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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 between the expression of inducible nitric oxide synthase (iNOS), the serum testosterone (T) and the apoptosis of spermatogenic cells in the rat model of fluorosis with fluorosis, and to explore the effect of coal-induced fluorosis on testicular reproductive function Damage mechanism. Methods: Forty male SD rats were randomly divided into 4 groups: control group, low fluoride group, middle fluoride group and high fluoride group. The rats in each group were fed diets containing coal and corn with different proportions of coal-fired fluorosis area to establish a coal-burning fluorosis model in male rats. Rats were sacrificed at 4 and 6 months after exposure to the femoral artery, and 5 rats were sacrificed at each time point. Serum T level was measured by RIA method. The activities of superoxide dismutase (SOD), malondialdehyde (MDA) and iNOS activity in serum were detected by enzyme linked immunosorbent assay (ELISA). The apoptosis of spermatogenic cells in testis was detected by TUNEL The apoptosis index (AI) was calculated and the changes of iNOS protein expression were observed by immunohistochemistry. Results: Fluorosis model was successfully established in male rats. The level of serum T in rats was significantly lower at 4 and 6 months (28.37 ± 0.19 nmol / L, 14.68 ± 0.36 nmol / L, respectively, and 25.85 ± 0.23 nmol / L and (29.77 ± 0.43) nmol / L, (29.97 ± 0.36) nmol / L, (4.89 ± 0.54) nmol / L, L] (P <0.05 or P <0.01), and there was a significant difference between each fluoride group (P <0.05 or P <0.01). Testicular spermatogenic AI in low fluoride group (8.83 ± 1.64, 10.40 ± 2.70), intermediate fluoride group (17.24 ± 3.96, 22.20 ± 3.96) and high fluoride group (44.21 ± 7.85, 49.60 ± 4.77) at 4 and 6 months (0.46 ± 0.11,0.54 ± 0.11) (P <0.01), and there was a significant difference between each fluoride group (P <0.05 or P <0.01). The OD value of iNOS protein expression in testis tissue was significantly lower at 4 and 6 months in the low fluoride group (0.073 3 ± 0.008 5 and 0.108 4 ± 0.012 6) and in the medium fluoride group (0.142 5 ± 0.027 2, 0.207 7 ± 0.020 4) The fluoride group (0.527 5 ± 0.079 1, 0.717 3 ± 0.065 6) increased (P <0.05 or 0.01) compared with the control group (0.001 3 ± 0.000 2 and 0.001 ± 6 ± 0.000 1) Significance (P <0.05 or P <0.01). The expression of iNOS protein was positively correlated with spermatogenic AI (r = 0.962, P <0.01). The serum T level was negatively correlated with spermatogenic AI (r = -0.744, P <0.01). Conclusion: The change of serum T level and the regulation of iNOS protein expression in testis may be involved in the process of testicular reproductive dysfunction caused by coal-induced fluorosis.