微波辐射对雄性大鼠生殖系统的昼夜时间毒性及分子机制

发布时间：2018-05-24 06:24

本文选题：微波辐射 + 雄性大鼠　；参考：《苏州大学》2014年博士论文

【摘要】：目的：微波辐射(Microwave Radiation, MWR)是一种重要的环境电磁污染物（EEPs），因无线通讯技术的快速发展，特别是全世界手机用户的急剧增加而备受关注。由于微波辐射可对雄性生殖系统产生不良的影响，明确其作用途径和损伤机制已成为迫切任务。已知哺乳动物的生理功能和行为存在昼夜节律，机体在24小时中不同时相对毒物及微波辐射敏感性不同。本课题采用时间生物学和生殖内分泌毒理学相结合的策略，在建立微波辐射动物模型的基础上，研究微波辐射对雄性生殖系统和褪黑素（Melatonin，MEL）分泌的昼夜时间毒性，以及褪黑素通过GATA-4/SF-1信号通路抑制睾酮合成的作用机制，为微波辐射对生殖功能损害的防治提供新的思路。方法：(1)微波辐射对雄性大鼠生殖系统的昼夜时间毒性。以褪黑素日节律筛选具有昼夜(L:D,12h:12h)节律的SD大鼠，分别在授时时间（zeitgeber time，ZT,以ZT0为光照开始）ZT0, ZT4, ZT8, ZT12, ZT16和ZT20进行微波辐射（1800MHz，205μw/cm2），每次2h，每天一次，连续32天。对应的假辐射组（Sham）同样处理但不给予微波信号。辐射后24h内每隔4h尾静脉采血一次，ELISA法测定睾酮含量；同时Sham和MWR各时点组在辐射结束时点采集睾丸和附睾组织。测定附睾精子活动度；制作睾丸HE病理切片，测定睾丸每日精子生成量、睾丸标志酶ACP和γ-GT活性，Real-time PCR检测睾酮合成基因StAR和p450cc mRNA表达。利用余弦节律分析软件，根据方程式F(t)=M+Acos(ωt+Φ)将各指标数据拟合为余弦曲线，分析节律参数。(2)微波辐射改变大鼠褪黑素分泌的昼夜节律。微波辐射方法同上，辐射32天后，24h内每隔4h尾静脉采血一次，ELISA法检测血浆褪黑素浓度，同时Sham和MWR各时点组在辐射结束时点采集松果体，Real-time PCR测定褪黑素合成酶NAT mRNA的表达。节律分析同上。(3)褪黑素抑制睾酮分泌的信号通路及微波辐射对睾丸间质细胞睾酮分泌的影响。以小鼠睾丸间质细胞系（TM3）细胞作为研究对象，添加不同浓度(0,10-6,10-8,10-10和10-12mol/L)的褪黑素，或先添加褪黑素受体阻断剂（Luzindole，1μmol/L）再加褪黑素，MTT法检测细胞增殖、流式细胞仪检测线粒体膜电位、ELISA法检测睾酮和cAMP，Real-time PCR和Western blotting测定褪黑素受体和睾酮相关基因GATA-4，SF-1，StAR，P450cc和3β-HSD的mRNA和蛋白的表达。通过睾丸注射褪黑素，利用免疫组化和免疫荧光方法以整体动物实验验证褪黑素抑制睾酮合成的信号通路。采用TM3细胞进行体外培养，在1800MHz微波辐射（205μw/cm2）2h后，流式细胞仪检测线粒体膜电位，ELISA法测定睾酮和cAMP含量，Real-time PCR测定睾酮代谢调节因子相关基因SF-1、StAR和P450cc mRNA表达的改变。结果：(1)微波辐射可使雄性大鼠睾丸组织发生病理改变，降低睾丸每日精子生成量、精子活动度、睾丸标志酶ACP和γ-GT活性，其中ACP和γ-GT活性以及每日精子生成量的下降在ZT0时辐射的MWR0组最为明显。微波辐射还可改变血浆睾酮的昼夜节律，其中MWR0、MWR8、MWR12和MWR20组的昼夜节律消失，同时MWR0组大鼠血浆中睾酮的日均值水平下降最大。睾酮合成调控因子StAR和P450cc的mRNA表达在微波辐射后失去昼夜节律，且MWR0变化最大，提示大鼠雄性生殖系统对微波辐射最敏感的时点为ZT0:00。(2)微波辐射降低褪黑素的日均分泌量，其中以MWR16组最为明显。微波辐射扰乱褪黑素合成的昼夜节律，其中MWR8，MWR16和MWR20组的血浆褪黑素昼夜节律被破坏，而振幅变化在MWR16组最为明显，表明在ZT16:00时微波辐射对大鼠血浆褪黑素节律的影响最大。MWR后松果体NAT基因表达的昼夜节律改变，以ZT16时下调最为明显，同时表达的中值和振幅明显改变，峰值相位明显滞后。(3)褪黑素在体外可抑制TM3细胞增殖，降低睾酮含量，减少细胞内cAMP浓度，改变线粒体膜电位，下调核转录因子GATA-4及其目标基因SF-1，，StAR，Cyp11A，HSD3β的基因和蛋白表达。褪黑素的这些抑制作用可被褪黑素受体阻断剂Luzindole所拮抗，特别是在褪黑素高水平作用时。大鼠睾丸组织注射褪黑素后0.5h、1h、2h，血浆中睾酮含量降低，睾丸间质细胞膜受体Mel1a，GATA-4和SF-1表达减弱，4h后上述效应消失。微波辐射后TM3细胞上清睾酮含量降低，细胞内cAMP浓度和线粒体膜电位水平下降，睾酮合成调节因子SF-1，StAR，P450cc基因表达下调。结论： 1、建立了微波辐射大鼠的动物模型，发现微波辐射对雄性大鼠的生殖功能和睾酮内分泌具有昼夜时间毒性。 2、微波辐射可改变褪黑素分泌和褪黑素合成酶NAT mRNA的昼夜节律，并通过褪黑素介导对雄性大鼠生殖功能的时间毒性。 3、体内外实验证实褪黑素通过GATA-4/SF-1信号通路抑制睾酮分泌，其中GATA-4因子起着关键性的作用。
[Abstract]:Objective: Microwave Radiation (MWR) is an important environmental electromagnetic pollutant (EEPs). It has attracted much attention due to the rapid development of wireless communication technology, especially the rapid increase of mobile phone users all over the world. The effect of microwave radiation on the male reproductive system is undesirable, and its mechanisms of action and damage mechanism have been made clear. For the urgent task. The physiological function and behavior of mammals are known to be circadian rhythms, and the body has different sensitivity to toxic substances and microwave radiation at the same time in 24 hours. This subject uses a strategy of combining time biology with reproductive endocrine toxicology to study microwave radiation on males based on the establishment of a microwave radiation model. The diurnal time toxicity of the reproductive system and melatonin (Melatonin, MEL), as well as the mechanism of the inhibition of testosterone synthesis by melatonin through the GATA-4/SF-1 signaling pathway, provides a new idea for the prevention and control of reproductive function damage by microwave radiation.
Methods: (1) the diurnal time toxicity of microwave radiation on the reproductive system of male rats. The SD rats with circadian (L:D, 12h:12h) rhythm were screened with melatonin diurnal rhythm, and ZT0, ZT4, ZT8, ZT12, ZT16, and ZT12, each time, once every day, at the time of time (zeitgeber time, ZT, ZT0 as light), respectively. For 32 consecutive days, the corresponding false radiation group (Sham) was treated with the same treatment but no microwave signal was given. Blood samples were collected every 4H tail vein in 24h after radiation, ELISA method was used to determine testosterone content. At the same time, the testis and epididymis were collected at each time point of Sham and MWR at the end of radiation. The motility of epididymis spermatozoa was measured; testicular HE pathological section was made and testicle was determined for each testicular test. Diurnal sperm production, testicular marker enzyme ACP and gamma -GT activity, Real-time PCR to detect the expression of testosterone synthesis gene StAR and p450cc mRNA. Using cosine rhythm analysis software, according to the equation F (T) =M+Acos (omega t+), each index data is fitted to cosine curve, and rhythmic parameters are analyzed. (2) microwave radiation changes the circadian rhythm of melatonin secretion in rats. The method of wave radiation was the same. 32 days after radiation, blood was collected once every 4H tail vein in 24h. ELISA method was used to detect the concentration of melatonin in plasma. At the same time, the pineal body was collected at the end point of radiation and Sham and MWR. Real-time PCR was used to determine the expression of melatonin synthase NAT mRNA. Rhythmic analysis was same. (3) melatonin inhibits the signaling pathway and micro of testosterone secretion. The effect of wave radiation on testosterone secretion in Leydig cells of testis. The mouse Leydig cell line (TM3) cells were used as the research object, adding melatonin of different concentrations (0,10-6,10-8,10-10 and 10-12mol/L), or adding melatonin receptor blocker (Luzindole, 1 mu mol/L) with melatonin, MTT method to detect cell proliferation, and flow cytometry to detect the line particles Testosterone and cAMP, Real-time PCR and Western blotting were used to detect the expression of melatonin receptor and testosterone related genes, GATA-4, SF-1, StAR, P450cc and 3 beta -HSD mRNA and protein, by ELISA method. Melatonin was injected through the testis, and immunohistochemistry and immunofluorescence methods were used to test the inhibition of testosterone synthesis by melatonin in whole animal experiments. The signal pathway was cultured in vitro by TM3 cells. After 1800MHz microwave radiation (205 u w/cm2) 2h, the mitochondrial membrane potential was detected by flow cytometry. The content of testosterone and cAMP was measured by ELISA. Real-time PCR measured the changes in the expression of testosterone metabolic regulator related genes SF-1, StAR and P450cc mRNA.
Results: (1) microwave radiation can make the testicular tissue of male rats pathological changes, reduce the daily sperm production, sperm motility, testicular marker enzyme ACP and gamma -GT activity, in which the activity of ACP and gamma -GT and the decrease of daily sperm production at ZT0 are most obvious. Microwave radiation can also change the day and night of plasma testosterone. The circadian rhythm of MWR0, MWR8, MWR12 and MWR20 disappeared, while the daily average level of testosterone in the plasma of MWR0 rats decreased most. The mRNA expression of the testosterone synthesis regulator StAR and P450cc lost the circadian rhythm after microwave radiation, and the MWR0 changes were the most, suggesting that the most sensitive time point of the male reproductive system to microwave radiation was the time point for the male reproductive system of rats. ZT0:00. (2) microwave radiation reduced the daily average secretion of melatonin, which was the most obvious in the MWR16 group. Microwave radiation disrupted the circadian rhythm of melatonin synthesis, in which the plasma melatonin circadian rhythm was destroyed in MWR8, MWR16 and MWR20 groups, and the amplitude changes were most obvious in the MWR16 group, indicating that microwave radiation at ZT16:00 was used in the plasma melatonin node in rats. The circadian rhythm of the NAT gene expression in the pineal body after the maximum effect of.MWR was the most obvious, and the median and amplitude of the expression were obviously changed, and the peak phase was obviously lagged. (3) melatonin could inhibit the proliferation of TM3 cells, reduce the content of testosterone, reduce the cAMP concentration in the cells, change the mitochondrial membrane potential and down regulate the nuclear transcription. The gene and protein expression of factor GATA-4 and its target gene SF-1, StAR, Cyp11A, HSD3 beta. These inhibitory effects of melatonin can be antagonized by melatonin receptor blocker Luzindole, especially at the high level of melatonin. After injection of melatonin in rat testicular tissue, 0.5h, 1H, 2h, plasma testosterone levels are reduced, and the membrane receptors of Leydig cells The expression of Mel1a, GATA-4 and SF-1 decreased. After 4h, the above effect disappeared. After microwave radiation, the content of testosterone in the supernatant of TM3 cells decreased, the concentration of cAMP in the cells and the level of mitochondrial membrane potential decreased, and the expression of testosterone synthesis regulator SF-1, StAR, and P450cc genes were downregulated.
Conclusion:
1, a rat model of microwave radiation was established. It was found that microwave radiation had a diurnal toxicity on reproductive function and testosterone secretion in male rats.
2, microwave radiation can change the circadian rhythm of melatonin secretion and melatonin synthetase NAT mRNA, and the time toxicity of Guro Susuke conductance to reproductive function in male rats.
3, in vivo and in vitro tests confirmed that melatonin inhibited the secretion of testosterone through the GATA-4/SF-1 signaling pathway, of which GATA-4 factor played a key role.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114

【参考文献】