褪黑素对Ctnnd2敲除小鼠的自闭症样行为及突触相关蛋白的影响

The effects of melatonin on autism-like behaviors and synaptic proteins in Ctnnd2 knockout mice

目的:观察褪黑素(MT)对Ctnnd2基因敲除(Ctnnd2^(-/-))小鼠自闭症样行为、丝裂原活化蛋白激酶(MEK)/细胞外信号调节蛋白激酶(ERK)通路蛋白和突触相关蛋白的影响。方法:将小鼠分为四组:野生型组(WT)、Ctnnd2基因敲除组(Ctnnd2^(-/-))、Ctnnd2基因敲除+10 mg/kg褪黑素组(Ctnnd2^(-/-)+10 mg/kg MT)及Ctnnd2基因敲除+50 mg/kg褪黑素组(Ctnnd2^(-/-)+50 mg/kg MT)。不同剂量MT给出生21 d Ctnnd2^(-/-)小鼠连续灌胃28 d,行为学实验评价小鼠的自闭症样行为,Western Blot检测MEK1/2、ERK1/2和突触素蛋白(Syn)的磷酸化水平以及突触后致密蛋白95(PSD95)的表达。接下来为了验证MT是否通过与MT-R结合进而上调体感皮层MEK/ERK通路和引起突触相关蛋白的变化,在Ctnnd2^(-/-)小鼠体感皮层局部微量注射褪黑素受体(MT-R)阻断剂,30 min后再注射MT,Western Blot再次检测MEK1/2、ERK1/2和Syn蛋白的磷酸化水平以及PSD95蛋白表达。结果:(1)与WT组比,Ctnnd2^(-/-)组与陌生鼠社会互动时间明显减少(P<0.05),自我捋毛时间增加(P<0.05),新事物偏好降低(P<0.05),探索及自主行为减少(P<0.05),提示Ctnnd2^(-/-)小鼠存在自闭症样行为;MT干预后,Ctnnd2^(-/-)小鼠自闭症样行为明显改善(P<0.05)。(2)Western Blot检测结果显示Ctnnd2^(-/-)小鼠体感皮层PSD95蛋白表达、MEK1/2蛋白的磷酸化水平、ERK1/2蛋白的磷酸化水平和Syn蛋白的磷酸化水平均降低(P<0.05),MT干预后能上调这些蛋白(P<0.05),MT-R阻断剂可以使MT上调PSD95蛋白表达、MEK1/2蛋白的磷酸化水平、ERK1/2蛋白的磷酸化水平和Syn蛋白的磷酸化水平的作用降低。结论:MT可改善Ctnnd2^(-/-)小鼠的自闭症样行为,同时可以通过上调体感皮层MEK/ERK通路引起突触相关蛋白的变化。

Objective:To investigate the effects of melatonin(MT)on autism-like behaviors,mitogen-activated protein kinase(MEK)/extracellular signal-regulated protein kinase(ERK)pathway and synapse-related proteins in cateninδ2(Ctnnd2)gene knockout(Ctnnd2^(-/-))mice.Method:The mice were divided into four groups:wild-type(WT)group,Ctnnd2^(-/-)group,Ctnnd2^(-/-)+10 mg/kg MT group and Ctnnd2^(-/-)+50 mg/kg MT group.The Ctnnd2^(-/-)mice were treated by melatonin with different concentrations for 28 d from postnatal day 21.The behavioral tests were used to evaluate the autism-like behaviors.Western Blot was used to detect MEK1/2 protein phosphorylation level,ERK1/2 protein phosphorylation level,synapsin(Syn)protein phosphorylation level and post-synaptic dense 95(PSD95)protein expression in somatosensory cortex.Next the experiment was designed to examine whether the binding of MT and MT-R could promote the activity of MEK/ERK pathway and cause the changes of synaptic-associated proteins.The Ctnnd2^(-/-)mice were microinjected with luzindole(MT-R antagonist)in somatosensory cortex,were followed microinjected with MT after 30 min.Then,the MEK1/2 protein phosphorylation level,ERK1/2 protein phosphorylation level,Syn protein phosphorylation level and PSD95 protein expression were examined by Western Blot.Results:(1)Compared with WT mice,Ctnnd2^(-/-)mice for behavioral tests showed prolonged self-grooming time(P<0.05),reduced interaction time(P<0.05),low novelty preference(P<0.05),decreased exploratory behaviors and reduced autonomous behaviors(P<0.05).The administration of MT could improve autistic-like behaviors of the Ctnnd2^(-/-)mice(P<0.05).(2)Western Blot showed that the expression of PSD95 protein,the phosphorylation levels of MEK1/2,ERK1/2 and Syn were decreased in somatosensory cortex of Ctnnd2^(-/-)mice(P<0.05).Treatment with MT could upregulate these proteins(P<0.05),while MT-R antagonist could diminish the effect of melatonin on changing these proteins in somatosensory cortex of Ctnnd2^(-/-)mice.Conclusion:MT can alleviate the autistic-like behaviors of Ctnnd2^(-/-)mice,and it also can activate MEK/ERK signaling pathway to bring the changes of synapse-associated proteins in somatosensory cortex.