摘要
群勃龙醋酸酯（17β-hydroxyestra-4,9,11-trien-3-one17-acetate）是一种高效的甾类蛋白同化激素，它从20世纪70年代开始便在美国被作为促生长剂用于农场牲畜。群勃龙醋酸酯在体内释放后，便在动物血液中被迅速地水解成17β-群勃龙（17β-trenbolone,17β-hydroxyestra-4,9,11-trien-3-one）。17β-群勃龙与雄激素有着类似的结构，且是一种有效的雄激素受体（AR）激动剂。人类与17β-群勃龙的可能接触途径有以下几种：（1）有意注射。作为一种同化类雄激素类固醇，群勃龙被健身者作为用来增加肌肉和力量的药品。在已经发表的调查结果中，群勃龙是最受欢迎的注射药物之一。多数注射者为业余健身爱好者，他们以较大的剂量长期注射群勃龙。（2）食用肉残留。（3）食物链富集。17β-群勃龙被牲畜和人排出体外后，饲养场的废水、饲养场旁边的河流以及城市污水排放的下游均可能被17β-群勃龙污染，而且饲养场的粪便常被用作农田的肥料。17β-群勃龙在环境中性质稳定，具有较长的半衰期（在粪水中半衰期为260天）。17β-群勃龙可以进入水生生物和植物体内，经过食物链的层层累积，最终可能进入到人体内。（4）可能的临床应用。虽然17β-群勃龙与内源雄激素睾酮（T）及二氢睾酮（DHT）结构类似，但是其不能被5α-还原酶还原，也不能被芳香化，它所引起的雄激素样和/或雌激素样的副作用比一般的雄激素给药产生的副作用要小，因此17β-群勃龙作为药物与T及DHT相比有明显的优势。目前已有研究考虑将其应用于临床治疗。
因为17β-群勃龙是一种合成的雄激素，因此对其生殖毒性的研究一直都是热点，其它方面较少有人研究。目前的研究主要采用动物模型研究17β-群勃龙的生殖毒性。17β-群勃龙的生殖毒性包括破坏下丘脑-垂体-性腺轴（HPG axis）、干扰动物体内源激素的水平及影响动物生殖器官的发育和生殖能力。17β-群勃龙既会引起不同物种中雌性个体的雄性化，也可能会使发育中的雄性个体去雄性化。17β-群勃龙的神经毒性尚未见报道。在神经退行性病变（如阿尔茨海默症，AD）的发病机理中，除了遗传因素，环境因素也起着至关重要的作用。中枢神经系统（CNS）是内源雄激素的重要作用靶点之一，而17β-群勃龙作为一种合成雄激素也有可能作用于神经系统。
本研究分别用动物实验和细胞培养实验研究17β-群勃龙的神经毒性作用。在动物实验中，将17β-群勃龙经肌肉注射给正常成年Wistar大鼠及孕鼠，17β-群勃龙的注射剂量分别为0.2、1、5mg/kg体重，药物处理时间为0.5、2、6、12、24、48h。检测大鼠脑组织、肌肉、血液及脑脊液（CSF）中17β-群勃龙的浓度及β-淀粉样蛋白42（Aβ42）的水平，并分析大鼠血清激素水平的变化。在细胞培养实验中，研究了1-100nmol/L浓度的17β-群勃龙对原代培养大鼠海马神经元的影响，包括对其细胞活力、神经元凋亡的影响，及对AD相关蛋白早老素-1（PS1）和Aβ42的影响，探索了AR和雌激素受体（ER）及基因组效应和非基因组效应在17β-群勃龙的作用中所扮演的角色。
主要实验结果如下：
1.17β-群勃龙可以穿过大鼠血脑屏障，聚集于大脑，尤其是海马中。当17β-群勃龙注射剂量分别为0.2、1、5mg/kg体重时，在给药48h后海马中17β-群勃龙的浓度分别为：雄鼠5.42±0.27、6.12±0.21、7.03±0.17ng/g，雌鼠2.07±0.10、2.99±0.39、6.43±0.55ng/g。相对应的除海马外剩余脑组织中的17β-群勃龙浓度分别为雄鼠2.64±0.25、2.85±0.30、4.44±0.24ng/g，雌鼠1.55±0.20、1.89±0.22、3.74±0.26ng/g。雄鼠三个剂量组及雌鼠最高剂量组（5mg/kg体重）中的海马中17β-群勃龙的浓度均显著高于对应的除海马外剩余脑组织中的17β-群勃龙浓度。17β-群勃龙可以影响大鼠的血清激素水平，引起T、雌二醇（E2）及孕酮（PROG）水平的波动。17β-群勃龙也可以穿过胎盘屏障，并可在胎鼠脑中检出。给予孕鼠注射5mg/kg体重的群勃龙48h后，胎鼠脑中17β-群勃龙的浓度为2.59±0.21ng/g。17β-群勃龙可以引起雄性大鼠脑及海马中Aβ42的过量积累，胎鼠脑中Aβ42也显著增加。
2.为更好地研究17β-群勃龙对海马损伤的机制，本研究采用原代培养的海马神经元作为实验对象，进行体外实验。发现17β-群勃龙对原代培养的海马神经元有细胞毒性作用，可以引起细胞活力下降，引起其形态学改变、染色质凝集、凋亡小体形成、磷脂酰丝氨酸外翻、线粒体膜电位下降及caspase-3活力的增强。说明17β-群勃龙可以引起海马神经元的凋亡。17β-群勃龙能够抑制T对神经元的神经保护作用。研究中采用不同的激动剂和抑制剂处理，探究17β-群勃龙的细胞毒性作用机制。17β-群勃龙使原代培养的海马神经元caspase-3活性及Aβ42产生增加，并引起PS1蛋白表达水平的下降，这三种过程均是AR和ER依赖性的，其作用过程同时有基因组效应和非基因组效应的参与。
大鼠血清激素水平的改变表明其HPG axis可能受到影响。当孕鼠接触17β-群勃龙时，子代个体的CNS在胎儿时期可能就已经受到损伤。脑中Aβ42的增加将会影响胚胎CNS的正常发育及神经元的正常功能。根据文献中所提到的生殖毒性在子代出生后的发育过程中显现出来的结果，CNS所受的损害可能是不可逆转的。海马在学习和记忆等行为中发挥着重要作用，17β-群勃龙在海马中的累积及其对海马神经元的细胞毒性作用可能会产生严重的后果，如认知障碍、记忆损伤等，这些症状最后会引发痴呆。17β-群勃龙具有神经毒性，可以引起海马神经元凋亡，影响AD相关蛋白Aβ42和PS1的表达，它在神经退行性病变中起到了一定的作用。目前17β-群勃龙作为一种同化类雄激素类固醇被健身爱好者广泛使用，其使用剂量、使用周期、使用时间长短等应当受到监管。另外，17β-群勃龙是一种环境雄激素，使除了健身爱好者之外的更多的人也处在暴露于17β-群勃龙的危险中。神经退行性病变（如AD）的发病是受基因因素和环境因素共同影响的。目前大量的人力、物力、财力都放在了对基因因素的研究上，但是相当多的AD病人并没有携带如PS1、APP等的突变基因，因此环境因素在AD的发病中扮演着重要的角色。因为CNS是激素作用的重要靶点之一，因此该研究的意义不只在于对17β-群勃龙毒性作用的发现。环境激素在我们的生存环境中无处不在，它们有着与人体内源激素相似的结构和/或性质，因此它们对神经系统的影响值得关注。发现这些危险因素并采取预防措施，将对延缓神经退行性病变有重要意义。
本文主要创新点：
1.给成年大鼠肌肉注射17β-群勃龙后，17β-群勃龙会分布于脑中，尤其是海马中。给孕鼠注射后，可以在胎鼠脑中检出17β-群勃龙。神经组织是17β-群勃龙的一个作用靶点。
2.17β-群勃龙有神经毒性，会引起原代培养的海马神经元凋亡，引起AD相关蛋白Aβ42和PS1的表达水平变化。由神经元产生的Aβ42的增加，而PS1的蛋白表达水平下降，并且这些过程由激素受体介导。
3.17β-群勃龙作为一种同化类雄激素类固醇和一种环境雄激素，在神经退行性病变中发挥了一定作用，这对于17β-群勃龙的使用起到了指导作用，并启发研究者对环境激素与神经退行性病变之间关系的思考。
Trenbolone acetate (17β-hydroxyestra-4,9,11-trien-3-one17-acetate) is asynthetic anabolic steroid that has been used extensively since the1970s as a growthpromoter for livestocks in the USA. After being released, trenbolone acetate is rapidlyhydrolyzed to17β-trenbolone (17β-hydroxyestra-4,9,11-trien-3-one) in blood streamof the animals.17β-trenbolone shares similar molecular structure with endogenousandrogens. Humans are exposed to17β-trenbolone through several possible routes.(1)Humans contact trenbolone through deliberate injection. As an anabolic-androgenicsteroid, trenbolone is used by a large portion of recreational exercisers to increasemuscle size and strength. The exercisers often inject trenbolone in large doses for longtimes.(2)17β-trenbolone may remains in meat consumed by humans.(3)17β-trenbolone can be excreted by the animals and humans and the feedlot effluent,the river near the feedlot, and downstream discharges from urban wastewater can allbe contaminated by17β-trenbolone. Besides, manure from livestock feedlot iscommonly applied to agricultural fields as an alternative to commercial fertilizers.17β-trenbolone has long half-life (260d) and stable properties in the environment. Itmay be absorbed by aquatic animals as well as plants and can be incorporated intofood chains.(4)17β-trenbolone is regarded as a promising candidate in clinicalapplication. The properties that17β-trenbolone cannot be reduced or aromatized andis metabolized to less potent androgens and reduces serum testosterone anddihydrotestosterone levels make17β-trenbolone to be able to reduce incidence ofandrogenic and/or estrogenic side effects associated with androgen administration.
Because17β-trenbolone is a synthetic androgen, its reproductive toxicity has beenthe research hotspot. Previous studies using different animal models reported that17β-trenbolone exposure altered the endogenous hormone level, disruptedhypothalamic-pituitary-gonadal axis, and influenced development of reproductiveorgan and reproductive capacity.17β-trenbolone could cause masculinization of females of different animal species and demasculinization developing maleindividuals. So far no literature has reported on the neurotoxicity of17β-trenbolone.Both genetic and environmental factors contribute to neurodegenerative disorders. Ina large number of neurodegenerative diseases, for example Alzheimer's disease (AD),patients do not carry the mutant genes. Other risk factors, for example theenvironmental factors, should be evaluated. Brain is an androgen-sensitive tissue and17β-trenbolone is a synthetic androgen, thus17β-trenbolone might have effects onnervous system.
In the present study, both animal experiment and cell culture experiment werecarried to evaluate the neurotoxicity of17β-trenbolone. In the animal experiment,17β-trenbolone was administered to adult and pregnant rats, with different doses (0.2,1, and5mg/kg body weight) and different treatment time (0.5,2,6,12,24, and48h).17β-trenbolone distribution in the adult rats and fetal rats were determined. Its effectson hormone levels and Aβ42accumulation in vivo were evaluated. In the in vitrostudies, effects of17β-trenbolone on primary cultured hippocampal neurons weretested.17β-trenbolone's concentrations were from1-100nmol/L and the treatmenttime was48h. Cell viability and neuron apoptosis were assessed. Changes ofAD-related proteins β-amyloid (Aβ42) and presenilin-1(PS) were determined byELISA and western blot analysis, respectively. Underlying mechanisms were studied.
The main results were listed below.
1.17β-trenbolone could be detected in brain, hippocampus, cerebrospinal fluid,plasma, and muscle of male and female rats, and in fetus brain. It crossed the bloodbrain barrier and placental barrier.17β-trenbolone accumulated in adult rat brain,especially in hippocampus. When the injection doses were0.2,1, and5mg/kg bodyweight, as for male rats,17β-trenbolone concentrations in the hippocampus were5.42±0.27,6.12±0.21, and7.03±0.17ng/g, respectively.17β-trenbolone concentrationsin the rest of the brain were2.64±0.25,2.85±0.30, and4.44±0.24ng/g,respectively. As for female rats, when the injection doses were0.2,1, and5mg/kgbody weight,17β-trenbolone concentrations in the hippocampus were2.07±0.10,2.99±0.39, and6.43±0.55ng/g, respectively, while in the rest of the brain were1.55±0.20,1.89±0.22, and3.74±0.26ng/g, respectively. After being treated with17β-trenbolone at the dose of5mg/kg body weight for48h,17β-trenbolone concentration in fetus brain was2.59±0.21ng/g.17β-trenbolone administrationaltered the serum hormone levels, causing fluctuation of testosterone, estradiol, andprogesterone levels.17β-trenbolone administration led to Aβ42accumulation in thebrain (especially the hippocampus) of adult male rats. Increase of Aβ42was alsoobserved in fetus brain. Excessive Aβ42would influence the development and normalfunction of nervous system.
2. To investigate the effects of17β-trenbolone on hippocampal neurons andunderlying mechanisms,17β-trenbolone was administered to primary culturedhippocampal neurons.17β-trenbolone exerted cytotoxicity on hippocampal neurons.Cell viability was reduced and17β-trenbolone induced neuron morphological changes,chromatin condensation, nuclear fragmentation, translocation of phosphatidylserine,and mitochondrial membrane potential decrease in primary hippocampal neurons,indicating induction of apoptosis of the neurons.17β-trenbolone resistedneuroprotective function of testosterone. Presenilin-1protein expression wasdown-regulated while Aβ42production and caspase-3activities were increased. Bothandrogen and estrogen receptors mediated the processes, so did the genomic andnon-genomic signaling pathways.
In conclusion,17β-trenbolone had neurotoxicity. It can induce apoptosis ofhippocampal neurons and regulate the expression of AD-related proteins Aβ42andpresenilin-1.17β-trenbolone played roles in neurodegeneration.17β-trenbolone couldinfluence brain, especially the hippocampus, and the damage even occurs indevelopmental brains if the mother has been exposed to17β-trenbolone. As ananabolic-androgenic steroid, it is used widely in large doses for long times byexercisers. As an environmental androgen, common people may also be exposed to17β-trenbolone through various ways. Since damages of neurons may occur muchearlier than the clinical symptoms of neurodegenerative disorders, exposure to17β-trenbolone should be regarded as a high risk environmental factor in AD onset.Thus, the use of17β-trenbolone should be monitored. In addition, environmentalhormone may be at anywhere around us. Inspired by17β-trenbolone, when wepouring large amount of funds and efforts on research over genetic factors of AD, wecan spare some time to discover the environmental factors which may be influencingmore people.
Innovations:
1.17β-trenbolone distributed in the brain, especially in the hippocampus of theadult rats after intramuscular injection. It can also be detected in the fetus brain whenthe pregnant rats were exposed to17β-trenbolone. Nervous system is one of thetargets of17β-trenbolone.
2.17β-trenbolone is neurotoxic. It induced apoptosis of the primary hippocampalneurons and changed the expression of two AD-related proteins, Aβ42andpresenilin-1. Production of Aβ42was promoted while the protein expression ofpresenilin-1was down-regulated. The processes were mediated by hormone receptors.
3. As both an anabolic-androgenic steroid and an environment androgen,17β-trenbolone played roles in neurodegeneration. The results guided the use of17β-trenbolone. Moreover, they inspired the thinking of the relationship ofenvironmental hormones and neurodegenerative diseases.
引文
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