艾司氯胺酮对小鼠抗抑郁作用的机制与海马GABAB受体的关系

Relationship between mechanism underlying antidepressant effect of S-ketamine and hippocampal GABABR in mice

目的评价艾司氯胺酮对小鼠抗抑郁作用的机制与海马γ-氨基丁酸B型受体(GABA_(B)R)的关系。方法雄性C57BL/6J(B6)小鼠54只,8周龄,体质量25~30 g,取40只小鼠采用慢性社会挫败应激法建立抑郁模型,造模后第11天时采用社交回避实验筛选出26只抑郁易感小鼠,采用随机数字表法分为2组(n=13):抑郁易感组(Sus组)和抑郁易感+艾司氯胺酮组(Sus+S-ket组);剩余的14只小鼠作为对照组(C组)。造模后第12天开始,Sus+S-ket组连续3 d每天腹腔注射艾司氯胺酮10 mg/kg,C组和Sus组连续3 d每天腹腔注射等容量生理盐水。最后一次腹腔注射后1 h时行旷场实验,记录运动总距离;旷场实验结束后1 d时行强迫游泳实验,记录不动时间;强迫游泳实验结束后1 d时行糖水偏好实验,计算糖水消耗比例。行为学测试结束后1 h时深麻醉下处死小鼠,取海马组织,采用Western blot法检测GABA_(B)R1、GABA_(B)R2、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化mTOR(p-mTOR)、脑源性神经营养因子(BDNF)、酪氨酸激酶受体B(TrkB)、磷酸化TrkB(p-TrkB)、谷氨酸受体1(GluR1)和突触后致密蛋白95(PSD95)的表达,计算p-mTOR/mTOR比值和p-TrkB/TrkB比值;免疫荧光法检测海马CA1区BDNF荧光强度;高尔基染色法测量海马CA1区树突棘数量。结果3组旷场实验运动总距离比较差异无统计学意义(P>0.05)。与C组比较,Sus组强迫游泳不动时间延长,糖水消耗比例降低,海马GABA_(B)R1、GABA_(B)R2、BDNF、GluR1、PSD95表达水平及p-mTOR/mTOR比值和p-TrkB/TrkB比值降低,海马CA1区BDNF荧光强度降低,树突棘数量减少(P<0.05),Sus+S-ket组上述指标差异无统计学意义(P>0.05)。与Sus组比较,Sus+S-ket组强迫游泳不动时间缩短,糖水消耗比例升高,海马GABA_(B)R1、GABA_(B)R2、BDNF、GluR1、PSD95表达水平及p-mTOR/mTOR比值和p-TrkB/TrkB比值升高,海马CA1区BDNF荧光强度升高,树突棘数量增加(P<0.05)。结论艾司氯胺酮对小鼠抗抑郁作用的机制可能与上调海马GABA_(B)R的表达,激活mTOR-BDNF信号通路,改善突触可塑性有关。

Objective To evaluate the relationship between the mechanism underlying the antidepressant effect of S-ketamine and hippocampal gamma-aminobutyric acid B receptor(GABA_(B)R)in mice.Methods A total of 54 male C57BL/6(B6)mice,aged 8 weeks,weighing 25-30 g,were used in this study.Forty mice were selected to develop the depression model by chronic social defeat stress.Twenty-six depression-susceptible mice were screened out by social avoidance test at day 11 after developing the model and divided into 2 groups(n=13 each)by a random number table method:depression-susceptible group(Sus group)and depression-susceptible+S-ketamine group(Sus+S-ket group).The remaining 14 mice served as control group(C group).Starting from day 12 after developing the model,S-ketamine 10 mg/kg was intraperitoneally injected every day for 3 consecutive days in Sus+S-ket group,while the equal volume of normal saline was given instead in C group and Sus group.The open field test was performed at 1 h after the last administration,and the total distance of movement was recorded.The forced swimming test was performed at 1 day after the open field test,and the immobile time was recorded.The sucrose preference test was performed to calculate the proportion of sucrose consumption at 1 day after the forced swimming test.One hour after the end of behavioral test,mice were sacrificed,and the hippocampal tissues were removed.Western blot was used to detect the expression of GABA_(B)R1,GABA_(B)R2,mammalian target of rapamycin(mTOR),phosphorylated mTOR(p-mTOR),brain-derived neurotrophic factor(BDNF),tyrosine kinase receptor B(TrkB),phosphorylated TrkB(p-TrkB),glutamate receptor 1(GluR1)and postsynaptic dense protein 95(PSD95).The p-mTOR/mTOR ratio and p-TrkB/TrkB ratio were calculated.The fluorescence intensity of BDNF in hippocampal CA1 region was detected by immunofluorescence.The number of dendritic spines in hippocampal CA1 region was measured by Golgi staining.Results In the open field test,no statistically significant difference in the total distance was detected among the three groups(P>0.05).Compared with C group,the immobile time in the forced swimming test was significantly prolonged,the proportion of sucrose consumption was decreased,the expression of hippocampal GABA_(B)R1,GABA_(B)R2,BDNF,GluR1 and PSD95 was down-regulated,and the ratios of p-mTOR/mTOR and p-TrkB/TrkB were decreased,the fluorescence intensity of BDNF and total number of dendritic spines in the hippocampal CA1 region were decreased in Sus group(P<0.05),and no significant change was found in the parameters mentioned above in Sus+S-ket group(P>0.05).Compared with Sus group,the immobile time in the forced swimming test was significantly shortened,the proportion of sucrose consumption was increased,the expression of hippocampal GABA_(B)R1,GABA_(B)R2,BDNF,GluR1 and PSD95 was up-regulated,the ratios of p-mTOR/mTOR and p-TrkB/TrkB were increased,and the fluorescence intensity of BDNF and total number of dendritic spines in the hippocampal CA1 region were increased in Sus+S-ket group(P<0.05).Conclusions The mechanism underlying the antidepressant effect of S-ketamine may be related to up-regulation of hippocampal GABA_(B)R expression,activation of mTOR-BDNF signaling pathway,and improvement in synaptic plasticity in mice.