电针对昼夜节律紊乱C57BL/6J小鼠肝脏基因Clock、Bmal1表达的影响

Effect of Electroacupuncture on Clock and Bmal1 Gene Expressions in Liver of C57BL/6J Mice with Circadian Rhythm Disorder

目的 观察电针肝俞对昼夜节律紊乱小鼠外周时钟基因的影响,分析各实验条件下,不同时间点Clock、Bmal1基因的分布规律,为外周生物钟基因时间分布提供参考,并探讨电针治疗睡眠节律紊乱的潜在机制。方法 将72只C57BL/6J小鼠按随机数字表法分成空白组、模型组、肝俞组和非经非穴组,每组18只。空白组常规饲养;其余3组采取光暗交替法L(光)∶D(暗)=2 h∶2 h造模法持续5 d,模型组只捆绑不针刺;肝俞组针刺肝俞穴;非经非穴组选取非经非穴部位进行针刺。电针干预5 d后,于第6天02∶00开始,每组每4 h取材1次,共取材6次。采用PCR法以及Western blot法检测各组小鼠肝脏内的Clock和Bmal1基因mRNA的表达水平和蛋白表达含量,同时用余弦函数计算出各组基因的节律性。结果 与模型组比较,肝俞组中Clock与Bmal1基因表达恢复时间节律(P<0.05)。与模型组比较,非经非穴组内Clock与Bmal1基因表达无时序性(P>0.05);肝俞组Clock、Bmal1的蛋白表达量较模型组明显降低且恢复节律性(P<0.05)。结论 小鼠肝脏中Clock和Bmal1时钟基因呈时序性变化,电针肝俞穴可改善小鼠睡眠,其机制可能与调节时钟基因Clock和Bmal1,降低两种基因及蛋白表达,恢复正常睡眠节律有关。

Objective To observe the effect of electroacupuncture(EA)at Ganshu(BL18)on peripheral Clock genes in mice with circadian disorder,analyze the distribution of Clock and Bmal1 genes at different time points under various experimental conditions and explore the potential mechanism of EA in restoring circadian rhythm and sleep.Methods A total of 72 C57BL/6J mice were randomly divided into four groups including control group,model group,Ganshu(BL18)group and non-meridian and non-acupoint group,with 18 in each group.The control group was routinely fed.The other three groups were modeled for 5 days with alternating light and dark method(L∶D=2 h∶2 h).The control group was only fixed without EA and Ganshu(BL18)group was applied EA at Ganshu(BL18).The non-meridian and non-acupoint group was given EA at non-meridian and non-acupoint areas.After 5 days of EA,starting at 02∶00 on the 6th day,each group took samples every 4 hours,a total of 6 times.PCR and Western Blot were used to detect the mRNA expression levels and protein contents of Clock and Bmal1 in the liver of mice in each group,and the rhythm of genes in each group was calculated using cosine function.Results Compared with those of the model group,mRNA expressions of Clock and Bmal1 in Ganshu(BL18)group recovered the time rhythm(P<0.05).Compared with those of the model group,the mRNA expressions of Clock and Bmal1 in the non-meridian and non-acupoint group had no time sequence(P>0.05).The protein expression levels of Clock and Bmal1 in Ganshu(BL18)group were significantly lower than those in model group(P<0.05).Conclusion Clock and Bmal1 genes in the liver of mice showed sequential changes,and EA at Ganshu(BL18)could improve the sleep of mice.The mechanism may be related to regulating Clock and Bmal1,reducing the protein expressions of the two genes and restoring the normal sleep rhythm.