AMPK-SIRT1通路介导的热量限制对成年单眼剥夺弱视小鼠视皮层可塑性的再激活作用

Reactivation of visual cortical plasticity by caloric restriction mediated by AMPK-SIRT1 pathway from monocular deprivation in adult mice

目的 探讨热量限制对成年单眼剥夺（MD）弱视小鼠视皮层可塑性的调节作用及对弱视治疗的促进作用,并探讨其可能的分子机制.方法 采用随机数字表法将50只清洁级健康新生昆明小鼠随机分为正常对照组（n=14）、MD＋自由进食组（n=18）和MD＋热量限制组（n=18）.选取MD＋自由进食组和MD＋热量限制组小鼠建立MD弱视模型,分别以自由进食和热量限制的方式进行饲养.检测各组小鼠视敏度和闪光视觉诱发电位（F-VEP）,透射电子显微镜下观察视皮层神经元突触超微结构,Western blot法检测视皮层中磷酸化腺苷酸激活蛋白激酶α（p-AMPKα）、沉默信息调节因子1（SIRT1）蛋白的表达. 结果 从第1周开始,MD＋热量限制组小鼠体质量增加的百分比明显低于MD＋自由进食组.与MD＋自由进食组比较,MD＋热量限制组小鼠视敏度明显恢复,F-VEP示P100波潜伏期缩短、振幅增加,差异均有统计学意义（均P＜0.05）.与正常对照组相比,MD＋自由进食组小鼠视皮层神经元突触间隙明显增宽,差异有统计学意义（P＜0.05）;MD＋热量限制组视皮层神经元突触间隙明显窄于MD＋自由进食组,差异有统计学意义（P＜0.05）.MD＋自由进食组视皮层神经元突触后致密物厚度明显薄于正常对照组和MD＋热量限制组,差异均有统计学意义（均P＜0.05）.Western blot检测结果显示,正常对照组、MD＋热量限制组和MD＋自由进食组p-AMPKα蛋白表达水平分别为0.89±0.03、0.94±0.02和0.74±0.02,SIRT1蛋白的表达水平分别为0.97±0.11、0.95±0.14和0.58±0.13,差异均有统计学意义（F=14.57,P=0.00;F=23.91,P=0.00）,其中MD＋热量限制组较MD＋自由进食组小鼠视皮层中p-AMPKα及SIRT1蛋白的表达显著升高,差异均有统计学意义（均P＜0.05）. 结论 热量限制能重塑成年MD小鼠视皮层神经元突触的超微结构,重新激活视皮层结构可塑性,改善其视觉功能,其机制可能与激活AMPK-SIRT1通路有关.

Objective This study was to investigate the effect of caloric restriction on the plasticity of visual cortex in adult monocular deprivation （MD） amblyopic mice,as well as to promote the treatment of amblyopia,and to explore the possible molecular mechanism of this benefical effect.Methods Fifty healthy newborn Kunming mice of clean grade were randomly divided into 3 groups using a random number table method：normal control group （n =14）,MD＋ ad libitum group （n=18） and MD＋ caloric restriction group （n=18）.A mouse model of adult MD amblyopia was established,and caloric restriction intervention and ad libitum were performed on MD ＋ caloric restriction group and MD＋ ad libitum group,respectively.The visual acuity and flash visual evoked potential （F-VEP） of each group were detected.The synaptic structure of visual cortex neurons was observed by transmission electron microscope,and the expression of phosphorylated AMP-activated protein kinase-α（p-AMPKα） and silent information regulator 1 （SIRT1） in visual cortex were detected by Western blot.The animal feeding and use was in accordance with the standards set by the ARVO.Results The weight of mice in MD＋ caloric restriction group increased from the beginning of the first week,and was significantly lower than that in the MD ＋ ad libitum group （P〈0.05）.Compared with the MD＋ ad libitum group,the visual acuity was restored,the latency was shortened,and the amplitude of F-VEP was increased in the deprived eyes of MD＋ caloric restriction group （all at P〈0.05）.Transmission electron microscope observation showed that the width of synaptic gap of visual cortical neurons was significantly narrower,and the thickness of postsynaptic density was significantly thicker in MD＋ caloric restriction group than that in the MD＋ ad libitum group （both at P〈0.05）;compared with the normal control group,the synaptic gap was widened and the postsynaptic density was significantly thicker than that in the MD＋ ad libitum group （both at P〈0.05）.Western blot showed that the expression of p-AMPKα in visual cortex in the normal control group,MD＋ caloric restriction group and MD＋ ad libitum group was 0.89±0.03,0.94±0.02 and 0.74 ±0.02,and the expression of SIRT1 was 0.97±0.11,0.95±0.14 and 0.58±0.13,respectively,showing significant differences among the three groups （F =14.57,P =0.00;F=23.91,P=0.00）,the expressions of p-AMPKα and SIRT1 in visual cortex were increased in MD＋ caloric restriction group than those in M D＋ ad libitum group （both at P〈0.05）.Conclusions Caloric restriction can restore the ultrastructure of synapses and improve the visual cortical plasticity in adult MD mice,so that help to improve visual function.Its mechanism may be related to the activation of AMPK-SIRT1 pathway.