亚麻醉剂量氯胺酮对抑郁小鼠肠道菌群的影响

Effect of subanesthetic dose of ketamine on gut microbiota in depressed mice

目的:探究亚麻醉剂量氯胺酮对雄性和雌性抑郁样小鼠肠道菌群的影响。方法:SPF级健康雄性和雌性C57BL/6小鼠各20只,随机分为8组(n=5):雌性对照组(FC组),雌性氯胺酮组(FCK组),雌性抑郁组(FD组)和雌性抑郁+氯胺酮组(FDK组);雄性对照组(MC组),雄性氯胺酮组(MCK组),雄性抑郁组(MD组)和雄性抑郁+氯胺酮组(MDK组)。FCK组和MCK组经尾静脉注射质量分数为10 mg/kg的氯胺酮,FC组和MC组经尾静脉注射等量生理盐水;FD组、FDK组、MD组和MDK组采用慢性不可预知温和应激(chronic unpredictable mild stress,CUMS)造模,造模后FDK组和MDK组分别经尾静脉注射10 mg/kg氯胺酮,FD组和MD组注射等量生理盐水。采用强迫游泳实验、糖水偏好实验和旷场试验评价小鼠抑郁行为变化,接着采集不同组别小鼠粪便进行粪便微生物16S rDNA序列定性分析及功能预测分析。采用单因素方差分析不考虑性别时各组肠道菌群的变化,采用双因素方差分析性别因素和氯胺酮处理对肠道菌群的影响,必要时进一步采用Tukey校正的多重比较对实验结果进行后检验。检验水准α=0.05。结果:分别与不同性别的对照组比较,使用CUMS方法处理小鼠后,强迫游泳不动时间明显延长(P<0.05),糖水偏好度明显下降(P<0.05),旷场试验水平运动得分和垂直运动得分明显减少(P<0.05)。经亚麻醉剂量氯胺酮处理后,行为学检测结果表明小鼠均有不同程度的改善(P<0.05)。物种组成分析结果显示,在经过CUMS造模处理后,各组小鼠肠道菌群多样性和丰度未发生明显改变。物种差异分析发现,亚麻醉剂量氯胺酮可以改善抑郁小鼠放线菌的降低程度,使肠道菌群的组成发生变化,并使雌性抑郁小鼠Bacteroidaceae相对丰度降低程度得到缓解,而雄性抑郁小鼠Alistipes相对丰度增加。结论:不同性别小鼠对CUMS刺激和亚麻醉剂量氯胺酮处理表现出不同的肠道菌群变化。亚麻醉剂量氯胺酮可以改善抑郁小鼠放线菌的降低程度,并缓解雌性抑郁小鼠Bacteroidaceae相对丰度降低程度,增加雄性抑郁小鼠Alistipes相对丰度,进而改善小鼠的抑郁样行为。

Objective:To investigate the effect of subanesthetic dose of ketamine on gut microbiota among male and female depressed mice. Methods:The study included in 20 healthy male SPF mice and 20 female C57BL/6 mice,and they were randomly assigned to eight groups(n=5):female control group(FC group),female control with ketamine treatment group(FCK group),female depression group(FD group),female depression with ketamine treatment group(FDK group),male control group(MC group),male control with ketamine treatment group(MCK group),male depression group(MD group) and male depression with ketamine treatment group(MDK group). The ketamine(10 mg/kg) or saline(0.9% NaCl) were administrated by intravenation in each group. FD group,FDK group,MD group and MDK group were modeled by the chronic unpredictable mild stress(CUMS). The behavioral tests included force swimming test,sucrose preference test and open filed test. The gut microbiota of mice was detected by 16S rDNA amplicon sequencing.Qualitative analysis of bacteria in the specimen and predictive analysis of 16S r DNA function genes were conducted. The results were reported as mean values with their standard errors,and submitted to one-way ANOVA regardless sex difference and two-way ANOVA to analyze the effect of sex and ketamine on ameliorate depressive-like behaviors in mice,followed by the Tukey multiple comparisons test,with a statistical significance(P<0.05).Results:Compared with control group,the scores of forced swimming test were significantly prolonged,the scores of the sucrose preference test was significantly decreased,and the scores of open field test were significantly decreased(all P<0.05). These results were significantly ameliorated to varying degrees after subanesthetic dose of ketamine treatment(P<0.05). The diversity and abundance of intestinal flora in each group did not change significantly after CUMS and ketamine treatment. The reduction of Actinobacteria could be improved by subanesthetic dose of ketamine in depressed mice,regardless of sex difference. Furthermore,the relative abundance of Bacteroidaceae could be alleviated in depressed female mice,and Alistipes increased in depressed male mice. Conclusion:Mice of different genders showed different changes of intestinal flora in response to CUMS stimulation and subanesthetic ketamine treatment.The depressive-like behaviors can be ameliorated through increasing the abundance of Actinobacteria,alleviating the abundance of Bacteroidaceae in depressed female mice,and increasing Alistipes in depressed male mice after ketamine treatment.