燃煤型氟中毒大鼠诱导型一氧化氮合酶蛋白表达、血清睾酮与生精细胞凋亡的相关性研究

Correlation of iNOS expression and serum testosterone level with the apoptosis of spermatogenic cells in rats following endemic fluorosis from coal burning

目的:研究燃煤型氟中毒大鼠睾丸组织诱导型一氧化氮合酶(iNOS)蛋白表达、血清睾酮(T)与生精细胞凋亡的相互关系,探索燃煤型氟中毒对睾丸生殖功能损伤的机制。方法:将40只雄性SD大鼠随机分为4组:对照组、低氟组、中氟组、高氟组。各染毒组喂饲含不同比例的燃煤型氟中毒病区煤烘玉米的饲料,建立雄性大鼠燃煤型氟中毒模型。分别于染毒4和6个月后以股动脉放血法处死大鼠,各时间点每组处死大鼠5只。采用RIA法测定大鼠血清T水平;检测血清中超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量、iNOS活性;原位缺口末端标记(TUNEL)法检测大鼠睾丸生精细胞凋亡情况并计算凋亡指数(AI);免疫组织化学染色SP法观察iNOS蛋白表达的变化。结果:成功建立雄性大鼠氟中毒模型。大鼠血清T水平在4和6个月时低氟组[(28.37±0.19)nmol/L、(14.68±0.36)nmol/L]、中氟组[(25.85±0.23)nmol/L、(9.08±0.31)nmol/L]、高氟组[(23.63±0.29)nmol/L、(4.89±0.54)nmol/L]较对照组[(29.77±0.43)nmol/L、(29.97±0.36)nmol/L]均降低(P<0.05或P<0.01),且各染氟组间差异有统计学意义(P<0.05或P<0.01)。睾丸生精细胞AI在4和6个月时低氟组(8.83±1.64、10.40±2.70)、中氟组(17.24±3.96、22.20±3.96)、高氟组(44.21±7.85、49.60±4.77)较对照组(0.46±0.11、0.54±0.11)升高(P均<0.01),且各染氟组间差异有统计学意义(P<0.05或P<0.01)。睾丸组织iNOS蛋白表达的OD值在4和6个月时低氟组(0.073 3±0.008 5、0.108 4±0.012 6)、中氟组(0.142 5±0.027 2、0.207 7±0.020 4)、高氟组(0.527 5±0.079 1、0.717 3±0.065 6)较对照组(0.001 3±0.000 2、0.001 6±0.000 1)增加(P<0.05或0.01),且各染氟组间差异有统计学意义(P<0.05或P<0.01)。iNOS蛋白表达量与生精细胞AI呈正相关(r=0.962,P<0.01);血清T水平与生精细胞AI呈负相关(r=-0.744,P<0.01)。结论:血清T水平改变和睾丸组织iNOS蛋白表达调控改变可能参与燃煤型氟中毒对睾丸生殖功能损害的过程。

Objective： To investigate the relationship of the expression of inducible nitric oxidase synthase（iNOS） and the level of serum testosterone（T） with the apoptosis of spermatogenic cells in the rat testis following endemic fluorosis from coal burning,and explore the mechanism of the damage to male reproduction induced by endemic fluorosis from coal burning. Methods： Forty male SD rats were randomly divided into four groups of equal number： normal control,low fluorosis,moderate fluorosis,and high fluorosis.The model of fluorosis was established by feeding the rats with corn dried by burning coal from endemic fluorosis areas. After 4 and 6months,the rats were sacrificed,the serum T levels measured by radio-immuno-assay,the activities of superoxide dismutase（SOD）and the expressions of malonaldehyde（ MDA） and iNOS determined by biochemical methods,and the apoptotic index of spermatogenic cells detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling（ TUNEL）. Results： The rat model of fluorosis was successfully established. At 4 and 6 months,the serum T levels were（ 28. 37 ± 0. 19） and（ 14. 68 ± 0. 36） nmol /L in the low fluorosis group,（ 25. 85 ± 0. 23） and（ 9. 08 ± 0. 31） nmol /L in the moderate fluorosis group,and（ 23. 63 ± 0. 29） and（ 4. 89 ±0. 54） nmol /L in the high fluorosis group,significantly lower than（ 29. 77 ± 0. 43） and（ 29. 97 ± 0. 36） nmol /L in the normal control（ P 〈0.05 or P 〈0.01） and with significant differences among the three model groups（ P〈 0.05 or P 〈0.01）; the apoptotic indexes of spermatogenic cells were 8. 83 ± 1. 64 and 10. 40 ± 2. 70 in the low fluorosis group,17. 24 ± 3. 96 and 22. 20 ± 3. 96 in the moderate fluorosis group,and 44. 21 ± 7. 85 and 49. 60 ± 4. 77 in the high fluorosis group,markedly higher than 0. 46 ± 0. 11 and 0. 54 ± 0. 11 in the control rats（ all P〈 0. 01） and with significant differences among the three model groups（ P 〈0. 05 or P 〈0. 01）; the optical densities of the iNOS expression were 0. 073 3 ± 0. 008 5 and 0. 108 4 ± 0. 012 6 in the low fluorosis group,0. 142 5 ± 0. 027 2 and0. 207 7 ± 0. 020 4 in the moderate fluorosis group,and 0. 527 5 ± 0. 079 1 and 0. 717 3 ± 0. 065 6 in the high fluorosis group,remarkably higher than 0. 001 3 ± 0. 000 2 and 0. 001 6 ± 0. 000 1 in the control（ P 〈0. 05 or P 〈0. 01） and with significant differences among the model groups（ P 〈0. 05 or P〈 0. 01） except between the moderate and high fluorosis groups. The apoptotic index of spermatogenic cells was correlated positively with the expression of iNOS（ r = 0. 962,P〈 0. 01）,but negatively with the serum T level（ r =-0.744,P 0.01）. Conclusion： Changes in the serum T level and iNOS expression in the rat testis may be involved in the damage to the reproductive function by coal burning-induced endemic fluorosis.