银杏叶提取物对抑郁模型小鼠内侧前额叶皮质氧化应激及锥体神经元兴奋/抑制平衡的影响

Effects of ginkgo biloba extract on oxidative stress and the balance of excitation/inhibition of pyramidal neurons in the medial prefrontal cortex of depressive model mice

目的:探讨银杏叶提取物(ginkgo biloba extract,GBE)对慢性不可预见性温和性应激(chronic unpredictable mild stress,CUMS)所致抑郁模型小鼠内侧前额叶皮层(medial prefrontal cortex,mPFC)氧化应激及锥体神经元兴奋/抑制(excitatory/inhibitory,E/I)平衡的影响。方法:48只SPF级7周龄雄性C57BL/6J小鼠按照随机数字表法分为4组:对照+生理盐水组(CTRL+Veh组)、对照+GBE组(CTRL+GBE组)、模型+生理盐水组(CUMS+Veh组)、模型+GBE组(CUMS+GBE组),每组12只小鼠。CUMS+Veh组和CUMS+GBE组小鼠采用CUMS方法建立抑郁模型,造模期间CTRL+GBE组和CUMS+GBE组小鼠给予腹腔注射GBE(70 mg/kg,1次/d),CTRL+Veh组和CUMS+Veh组小鼠无应激、腹腔注射等体积0.9%氯化钠溶液。造模结束后采用糖水偏爱实验、强迫游泳实验、悬尾实验检测小鼠抑郁行为,采用旷场实验检测小鼠自主运动、探索能力、焦虑行为。采用分光光度计法检测mPFC超氧化物歧化酶(superoxide dismutase,SOD)与丙二醛(malondialdehyde,MDA)水平,全细胞记录检测神经元E/I平衡,包括自发性兴奋性突触后电流(spontaneous excitatory postsynaptic currents,sEPSC)与自发性抑制性突触后电流(spontaneous inhibitory postsynaptic currents,sIPSC)。采用SPSS 23.0进行数据管理和统计分析,所有数据采用双因素(是否给予GBE,是否给予CUMS造模,用GBE×CUMS表示)方差分析进行统计。结果:(1)行为学结果显示:小鼠旷场中央区活动时间及运动总距离、糖水偏爱率比较,GBE×CUMS交互作用显著(F=24.90,4.82,3.91,均P<0.05)。简单效应分析结果显示,CUMS+Veh组小鼠旷场中央区活动时间[(47.15±3.58)s]及运动总距离[(19.33±0.86)m]、糖水偏爱率[(59.11±8.79)%]均低于CTRL+Veh组[(61.55±2.49)s、(23.24±1.21)m、(84.02±7.45)%](均P<0.01),CUMS+GBE组[(56.51±3.53)s、(20.75±1.31)m、(70.80±11.79)%]均高于CUMS+Veh组(均P<0.05)。各组小鼠的强迫游泳不动时间和悬尾不动时间比较,GBE×CUMS交互作用显著(F=85.53,83.39,均P<0.01),CUMS+Veh组强迫游泳与悬尾实验的不动时间均高于CTRL+Veh组(均P<0.01),CUMS+GBE组均低于CUMS+Veh组(均P<0.05)。(2)ELISA结果显示:各组小鼠mPFC中SOD比较,GBE×CUMS交互作用不显著(F=3.52,P=0.07),GBE和CUMS主效应均显著(F=4.69,46.93,均P<0.05);MDA比较,GBE×CUMS交互作用显著(F=16.61,P<0.01)。CUMS+Veh组SOD的表达量低于CTRL+Veh组(P<0.01),CUMS+GBE组SOD的表达量高于CUMS+Veh组(P<0.05);CUMS+Veh组MDA表达量高于CTRL+Veh组(P<0.01),CUMS+GBE组低于CUMS+Veh组(P<0.01)。(3)全细胞记录结果显示:sEPSC频率、电容量比较,4组小鼠GBE×CUMS交互作用显著(F=5.45,6.94,均P<0.05)。CUMS+Veh组sEPSC频率、电容量均低于CTRL+Veh组(均P<0.01),CUMS+GBE组均高于CUMS+Veh组(均P<0.05);sIPSC频率、电容量比较,4组小鼠GBE×CUMS交互作用显著(F=7.78,8.96,均P<0.01)。CUMS+Veh组sIPSC频率、电容量均高于CTRL+Veh组(均P<0.01);CUMS+GBE组均低于CUMS+Veh组(均P<0.01);sEPSC/sIPSC比值,4组小鼠GBE×CUMS交互作用显著(F=5.45,P=0.02)。CUMS+Veh组sEPSC/sIPSC比值(0.09±0.01)低于CTRL+Veh组(0.28±0.04)(P<0.01),CUMS+GBE组sEPSC/sIPSC比值(0.14±0.03)高于CUMS+Veh组(P<0.05)。结论:银杏叶提取物可以改善CUMS所致小鼠抑郁样行为,同时降低抑郁小鼠内侧前额叶皮层氧化应激、提高锥体神经元兴奋/抑制平衡。

Objective To investigate the effect of ginkgo biloba extract(GBE)on oxidative stress in medial prefrontal cortex and excitatory/inhibitory balance of pyramidal neurons in chronic unpredictable mild stress(CUMS)-induced depressive model mice.Methods Totally 48 SPF grade 7-week-old male C57BL/6J mice were divided into 4 groups according to random number table method:control+saline group(CTRL+Veh),control+GBE group(CTRL+GBE),model+saline group(CUMS+Veh),model+GBE group(CUMS+GBE),with 12 mice in each group.Mice in CUMS+Veh group and CUMS+GBE group were established by CUMS method,and mice in CTRL+GBE group and CUMS+GBE group were intraperitoneally injected with GBE(70 mg/kg)once a day,and mice in CTRL+Veh group and CUMS+Veh group were injected intraperitoneally with 0.9%sodium chloride solution.Then,the sucrose preference test,forced swimming test(FST)and tail suspension test(TST)were performed to evaluate the depressive-like behavior of mice,and open field test(OFT)was performed to evaluate the autonomous locomotion and exploration ability and anxiety-like behavior.The content of malondialdehyde(MDA)and activity of superoxide dismutase(SOD)in mPFC were determined by ELISA.Spontaneous excitatory postsynaptic currents(sEPSC)and spontaneous inhibitory postsynaptic currents(sIPSC)were detected by whole-cell recording.SPSS 23.0 was used for data analysis and two-factor analysis of variance(whether to get GBE,whether to mold,show as GBE×CUMS)was used for statistical analysis.Results(1)Behavioral results:the the time spent in center and total distance of OFT and sugar preference rate of the four groups of mice were compared,and the interaction of GBE×CUMS was significant(F=24.90,4.82,3.91,all P<0.05).The results of simple effect analysis showed that the time spent in center((47.15±3.58)s),the total distance((19.33±0.86)m)and the sugar preference rate((59.11±8.79)%)of the mice in CUMS+Veh group were lower than those in the CTRL+Veh group((61.55±2.49)s,(23.24±1.21)m,(84.02±7.45)%)(all P<0.01),and the above indexes in CUMS+GBE group((56.51±3.53)s,(20.75±1.31)m,(70.80±11.79)%)were higher than those in CUMS+Veh group(all P<0.05).In the immobility time of FST and TST of mice in the 4 groups,the interaction of GBE×CUMS were significant(F=85.53,83.39,both P<0.01).The immobility time of FST and TST in CUMS+Veh group were higher than those in CTRL+Veh group(both P<0.01),and the above indexes in CUMS+GBE group were lower than CUMS+Veh group(both P<0.05).(2)The results of ELISA showed that the interaction of GBE×CUMS of SOD level of mice in the 4 groups was not significant(F=3.52,P=0.07),but the main effects of GBE factor and CUMS factor were both significant(F=4.69,46.93,both P<0.05).The interaction of GBE×CUMS of MDA level was significant(F=16.61,P<0.01).The level of SOD in the CUMS+Veh group was lower than that in the CTRL+Veh group(P<0.01),and the level of SOD in the CUMS+GBE group was higher than that in the CUMS+Veh group(P<0.05).The level of MDA in the CUMS+Veh group was higher than that of the CTRL+Veh group(P<0.01),and the level of MDA in CUMS+GBE group was lower than that of the CUMS+Veh group(P<0.01).(3)The results of whole-cell recording showed that the interaction of GBE×CUMS of frequency and quantification of sEPSC in the four groups were significant(F=5.45,6.94,both P<0.05).The sEPSC frequency and quantification in the CUMS+Veh group were lower than those in the CTRL+Veh group(both P<0.01),and the sEPSC frequency and quantification in CUMS+GBE group were higher than those of CUMS+Veh group(both P<0.05).The interaction of GBE×CUMS of frequency and quantification of sIPSC in the four groups were significant(F=7.78,8.96,both P<0.01).The sIPSC frequency and quantification of the CUMS+Veh group were higher than those of CTRL+Veh group(both P<0.01),and the above indexes of CUMS+GBE group were lower than those of CUMS+Veh group(both P<0.01).As for the sEPSC/sIPSC ratio,GBE×CUMS interaction was significant(F=5.45,P=0.02).The sEPSC/sIPSC ratio of CUMS+Veh group(0.09±0.01)was lower than that of CTRL+Veh group(0.28±0.04)(P<0.01),and the sEPSC/sIPSC ratio of CUMS+GBE group(0.14±0.03)was higher than that of CUMS+Veh group(P<0.05).Conclusion Ginkgo biloba extract can improve the depression-like behavior of mice induced by CUMS,reduce the oxidative stress of mPFC and improve the excitation/inhibition balance of pyramidal neurons in depressive model mice.