择时电针对肝癌小鼠生物节律及其视交叉上核表观遗传学修饰的影响

Effect of electroacupuncture at different time on circadian rhythm and SCN epigenetic modification in mice with hepatocellular carcinoma

目的:以原位肝癌(hepatocellular carcinoma,HCC)小鼠为模型,观察电针对肝癌小鼠转轮活动节律的调节作用及其对视交叉上核(suprachiasmatic nucleus,SCN)内表观遗传学相关基因表达的影响,探讨电针调整肝癌患者昼夜节律的表观遗传学机制。方法:将筛选合格的108只雄性C57BL/6J小鼠按6个授时因子时间(zeitbeger time,ZT)即ZT0(7:00)、ZT4(11:00)、ZT8(15:00)、ZT12(19:00)、ZT16(23:00)和ZT20(3:00)实施干预,每个时间点设空白组、模型组和电针组,每组6只。肝脏注射H22癌细胞悬液制备肝癌小鼠模型。造模后第11天开始干预,电针组在各相应时间点给予电针小鼠"肝俞"和"至阳"穴(电针参数:2 Hz/15 Hz,0.2 m A),每天1次,每次15 min,连续电针10 d。空白组和模型组在同时间、同等条件下捆绑固定。全程采用Clock Lab(ACT-500)软件不间断记录小鼠活动节律,干预10 d后采用MATLAB(R2007b)导出小鼠转轮活动节律图谱,分析小鼠活动振幅与峰相位。选择最佳时间点干预组,采用高通量表观遗传学PCRarray阵列检测小鼠SCN部表观遗传学相关基因表达。结果:(1)造模后,与对应时间点空白组比较,模型组和电针组小鼠活动振幅下降、峰相位滞后(均P<0.05),模型组与电针组节律参数比较差异无统计学意义(均P>0.05);(2)干预后,与ZT8模型组比较,ZT8电针组小鼠活动振幅升高、峰相位前移(均P<0.05),ZT0、ZT4、ZT12、ZT16、ZT20电针组与相应时间点模型组比较活动振幅、峰相位差异无统计学意义(均P>0.05);ZT8电针组与ZT0、ZT4、ZT12、ZT16、ZT20电针组比较,活动振幅升高、峰相位前移(均P<0.05);(3)表观遗传PCRarray阵列(Epigenetic PCRarray)结果示:最佳时间点ZT8干预结束后,与空白组比较,肝癌小鼠SCN内48个表观遗传学相关基因表达上调;与模型组比较,电针下调SCN内49个表观遗传学相关基因相对表达量;3组小鼠SCN内表观遗传学相关差异表达基因有23个。结论:电针能够对肝癌小鼠转轮活动节律产生良性调节,且以15:00最佳;该时间点电针能下调肝癌小鼠SCN内异常高表达的表观遗传学相关基因。

Objective To observe the effects of electroacupuneture （EA） on the circadian rhythm and suprachiasmatic〈, nueleus （SCN） epigenetic modification in mice with hepatoeellular carcinoma （HCC）, and to explore the epigeneties mechanism of EA on circadian rhythm in patients with HCC. Methods Aeenrding to six zeitheger lime （ZT） of ZT0 （7：00）, ZT4 （11：00））, ZT8 （15：00）, ZTI2 （19：00）, ZT16 （23：00） and ZT20 （3：00）, a total of 108 eligible male C57BL/6J mice were divided into a blank group, a model group and an EA group at each ZT, 6 mice each group. Injection of H22 cancer cell suspension was used to establish the HCC modeh Alter 11 days, EA （2 Hz/15 Hz, 0.2 mA） for 10 days was applied at ＂Ganshu＂ （BL 18） and ＂Zhiyang＂ （GV 9） in the＂ EA group at eaeh ZT, once a day, 15 min a time： the rats in the blank group and model group were treated with immobilizalion at the same time and under the same conditions. CloekLab （ACT-500） software was used to record the activity rhythm of mice. After 10 days intervention, MATLAB （R2007b） was used to export the eircadian rhythm of mice, and the amplitude and peak phase of the mice were analyzed. The high-throughput epigenelies PCRarray array was applied to detect epigenelies-related gene expression in SCN. Results （1） After modeling, compared with the hlank group, the amplitude of activity was decreased and peak phase was delayed in the model group and EA group at each ZT （all P〈0.05）, but the difference of rhythm parameters between the model group and EA group was not significant （all P〉 0.05）. （2） After intervention, compared with the model group, the amplitude of activity in the EA group at ZT 8 was increased and peak phase was advanced （both P〈0.05）; the difference of the activity amplitude and peak phase between the EA group and model group at ZT0, ZT4, ZT12, ZT16 and ZT20 was not significant （all P〉0.05）; compared with the ZT0, ZT4, ZTI2, ZTI6 and ZT20, the amplitude of activity in the EA group at ZT 8 was increased and peak phase was advanced （all P〈0.05）. （3） The results of epigenetic PCRarray array showed that after intervention at ZT 8, compared with the blank group, the expression of 48 epigenetic-related genes in SCN of HCC mice was up-regulated; compared with the model group, the relative expression of 49 epigenetic-related genes in the SCN was down-regulated in the EA group; there were 23 epigenetic-related genes diferentially expressed among the three groups. Conclusion EA has benign regulation on eircadian rhythm of HCC mice, and achieves the best efficacy at ZT 8. EA at ZT 8 could down-regulate the overexpression of epigenetic-related genes.