多种营养元素和强化大豆对氟中毒雌鼠性腺轴内分泌功能的作用

Combined effects of multiple nutrient elements and enriched soybean on damage of ovarian axis of female rats induced by fluorosis exposure

目的了解钙、镁、铜、锌、维生素D、蛋白质等多种营养元素对氟中毒雌鼠性腺轴内分泌功能的作用。方法2012年10月采集贵州省织金病区原煤烘烤的玉米喂养SD雌鼠，复制燃煤型氟中毒动物模型。采用随机数字表法将雌鼠分为4组：对照组（食用含氟5．2mg／kg，蛋白含量18％的饲料）、染氟组（食用含氟96mg／kg，蛋白含量18％的饲料）、多维元素干预组（食用含氟96mg／kg，多维元素添加片2．08g／kg．蛋白含量18％的饲料）、多维元素加强化大豆干预组（食用含氟96mg／kg，多维元素添加片2．08异／k，蛋白含量30％的饲料）。于180d动情期麻醉后处死雌鼠，股动脉取血分离血清，检测促性腺释放激素（GnRH）、促卵泡凝集（FSH）、黄体生成素（LH）、睾酮（T）、雌二醇（E2）、黄体酮（P）、抑制素（INH）含量；光镜下观察下丘脑、垂体、卵巢组织形态，透射电镜下观察卵巢颗粒细胞超微结构。结果光镜观察显示，各组雌鼠下丘脑、垂体均未出现明显组织损伤；染氟组雌鼠卵巢组织颗粒细胞出现中、重度水肿，细胞结构和细胞轮廓变得模糊，闭锁的卵泡和黄体退化增多，成熟卵泡偶见；两干预组组织结构基本正常，仅见轻度水肿。电镜观察发现，雌鼠染氟组卵巢组织超微结构出现颗粒细胞凋亡；两干预组雌鼠卵泡颗粒细胞超微结构均基本正常，可见胞质内多少不等的脂滴，未见凋亡样改变，其中多维元素加强化大豆干预组脂滴明显少于多维元素干预组。雌鼠血清GnRH、FSH、LH、T、E，、P、INH含量组间比较差异均有统计学意义（F=18．87、13．84、55．29、215．87、7．35、12．55、117．15，P均〈0．05）。其中染氟组血清GnRH、FSH、LH、T水平[（21．65±3．94）、（29．88±7．71）、（53．89±6．97）、（25．06±2．39）mg／L。曲高于对照组[（10．58±2．65）、（19．92±6．12）、（30．21±4．45）、（4．88±1．77）mg／L，P均〈0．05]；染氟组血清E2、P、INH水平[（40．72±7．65）、（22．15±4．32）、（48．08±3．53）mg／L]均低于对照组[（28．26±8．02）、（14．14±2．27）、（18．93±3．47）mg／L，P均〈0．05]。多维元素干预组[（15．69±3．02）、（31．70±2．35）、（35．65±9．73）、（23．32±4．13）mg／L]、多维元素加强化大豆干预组[（17．79±2．86）、（26．23±5．88）、（38．25±9．60）、（22．18士4．41）mg／L]血清FSH、LH、E2、P水平与染氟组[（29．88±7．71）、（53．89±6．97）、（28．26±8．02）、（14．14±2．27）mg／L]比较差异有统计学意义（P均〈0．05）。结论钙、镁、铜、锌、维生素D等多种营养元素对氟中毒所致雌鼠性腺轴内分泌损伤有明显的保护作用。且加入强化大豆后对这种保护作用有一定的增强。

Objective To observe the combined effects of multiple nutrient elements, including calcium, magnesium, copper, zinc, vitamin D and protein, on damage of hypothalamic-pituitary-ovary axis of female rats induced by fluorosis exposure. Methods In order to establish fluorine poisoning model, all rats in the experimental groups were fed with corn baked by high fluorine coal from Zhijin Guizhou Province in October 2012. Forty healthy female SD rats were randomized by weight and divided into control group （fed with food containing 5.2 mg/kg fluoride, and 18 percent of protein）, fluoride group （fed with food containing 96 mg/kg fluoride, and 18 percent of protein）, multiple nutrient elements group （fed with food containing 96 mg/kg fluoride, additives 2.08 mg/kg, and 18 percent of protein）, multiple nutrient elements and enriched soybean group （fed with food containing 96 mg/kg fluoride, additives 2.08 mg/kg, and 30 percent of protein）. After 180 days the rats were executed for femoral artery blood collection during the estrous period and serum concentration of gonadotropin-releasing hormone （GnRH）, follicle- stimulating hormone （FSH）, luteinizing hormone （LH）, testosterone （T）, estradiol （E2）, progesterone （P） and inhibin （INH） were detected. The pathological morphology of hypothalamus, pituitary, ovary were observed with microscopy and the uhra-structure of ovarian granulosa cells were observed with transmission electron microscopy. Results The results of microscope showed that there were no severe damage in hypothalamus and pituitary tissues of all groups. In the fluoride group, edema of ovarian granulosa cell was moderate to severe, cell structure and cell contours were blurred and unclear, and there was an increase in the number of atretic follicles and regressed luteal cells, mature follicles occasionally, while the two intervention groups showed mild edema. According to the results of transmission electron microscopy, apoptosis presented in granulosa ceils of ovarian tissue in the fluoride group, ultra- structure of follicular granulosa cells were essentially normal in the two intervention groups, but varying amounts of lipid droplets were observed in cytoplasm, moreover, lipid droplets of multiple nutrient elements and enriched soybean group were significantly less than those of the multiple nutrient elements group. The contents differences of GnRH, FSH, LH, T, E2, P and INH in serum of female rats between groups were statistically significant （F = 18.87, 13.84, 55.29, 215.87, 7.35, 12.55, 117.15, all P 〈 0.05）. Compared to the control groups [（10.58±2.65）, （19.92±6.12）, （30.21±4.45）, （4.88±1.77） mg/L], the serum GnRH, FSH, LH and T levels of fluoride groups were significantly increased [（21.65±3.94）, （29.88±7.71）, （53.89±6.97）, （25.06±2.39） rag/L, all P 〈 0.05]; compared to the control groups [（28.26±8.02）, （14.14±2.27）, （18.93±3.47） mg/L], the serum E2, P and INH levels of fluoride groups were significantly decreased [（40.72 ±7.65）, （22.15± 4.32）, （48.08±3.53） mg/L, all P 〈 0.05]. There were statistically significant differences of serum FSH, LH, E2 and P levels between the fluoride groups [（29.88±7.71）, （53.89±6.97）, （28.26±8.02）, （14.14±2.27） mg/L] and the two intervention groups [（15.69±3.02）, （31.70±2.35）, （35.65 ± 9.73）, （23.32±4.13） mg/L; （17.79±2.86）, （26.23±5.88）, （38.25± 9.60）, （22.18 ±4.41） mg/L]. Conclusion It is concluded that multiple nutrient elements, such as calcium, magnesium, copper, zinc and vitamin D, have displayed an obvious antagonistic effects on disorder of hormone secretion and pathological damage of ovaries induced by fluorosis exposure, and the influences are more significant in the presence of enriched soybean.