甲状腺激素通过调节甲亢大鼠卵巢葡萄糖转运影响黄体增殖和血管生成、凋亡的机制

Mechanism by which thyroid hormone affects the proliferation,angiogenesis and apoptosis of the corpus luteum by regulating ovarian glucose transport in hyperthyroid rats

目的探究甲状腺激素通过调节甲亢大鼠卵巢葡萄糖转运影响黄体增殖和血管生成、凋亡的机制。方法将无特定病原体(SPF)级3周龄Sprague-Dawley大鼠根据随机数字表法分为两组:对照组(大鼠每日灌服5 mL蒸馏水,n=5)和甲亢组(L-甲状腺素250μg/kg溶于5 mL蒸馏水后灌胃,诱导大鼠甲亢,n=5)。甲亢诱导第14天处死大鼠并对身体和卵巢称重。通过放射免疫分析试剂盒分析检测大鼠血清三碘甲状腺原氨酸(T3)、甲状腺素(T4)和促甲状腺激素(TSH)的浓度。通过蛋白质印迹法分析大鼠卵巢组织中Glut1、Glut4的蛋白表达。通过免疫组织化学染色分析黄体细胞增殖标记蛋白CDC47的蛋白表达和血管内皮生长因子(VEGF)的蛋白表达。通过TUNEL分析黄体细胞凋亡。结果甲亢组较对照组卵巢重量减轻[(18.79±1.24) g vs.(38.71±4.25) g],差异有统计学意义(P <0.05),体重[(156.58±24.77) g vs.(147.39±22.15) g]差异无统计学意义(P> 0.05)。甲亢组较对照组T3[(2.65±0.27)ng/mL vs.(0.42±0.01) ng/mL]和T4[(48.42±6.37) ng/mL vs.(154.82±39.58) ng/mL]浓度升高,差异均有统计学意义(P <0.05),而TSH浓度(0.54±0.05) ng/mL vs.(0.51±0.04) ng/mL比较差异无统计学意义(P> 0.05)。甲亢组较对照组Glut1蛋白表达降低(1.86±0.17 vs. 1.86±0.17),而Glut4蛋白表达升高(2.16±0.21 vs. 1.16±0.02),差异均有统计学意义(P <0.05)。甲亢组较对照组的CDC-47表达的总细胞数[(15.71±2.39)个vs.(22.43±4.18)个]、CDC-47表达的黄体细胞数[(9.55±1.03)个vs.(7.24±0.88)个]和CDC47表达的内皮细胞/周细胞的百分比[(42.38±2.11)%vs.(55.63±3.96)%]降低,差异均有统计学意义(P <0.05)。在上一周期的黄体中,甲亢组较对照组VEGF表达的百分比[(66.32±5.44)%vs.(50.63±4.13%)]升高,差异均有统计学意义(P <0.05),新形成的黄体中VEGF表达的百分比[(76.58±7.18)%vs.(73.24±6.88)%]比较差异无统计学意义(P> 0.05)。甲亢组较对照组细胞凋亡量增加(0.58±0.03 vs. 0.31±0.01),差异有统计学意义(P <0.05)。结论甲状腺激素通过改变黄体增殖、凋亡和血管生成活性来影响大鼠卵巢的寿命和功能,实验性甲亢降低了大鼠黄体、血管的增殖活性,同时促进黄体细胞凋亡。

Objective To explore the mechanism by which thyroid hormone affects the proliferation,angiogenesis and apoptosis of the corpus luteum by regulating the ovarian glucose transport in hyperthyroid rats. Methods Specific pathogen free Sprague-Dawley rats of 3 weeks old were divided into two groups according to the random number table method: control group( rats take 5 mL distilled water daily,n = 5) and hyperthyroidism group( L-thyroxine 250 μg/kg dissolved in 5 mL distilled water and then gavage,induce hyperthyroidism in rats,n = 5). Rats were sacrificed on the 14 th day of hyperthyroidism induction,and their bodies and ovaries were weighed. The concentration of triiodothyronine( T3),thyroxine( T4),thyroid stimulating hormone( TSH) in rat serum was analyzed by the radioimmunoassay kit. The protein expression of Glut1 and Glut4 in rat ovarian tissue was analyzed by Western blotting. The protein expression of luteal cell proliferation marker protein CDC47 and the protein expression of vascular endothelial growth factor( VEGF) were analyzed by immunohistochemistry. The apoptosis of luteal cells was analyzed by TUNEL. Results The ovarian weight of the hyperthyroidism group was less than that of the control group [( 18. 79 ± 1. 24) g vs.( 38. 71± 4. 25) g],the difference was statistically significant( P < 0. 05),but there was no statistically significant difference in body weight [( 156. 58± 24. 77) g vs. 147. 39 ± 22. 15) g]( P > 0. 05). The T3 [( 2. 65 ± 0. 27) ng/mL vs. 0. 42 ± 0. 01) ng/mL] and T4 [( 48. 42 ± 6. 37) ng/mL vs.( 154. 82 ± 39. 58) ng/mL) ] concentrations of the hyperthyroidism group were higher than those of the control group,the differences were statistically significant( P < 0. 05),and there was no difference in the TSH concentration [( 0. 54 ± 0. 05) ng/mL vs.( 0. 51 ± 0. 04) ng/mL]( P >0. 05). Compared with the control group,the expression of Glut1 protein was lower in the hyperthyroidism group( 1. 86 ± 0. 17 vs. 1. 86 ± 0. 17),while the expression of Glut4 protein( 2. 16 ± 0. 21 vs. 1. 16 ± 0. 02) was higher,the differences were statistically significant( P < 0. 05). Compared with the control group,the total number of cells expressed by CDC-47 in the hyperthyroidism group( 15. 71 ± 2. 39 vs. 22. 43 ± 4. 18),the number of luteal cells expressed by CDC-47( 9. 55 ± 1. 03 vs. 7. 24 ± 0. 88) and the percentage of endothelial cells/pericytes expressing CDC47[( 42. 38 ± 2. 11) % vs.( 55. 63 ± 3. 96) %]decreased,the differences were statistically significant( P < 0. 05). In the corpus luteum of the previous cycle,the percentage of VEGF expression [( 66. 32 ± 5. 44) % vs.( 50. 63 ± 4. 13) %]in the hyperthyroidism group was higher than that in the control group( P < 0. 05),and there was no difference in the percentage of VEGF expression[( 76. 58 ± 7. 18) % vs.( 73. 24 ± 6. 88) %]in the newly formed corpus luteum( P > 0. 05). The amount of apoptosis in the hyperthyroidism group was higher than that in the control group( 0. 58 ± 0. 03 vs. 0. 31 ± 0. 01),the difference was statistically significant( P < 0. 05). Conclusion Thyroid hormone affects the lifespan and function of rat ovaries by changing the proliferation,apoptosis and angiogenesis activity of the corpus luteum. Experimental hyperthyroidism reduces the proliferation activity of the rat corpus luteum and blood vessels,and at the same time promotes the apoptosis of corpus luteum cells.