摘要
目的观察成年期甲状腺功能减退症(简称甲减)大鼠海马内突触小体相关蛋白-25(synaptosomal associated protein of 25KD, SNAP-25)和突触结合蛋白1(synaptotagmin 1,syt1)改变及不同剂量甲状腺素治疗后的恢复状况,探讨甲减脑损伤及恢复可能的分子机制。
方法健康3月龄成年Sprague-Dawley雄性大鼠随机分为:(1)甲减组大鼠每日腹腔注射PTU 1mg/100g体重(body weight, BW);(2)小剂量替代治疗组(T4-5组)大鼠每日腹腔注射PTU 1mg/100g BW,四周后每日给予左旋T4 5μg/100g BW;(3)大剂量替代治疗组(T4-20组)大鼠每日腹腔注射PTU 1mg/100g BW,四周后每日给予左旋T4 20μg/100g BW;(4)正常对照组大鼠每日腹腔注射等体积生理盐水。造模六周结束后,采血取脑,应用放射免疫法检测大鼠血清T3、T4水平;提取背侧海马突触小体,采用Western blot方法获得显影条带,对条带进行光密度分析,通过SNAP-25及syt 1与内参照GAPDH的条带光密度值的比值间接反映SNAP-25及syt 1的表达量。所有数据采用单因素方差分析(one way ANOVA)。
结果造模前各组大鼠体重无统计学差异,造模结束后甲减组及各替代治疗组大鼠体重低于正常对照组(P<0.05);甲减大鼠血清T3、T4水平显著低于正常对照组(P<0.05),背侧海马突触小体内SNAP-25的表达水平显著高于正常对照组(P<0.05),syt 1的蛋白表达水平显著低于正常对照组(P< 0.05);小剂量替代治疗组大鼠血清T3、T4恢复至正常水平,SNAP-25及syt 1蛋白表达水平与甲减组比较未见明显改变(P>0.05);经大剂量替代治疗后血清T3、T4高于正常;SNAP-25及syt 1的表达恢复到正常水平(P<0.05)。
结论(1)成年期甲减大鼠海马内SNAP-25表达增加,syt 1的蛋白表达水平降低。(2)甲状腺素治疗能使其恢复,大剂量替代治疗使SNAP-25及syt 1表达恢复至正常水平。
Objective To observe the expression of SNAP-25 and synaptotagmin 1 in adult onset hypothyroidism rat hippocampus and learn the effect of Levothyroxin treatment.
Methods All rats were separated into 4 groups randomly: (1) hypothyroidism rats (Hypo group). Twelve hypothyroid rats were induced by daily intraperitoneal injection of PTU (dissolved in saline solution, 1 mg/100g body weight (BW)) for six weeks; (2) T4-5 group. Eleven rats were treated with PTU for six weeks as described above. But from the fifth week, they were also treated with intraperitoneally injected L-T4 (dissolved in saline solution, 50μg/kg BW) every day for two weeks; (3) T4-20 group. L-T4 treatment rats with high dose were treated as the same protocol as T4-5 group but the dose of L-T4 (200μg/kg BW); (4) C group. Control rats were treated with saline solution for six weeks. The radioimmunoassay kits were applied to assay the levels of serum T3 and T4, and west blot method was used to determine the levels SNAP-25 and synaptotagmin 1in dorsal hippocampi of rats from different groups.
Results Body weight levels in the hypothyroidism group and therapy groups were significantly lower than those in the control group rats(P<0.05); In the hypothyroidism group rats, serum T3、T4 levels were significantly lower than those in the control group rats(P<0.05); and the level of SNAP-25 protein were significantly increased and the level of syt 1 protein was significantly decreased in the crude synaptosome of dorsal hippocampus compared to the control group(P<0.05); after giving common dosage of L-T4 treatment, the levels of serum T3、T4 were returned to the normal, the level of SNAP-25 and synaptotagmin 1 protein was ameliorated, but there was no significant alteration compared to the hypothyroidism group( P>0.05). The high dosage of L-T4 could restore the increase of SNAP-25 protein to the normal and normalize the hypoexpression of syt 1 protein, while the levels of serum T3 and T4 were higher than the C group.
Conclusion These results suggested that adult-onset hypothyroidism can increase the expression of SNAP-25 and decrease the expression of syt 1 in the dorsal hippocampus. Levothyroxin could reverse this alteration, and high dose is superior to common dose in recorverying SNAP-25 and syt 1 expression.
引文
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