电针早期干预对创伤后应激障碍模型小鼠行为及肠道菌群的影响

Effect of early intervention with electroacupuncture on behavior and gut microbiota in mice with post-traumatic stress disorder

目的探讨电针(electroacupuncture,EA)早期干预对创伤后应激障碍(post-traumatic stress disorder,PTSD)模型小鼠的焦虑恐惧样行为及肠道菌群的影响。方法将32只雄性SPF级C57BL/6小鼠按照随机数字表法分为对照组(Control组)、电针组(EA组)、模型组(PTSD组)、电针干预组(PTSD+EA组),每组8只。适应性饲养7 d后,PTSD组和PTSD+EA组小鼠接受改良的单次延长应激造模。造模结束后,给予PTSD+EA组和EA组小鼠电针刺激百会穴(2/15 Hz,1 mA,疏密波),30 min/d,连续干预7 d,给予Control组和PTSD组小鼠假刺激(只针刺百会穴,不通电)干预7 d。干预结束7 d后,收集各组小鼠粪便并采用高架十字迷宫实验和恐惧条件实验检测小鼠行为学变化,收集的粪便通过16S rRNA测序检测分析其肠道菌群变化。采用SPSS 19.0进行统计分析,多组间比较采用单因素方差分析,两两比较采用Bonferroni检验。结果(1)4组小鼠在高架十字迷宫实验中的开放臂活动时间和条件恐惧测试中的环境诱发和事件诱发僵直时间均差异有统计学意义(F=6.93,5.26,14.51,均P<0.01)。高架十字迷宫测试中,PTSD组小鼠在开臂区活动的时间[(60.17±15.52)s]明显少于Control组[(96.37±14.62)s]和PTSD+EA组[(86.89±15.02)s](均P<0.05)。条件恐惧测试中,PTSD组小鼠的环境诱发僵直时间[(121.99±29.67)s]和事件诱发僵直时间[(130.82±29.11)s]均明显多于Control组[(74.50±26.65)s,(39.50±23.52)s]和PTSD+EA组[(76.77±22.60)s,(102.17±3.39)s](均P<0.05)。(2)4组小鼠肠道菌群α多样性指标均差异无统计学意义(F=0.79~2.45,均P>0.05)。(3)相关性分析显示,有13个菌属与环境诱发僵直时间呈负相关,2个菌属与环境诱发僵直时间呈正相关;有7个菌属与事件诱发僵直时间呈负相关,1个菌属与事件诱发僵直时间呈正相关;另外有3个菌属与高架十字开放臂活动时间呈负相关。结论电针早期干预可以改善PTSD模型小鼠的焦虑恐惧样行为和肠道菌群紊乱。

Objective To investigate the effect of early intervention with electroacupuncture(EA)on the gut microbiota in a mouse model of post-traumatic stress disorder(PTSD).Methods Totally 32 C57BL/6 mice were randomly assigned to the following 4 groups(n=8 for each group):Control group,EA group,PTSD group and PTSD+EA group.After 7 days acclimation,mice in the PTSD group and PTSD+EA group were subjected to modified single prolonged stress(mSPS).Mice in the EA group and PTSD+EA group received EA(2/15 Hz,1 mA,dilatational wave,30 min/d)on"Baihui"for 7 days.Mice in the Control group and PTSD group received false stimulation(stimulated the same acupiont without electricity)for 7 days.Seven days after the last stimulation,elevated plus maze test and fear conditioning test were conducted to observe the effect of EA on PTSD-like behavior of mice.At the same time,feces of the mice were collected for gut microbiota detection by 16S rRNA sequencing.SPSS 19.0 was used for statistical analysis.One-way ANOVA was used for multiple group comparison and Bonferrani test was done for further pairwise comparision.Results(1)There were statistically differences in the open arm activity time of the elevated plus maze test and the immobility time in contextual and cued fear conditioning test among the four groups(F=6.93,5.26,14.51,all P<0.01).In the elevated plus maze test,mice in PTSD group((60.17±15.52)s)showed significant less time in the open arms than mice in Control group((96.37±14.62)s)and PTSD+EA group((86.89±15.02)s)(both P<0.05).In the fear conditioning test,mice in PTSD group((121.99±29.67)s,(130.82±29.11)s)showed significant increased immobility time both in contextual and cued fear conditioning tests than mice in Control group((74.50±26.65)s,(39.50±23.52)s)and PTSD+EA group((76.77±22.60)s,(102.17±3.39)s)(both P<0.05).(2)There were no significant differences among the four groups in the alpha diversity of gut microbiota(F=0.79-2.45,all P>0.05).(3)Correlation analysis showed that 13 gut microbiotas were negatively correlated with the immobility time in contextual fear conditioning test,2 gut microbiotas were positively correlated with it;7 gut microbiotas were negatively correlated with the immobility time in cued fear conditioning test,1 gut microbiota was positively correlated with it;3 gut microbiotas were positively correlated with time spent in open arms of elevated plus maze test.Conclusion Early intervention with EA can improve anxiety-fear like behaviors and gut microflora disorder in PTSD model mice.