交泰丸水煎液腹腔注射对失眠小鼠昼夜节律紊乱的改善作用及其机制

Improvement of Jiaotai pill water decoction on circadian rhythm disorders in insomnia mice

目的观察交泰丸水煎液对失眠小鼠昼夜节律紊乱的改善作用,并探讨其作用机制。方法30只雄性ICR小鼠随机分为交泰丸组、模型组和对照组。交泰丸组按450 mg/kg腹腔注射对氯苯丙氨酸(PCPA),每天1次、连续2 d,建立小鼠失眠模型;然后灌胃交泰丸水煎液0.4 mL/次,连续7 d。模型组参照上述方法建立小鼠失眠模型后,灌胃交泰丸水煎液等量蒸馏水,连续7 d;对照组腹腔注射PCPA等量生理盐水每天1次、连续2 d后,灌胃交泰丸水煎液等量蒸馏水,连续7 d。然后采用旷场实验、跑台实验评价小鼠活跃度及运动能力(反映昼夜节律紊乱情况),RT-qPCR法检测小鼠脑组织时钟基因CLOCK、PER1、BMAL1及食欲素前体(HCRT),ELISA法检测小鼠外周血清DNA甲基转移酶1(DNMT1)和组蛋白去乙酰化酶(HDAC)活性,Western blotting法检测小鼠脑组织中转录调控轴SIRT1和FOXO1蛋白。结果与对照组相比较,模型组小鼠被电击次数增加、夜间总活动距离降低,脑组织中CLOCK、BMAL1、HCRT mRNA相对表达量下降,血清DNMT1水平上升、HDAC水平下降,脑组织SIRT1蛋白水平下降、FOXO1蛋白水平上升(P均<0.05)。与模型组相比较,交泰丸组小鼠在夜间总活动距离增加、被电击次数减少(P均<0.05),脑组织中CLOCK、HCRT mRNA相对表达量升高(P均<0.05),血清DNMT1活性下降、SIRT1蛋白水平升高、FOXO1蛋白水平降低(P均<0.05)。结论交泰丸水煎液能使失眠小鼠昼夜节奏紊乱改善,其作用可能是通过SIRT1/FOXO1轴上调CLOCK、BMAL1等时钟基因以及HCRT的转录水平,并抑制DNMT1活性而实现的。

Objective To observe the improvement effects of Jiaotai pill water decoction on circadian rhythm disor⁃ders in insomnia mice and to explore its mechanism.Methods Thirty male ICR mice were randomly divided into the Ji⁃aotai pill group,model group and control group.Mice in the Jiaotai group were injected intraperitoneally with 450 mg/kg of p-chlorophenylalanine(PCPA)once a day for 2 consecutive days to establish the insomnia models,followed by intragas⁃tric administration of Jiaotai pill water decoction 0.4 mL/time for 7 consecutive days;the mice in the model group were ga⁃vaged with the same amount of normal saline for 7 consecutive days after the establishment of insomnia models according to the above method;the mice in the control group were injected intraperitoneally with an equal amount of PCPA normal sa⁃line once a day for 2 d and then were gavaged with an equal amount of distilled water for 7 consecutive days.Then,the mice were evaluated for activity and locomotor ability by open-field test and table-running test,and the clock genes CLOCK,PER1,BMAL1,and appetitive hormone precursor(HCRT)were detected by RT-qPCR,and the activities of epigenetic modifier DNA methyltransferase 1(DNMT1)and histone deacetylase(HDAC)in the peripheral blood of mice were detected by ELISA,and the protein levels of silent information regulator 1(SIRT1)and Forkhead box O1(FOXO1)in the brain tissues were detected by Western blotting.Results Compared with the control group,the frequency of elec⁃tric shock increased,the total activity distance at night decreased,the relative expression levels of CLOCK,BMAL1 and HCRT mRNA in the brain tissues decreased,the level of serum DNMT1 increased,and the activity of HDAC decreased,and the level of SIRT1 protein decreased and the level of FOXO1 protein increased in the model group(all P<0.05).Com⁃pared with the model group,the total activity distance of mice increased and the number of electric shock decreased(both P<0.05),and the relative expression levels of CLOCK and HCRT mRNA in the brain tissues increased,the activity of DN⁃MT1 in serum decreased,the level of SIRT1 protein increased,and the level of FOXO1 protein decreased in the Jiaotai pill group(all P<0.05).Conclusion Jiaotai pill water decoction can improve the circadian rhythm disorder of insomni⁃ac mice,which may be achieved by up-regulating the transcription levels of clock genes such as CLOCK and BMAL1 and HCRT through SIRT1/FOXO1 axis and inhibiting the activity of DNMT1.