摘要
目的:探讨四逆散通过调控糖原合成酶激酶-3β(GSK-3β)/肿瘤坏死因子-α诱导蛋白3(A20)/CCAAT增强子结合蛋白β(C/EBPβ)抑制小胶质细胞激活发挥抗抑郁作用。方法:将72只雄性C57/6J小鼠随机分出正常组、模型组、氟西汀组(5.0 mg·kg^(-1))、四逆散低、中、高剂量组(4.9、9.8、19.6 g·kg^(-1)),每组12只。适应性喂养1周后,采用慢性不可预知温和应激(CUMS)建立抑郁模型,第5周开始进行药物治疗4周。第9周进行行为学实验,包括蔗糖偏好实验、旷场实验、高架十字迷宫实验和强迫游泳实验。采用蛋白免疫印迹法(Western blot)检测皮质白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、一氧化氮合酶(iNOS)炎症蛋白的表达水平和GSK-3β、A20、C/EBPβ的表达水平。利用免疫荧光法检测皮质小胶质细胞的M1型极化标志物离子钙结合衔接分子1(Iba1)和巨噬细胞抗原(CD68)的荧光表达。结果:CUMS造模8周后,与正常组比较,模型组小鼠蔗糖偏好率显著降低(P<0.01),旷场实验中央区域活动显著减少(P<0.01),在高架十字迷宫实验开臂区域的活动明显减少(P<0.05),强迫游泳的不动时间显著增加(P<0.01);炎症蛋白IL-1β、IL-6、iNOS的表达水平显著升高(P<0.01),Iba1和CD68的荧光共定位的指数升高(P<0.05);GSK-3β蛋白表达水平升高(P<0.05),C/EBPβ蛋白表达水平显著升高(P<0.01)。药物干预4周后,与模型组比较,四逆散组小鼠糖水偏好率显著提高(P<0.01),旷场实验和高架十字迷宫实验中在中央区域和开臂区域的活动明显增加(P<0.05),四逆散中、高剂量组小鼠在强迫游泳实验中不动时间显著减少(P<0.01);IL-1β、IL-6、iNOS的蛋白表达水平明显下降(P<0.05),四逆散高剂量组Iba1和CD68的荧光共定位的指数明显下降(P<0.05);四逆散中、高剂量组GSK-3β、C/EBPβ的蛋白表达水平显著下降(P<0.01),A20的蛋白表达显著上调(P<0.01)。结论:四逆散抗抑郁作用与调控GSK-3β/A20/C/EBPβ蛋白的表达,抑制小胶质细胞M1型激活有关。
Objective To investigate the antidepressant effect of Sinisan(SNS)by regulating glycogen aynthase kinase-3β(GSK-3β)/tumor necrosis factor alpha-induced protein 3(A20)/CCAAT enhancer binding proteinβ(C/EBPβ)to inhibit the activation of microglia.Method A total of 72 male C57/6J mice were randomly divided into the normal group,model group,fluoxetine group(5.0 mg·kg^(-1)),low-dose Sinisan group(4.9 g·kg^(-1)),medium-dose Sinisan group(9.8 g·kg^(-1)),and high-dose Sinisan group(19.6 g·kg^(-1)),with 12 mice in each group.After one week of adaptive feeding,chronic unpredictable mild stress(CUMS)was performed to establish the depression model.In the fifth week,drug treatment was conducted for four weeks.In the ninth week,behavioral tests were performed,including sucrose preference test(SPT),open field test(OPT),elevated plus maze(EPM)test,and forced swimming test(FST).Western blot was used to detect the expression levels of interleukin-1β(IL-1β),interleukin-6(IL-6),nitric oxide synthase(iNOS),GSK-3β,A20,and C/EBPβin the cortex.The expression of M1-polarized ionized calcium-binding adapter molecule 1(Iba1)and cluster of differentiation 68(CD68)in microglia was detected by immunofluorescence.Result After eight weeks of CUMS,compared with the normal group,the mice in the model group had a significantly reduced sucrose preference rate(P<0.01),and the activity in the central area of the OPT was significantly reduced(P<0.01).The activity in the open arm area of the EPM test was significantly reduced(P<0.05),and the immobility time of FST was increased(P<0.01).The expression levels of inflammatory proteins IL-1β,IL-6,and iNOS were increased(P<0.01),and the fluorescence co-localization index of Iba1 and CD68 was increased(P<0.05).The protein expression levels of GSK-3βand C/EBPβwere significantly increased(P<0.05,P<0.01).After four weeks of SNS intervention,compared with the model group,the mice in the SNS group had significantly increased sucrose preference rate(P<0.01),significantly increased activities in the central area and the open arm area in the OPT and the EPM test(P<0.05),and significantly reduced immobility time in the FST(P<0.01).The protein expression levels of IL-1β,IL-6,and iNOS were significantly decreased(P<0.05),and the fluorescence co-localization index of Iba1 and CD68 was decreased in the high-dose SNS group(P<0.05).The protein expression levels of GSK-3βand C/EBPβin the medium-dose and high-dose SNS groups were significantly decreased(P<0.01),and that of A20 was significantly increased(P<0.01).Conclusion The antidepressant effect of SNS is related to the regulation of GSK-3β/A20/C/EBPβprotein expression and the inhibition of M1-type activation of microglia.
作者
陈虹云
杨东英
廖回庆
曾妍妍
潘琳可
白莎莎
邓迪
史亚飞
张荣
杨蕾
CHEN Hongyun;YANG Dongying;LIAO Huiqing;ZENG Yanyan;PAN Linke;BAI Shasha;DENG Di;SHI Yafei;ZHANG Rong;YANG Lei(Chinese Materia Medica,Guangzhou University of Chinese Medicine,Guangzhou 510006,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第12期16-23,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
广东省自然科学基金项目(2018A0303130072)。
