous study, we also found that OR could increase the activity of superoxide dismutase(SOD), inhibit free radical accumulation in the reproductive organs and resist the aging of cells and degeneration of tissues and significantly increased estrogen levels organs in aged mouse induced by D-galactose. It could increase the ovary index and the uterus index, ameliorate their pathological change to improve the decaying ovary and uterus. We had made the YiFuling Soft Gelatin Capsules (YFL) with OR as principal drug for Climacteric Syndrome. Animal experiments showed that YFL could significantly elevate estrogens content in vivo and significantly improve uterus index, adrenal gland index. YFL could improve the uterine morphological structures in pathological condition and increase the number and secretion of gland and make uterine wall thickening. YFL had already become a hospital pharmaceutics (licensed No. XZ20010003).YFL was applicated in clinical treatment for many years and found it had better treatment effectiveness. We had also developed Kun Li tablet with OR, Ginseng, Angelica as healthy food (register No. G20090379). Kun Li tablet had obviously effect of reducing chloasma. We also obtained Chines patent of invention (patent number ZL01107471.X).Free radical theory was the best important theory in numerous causes of senescence. Free radical theory was supported by abundant experimental evidences. Although others causes of senescence had individual strong point, only free radical theory could contact with others theories and became a center of causes of senescence. Free radical theory was one of persuasivest theories to be verificated by theory and experiments study in 50 years. In recently years, researchers showed that as a normal and complicated life process, senescence was involved in a series of routine changing in various tissues and cells. Many studies indicated that cellular senescence was implemented via some signaling transduction pathways which a lot of cytokines were involved in. The most classical cell senescence pathways were P161NK4a-Rb and p53-p21WAF1/ClP1-Rb. When the key regulating factors (p16, p21, cyclin, CDK and Rb, et al), in two pathways were changed, cellular senescence would be delayed. Now some scientists found inhibition of p21-mediated reactive oxygen species (ROS) accumulation could rescue p21-induced senescence. They also showed that the p16INK4A-Rb pathway cooperates with mitogenic signals to induce elevated intracellular levels of ROS in human senescent cells. Phospho-Rb and its regulating factors (cyclin and CDK) might be playing a significant role in the senescence process.References analysis of OR discovered mechinsm of anti-aging in OR was related to antioxidation. Therefore we thought while OR inhibited ROS accumulation to anti-aging, OR regulated key factors expression of the two cell aging pathways was another possible anti-aging role of OR. It was reported that correlation of antioxidant effect of OR with regulate key factors expression effect of OR. In this study, we were going to investigat the changes of some key regulators of cellular senescence signal transduction pathway p53-p21-Rb and pl6-Rb, including p21, p16, cyclinDl, cyclinD3, CDK6,pRb,and detect the activity level changes of superoxide dismutase (SOD),CuZnSOD, peroxidase(POD) and total antioxidation capacity(T-AOC) in rats serum and liver, and observe changes of pathology of liver, uterus, ovary after taking OR by using the sub-acutely aging model rats induced D-galactose (D-gal), so as to find the mechanism of OR in anti-aging and correlation of antioxidation and these key regulating factors,to observe effect of OR in female aged rats estrus cycle, to investigate the mechanism of OR in delaying reproductive senescence with molecular biology techniques. This study could provide theoretical and experimental basis for further studying the effect of OR on anti-aging.Objective:①To observe the changes of weight and oestrous cycle in aged rats after taking OR, and analysis the changes of pathology in liver,uterus,ovary,and detecte activity level of SOD,CuZnSOD,POD andT-AOC in aged rats serum and liver.②To detect the level of mRNA cyclinDl and p21 by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and evaluated the expression of p16 and p21 by immunohistochemistry(IHC) in liver,uterus,ovary of aged rats, and examined the expression of cyclinDl,cyclinD3,CDK6 and pRb protein of liver and uterus by Westernblotting, and to further find out the mechanism of OR in anti-aging and the mechanism of OR in delaying reproductive senescence.③To demonstrate correlations of antioxidant effect of OR with expression of regulate key factors in cell proliferation.Methods:①Choosing of the experimental animal and establishment of the aging model.Young female SD rats(n=48)were separated into six groups at random:Normal group,D-gal group(350mg/kg/), positive control group,OR group.The rats in positive control group was Vitamin E(VE,30mg/kg) group.The rats in OR group were redivided into three group:OR high dosage group(OR-H,1.8g/kg),OR medium dosage group(OR-M,0.9g/kg),OR low dosage group(OR-L,0.45g/kg).8 rats were in each group. Young male SD rats (n=32) were separated into four groups at random: Normal group,D-gal group(350mg/kg),VE group(30mg/kg),OR group(0.9g/kg).8 rats were in each group. The sub-acutely aging model rats were induced by D-galactose,and the rats in VE and OR group accepted intragastric administration of VE and OR while accepting D-galactose neck back injection for 42 days.②Experimental procedures. To observe the weight change of aged rats during taking OR. The change of vaginal epidermis keratosis was observed by Wright-Giemsas staining in two weeks before rats were killed.Serum,liver,uterus and ovary tissues of each group were obtained after the rats undergoing experiment for 30d with taking OR. We detected activity level of SOD, CuZnSOD, POD and T-AOC in serum and liver by UV spectrophotometry, and observed the pathological changes of liver, uterus,ovary tissues with hematoxylin and eosin staining by microscope. the gene mRNA expression of cyclinDl and p21 were detected by qRT-PCR in liver, uterus, ovary tissues, and evaluated the expression of p16 and p21 by IHC in liver, uterus, ovary tissues, and examined the protein expression of cyclinDl,cyclinD3,CDK6 and pRb by Westernblotting in liver and uterus. To demonstrate correlations of SOD,POD and T-AOC activity level with expression of regulate key factors p16,p21,cyclinD1, CDK6 and pRb proteins in liver.Results:1、Effect of OR on senescent signs,histomorphology and anti-oxidant index in aging rats.1.1 Result of animal experiment:Rat’s general signs:Appearance of D-gal group was dispiriteder, less activity, loss of hair, yellowishewithered hair than Normal group. OR could improve aged rats appearance, activity and its hair color and luster. The weight of Normal group has no difference with D-gal group (P=0.152). The weight of OR-H、M group was significantly decreased than D-gal group (P=0.000, P=0.001), but such effect was not shown in aged male rats (P=0.648). OR could also make shorten female aged rats estrus cycle, lengthen oestrus time (P=0.000).1.2 Result of Pathology:Change of macroscopic anatomy, surfaces of liver in Normal group was smoot and glossy. Color of liver was brownish red and substance was soft and crisp. It was no abnormality seen. Surfaces of liver in D-gal group had white necrotic zone and color of liver was partital white. Compare to Normal group, uterus of D-gal group metratrophia and thinning. Ovary was decreased size and its surfaces had fewer punctiform apophysis. Its color was pallor. Shape of liver in OR group was normal and its color was reder than D-gal group. Uterus of OR group was hypertrophy and ovary enlargement than D-gal group. Color of ovary was reder than D-gal group and surfaces of ovary had many white punctiform apophysis.By microscope, we found structure of liver cell in normal group was normal. Appearance of hepatic cord was regular pattern. Hepatic sinusoid and central veins were normal. Liver cell of D-gal group was loss of volume and cell nuclei. Acidophilia of cytoplasm was higher than Normal group. Some liver cell junction of D-gal group was surface loosening. Hepatic sinusoid was obviously extension and hepatic cord became stenosis. Hepatic sinusoid,hepatic cord and structure,size of liver cell and cell muclei in OR-H group were normal. Liver cell and cell nuclei of volume became large and some liver cell had conjugate nuclei. Hepatic sinusoid,hepatic cord had no obviously abnormality. Structure of endometrium, muscularlayer and perimetrium in Normal group were clearness and epithelial cells aligned regularity. There were abundance glandular organ in uterus. Endometrium of D-gal group had epithelial proliferation and became thinning. Excretion of glandular organ lessened or unobviously. There wwas a great quantity of eosinophile granulocyte infiltrating in intercellular substance. All OR groups had analogical atrophic pathplogy change in uterus with D-gal group. But endometrium pathological change got better than D-gal group. Glandular organ hyperplasia and excretion in uterus were obviously in OR-H and OR-M. Its stromal cells became hypertrophy and hyperplasia. Glandular organ and epithelial in endometrium proliferation in OR-L group were lower than OR-H and OR-M groups. Various developmental stage ovarian follicle and multiple corpus luteum was seen by microscope in Normal group. Multilayer granular cell in ovarian follicle was seen. All layer cells in mature follicle of Normal group were well distributed and regularity. Ovarian follicle of D-gal group was impaired development and number of primary follicle increased, and number of mature follicle and corpus luteum decreaed. Different levels ovarian follicle and mature follicle in OR groups were seen by microscope. Corpus luteum volume became large and multilayer granular cells in ovarian follicle were seen in OR groups.1.3 Result of anti-oxidant index:The activity of SOD, CuZn-SOD, POD and T-AOC of serum and liver tissue in aged rats were significantly decreased than Normal group (P=0.000). All OR groups could significantly increase these indexes of serum and liver tissue (P=0.000), OR-H group was the best in all OR groups.2 Expression of gene mRNA cyclinDl was lower expression and gene mRNA p21 was overexpression in liver, uterus, ovary tissues of aged rats, and OR could up-regulate the expression of cyclinDl and retrieve the overexpression of p21 so as to cell proliferation.2.1 Expression of gene mRNA cyclinDl in liver, uterus, ovary tissues of aged rats.2.1.1 The result of qRT-PCR had shown that gene expression of cyclinDl in liver of D-gal female rat group was marked decreased than normal group (P=0.028). All OR groups could notable increased cyclinD1 mRNA’s expression than D-gal group(P=0.016,P=0.007,P=0.000). OR-M group was was the best in all OR groups.2.1.2 The result of qRT-PCR had shown that gene expression of cyclinD1 in liver of D-gal male rats group was significantly lower than normal group (P=0.040). cyclinD1 mRNA’s expression of OR group had significantly higher than D-gal group(P=0.000).2.1.3 The result of qRT-PCR had shown that gene expression of cyclinD1 in uterus of D-gal group was marked lower than normal group (P=0.000). Expression of cyclinD1 mRNA in all OR groups was significantly higher than D-gal group (P=0.000, P=0.002, P=0.004). The more dose of OR, the more expression of cyclinD1.2.1.4 The result of qRT-PCR had shown that gene expression of cyclinD1 in ovary of D-gal group was prominently lower than normal (P=0.002). Expression of cyclinD1 in all OR groups was significantly higher than D-gal group (P= 0.000).2.2 Expression of gene mRNA p21 in liver, uterus, ovary tissues of aged rats2.2.1 The result of qRT-PCR had shown that gene expression of p21 in liver of D-gal female group was marked increased than normal group (P=0.000). All OR groups could decrease expression of p21 mRNA than D-gal group (P=0.000).2.2.2 The result of qRT-PCR had shown that gene expression of p21 in liver of D-gal male group was marked increased than normal group (P=0.018). OR group could decreaed expression of p21 mRNA than D-gal group (P=0.001).2.2.3 The result of qRT-PCR had shown that gene expression of p21 in uterus of D-gal female group was marked increased than normal. All OR groups were significantly lower than D-gal group(P=0.000).2.2.4 The result of qRT-PCR had shown that gene expression of p21 in ovary of D-gal female group was significantly increased than normal group (P=0.000). All OR groups could markedly decreased expression of p21 mRNA than D-gal group (P=0.000).3 The expression of p16 and p21 were overexpression in liver, uterus, ovary tissues of aged rats by IHC, and the expression of these regulators proteins could be retrieved by using OR so as to cell proliferation.3.1 The result of IHC had shown the positive production of p16 and p21 were mainly multifocal located in endochylema of liver cell and their distribution were diffuse and focus. Positive cell integral of p16 and p21 in liver tissue of D-gal female aged rats were significantly increased than normal group (P=0.000). After taking OR, the integral of positive cell of p16 in OR-M and OR-L groups was significantly decreased than D-gal group(P=0.001,P=0.000). The integral of positive cell of p21 in OR-H,OR-M and OR-L groups was significantly decreased than D-gal group (P=0.000).3.2 The result of IHC had shown that the positive expression of p16 and p21were mainly multifocal located in endochylema of liver cell, and their distribution was diffuse and focus. Positive cell integral of p16 and p21 in liver tissue of D-gal male aged rats was significantly increased than Normal group (P=0.002). After taking OR, the positive cell integral of p16 and p21 in OR group were significantly decreased than D-gal group (P=0.002,P=0.008).3.3 The result of IHC had shown that the positive staining of the P16 and P21 protein in uterus tissue of D-gal rats was mainly located in cytoplasm, which were on the endometrium, the cytoplasm of epithelial cells and interstitial glands and a few in uterine glandular epithelium, and their distribution was diffuse and focus. The expression of P16 positive cells integral in D-gal group was significantly increased than normal group (P=0.009). Positive cell integral of p21 in D-gal group was significantly increased than Normal group (P=0.000). Positive cell integral of P16 and P21 in all OR groups were significantly decreased than D-gal group (P=0.000, P = 0.001; P= 0.002, P= 0.001; P= 0.000, P= 0.000).3.4 The result of IHC had shown that the positive staining of the p16 and p21 protein in ovary tissue of D-gal rats was mostly expressed diffuse staining in the follicle, oocyte, corpus luteumn, with expression in luteal higher than that in the follicle, and the positive expression was also observed in some mesenchymal cells. The positive cell integral of p16 and P21 protein of ovary tissue in D-gal group was significantly higher than normal group (P= 0.019, P=0.000). Positive cell integral of p16 in ovary of OR-M and OR-L groups was prominently lower than D-gal group.(P=0.004,P=0.002). Positive cell integral of p21 in all OR groups was significantly lower than D-gal group(P=0.000, P=0.001, P=0.000).4 The expression of cyclinDl,cyclinD3,CDK6 and pRb were lower in liver, uterus tissues of aging model by Westernblotting, and the expression of these proteins could be up-regulated by using OR so as to cell proliferation.4.1 The result of Westernblotting had shown that expression of cyclinDl protein in liver of D-gal female rats was decreased than normal group and the difference was significant (P=0.000). Expression of cyclinD1 in all OR groups was increased compared with D-gal group and the difference was significant (P=0.000). Expression of cyclinDl in OR-H group was the highest in all OR groups.Expression of cyclinD3 in liver of D-gal group was markedly lower than normal group(P=0.000). Expression of cyclinD3 in all OR groups was increased compared with D-gal group and the difference was significant (P=0.000). Expression of cyclinD3 in OR-H group was the highest in all OR groups.Expression of CDK6 in liver of D-gal group was significantly lower than normal group(P=0.009).Expreesion of CDK6 in OR-H was significantly higher than D-gal group(P=0.014). Expression of CDK6 in OR-M and OR-L groups was lower than D-gal group.(P=0.000). OR-H group was the highest in all OR groups.Expression of pRb in liver tissue in D-gal group was decreased compared with normal group and the difference was significant (P=0.000). pRb expression in all OR groups was higher than D-gal group (P=0.000). pRb expression of OR-H group was the highest in all OR groups.4.2 Expressions of cyclinDl, cyclinD3, CDK6 and pRb in liver tissue of D-gal male group were significantly lower than normal group (P=0.000). Expressions of cyclinD1, cyclinD3, CDK6 and pRb in OR group were significantly higher than D-gal group (P=0.000).4.3 Expression of cyclinDl in uterus tissue of D-gal group had no difference with normal group(P=0.257). Expression of cyclinDl in OR-H, L group were higher than that in D-gal group and the difference was significant (P=0.000, P=0.001), while expression of cyclin D1 in OR-M group had no difference with D-gal group(P=0.999). Expression of cyclinDl in OR-H group was the highest.Expression of cyclinD3 in uterus of D-gal group was significantly lower than normal group (P=0.000). Compared with D-gal group, all OR groups could elevate cyclinD3 expression and the difference was significant (P=0.000). Expression of OR-L was the highest.CDK6 expression in D-gal group was decreased compared with the normal group and the difference was significant (P=0.000). Expression of CDK6 protein in all OR groups were significantly higher than D-gal group (P=0.000). Expression of OR-L was the highest.Expression of pRb in uterus tissue in D-gal group was decreased compared with normal group which was significant (P=0.000). pRb expression in all OR groups was increased compared with D-gal group(P=0.001,P=0.002, P=0.000).5 Correlations were analyzed between antioxidant indexes (activity of SOD, POD and T-AOC) and expression of cell proliferation regulating factors (p16, p21, cyclinDl, CDK6 and pRb protein) in liver tissue.5.1 The negative correlations were demonstrated between the activity of SOD and the expression of p16 protein (Spearman r=-0.487, P=0.000), between the activity of SOD and the expression of p21 protein (Pearson r=-0.849, P=0.000) in liver of female aged rats. The positive correlations were showed between the activity of SOD and the expression of cyclinDl protein (Pearson r=0.752,P=0.000), between the activity of SOD and the expression of CDK6 protein (Spearman r=0.484, P=0.000), also existed activity of SOD and expression of pRb protein in liver tissue of female aged rats (Pearson r=0.740,P=0.000).The negative correlations were demonstrated between the activity of SOD and the expression of p16 protein (Pearson r=-0.719, P=0.000), between the activity of SOD and the expression of p21 protein (Pearson r=-0.680, P=0.000) in liver of male aged rats. The positive correlations were showed between the activity of SOD and the expression of cyclinDl protein (Pearson r=0.755,P=0.000), between the activity of SOD and the expression of CDK6 protein (Spearman r=0.687,P=0.000), also existed activity of SOD and expression of pRb protein in liver tissue of male aged rats (Pearson r=0.863,P=0.000)5.2 The negative correlations were demonstrated between the activity of POD and the expression of p16 protein (Spearman r=-0.403,P=0.000), between the activity of POD and the expression of p21 protein (Pearson r=-0.712, P=0.000) in liver of female aged rats. The positive correlations were showed between the activity of SOD and the expression of cyclinD1 protein (Pearson r=0.760,P=0.000), between the activity of POD and the expression of CDK6 protein (Spearman r=0.396, P= 0.000), also existed activity of POD and expression of pRb protein in liver tissue of female aged rats (Spearman r=0.806,P=0.000).The negative correlations were demonstrated between the activity of POD and the expression of p16 protein (Pearson r=-0.765, P=0.000), between the activity of POD and the expression of p21 protein (Pearson r=-0.774, P=0.000) in liver of male aged rats. The positive correlations were showed between the activity of POD and the expression of cyclinD1 protein (Pearson r=0.869,P=0.000), between the activity of POD and the expression of CDK6 protein (Spearman r=0.716, P=0.000), also existed activity of POD and expression of pRb protein in liver tissue of male aged rats (Pearson r=0.918,P=0.000).5.3 The negative correlations were demonstrated between the activity of T-AOC and the expression of p16 protein (Spearman r=-0.552,P=0.000), between the activity of T-AOC and the expression of p21 protein (Pearson r=-0.764, P=0.000) in liver of female aged rats. The positive correlations were showed between the activity of T-AOC and the expression of cyclinD1 protein (Pearson r=0.577,P=0.000), between the activity of T-AOC and the expression of CDK6 protein (Spearman r=0.357, P= 0.000), also existed activity of T-AOC and expression of pRb protein in liver tissue of female aged rats (Spearman r=0.724,P=0.000).The negative correlations were demonstrated between the activity of T-AOC and the expression of p16 protein (Pearson r=-0.702, P=0.000), between the activity of T-AOC and the expression of p21 protein (Pearson r=-0.703, P=0.000) in liver of male aged rats. The positive correlations were showed between the activity of T-AOC and the expression of cyclinDl protein (Pearson r=0.782,P=0.000), between the activity of T-AOC and the expression of CDK6 protein (Spearman r=0.615, P=0.000), also existed activity of T-AOC and expression of pRb protein in liver tissue of male aged rats (Pearson r=0.880,P=0.000).Conclusion:1. Cell morphologic and pathological structure liver, uterus, ovary tissue in aged rats is damaged. Estrouscycle of female rat is obviously prolonged and oestrus becomes shorten. Activity of SOD, CuZnSOD, POD and T-AOC in serum and liver are obviously lower in aged rats. After taking OR, impairment of pathology is able to be restored and OR can maintain cellular normal form, alleviates the pathological change of uterus and ovary. OR can shorten Estrous cycle and prolong oestrus in female aged rats. OR has definite antioxidative effects and enhances antioxidative indexes of serum and liver. These results indicate that OR delays senescence of organism and reproductive organs in aged rats.2. Result of qRT-PCR shows expression of gene cyclinDl mRNA in liver, uterus and ovary tissues of aged rats was lower. Expression of gene p21 mRNA in liver, uterus and ovary tissues of aged rats was overexpression. OR can increase expression of cyclinDl mRNA in liver, uterus and ovary tissues of aged rats. Meanwhile OR also can decrease over-expression of p21mRNA in liver, uterus and ovary of aged rats.3. Result of IHC shows expression of p16 and p21protein in liver, uterus and ovary tissues of aged rats was overexpression. OR can decrease over-expression of p16 and p21 protein in liver, uterus and ovary tissues of aged rats.4. Result of Westerblotting shows expression of cyclinD1, cyclinD3, CDK6 and pRb protein was lower in aged rats. OR can enhance expression of cyclinD1, cyclinD3, CDK6 and pRb in liver, uterus and ovary tissues of aged rats.5. Result of correlation analysis between antioxidation of OR and regulating factors of cell proliferation indicates there is a correlation in both sides, but it has difference in gender. In male aged rats, the negative correlations are demonstrated between the expression of p16 and SOD,POD and T-AOC, between the expression of p21 and SOD,POD and T-AOC. The positive correlations are shows between the expression of cyclinD1 and SOD,POD and T-AOC, between the expression of CDK6 and SOD,POD and T-AOC, also exists expression of pRb and SOD,POD and T-AOC.In female aged rats, the negative correlations are demonstrated between the expression of p16 and SOD,POD and T-AOC, between the expression of p21 and SOD,POD and T-AOC. According to coefficient correlation comparison, p21 correlation was better than p16’s. This is one of difference with male rats. The positive correlations are shows between the expression of cyclinDl and SOD,POD and T-AOC, between the expression of CDK6 and SOD,POD and T-AOC, also existed expression of pRb and SOD,POD and T-AOC. CDK6 correlation also is one of difference with male rats.6. We postulate one of the anti-aging mechanism of OR is as below:↓ROS accumulation,↑antioxidizing ability of organism→meanwhile↓p16 or p21 over-expression in aged tissues→↑positivie regulating factors of cell proliferation-cyclinDl,cyclinD3 and CDK6 protein→↑phosphorylation level of Rb protein→dissociation of E2F1 protein→bind to target gene promoter in intranuclear→cell differentiation and proliferation→anti-aging effect, which needs more evidences from experiments.更多还原