| Objective: To observe intervention effects of calcium, magnesium,copper, zinc, vitamin D, and enriched soybean to gonad axis injurious induced by coal-burning fluorosis on female rats and its mechanism.Methods:1. SD female rats were feeding on zhijin coal-baked corn feed in ward to replicate the coal-burning fluorosis animal models and divided into four groups:control group, fluoride group,multiple nutrient element intervention groups, multiple nutrient element and intensive soybean intervention groups. Control group received a normal feed, fluoride group feed on the food containing 100 mg/kg fluoride, the food of multiple nutrient element intervention groups was joined caltrate on the basis of fluoride group.The food of multiple nutrient element and intensive soybean intervention groups was joined caltrate and intensive soybean on the basis of fluoride group.2. Dynamic To observe the changes of female rat’s teeth during exposed to fluoride dynamiclly.3. Ion selective electrode method, high-temperature ashing-fluoride ion-selective electrode were used to determinate urinary fluoride level and bone fluoride content in female rats. 4. of emotional period Female rats in estrus were executed after anesthesia on different time(60d, and 120 d, and 180d).Serum level of FSH,LH,T,E2 P, were detected by electrochemical glow radiation immune method and GnRH, INH 5 with the method of ELISA.(1) Separate hypothalamus, pituitary and ovarian from Other organizations and weighing, then calculation coefficients of hypothalamus,pituitary and ovarian.(2) HE staining of hypothalamus, pituitary, ovary were observed under optical microscope.(3)Ultrastructure and apoptosis of granulosa cells of ovary were observed electron microscopic.Results:1. Animal models of fluoride group and intervention group were copied successfully 2. Apart from the control group, the remaining groups showed varying degrees of dental fluorosis, gradually increased with time, and that of two intervention groups were lesser than the fluoride group,moreover,between the two groups, multiple nutrient element and enriched soybean intervention groups were lesser than multiple nutrient element intervention groups;3. The fluoride content within urine and bone of the fluoride group were higher than the control group(p < 0.05)at the same time point(60d,120 d,180d);Urinary fluoride of fluoride group, multiple nutrient element intervention groups,multiple nutrient element and enriched soybean intervention groups were increased in turn(p<0.05), and bone fluoride reduced in turn(p<0.05).Over time exposed to Urinary fluoride content of fluoride group, multiple nutrient element interventiongroups and multiple nutrient element and enriched soybean intervention groups were increased(p < 0.05), bone fluoride content was increased(p < 0.05). There were no significant between bone fluoride content of multiple nutrient element intervention groups 60 d and 120d(p>0.05), while significance between others(p<0.05) 4. The pituitary, hypothalamus and ovary coefficients between the groups were not statistically significant(p > 0.05) 5. Under the light microscope, fluoride groups granulosa cell got a edema changes from mild to severe progressive,The cell structure and outline got blur, atretic follicles and degradation of corpus luteum increases gradually, mature follicle occasionally. Two intervention groups of rare mild edema of follicular granulosa cells, cell structure and outlines to normal, normal follicle is common and atretic follicles and degradation of corpus luteum was significantly lower than the fluoride group 6. granule cells and stromal cells of ovarian tissue with Fluorine group were occurred apoptosis Two intervention group can still be seen apparent ultrastructural damage in the early stage. With the intervention time was prolonged, two intervention group ultrastructure of follicular granulosa cells and stromal cells are basically normaland no apoptosis changes, compared with fluoride.After adding enriched soybean, multiple nutrient element and intensive soybean intervention groups showed ultrastructure in ovary lipid droplets less than multiple nutrient element intervention groups, 7.(1)The same time(60d, 120 d, 180d) the female GnRH, FSH, LH and t levels in serum fluoride group were higher than those in the control group, with the exception of 60 d fluoride group serum FSH level is slightly higher than in the control group was not statistically significant(p>0.05), the other was statistically significant(p<0.05);Two intervention groups compared with fluoride group, 60 d female rat serum GnRH, FSH, LH and T levels had no significant difference, in addition to LH 60 d fluoride group was higher than multiple nutrient element and intensive soybean intervention groups was statistically significant(p <0.05), the other was not statistically significant(p > 0.05),120 d, 180 d, content of hormone in serum are higher than the two intervention groups, and both intervention groups compared with the fluoride group were statistically significant;Comparison between the two intervention groups, with the exception of LH multiple nutrient element and intensive soybean intervention groups lower than multiple nutrient element intervention groups was statistically significant, the rest are not statistically significant;With the extension of time, in addition to serum FSH levels compared to60 d and 180 d and serum LH levels compared to 60 d,120d and 180 d in the control group was statistically significant(p < 0.05), the other showed no significant(p >0.05);The levels of GnRH, FSH, LH and T in serum extended gradually increased over time, with the exception of contents of serum LH compared to 60 d and 120 d, and GnRH compared in the group was no statistically significant(p>0.05), the rest werestatistically significant(p<0.05);Serum LH concentration compared to the 60 d, in multiple nutrient element intervention group,120 d and 180 d was statistically significant(p<0.05) the rest are not statistically significant(p>0.05), in multiple nutrient element and intensive soybean intervention group,content of serum FSH, LH compared 60 d with 180 d and content of serum GnRH, compared 60 d,120d with 180 d was statistically significant(p < 0.05) the rest are not statistically significant(p >0.05)(2)60d, 120 d, 180 d P, INH serum levels were lower than the control group, with the exception of fluoride group. P level in serum compared with the control group was not statistically significant(p>0.05) on the point of 60 d, the other showed a significant(p<0.05);compared with the two intervention groups, P and INH serum levels of fluoride group were similar with two interventions groups and no statistical significance when 60(p>0.05), 120 d, 180 d, the fluoride group compared with the two intervention groups were significant decrease and showed statistically significant(p <0.05);In the two interventions,INH of the multiple nutrient element and enriched soybean intervention groups were higher than multiple nutrient element intervention groups and statistically significant(p < 0.05);the Serum level of P between two intervention groups were similar.With the extension of time, P level in fluoride groups reduced gradually, in addition to, compared with 60 d and 120 d,180d,there was statistically significant,except the rest(p>0.05).Contents of INH in serum decreased gradually in fluoride groups, 60 d, 120 d, compared with 180 d,showed statistical significance(p > 0.05), the other three groups increased as time went by, 60 d and 180 d was statistically significant in Control group and multiple nutrient element intervention group.(3)60d E2 serum level in the control group,exposed to fluoride group, multiple nutrient element intervention groups, multiple nutrient element and intensive soybean intervention groups increased, except fluoride group and multiple nutrient element intervention groups showed no statistical significance(p>0.05) the other showed a significant(p<0.05). 120 d, 180 d, fluoride group were less than other groups, apart from the multiple nutrient element intervention groups compared with the fluoride group no significant results(p>0.05),the rest were statistically significant(p<0.05),multiple nutrient element and intensive soybean intervention groups results are higher than multiple nutrient element intervention groups, but without statistical significance(p>0.05). Serum E2 levels in the control group as time goes from low to high to low and 60 d and 120 d showed that there was significant(p<0.05) The serum levels of E2 gradually reduces with time extended and showed statistical significance in fluoride group. The serum levels of E2 had a decreasing trend in the two intervention groups and 60 d compared with 180 d was showed statistical significance(p < 0.05)Conclusion:1.There were a certain antagonizing action that Calcium, magnesium, copper, zinc, vitamin d and other nutrients could confront the reduction of P, E2, INH and increase of Tsignificantly caused by fluorosis and with intervention prolonged, hormones of ovarian related were tend to be normal. So that the feedback effects of the hormone GnRH hypothalamus and pituitary hormones FSH and LH wane, serum levels of GnRH, FSH and LH levels were becoming more normal 2. Calcium, magnesium,copper, zinc, vitamin d and other nutrients have significant antagonism on ovarian tissue damage of female rats induced by fluoride(follicular growth and Ultrastructure of ovarian granulosa cells and stromal cells) and with intervention prolonged, better results got. 3. Antagonism were much more significant after enriched soy added in calcium, magnesium, copper, zinc, vitamin D, and with time prolonged,better results got. |
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