甲状腺素联合多奈哌齐对成年期甲状腺功能减退症大鼠海马munc-18、乙酰胆碱的影响

摘要

目的观察甲状腺素联合多奈哌齐对成年期甲状腺功能减退症(简称甲减)大鼠海马munc-18表达及乙酰胆碱含量的影响，探讨甲减脑损伤及恢复的可能分子机制。方法55只SD大鼠随机均分为甲减组、甲状腺素治疗组、多奈哌齐治疗组、联合治疗组及对照组。丙基硫氧嘧啶( PTU)建立成年期大鼠甲减模型共6周。第5周起，在继续甲减造模的前提下，甲状腺素治疗组每日给予左旋甲状腺素(L-T4)6μg/100 g体重( BW)腹腔注射，多奈哌齐治疗组每日在饮用水中加入0.005%( w/v)多奈哌齐，联合治疗组每日给予L-T46μg/100 g BW腹腔注射以及在饮用水中加入0.005%( w/v)多奈哌齐，对照组每日给与等量的生理盐水腹腔注射。采用放射免疫法测定血清甲状腺激素水平，碱性羟胺比色法测定海马内乙酰胆碱含量，免疫组织化学法观察munc-18在大鼠海马各层的表达与分布。结果与对照组比较，甲减组中大鼠血清三碘甲状腺原氨酸( T3)、四碘甲状腺原氨酸(T4)水平减低(P<0.01)，促甲状腺激素(TSH)水平升高( P <0.01)， munc-18在 CA3区起始层( SO )、放射层( SR)、DG区分子层( ML)、DG区多形层( PL)表达明显减少(P<0.01)，海马内乙酰胆碱含量下降(P<0.05)；与对照组比较，甲状腺素治疗组及多奈哌齐治疗组中乙酰胆碱表达恢复，但munc-18表达仍然低下( P<0.05)；联合治疗组中乙酰胆碱含量及munc-18的表达与对照组相比差异无统计学意义。结论甲状腺素联合多奈哌齐可以改善成年期甲减造成的海马内突触蛋白损伤。%Objective To evaluate the thyroxine and donepezil ( DON) effect on the acetylcholine ( Ach) content and munc-18 expression in the hippocampus of adult hypothyroid rats and clarify the possible mechanism under the hypothyroidism-induced cognitive dysfunction. Methods 55 Sprague-Dawley rats were randomly divided into five groups:control, hypothyroid (PTU:0.05%; added to the drinking water), hypothyroid treated with T4 (6 μg/100 g body weight once daily;ip injection) , hypothyroid treated with DON (0.005%;added to the drinking wa-ter ) and a combination of both drugs ( T4:6μg/100 g;DON:0.005%) . Concentration of Ach was determined in the homogenated hippocampus of each animal and Ach content was measured by the modified method of Hestrin. Protein levels of munc-18 were determined by immunohistochemistry. The radioimmunoassay kits were applied to assay the levels of serum T3, T4 and TSH. Results Compared to the controls, the content of Ach in the hippo-campus of the hypothyroid rats was significantly decreased, munc-18 in the hippocampus was expressed at signifi-cantly lower levels in the stratum radiatum in CA3 as well as in dentate gyrus. A two-week treatment with T4 alone restored the content of Ach but failed to normalize the expression of munc-18 . Munc-18 was restored completely in all layers to normal levels by two-week T4 plus DON treatment. Conclusion Our findings suggest that the thyroid hormone may have a direct effect on the metabolism of hippocampal Ach in adult rats and the DON treatment can ameliorate the synaptic protein impairment induced by hypothyroidism to some extent.