巴戟天水提液萃取成分对微波辐射大鼠下丘脑—垂体—睾丸轴的影响

发布时间：2018-04-15 22:29

  本文选题：巴戟天 + 微波辐射　； 参考：《福建医科大学》2014年博士论文

【摘要】：【目的】通过微波辐射构建雄性SD大鼠生殖系统损伤模型,观察巴戟天水提液经乙酸乙酯萃取后水层和酯层修复微波辐射致生殖系统损伤的功效,探讨巴戟天活性成分对下丘脑-垂体-睾丸轴(Hypothalamic-Pituitary-Testis axis,HPTA)的可能作用机制,为巴戟天有效成分的进一步筛选和对生殖系统损伤的修复作用及机制提供依据。第一部分【方法】12只健康成年雄性SD大鼠随机分为2组,每小组6只。A组为空白对照组,B组为辐射组。B组应用频率为900MHz、辐射密度为218μW/cm2的微波信号发生器,辐射24h/d,持续10天,以获得微波辐射致生殖系统损伤模型。观察大鼠生长发育、性行为学差异,比较睾丸、附睾指数和组织形态学的改变,观察下丘脑Gn RH荧光表达,以评估模型是否成功建立。【结果】1.体重:微波辐射10天后,辐射组大鼠体重增加(19.83±12.32)g,体重较之空白对照组(80.17±29.01)g差别有显著性意义(P0.05)。2.性行为学:与空白对照组相比,辐射组大鼠扑捉潜伏期(CIP)显著延长(P0.05),扑捉次数(CT)显著减少(P0.01)。3.与空白对照组相比,辐射组大鼠睾丸和附睾指数均显著降低(P0.05)。睾丸和附睾HE染色可见辐射组睾丸生精小管管径减小,排列疏松,未见精原细胞、各级生精细胞排列松散、脱落、坏死,附睾管内充满脱落的细胞及细胞碎片,未见精子。4.免疫荧光组织化学染色法:Gn RH主要表达在位于第三脑室底两侧的弓状核;较之空白对照组,Gn RH荧光强度在辐射模型组表达有所减弱。【结论】以频率为900MHz、辐射密度为218μW/cm2的低功率微波持续辐射10天可成功建立雄性SD大鼠生殖损伤模型。第二部分【方法】66只健康成年雄性SD大鼠随机分为11组(其中D、E组各有4个不同剂量组10g/kg、20 g/kg、30 g/kg、40 g/kg),每小组6只。A组为空白对照组,B组为辐射未给药组,C组为辐射+生理盐水组,D组为萃取水层梯度浓度液治疗组,E组为萃取酯层梯度浓度液治疗组。制备巴戟天水提液,经乙酸乙酯萃取后得到萃取水层和萃取酯层的梯度浓度液(每ml各含2、4、6和8g生药量)。以电感耦合等离子体质谱法(Inductively coupled plasma massspectrometry,ICP-MS)测定和对比与HPTA轴修复作用相关的微量元素锌(Zinc,Zn)、锰(Manganese,Mn)、硒(Selenium,Se)、铁(Iron,Fe)、铜(Copper,Cu)在巴戟天水提液各萃取层的分布情况。辐射后C、D、E组分别给予等量生理盐水或相应浓度的巴戟天提取物萃取液持续灌胃12天。观察大鼠性行为学差异,同时在下丘脑、垂体水平检测Gn RH表达,血清LH、FSH、Gn RH和T变化;比较睾丸、附睾指数以及组织形态学的改变。【结果】1.微量元素分布:ICP-MS测定结果显示微量元素锌、锰、铁在巴戟天水提液和巴戟天水提液乙酸乙酯萃取水层中含量一致,在巴戟天水提液乙酸乙酯萃取酯层中含量极少(P0.05)。铜含量在以上三份样品中差别无显著性意义。硒含量差别亦无显著性意义。2.性行为学巴戟天治疗后,D3、D4组扑捉潜伏期/D4组扑捉次数与B组、C组差别有统计学意义,余治疗组性行为指标与B组、C组差别均无统计学意义。3.性腺水平:(1)B~E组睾丸指数均低于A组,差别有统计学意义;B组附睾指数低于A组,差别有统计学意义;D4组睾丸、附睾指数与B组、C组差别有统计学意义,余治疗组与B组、C组差别均无统计学意义。(2)血清睾酮水平比较:A组与B组差别无统计学意义。D3、D4组睾酮水平与B组、C组差别有统计学意义,余治疗组与B组、C组差别均无统计学意义。(3)睾丸和附睾组织形态学改变:辐射组形态学改变见第一部分。D组中,随着给药剂量从20~40g/kg逐渐增加,大鼠睾丸生精小管上皮各级生精细胞及生精小管内精子数量逐渐增多;B、C、E组形态学无明显差别。辐射组大鼠附睾管管腔空虚、未见精子,可见少量脱落的生精细胞。D3、D4组大鼠附睾管内可见密集的精子,但仍少于空白对照组数的精子量。4.血清FSH、LH含量差异:各组间FSH差别均无统计学意义。A组与B组LH差别无统计学意义。D3、D4组LH水平与B组、C组差别有统计学意义,余治疗组与B组、C组差别均无统计学意义。5.Gn RH表达量对比:(1)经ELISA法测定各组血清Gn RH含量,显示B组大鼠血清Gn RH含量低于A组;D3、D4组Gn RH水平与B组、C组差别有统计学意义,余治疗组与B组、C组差别均无统计学意义。(2)免疫荧光组织化学染色法标记可见Gn RH主要表达在位于第三脑室底两侧的弓状核;B组Gn RH水平较A组下降,但差别无统计学意义。D3、D4组Gn RH与B组差别有统计学意义,余治疗组与B组差别均无统计学意义。(3)Real-time PCR结果显示。D4组大鼠下丘脑Gn RH-m RNA表达较之B、C组降低(P0.05)。余治疗组Gn RH-m RNA表达与B、C组差距无统计学意义。【结论】1.巴戟天水提液及巴戟天水提液乙酸乙酯萃取水层中富含微量元素锌、锰、铁,可能与生殖功能保护有关。2.巴戟天水提液乙酸乙酯萃取水层可改善大鼠性行为学,其机制可能与调节HPTA下丘脑、垂体以及性腺各个水平上相关激素分泌有关,其中最佳治疗剂量为40g/kg。3.巴戟天水提液乙酸乙酯萃取酯层对微波辐射诱导的大鼠生精功能障碍无明显修复作用。
[Abstract]:[Objective] to construct the model of radiation injury of reproductive system in male SD rats by microwave induced by extract of Morindae Officinalis, observe efficacy of injury of reproductive system after extracted by ethyl acetate layer and the ester layer repair microwave radiation of Morinda active components on the hypothalamic pituitary testicular axis (Hypothalamic-Pituitary-Testis, axis, HPTA) the possible mechanism, to provide the basis for further screening the constituents of Morinda officinalis and on the reproductive system damage repair effects and mechanism of the first part. [Methods] 12 healthy adult male SD rats were randomly divided into 2 groups, 6 in each group group.A group B group.B group applied radiation frequency is 900MHz, the density of radiation microwave signal generator, 218 W/cm2 radiation 24h/d, lasting 10 days, in order to obtain the microwave radiation induced reproductive system injury model. To observe the growth of rat, differences in behavior, compared with testis, epididymis The morphological index and tissue changes were observed in the hypothalamus Gn RH expression, to assess whether the model was successfully established. [results] 1. weight: 10 days after the microwave radiation, the radiation group weight increase (19.83 + 12.32) g, body weight compared with the blank control group (80.17 + 29.01) had significant difference (P0.05 g.2.) of behavior: compared with the control group, radiation group rats Puzhuo latency (CIP) significantly prolonged (P0.05), capture times (CT) was significantly reduced (P0.01).3. compared with the blank control group, radiation group of rat testis and epididymis index were significantly lower (P0.05) in testis and epididymis. HE staining decreased, visible radiation group seminiferous tubule diameter were arranged loosely, spermatogonial stem cells, spermatogenic cells arranged loose, fall off, necrosis, cell and epididymis tube filled with falling debris, no sperm.4. immunofluorescence staining method: Gn RH was mainly expressed in the in The end of the third ventricle on both sides of the arcuate nucleus; compared with the control group, the Gn fluorescence intensity of RH was reduced in the model group. [Conclusion] the frequency of 900MHz, low power microwave radiation density was 218 W/cm2 irradiation for 10 days can be successfully established model of reproductive damage in male SD rats. The second part [method] 66 healthy adult male SD rats were randomly divided into 11 groups (D, E group each have 4 different doses of 10g/kg, 20 g/kg, 30 g/kg, 40 g/kg), each group 6.A group was control group, B group was not radiation dose group, C group and normal radiation saline group, D group to extract water concentration gradient liquid treatment group, E group for the extraction of the ester layer concentration gradient liquid treatment group. Preparation of Morindae Officinalis Extract, the concentration gradient of liquid extraction and water extraction ester layer was extracted by ethyl acetate (each containing 2,4,6 per ml and 8g crude drug) by inductively coupled. Plasma mass spectrometry (Inducti Vely coupled plasma massspectrometry, ICP-MS) determination of trace elements zinc and compared with HPTA axis repair related (Zinc, Zn), Mn (Manganese, Mn), Se (Selenium, Se), iron (Iron, Fe), copper (Copper, Cu). The distribution of each layer in the liquid extract from Morindae officinalis radiation. After C, D, E group were given normal saline or the corresponding concentrations of Morinda officinalis extract extract continuous gavage for 12 days. Observe the behavior of rats and the difference in the hypothalamus, pituitary Gn RH expression, detect the level of serum LH, FSH, Gn, RH and T change; comparison of testis, epididymis index and the change of morphology. [results] 1. trace element distribution: ICP-MS measurement results show that the trace elements of zinc, manganese, and iron in Morindae Officinalis Extract from Morindae Officinalis Extract ethyl acetate extract water content in Morindae Officinalis Extract, ethyl acetate ester layer with low content (P0.05). The content of copper in the above 涓変唤鏍峰搧涓�宸�鍒�鏃犳樉钁楁�ф剰涔，

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