二仙汤对迟发性抑郁症模型大鼠脑组织脉络丛叶酸转运-海马神经发生的影响

Effects of Erxian Decoction(二仙汤)on Choroid Plexus Folate Transport-Hippocampal Neurogenesis in Rat Brain Tissue of Late-Onset Depression Model

目的探讨二仙汤治疗迟发性抑郁症(LOD)的效果及可能作用机制。方法40只雄性7~8周龄Wistar大鼠分为正常组和青年抑郁组各20只。60只20月龄雄性Wistar大鼠分为衰老组、迟发抑郁组、二仙汤组各20只。青年抑郁组大鼠采用慢性不可预计应激刺激(CUMS)6周建立抑郁模型,迟发抑郁组、二仙汤组衰老大鼠采用CUMS 6周建立LOD模型,正常组和衰老组不进行任何干预。造模的同时进行灌胃给药,二仙汤组大鼠给予二仙汤8 g/(kg·d)灌胃,正常组、青年抑郁组、衰老组、迟发抑郁组给予4 ml/(kg·d)纯净水灌胃,各组均给药6周。采用糖水偏爱实验、强迫游泳实验评估大鼠抑郁样行为,旷场实验评价大鼠自发活动能力,T迷宫实验评估大鼠认知功能;Western Blot法检测海马组织神经元核心抗原(NeuN)、巢蛋白、双皮质素(DCX)及脉络丛组织中闭锁小带蛋白1(ZO-1)、叶酸α受体(FRα)、还原型叶酸转运体(RFC)、质子耦合叶酸转运体(PCFT)蛋白表达;采用免疫荧光法检测海马齿状回Ki-67/巢蛋白双阳性细胞数、5-溴脱氧尿嘧啶核苷(BrdU)/DCX双阳性细胞数及脉络丛组织中ZO-1、FRα、RFC、PCFT蛋白表达;ELISA法检测血浆、脑脊液、海马组织中5-甲基四氢叶酸(5-MTHF)含量;采用Pearson相关分析对各组大鼠脑脊液、海马组织中5-MTHF含量与海马组织中巢蛋白、DCX、NeuN蛋白表达进行相关性分析。结果与正常组比较,青年抑郁组及迟发抑郁组大鼠糖水偏爱度降低,旷场总路程、穿格次数减少,强迫游泳不动时间延长,海马组织NeuN、巢蛋白、DCX蛋白表达均降低,海马齿状回Ki-67/巢蛋白双阳性细胞数、BrdU/DCX双阳性细胞数减少,ZO-1、FRα蛋白表达及荧光强度减少(P<0.05或P<0.01);青年抑郁组大鼠第3、4天及迟发抑郁组大鼠第2~4天T迷宫正确率均明显降低,迟发抑郁组大鼠脑脊液、海马组织中5-MTHF含量明显降低(P<0.05或P<0.01)。与青年抑郁组相比,迟发抑郁组大鼠旷场总路程、穿格次数减少,第1~4天T迷宫正确率均显著降低,海马组织NeuN、巢蛋白、DCX蛋白表达及海马齿状回Ki-67/巢蛋白双阳性细胞数、BrdU/DCX双阳性细胞数减少(P<0.05或P<0.01)。与迟发抑郁组比较,二仙汤组大鼠糖水偏爱度及旷场总路程、穿格次数均升高,强迫游泳不动时间缩短,第3~4天T迷宫正确率明显升高,海马组织NeuN、巢蛋白、DCX蛋白表达升高,海马齿状回Ki-67/巢蛋白双阳性细胞数、BrdU/DCX双阳性细胞数增加,脑脊液、海马组织中5-MTHF含量明显升高,脉络丛中ZO-1、FRα、RFC、PCFT蛋白表达及荧光强度升高(P<0.05或P<0.01)。相关性分析结果显示,脑脊液(r=0.466)和海马组织(r=0.522)中5-MTHF含量与海马组织中巢蛋白表达存在正相关(P<0.05)。结论二仙汤可改善LOD模型大鼠抑郁样行为及认知障碍,其机制可能通过减轻脑组织脉络丛结构损伤,改善脉络丛叶酸转运障碍,从而促进海马神经发生。

Objective This study aims to investigate the effectiveness and potential mechanism of Erxian Decoction(二仙汤)for late-onset depression(LOD).Methods Forty Wistar male rats of 7-8 weeks were divided into 20each of normal group and youth depression group.Sixty Wistar male rats of 20 months were divided into 20 each of elder group,LOD group and Erxian Decoction group.The rats in youth depression group were modelled with chronic unpredictable mild stress(CUMS)for 6 weeks to build a depression model,and the elder rats in LOD group and Erxian Decoction group were modelled with CUMS for 6 weeks to build a LOD model,with no interventions in the normal group and the elder group.Gastric administration was carried out at the same time of modelling,rats in Erxian Decoction group were given 8 g/(kg·d)of Erxian Decoction by gavage,and rats in the normal group,youth depression group,elder group,and LOD group were given 4 ml/(kg·d)of pure water by gavage,for 6 weeks in each group.Sugar water preference experiment and forced swimming experiment were used to evaluate the depression-like behaviour of rats,absent field experiment was used to evaluate the spontaneous activity ability of rats,and T-maze experiment was used to evaluate the cognitive function of rats;Western blot assay was used to detect neuronal nuclei(NeuN),nestin,doublecortin(DCX)in hippocampal tissue,and zonula occludens-l(ZO-1),folate receptorα(FRα),reduced folate carrier(RFC),and protoncoupledfolate transporter(PCFT)protein expression in choroid plexus;immunofluorescence was used to detect the number of double-positive cells for Ki-67/nestin,the number of double-positive cells for 5-bromodeoxyuracil nucleoside(BrdU)/DCX in hippocampal dentate gyrus,and the protein expression of ZO-1,FRα,RFC,and PCFT in choroid plexus tissues;ELISA technique was used to detect plasma,cerebrospinal fluid,5-methyltetrahydrofolate(5-MTHF)in hippocampal tissues;Pearson correlation analysis was used to correlate the 5-MTHF content in cerebrospinal fluid and hippocampal tissues and the expression of nestin,DCX,and NeuN proteins in hippocampal tissues of rats in youth depression group and LOD group.Results Compared with normal group,rats in youth depression group and LOD group showed reduced sugar water preference,reduced total distance travelled in the absent field,reduced number of times through the grid,prolonged forced swimming immobilisation,reduced hippocampal NeuN,nestin and DCX protein expression,reduced number of Ki-67/nestin double-positive cells in hippocampal dentate gyrus,reduced number of BrdU/DCX double-positive cells in hippocampal dentate gyrus,and reduced expression of ZO-1 and FRαproteins and fluorescence intensity(P<0.05 or P<0.01);the correct rate of T-maze on days 3 and 4 in the rats of youth depression group and on days 2 to 4 in the rats of LOD group significantly decreased,and the content of 5-MTHF in the cerebrospinal fluid and hippocampal tissues of the rats of LOD group significantly decreased as well(P<0.05 or P<0.01).Compared with youth depression group,rats in LOD group showed a decrease in total distance travelled in absenteeism,number of times through the grid,a significant decrease in the correct rate of the T-maze on day 1-4,a decrease in NeuN,nestin,DCX protein expression of hippocampal tissue and the number of Ki-67/nestin double-positive cells,BrdU/DCX double-positive cells of hippocampal dentate gyrus(P<0.05 or P<0.01).Compared with LOD group,rats in Erxian Decoction group had elevated sugar-water preference and total distance travelled in absenteeism and number of times through the grid,shorter forced swimming immobility time,significantly higher correct rate of the T-maze on day 3-4,elevated expression of NeuN,nestin,and DCX proteins in hippocampal tissues,increased number of Ki-67/nestin double-positive cells and BrdU/DCX doublepositive cells in hippocampal dentate gyrus,and increased 5-MTHF content in cerebrospinal fluid and hippocampal,and ZO-1,FRα,RFC,PCFT protein expression and fluorescence intensity increased in choroid plexus(P<0.05 or P<0.01).Correlation analysis showed that there was a positive correlation between 5-MTHF content in cerebrospinal fluid(r=0.466),hippocampal tissue(r=0.522)and nestin expression in hippocampal tissue(P<0.05).Conclusion Erxian Decoction could improve depressive-like behaviour and cognitive impairment in LOD model rats,and its mechanism may promote hippocampal neurogenesis by alleviating structural damage to the choroid plexus of the brain tissue,and improving impaired choroid plexus folate transport.