桔梗皂苷D杀精机制及初步成药性研究

发布时间：2018-02-27 09:19

本文关键词： 桔梗皂苷D 精子杀精剂凋亡避孕阴道刺激性　出处：《第三军医大学》2013年硕士论文　论文类型：学位论文

【摘要】：背景与目的：人口爆炸已成为全球重大问题。在全球范围内，每一年有超过2亿个妊娠发生，而其中50%是意外妊娠。我国是人口大国，计划生育是我们的基本国策，避孕药的研发一直备受关注。杀精剂是用于避孕的药物，在性交前用于阴道以避免妊娠，杀精剂配合其他屏障性避孕措施使用是一种非常有效的避孕措施。目前广泛使用的杀精剂壬苯醇醚-9（nonoxynol-9，N-9）可能增加艾滋病感染可能性，因而亟需研发新的杀精剂。中药是我们的传统药物，是药物研发的宝贵资源库，山东省计划生育科学技术研究所的前期研究从数百种单味中药中已发现桔梗、远志、公丁香具有快速体外杀精子活性，其中桔梗皂苷类提取物的杀精活性最强。有研究显示来自植物的天然皂苷类是一类很有发展前景的体外杀精剂。本研究通过预实验证实桔梗中含量最丰富的一种皂苷-桔梗皂苷D（Platycodin D，PD）具有体外快速杀精子活性，进一步通过与其他数种皂苷单体比较，证实PD的体外杀精活性，并通过精子凋亡检测、低渗肿胀实验、透射及扫描电镜等探索其杀精机制，同时通过DNA损伤实验、体内避孕实验、阴道刺激实验等初步研究其成药可能性，以期望获得具有杀精作用的临床前候选药物。方法和结果： 1.伊红染色检测精子活性（远志酸或桔梗皂苷D）1μl+伊红49μl，混匀，取混合液5μl+5μl精液，载玻上混匀，观察精子着色情况。0.25mmol/L桔梗皂苷D（PD）在瞬间杀灭全部精子（20s），同样条件下，远志酸（Polygalacic acid，PA）杀灭精子所需浓度为7.5mmol/L。PA有效杀精浓度太高被淘汰，0.2mmol/LPD可有效杀灭精子，作为进一步实验的作用浓度。 2.计算机辅助精子分析系统（CASA）筛选备选药物并分析目标药物杀精活性将同一健康男性的精液均分为6份，分别用0.9%NaCl对照，1%DMSO，0.2mmol/l远志皂苷B（OnjisaponinB，OB），0.2mmol/L常春藤皂苷B（Hederasaponin B，HB），0.2mmol/L去芹菜糖桔梗皂苷D（Deapio platycodin D，DPD）处理精子2min。用计算机辅助的精液分析系统（CASA）检测备选药物对人精子的杀伤效果。筛选出PD为活性最强的单体，将同一健康男性的精液均分为4份，分别用0.9%NaCl对照，0.15mmol/LPD，0.20mmol/LPD，0.20mg/ml N-9(=0.308mmol/L)阳性对照处理2min。CASA检测0.15mmol/LPD精子活率（a+b+c%）为47.15±12.44，0.20mmol/LPD精子活率21.38±10.69，0.25mmol/LPD精子瞬间完全失活。得出PD最佳杀精浓度是0.20mmol/LPD，用于后继实验。 3.杀精机制研究健康男性的精液上游后，用凋亡试剂盒检测PD对人精子的凋亡诱导作用；用低渗肿胀检测试剂盒，LIVE/DEAD Sperm Viability Kit检测PD对人精子细胞膜的完整性及存活状态的影响。结果发现0.20mmol/LPD引起大量精子发生晚期凋亡，精子肿胀率下降，LIVE/DEAD检测发现大量死亡精子；扫描和透射电镜结果显示，精子头颈处断裂，精子膜破裂，线粒体肿胀及膜丢失； CometAssay结果显示0.20mmol/L PD不会对精子DNA造成损伤。 3.1精子凋亡检测：0.10mmol/L,0.15mmol/L,0.20mmol/L,0.25mmol/L PD使大量精子发生凋亡，以晚期凋亡和坏死（即FITC+PI+）为主；0.25mmol/L PD晚期凋亡及坏死精子比例占精子总数的75.84±11.12%；各剂量组PD对精子的早期凋亡（FITC+PI-）均没有影响。表明PD破坏了精子膜。 3.2低渗肿胀实验：0.9%NaCl对照组多数精子发生了膨胀（尾部卷曲），而PD组精子膨胀不明显，低渗肿胀反应消失，膨胀率从76.5±6.12%下降28.0±6.13%，两组间膨胀率有显著差异。结果显示精子膜受到PD损伤。 3.3LIVE/DEAD检测：对照组精子以绿色荧光（活精子）为主，PD处理后橙色荧光（频死精子）、红色荧光（死亡精子）明显增加，阳性对照组（0.2mmol/L N-9）与0.1mmol/L PD组杀精效果相近，0.2mmol/L PD组精子几乎都是红色荧光，杀精率95%。 3.4扫描电镜及透射电镜：在扫描电镜下，对照组精子形态正常，顶体清楚，用药后，可见大量精子头颈处断裂。在透射电镜下，对照组精子细胞膜完整，线粒体结构完整。用药后，，精子膜破裂，线粒体肿胀，甚至膜脱落。 3.5Comet Assay实验：阴性对照组，PD0.2mmol/L组的所有实验用精子，均没有发现DNA移动尾巴，阳性对照组有明显的DNA移动尾巴。 4.动物实验 4.1避孕实验：雌性SD大鼠右侧子宫注入0.9%NaCl100μl（对照组），在左侧子宫给予3mg/ml PD100μl(剂量为0.3mg)，术后与雄鼠2:1合笼；第二天发现阴栓后分笼，10天后处死老鼠，发现注射PD侧（左侧）子宫无孕囊，而注射生理盐水侧（右侧）子宫正常妊娠。 4.2阴道刺激实验：雌性大鼠随机分为5组，每天定时给予0.2ml（约0.5g)PD凝胶阴道局部给药，连续给药14天，末次给药后处死动物，取出阴道组织，用甲醛固定，进行组织病理学检查及阴道刺激评分[40]。自然对照组，空白凝胶组，3mg/g PD组，8mg/gPD组，8mg/g N-9组的阴道刺激评分分别为2.11±2.01，3.22±1.07，4.89±0.69，7.44±1.58，3.67±1.53。阴道刺激试验评分低于8分，刺激性在可以接受范围内。结论： PD对人精子有显著的瞬间杀灭效应，能引起精子晚期凋亡，杀精机制研究显示PD直接损伤精子膜。杀精浓度的PD对DNA无损伤，大鼠避孕实验证实PD在体内可有效避孕，对阴道刺激性在可接受范围内。PD将来可能作为一种有潜力的临床杀精剂用于避孕。
[Abstract]:Background and purpose:
The population explosion has become a major problem in the world. Globally, every year there are more than 200 million pregnancy, of which 50% is pregnant. China is a populous country, family planning is our basic national policy, developing contraceptives has attracted much attention. The spermicidal agent is used for contraceptive drugs for vaginal intercourse in to avoid pregnancy, spermicide with other barrier contraceptive use is a very effective contraceptive measures. The widely used spermicide nonoxynol-9 -9 (nonoxynol-9, N-9) may increase the possibility of HIV infection, and therefore need to develop new spermicide. Traditional Chinese medicine is our drug is. The precious resources for drug development, preliminary study of family planning in Shandong province science and Technology Research Institute from hundreds of Chinese herbs have been found in Platycodon grandiflorum, Polygala tenuifolia, clove has rapid spermicidal activity in vitro, the saponins of Platycodon grandiflorum Extract spermicidal activity was the strongest. Studies have shown that natural saponins from plants is a kind of promising spermicide. This study through the preliminary study confirmed a Platycodon saponins - the most abundant content of platycodin D (Platycodin D PD) has in vitro rapid spermicidal activity, further through compared with several other kinds of saponin monomer, confirmed the spermicidal activity of PD in vitro, and the sperm apoptosis detection, hypoosmotic swelling test, transmission and scanning electron microscopy to explore the spermicidal mechanism, at the same time through the DNA damage test in vivo contraceptive experiment, preliminary study on vaginal irritation experiment to obtain the possibility of medicine, has killed the role of the fine preclinical drug candidates.
Methods and results:
Detection of sperm activity by 1. eosin staining
(Polygalic acid or platycodin D) 1 l+ eosin 49 L, mixing, mixing liquid 5 l+5 l glass loading on semen, mixing, observe the sperm coloring of.0.25mmol/L platycodin D (PD) in an instant kill all sperm (20s), under the same conditions, Polygalic acid (Polygalacic acid, PA) required to kill sperm concentration is 7.5mmol/L.PA effective spermicidal concentration is too high to be eliminated, 0.2mmol/LPD can effectively kill sperm, as the concentration of further experiments.
2. computer assisted sperm analysis system (CASA) screening alternative drugs and analysis of target drug spermatozoon activity
Sharing the same semen in healthy men was 6, respectively, with 0.9%NaCl control, 1%DMSO, 0.2mmol/l Tenuigenin B (OnjisaponinB, OB), 0.2mmol/L B (Hederasaponin B hederin, HB, 0.2mmol/L) to apiose platycodin D (Deapio platycodin D, DPD) system of sperm 2min. using computer assisted semen analysis (CASA) the killing effect of detection of candidate drug on human sperm. Selected from the PD monomer is the most active, sharing the same semen in healthy men for 4 copies, respectively with 0.9%NaCl control, 0.15mmol/LPD, 0.20mmol/LPD, 0.20mg/ml N-9 (=0.308mmol/L) positive control 2min.CASA detection of 0.15mmol/LPD sperm motility (a+b+c%) was 47.15. 12.44,0.20mmol/LPD 21.38 + 10.69,0.25mmol/LPD sperm motility instantly completely inactivated. The PD optimal spermicidal concentration is 0.20mmol/LPD, used for experiments.
3. study on the mechanism of sperm killing
Semen upstream of healthy men, with apoptosis kit for detection of PD induced apoptosis on human sperm; swelling test kit with low permeability, impact on the integrity of human sperm membrane and LIVE/DEAD Sperm Viability Kit to detect the survival status of PD. The results showed that 0.20mmol/LPD induced apoptosis caused by a large number of sperm, sperm swelling rate decreased. LIVE/DEAD detected that the death of a large number of sperm; scanning and transmission electron microscopy results show that the fracture of head and neck of sperm, sperm membrane rupture, mitochondrial swelling and loss of membrane; CometAssay results showed that 0.20mmol/L PD will not cause damage to sperm DNA.
Detection of 3.1 sperm apoptosis: 0.10mmol/L, 0.15mmol/L, 0.20mmol/L, 0.25mmol/L and PD so that a large number of sperm apoptosis in late apoptosis and necrosis (FITC+PI+); 0.25mmol/L PD late apoptosis and necrosis accounted for 75.84 of the total number of sperm sperm + 11.12%; early apoptosis in each dose group PD on sperm (FITC+PI-) showed that PD had no effect. Destroy the sperm membrane.
3.2 hypotonic swelling test: most of the 0.9%NaCl control group had swelling (tail curl), while the PD group had no obvious expansion of sperm and low permeability and swelling reaction disappeared. The expansion rate decreased from 28 to 6.13% of 76.5 + 6.12%, and the expansion rate between the two groups was significantly different. The results showed that the sperm membrane was damaged by PD.
3.3LIVE/DEAD detection: the control group with green fluorescence (sperm live sperm), PD treated orange fluorescence (frequency of dead sperm), red fluorescence (dead sperm) increased significantly, the positive control group (0.2mmol/L N-9) and 0.1mmol/L PD group spermicidal effect similar to that of 0.2mmol/L PD group of sperm is almost red fluorescence, spermicide the rate of 95%.
3.4 scanning electron microscopy and transmission electron microscopy in scanning electron microscope, the control group of normal sperm morphology and acrosome clearly, after treatment, showed a large number of sperm head and neck fracture under TEM. The control group, sperm cell membrane integrity, mitochondrial structural integrity. After treatment, the sperm membrane rupture, mitochondrial swelling, membrane or even fall off.
3.5Comet Assay experiment: negative control group, all experimental spermatozoa in group PD0.2mmol/L did not find DNA mobile tail, and the positive control group had obvious DNA mobile tail.
4. animal experiments
4.1 contraceptive effect: female SD rats were injected 0.9%NaCl100 l right uterus (control group), 3mg/ml PD100 L in the left uterine (dose of 0.3mg), and postoperative 2:1 male rats cage; second days after vaginal plug found cage, 10 days after the mice were killed, found that the injection of PD (left) side of the uterus no gestational sac, and saline injection side (right) normal uterine pregnancy.
4.2 vaginal stimulation test: female rats were randomly divided into 5 groups, every time to give 0.2ml (about 0.5g) PD gel vaginal administration, continuous administration for 14 days, after the last administration were animal, remove the vaginal tissue, fixed with formaldehyde for histological examination and vaginal tissue pathological stimuli score [40]. natural control blank gel group, 3mg/g group, PD group, 8mg/gPD group, 8mg/g group N-9 vaginal irritation scores were 2.11 + 2.01,3.22 + 1.07,4.89 + 0.69,7.44 + 1.58,3.67 + 1.53. vaginal irritation test score less than 8 points, irritation in the acceptable range:
PD instantly kill effect on human sperm, can cause sperm apoptosis, study the spermicidal mechanism showed that PD damage of sperm membrane. Spermicidally concentration of PD damage in DNA rats, PD in vivo experiments confirmed that the contraceptive effective contraception, the vaginal irritation in the acceptable range of.PD as a possible future the clinical potential of spermicide for contraception.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R169.4;R285

【参考文献】