枸杞多糖对己烯雌酚致成年雄性仓鼠生精损害的缓解作用及机理研究

发布时间：2018-03-24 17:32

本文选题：己烯雌酚　切入点：枸杞多糖　出处：《河北农业大学》2014年硕士论文

【摘要】：本实验旨在研究己烯雌酚（Diethylstilbestrol，DES）对成年雄性仓鼠生殖损伤的作用机制及枸杞多糖（Lyciumbar barum polysaccharide，LBP）的缓解作用。将66只成年雄性仓鼠随机分为空白对照组（A），DES组（B），1mg/kg、10mg/kg及50mg/kg LBP组（C，D，E）及VC+VE组（F）（n=11）。除对照组外，其余5组均皮下注射己烯雌酚，对照组注射等剂量的橄榄油，连续用药7d，同时C、D、E组分别灌胃1、10、50mg/kg的LBP，F组灌服100mg/kg的VC及200IU/kg的VE，A、B组灌服等剂量的生理盐水。于末次给药24h后，乙醚麻醉仓鼠，心脏采血，分离血清，分别用于氧化指标SOD、GSH-Px、MDA及激素T、LH、FSH的检测；分别摘取两侧睾丸、附睾、精囊腺，迅速去除周围脂肪及结缔组织称量并计算脏器指数；于波恩氏液固定睾丸制成切片观察组织学变化，并进行免疫组化Bax、Fas、FasL及Caspase-3的检测；取睾丸迅速保存于液氮中进行RT-PCR及Western-blot检测，确定Bcl-2、Bax、Fas、FasL及Caspase-3的表达。结果显示DES显著降低仓鼠体重、器官重量及器官指数，而10/50mg/kg LBP及维生素对其有显著缓解作用（P0.05）；DES处理组仓鼠睾丸组织结构严重损伤，LBP作用后其损伤显著减轻，其中以10/50mg/kg LBP的作用最为明显，VC+VE也对其有的缓解作用，且50mg/kg LBP组与VC+VE组无显著差异。通过免疫组织化学、RT-PCR及Western-blot的方法检测睾丸组织Bcl-2、Bax、Fas、FasL及Caspase-3的表达，结果显示DES显著降低Bcl-2的表达（P0.01），提高Bax、Fas、FasL及Caspase-3的表达（P0.05）；给予不同剂量的LBP后，Bcl-2的表达呈剂量依赖性增加，以50mg/kg LBP增加最为显著（P0.01），Bax、Fas、FasL及Caspase-3的表达呈LBP剂量依赖性降低，以50mg/kg LBP组表达最低（P0.01）；与VC+VE组相比，50mg/kg LBP对凋亡蛋白的调节作用差异不显著；在抗氧化指标检测中，DES显著性降低血清SOD、GSH-Px水平（P0.01），增加MDA水平（P0.01）；补充不同剂量的LBP后，血清SOD、GSH-Px水平增加（P0.01），而MDA水平下降（P0.01），VC+VE组对机内抗氧化指标的作用要优于LBP组；对血清生殖激素的检测发现，，血清FSH、LH和T的水平在DES作用下降低（P0.01）；而LBP对血清中激素的水平有不同程度的提高，其中以50mg/kg作用最显著（P0.01）。结论：本实验证实，DES通过降低机体抗氧化酶活性，扰乱生殖内分泌系统，激活体内的生殖凋亡途径导致成年雄性仓鼠生殖损伤；补充不同剂量的LBP后，LBP降低睾丸组织的脂质过氧化水平，升高抗氧化酶活性；提高生殖激素FSH、LH和T的水平；抑制Bax、Fas、FasL及Caspase-3凋亡因子的表达，而促进Bcl-2的表达，结果提示10mg/kg或50mg/kg LBP能够有效缓解DES诱导的成年雄性仓鼠的生殖损伤。
[Abstract]:The aim of this study was to study the mechanism of diethylstilbestrol diethylstilbene (Diethylstilbene) on reproductive injury in adult male hamsters and the effect of Lycium barbarum barum polysaccharide on LBP.The 66 adult male hamsters were randomly divided into control group (1 mg / kg) and 50mg/kg LBP group (10 mg / kg) and VC. In VE group, there was no significant difference between the two groups, except for the control group. The other five groups were subcutaneously injected with diethylstilbestrol, while the control group was injected with olive oil of the same dose for 7 days. Meanwhile, the control group was given intragastric administration of VC of 100mg/kg and the group of 200IU/kg was given saline of the same dose 24 hours after the last administration. The rats were anesthetized with ether, the blood was collected from the heart, the serum was isolated and used for the detection of the oxidation index SODX GSH-PxX MDA and the hormone TLHH FSH, the testis, epididymis and seminal vesicle were removed, the surrounding fat and connective tissue were removed rapidly and the visceral index was calculated. The specimens of testis were fixed in Bonn's fluid to observe the histological changes, and the expression of FasL and Caspase-3 in Bax-2P Bax-Fas-FasL and Caspase-3 were determined by immunohistochemical staining, and the RT-PCR and Western-blot of the testis were rapidly preserved in liquid nitrogen to determine the expression of FasL and Caspase-3. The results showed that DES significantly reduced the body weight, organ weight and organ index of hamsters, and that 10/50mg/kg LBP and vitamin could significantly alleviate the damage to the testis of hamsters treated with P0.05DES. The effect of 10/50mg/kg LBP was the most obvious, and there was no significant difference between the 50mg/kg LBP group and the VC VE group. The expression of Bcl-2 LBP FasL and Caspase-3 in testicular tissue was detected by immunohistochemistry RT-PCR and Western-blot. The results showed that DES significantly decreased the expression of Bcl-2 and increased the expression of FasL and Caspase-3 in LBP, and increased the expression of Bcl-2 in a dose-dependent manner, especially in 50mg/kg LBP. Compared with VC VE group, the effect of 50 mg / kg LBP on apoptosis protein in 50mg/kg LBP group was lower than that in VC VE group, and DES significantly decreased serum SDS-GSH-Px level and increased MDA level (P0.01). The level of GSH-Px in serum increased P0.01, while the level of MDA in VC VE group was better than that in LBP group. The levels of serum FSH LH and T decreased under the action of DES, while LBP increased the levels of serum hormones to varying degrees, among which 50mg/kg played the most significant role in P0.01. Conclusion: this study demonstrated that des could induce reproductive injury in adult male hamsters by reducing the activity of antioxidant enzymes, disrupting the reproductive endocrine system and activating the pathway of reproductive apoptosis in vivo. After different doses of LBP supplementation, LBP decreased the level of lipid peroxidation in testis, increased the activity of antioxidant enzymes, increased the levels of FSH LH and T, inhibited the expression of FasL and Caspase-3 apoptosis factor, and promoted the expression of Bcl-2. The results suggest that 10mg/kg or 50mg/kg LBP can effectively alleviate the reproductive injury induced by DES in adult male hamsters.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：S853.7

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