抑制雷帕霉素靶蛋白复合物1信号通路可改善孤独症谱系障碍模型BTBR小鼠的症状

Improving the behavioral and neuroanatomical phenotypes in mouse models of autism spectrum disorder by inhibiting the mammalian target of rapamycin 1 signaling pathway

目的探讨抑制孤独症谱系障碍模型BTBR小鼠的中枢雷帕霉素靶蛋白复合物1(mTORC1)信号通路能否改善其交流和社交障碍。方法选择6只6周龄C57BL/6J(B6)小鼠和18只6周龄BTBR小鼠,每笼3只小鼠,自由进食和饮水。将6只B6小鼠(B6组)和6只BTBR(BTBR组)小鼠适应性饲养1周后,进行交流障碍实验和社交行为实验,实验结束后处死两组小鼠,提取其大脑额叶皮层组织抽提蛋白进行后续Western免疫印迹实验。将余12只6周龄BTBR小鼠分成干预组和对照组,每组6只,分别于第三脑室注射2\mL带有短发夹RNA(shRna)-Raptor的慢病毒和带有shRna阴性对照的慢病毒[滴度为1×10^(10)空斑形成单位(PFU)/mL\],注射2周后进行前述的行为实验和Western免疫印迹实验。观察并记录每只小鼠10min内吸嗅蘸有鼠尿、乙醇和0.9%氯化钠溶液的棉签所用的时间,以及社交行为(身体接触和跟随同伴)和非社交行为(自我理毛和区域探索)的发生次数;检测小鼠前额叶皮层Raptor蛋白或RNA,以及磷酸化蛋白S6(pS6)的蛋白表达情况。结果交流障碍实验结果显示,BTBR组小鼠吸嗅鼠尿的时间显著短于B6组小鼠(P<0.05),两组小鼠吸嗅0.9%氯化钠溶液和乙醇的时间的差异均无统计学意义(P值均>0.05)。社交行为实验结果显示,BTBR组小鼠的身体接触和跟随同伴的次数均显著少于B6组小鼠(P值均<0.05),自我理毛的次数显著多于B6组小鼠(P<0.05);两组小鼠区域探索次数的差异无统计学意义(P>0.05)。Western免疫印迹实验结果显示,BTBR组小鼠前额叶皮层mTORC1信号通路下游pS6的蛋白相对表达量显著高于B6组(P<0.05)。经慢病毒干预后,实时定量PCR结果显示,干预组小鼠前额叶皮层Raptor mRNA的相对表达量显著低于对照组(P<0.05);Western免疫印迹实验结果显示,干预组小鼠前额叶皮层Raptor蛋白和mTORC1信号下游pS6的蛋白相对表达量均显著低于对照组(P值均<0.05);交流障碍实验结果显示,干预组小鼠吸嗅鼠尿的时间显著长于对照组(P<0.05),两组小鼠吸嗅0.9%氯化钠溶液和乙醇的时间的差异均无统计学意义(P值均>0.05);社交行为实验结果显示,干预组小鼠的身体接触次数显著多于对照组(P<0.05),自我理毛次数显著少于对照组(P<0.05),两组小鼠跟随同伴和区域探索次数的差异均无统计学意义(P值均>0.05)。结论抑制孤独症谱系障碍模型BTBR小鼠的中枢mTORC1信号通路能明显改善其社交障碍和刻板动作,该小鼠的孤独症谱系障碍样表现与其中枢异常激活的mTORC1信号通路明显相关。

Objective To investigate whether central mammalian target of rapamycin 1 （mTORC1） signaling pathway is related to communication and social disorders in BTBR mice with autism spectrum disorder. Methods Six C57BL/6J mice aged 6 weeks old （B6 group） and six BTBR mice aged 6 weeks （BTBR group） were fed for one week. Then communication and social behavior disorder test were carried out. After the experiments, the mice were sacrificed and cortical tissue of frontal lobe of brain was collected for subsequent Western blot. The remaining 12 BTBR mice aged 6 weeks were divided into intervention group and control group, and injected respectively with 2 μL Lv-shRaptor and Lv- shNC in the third ventricle （1 × 1010 plaque forming unit[PFU]/mL）. The same behavioral experiment and Western blot were carried out in the intervention group and control group 2 weeks after the injection. The time when the mice smelt the cotton swabs containing mouse urine, alcohol or 0.9% sodium chloride solution was recorded. The number of body contact, following companion, self grooming and region exploration were also recorded. The level of Raptor mRNA and protein in the cortical tissue of frontal lobe of brain and the expression of phosphorylated S6 （pS6） protein were detected. Results The results of social behavior disorder test showed that the time of the mice smelling urine in BTBR group was significantly shorter than that in B6 group （P〈0.05）, 10ut there were no significant difference in the time of mice smelling alcohol or 0.9% sodium chloride solution between the groups （both P〉0.05）： The frequencies of body contact and following companion in BTBR group were significantly less than those in B6 group （both P〈0.05）, while the number of self grooming in BTBR group was significantly more than that in B6 group （P〈0.05）. There was no significant difference in the number of region exploration between B6 group and BTBR group （P^0.05）. The relative expression of pS6 protein in BTBR group was significantly higher than that in B6 group （P〈0.05）. The level of Raptor mRNA and protein and the relative expression of pS6 protein in the intervention group were significantly lower than those in control group （all P〈0.05）. The time of the mice smelling urine in the intervention group was significantly longer than that in the control group （P〈0.05）, but there was no significant difference in the time of mice smelling alcohol or 0.9% sodium chloride solution between the groups （both P〉0.05）. The frequency of body contact in the intervention group was significantly more than that in the control group （P〈0. 05）, while the number of self grooming in the intervention group was significantly less than that in the control group （P〈0.05）. There were no significant differences in the number of region exPloration or following companion between intervention group and cotrol group （both P〉0.05）. Conclusion The social disorder and stereotyped movement of BTBR mice can be improved by inhibiting the central mTORC1 signaling pathway.