逍遥散加味对青幼期抑郁模型大鼠抑郁行为及小胶质细胞活化的影响

Modified Xiaoyaosan Alleviates Depression-like Behaviors by Regulating Activation of Hippocampal Microglia Cells in Rat Model of Juvenile Depression

目的:探讨逍遥散加味百合、合欢皮(百欢逍遥汤)通过调节小胶质细胞活化改善青幼期大鼠抑郁样行为的作用机制。方法:应用慢性不可预见性温和应激法(CUMS)创建青幼期(3~8周龄)抑郁大鼠模型,将60只青幼期SD大鼠随机分成正常组、模型组、氟西汀组、百欢逍遥汤低、中、高剂量组(5.36、10.71、21.42 g·kg^(-1)),蔗糖偏好实验(SPT)检测大鼠糖水偏好值;强迫游泳实验(FST)检测大鼠不动时间;旷场实验(OFT)检测总路程、中心区域路程、水平穿越方格数及前肢离地垂直活动次数;Morris水迷宫实验(MWM)检测逃避潜伏期和穿梭平台次数;免疫荧光检测海马区M1型小胶质细胞极化标志物诱导型一氧化氮合酶(iNOS)和M2型小胶质细胞极化标志物巨噬细胞甘露糖受体(CD206)的表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测海马组织iNOS、CD206和促炎细胞因子肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、IL-6及抗炎细胞因子IL-4、IL-10的mRNA表达;蛋白免疫印迹法(Western blot)检测海马iNOS和CD206的蛋白表达;酶联免疫吸附测定法(ELISA)检测海马炎症因子IL-4、IL-6的含量。结果:与正常组比较,模型组大鼠蔗糖偏好值明显降低(P<0.05),不动时间明显增加(P<0.05),运动和探索行为明显减少(P<0.05),学习、空间记忆水平明显降低(P<0.05),iNOS的mRNA和蛋白水平均明显提高(P<0.05),促炎细胞因子IL-1β、IL-6、TNF-α的mRNA水平也明显提高(P<0.05)。与模型组比较,百欢逍遥汤干预后青幼期抑郁大鼠的蔗糖偏好值明显升高(P<0.05),不动时间显著缩短(P<0.01),运动和探索行为明显增加(P<0.05),学习和空间记忆能力显著提升(P<0.01),百欢逍遥汤高剂量组海马区iNOS阳性表达和蛋白含量显著降低(P<0.01),TNF-α、IL-1β、IL-6水平显著下降(P<0.01);百欢逍遥汤高剂量组CD206阳性表达显著增加(P<0.01),IL-4、IL-10水平显著上升(P<0.01)。与氟西汀组比较,百欢逍遥汤高剂量组蔗糖偏好率显著升高(P<0.01),不动时间显著减短(P<0.01),中心区域路程显著增多(P<0.01),学习和空间记忆能力显著增强(P<0.01),海马区iNOS阳性表达和mRNA水平明显降低(P<0.05),TNF-α、IL-1β、IL-6水平明显较低(P<0.05,P<0.01);CD206阳性表达显著更高(P<0.01),IL-4、IL-10水平明显升高(P<0.05,P<0.01)。结论:百欢逍遥汤对青幼期大鼠抑郁行为的改善作用与抑制海马区小胶质M1型极化,促进M2型极化有关。

Objective:To investigate the mechanism of Baihuan Xiaoyao Decoction(Xiaoyaosan added with Lilii Bulbus and Albiziae Cortex)in alleviating depression-like behaviors of juvenile rats by regulating the polarization of microglia.Method:Sixty juvenile SD rats were randomized into normal control,model,fluoxetine,and low-,medium-,and high-dose(5.36,10.71,21.42 g·kg^(-1),respectively)Baihuan Xiaoyao decoction groups.The rat model of juvenile depression was established by chronic unpredictable mild stress(CUMS).The sucrose preference test(SPT)was carried out to examine the sucrose preference of rats.Forced swimming test(FST)was carried out to measure the immobility time of rats.The open field test(OFT)was conducted to measure the total distance,the central distance,the number of horizontal crossings,and the frequency of rearing.Morris water maze(MWM)was used to measure the escape latency and the number of crossing the platform.The immunofluorescence assay was employed to detect the expression of inducible nitric oxide synthase(iNOS,the polarization marker of M1 microglia)and CD206(the polarization marker of M2 microglia).Real-time polymerase chain reaction was employed to determine the mRNA levels of iNOS,CD206,pro-inflammatory cytokines[tumor necrosis factor(TNF)-α,interleukin(IL)-1β,and IL-6]and anti-inflammatory cytokines(IL-4 and IL-10)in the hippocampus.Western blotting was employed to determine the protein levels of iNOS and CD206 in the hippocampus.The levels of IL-4 and IL-6 in the hippocampus were detected by enzyme-linked immunosorbent assay.Result:Compared with the normal control group,the model rats showed a reduction in sucrose preference(P<0.05),an increase in immobility time(P<0.05),decreased motor and exploratory behaviors(P<0.05),and weakened learning and spatial memory(P<0.05).In addition,the model rats showed up-regulated mRNA and protein levels of iNOS and mRNA levels of IL-1β,IL-6,and TNF-α(P<0.05).Compared with the model group,Baihuan Xiaoyao decoction increased the sucrose preference value(P<0.05),shortened the immobility time(P<0.01),increased the motor and exploratory behaviors(P<0.05),and improved the learning and spatial memory(P<0.01).Furthermore,the decoction down-regulated the positive expression and protein level of iNOS,lowered the levels of TNF-α,IL-1β,and IL-6(P<0.01),promoted the positive expression of CD206,and elevated the levels of IL-4 and IL-10(P<0.01)in the hippocampus of the high dose group.Moreover,the high-dose Baihuan Xiaoyao decoction group had higher sucrose preference value(P<0.01),shorter immobility time(P<0.01),longer central distance(P<0.01),stronger learning and spatial memory(P<0.01),higher positive expression and protein level of iNOS(P<0.01),lower levels of TNF-α,IL-1β,and IL-6(P<0.05,P<0.01),lower positive expression and mRNA level of iNOS(P<0.05),and higher levels of IL-4 and IL-10(P<0.05,P<0.01)than the fluoxetine group.Conclusion:Baihuan Xiaoyao decoction can improve the depression-like behavior of juvenile rats by inhibiting the M1 polarization and promoting the M2 polarization of microglia in the hippocampus.