【作者】 戴建国；

【导师】 黄玉芳；

【作者基本信息】 南京中医药大学 ， 中西医结合基础， 2012， 博士

【摘要】 研究目的依据中医临床“助阳入脑,降火宁神”的治疗抑郁症的新思想创制助阳宁神方,该方在临床上确具较好疗效。研究发现其作用机制可能不是通过影响单胺类神经递质而产生,而可能与星形胶质细胞可塑性改变有关。为此,本课题采用皮质酮注射制备抑郁动物模型,从非单胺类神经递质假说中的星形胶质细胞可塑性入手,观察应激和助阳宁神方(Zhuyang-ningshen Formula, ZNF)作用前后星形胶质细胞可塑性变化：包括GFAP蛋白的表达改变、星形胶质细胞形态、数量等,期望探究助阳宁神方在该模型的效应,阐明调节海马星形胶质细胞可塑性是助阳宁神方临床改善抑郁症的作用机制,从而使“助阳入脑,降火宁神”思想得到实验证实,为抑郁症防治和新药研发提供科学的实验理论依据。研究方法(1)助阳宁神方药效学研究采用利血平拮抗模型观察小鼠闭眼率；5-羟色氨酸(5-hydroxytryptophane,5-HTP)诱导的甩头行为模型观测甩头率及甩头次数；育亨宾毒性模型观测各组小鼠死亡率；悬尾实验观测行为学改变。(2)抑郁小鼠模型制备及助阳宁神方抗抑郁有效性验证实验研究皮质酮皮下注射制备抑郁动物模型,实验分为6组,分别是正常组、模型组、氟西汀组,助阳宁神方低剂量组(1.9g/kg)、中剂量组(3.8g/kg)和高剂量组(7.6g/kg),每组8只,共48只C57BL/6N小鼠；采用强迫游泳、强迫悬尾观察给药4周后小鼠行为学改变和体重变化,然后处死动物,快速取脑剥离海马,Western blot蛋白免疫印迹检测神经元NF-L、SY-P蛋白表达变化,同时,观测助阳宁神方对该模型的拮抗作用。(3)基于星形胶质细胞可塑性的助阳宁神方抗抑郁作用机制研究实验分6组,分别是正常组、模型组、氟西汀组,助阳宁神方低剂量组(1.9g/kg)、中剂量组(3.8g/kg)和高剂量组(7.6g/kg),每组12只C57BL/6N小鼠,共72只小鼠。给药4周后,采用Western blot及免疫组化技术观测海马胶质纤维酸性蛋白(GFAP)、BDNF和GDNF表达改变,体视学分析技术检测海马GFAP表达阳性细胞数。研究结果(1)助阳宁神方药效学研究结果1)利血平拮抗模型与模型组闭眼率100%比,氟西汀组闭眼率为30%,闭眼率显著降低(P<0.01),助阳宁神方高、中、低剂量组闭眼率依次为60%、50%、40%,闭眼率也显著性降低(P<0.05、P<0.05、P<0.01),与氟西汀组比,助阳宁神方高、中、低剂量组闭眼率随剂量下降,闭眼率逐渐下降,但无显著性差异(P>0.05)。2)5-HTP诱导的甩头行为模型连续给药第3天、第7天15min后,正常组、氟西汀组、助阳宁神方高、中、低剂量组甩头率分别为：40%、80%、50%、40%和30%；40%、90%、40%、30%和20%,与正常组比,氟西汀组甩头率明显增加(P<0.01),而ZNF高、中、低剂量组均未增加甩头率(P均>0.05)；与氟西汀组比,ZNF高、中、低剂量组均甩头率显著下降(P<0.01)；第7天15min后,各相应组甩头次数依次为70次、246次、53次、27次和34次；ZNF各组甩头次数与正常组比无显著上升(P均>0.05)。3)育亨宾毒性模型正常组、氟西汀组、ZNF高、中、低剂量组死亡小鼠依次为1只、5只、2只、1只、1只,与正常组比,氟西汀组小鼠死亡率显著升高(P<0.01),而ZNF各组小鼠死亡率未显著上升(P>0.05),与氟西汀组比,ZNF各组小鼠死亡率显著下降(P均<0.01)。4)悬尾实验与正常组比,氟西汀组、ZNF高、中、低剂量组悬尾不动时间显著缩短(p<0.01或p<0.05)。(2)皮质酮致抑郁小鼠模型建立及助阳宁神方有效性验证实验结果1)体重变化结果各实验组起始体重无显著性差异(P均>0.05),4周后,模型组动物较正常组动物体重明显减轻(P<0.05),但其他各组与模型组比,均具有上调体重趋势,但无统计学意义(P均>0.05)。2)行为学实验研究结果在悬尾实验、强迫游泳中,与正常组比,模型组不动时间明显延长(P<0.01,P<0.05),与模型组比,氟西汀组不动时间明显缩短(P<0.01,P<0.05),助阳宁神方中((P<0.01,P<0.05)、低剂量组(P均<0.05)不动时间也显著缩短,助阳宁神方高剂量组不动时间未见显著缩短(P>0.05)。3) westem blot免疫印迹实验结果与正常组比,模型组NF-L、SYP表达水平著下降(P<0.01、P<0.05)；与模型组比,氟西汀组NF-L、SYP表达水平显著上调(P<0.01、P<0.05),助阳宁神方高、中、低剂量组均能明显上调NF-L、SYP表达水平(P均<0.05)。(3)基于星形胶质细胞可塑性的助阳宁神方抗抑郁作用机制研究结果1) westem blot免疫印迹实验结果与正常组比,模型组GFAP、BDNF及GDNF的表达较正常组水平著下调(P<0.01、P<0.01、P<0.01),与模型组比,氟西汀组除GDNF未见显著上调外,其他指标均显著著上调(P<0.05或P<0.01),助阳宁神方中、低剂量组均能显著上调GFAP、BDNF及GDNF的蛋白表达水平(P<0.05或P<0.01),而ZNF高剂量组显著上调GFAP及GDNF (P<0.05或P<0.01),而对BDNF却无显著上调作用(P>0.05)。2)免疫组化表达结果在海马CA3区和CA1区,与正常组比,模型组GFAP阳性区域总光密度值和面积均明显下调(P<0.05或P<0.01),与模型组比,氟西汀组、助阳宁神方高、中、低剂量组均能显著上调GFAP阳性区域总光密度值(P<0.05或P<0.01)；在CA3区助阳宁神方高、中剂量组及在CA1区助阳宁神方中剂量组GFAP阳性表达区域面积明显上调(P<0.05或P<0.01)；在DG区,氟西汀组、助阳宁神方高、中、低剂量组GFAP阳性表达区域总光密度值显著上调(P<0.05或P<0.01),但GFAP阳性表达区域面积均无明显上调(P>0.05)。3)体视学分析结果与正常组比,模型组海马GFAP阳性细胞(星形胶质细胞)个数显著下调(P<0.05),与模型组比,氟西汀组、ZNF高、中剂量组能显著上调GFAP阳性细胞(P<0.05、P<0.01、P<0.05),ZNF低剂量组有上调GFAP阳性细胞的趋势(P>0.05)。结论(1)助阳宁神方具有抗抑郁样行为作用；(2)助阳宁神方抗抑郁作用可能不是通过影响单胺类神经递质产生；(3)助阳宁神方可能通过上调星形胶质细胞可塑性、神经可塑性、增加神经营养因子对神经元的损伤修复,改善海马可塑性,进而发挥抗抑郁作用。更多还原

【Abstract】 ObjectiveBased on the new TCM idea of "zhu yang ru nao, jiang huo ning shen", the formula of "zhu yang ning shen" was developed,which showed good curative effects on depression in clinic. We found that the underlying mechanisms might be associated with the astroglia plasticity instead of the monoamine transmitters. In this project, we will adopt corticosterone-induced animal model of depression, and observed effects of the formula on the downregulation of astroglia plasticity in the hippocampus of model animals (e.g. the number of astroglia and the expression of GFAP).Through above experimental design, we hope that we can prove the crucial role of astroglia plasticy improvement in the treatment of the "zhu yang ning shen" formula on depression, which will contribute to illustrate the value of the new TCM idea in the treatment of depression in clinic and the development of new antidepressants from the aspect of astroglia.Methods(1) Study on pharmacodynamics of Zhuyang-ningshen Formula(ZNF)Reserpine antagonistic model in mice was observed with eyes closed rate;The head-witching test models were established by injection of5-HTP,the rate and the number of head-witching were tested; Yohimbine toxicity model was uesd to observe the mortality of mice; tail suspension test were observed behavior change.(2) Preparation of mice depression model induced by corticosterone and antidepressive validation experiment of Zhuyang-ningshen FormulaMice depression model was developed by subcutaneous injection of corticosterone in48C57BL/6N, which were equally devided into6groups of model control, F(treated with fiuoxetine hydrochloride3.6mg/kg), ZNF1(treated with gastric gavage of ZNF1.9g/kg),ZNF2(treated with ZNF3.8g/kg), ZNF3(treated with ZNF7.6g/kg);4weeks later, observed behavior change by forced swimming test and tail suspension test and weight chang,then the mice were sacrificed by decapitation and the hippocampal tissues were rapidly collected for western blot analysis of neurofliament light chain protein(NF-L) and synaptic vescle protein(SYP), at the same time, the antagonistic effects of Zhuyang-ningshen Formula also were observed.(3) Antidepressive mechanism of Zhuyang-ningshen Formula on hippocampal astrocyte plasticityMice depression model was developed by subcutaneous injection of corticosterone in72C57BL/6N, which were equally devided into6groups of model control, F(treated with fiuoxetine hydrochloride3.6mg/kg), ZNF1(treated with gastric gavage of ZNF1.9g/kg), ZNF2(treated with ZNF3.8g/kg), ZNF3(treated with ZNF7.6g/kg); After drug delivery4weeks, we dectected the protein lever of GFAP and BDNF and GDNF with western blot, counted hippocampal GFAP positive cells by steorology analysis system.Results(1) Pharmacodynamics research of Zhuyang-ningshen Formula results1) In the reserpine antagonistic model, eyes closed rate of model control group,F(treated with fiuoxetine hydrochloride)group, ZNF1(treated with gastric gavage of ZNF1.9g/kg), ZNF2(treated with ZNF3.8g/kg), ZNF3(treated with ZNF7.6g/kg) each was100%,30%,60%,50%and40%, compared with model control group, eyes closed rate of F group and ZNF1and ZNF2and ZNF3was remarkable decreased (P<0.01, P<0.05、P<0.05、P<0.01); In comparison with F group, it was decreased to all dosage of ZNF,at the same time, as the dose increased,the eyes closed rate was stepped up,but no significant difference(P>0.05); In all dose of ZNF(P<0.01), no significant difference in each ZNF groups(P>0.05).2) In the head-switching test model by injection of5-HTP, the rate of head-switching behavior on the3rd and7th day15min after drug delivery each was40%,80%,50%,40%,30%and40%,90%,40%,30%,20%,the number of head-switching behavior was70,246,53,27and34,respectively, the rate of head-switching was higher in F group than that in normal group(P<0.01), but no significantly Increased in each ZNF,compared with F group, there was notable descended(P<0.01),and so for the number of head-switching behavior.3) In the yohimbine toxicity model, mouse death number each was1,5,2,1,1,the mortality rate of was higher in F group than that in normal group(P<0.01)and in all dose of ZNF(P<0.01), but no outstanding increased in each ZNF groups(P>0.05).4) In tail suspension test, compared of normal group, immobility time were noticeable shortened in F group (P<0.01) and ZNF1group(P<0.05) and ZNF2group(P<0.05) and ZNF3group (P<0.05).(2) Preparation of mice depression model induced by corticosterone and antidepressive validation experiment of Zhuyang-ningshen Formula1) Results of weight changeThere was no significant difference between the initial weight in each experimental group(P>0.05).Four weeks later, there was striking weight loss in model group than in normal groups(P<0.05), but if compared with model group,other groups had raised the weight trend, but no statistical significance(P>0.05).2) Results of behavior experimental studyCompared of normal group,immobility time in tail suspension test and in forced swimming of model group were noticeable lengthened (P<0.01,P<0.05); in comparison with the model group, immobility time were eminent shortened in F group (P<0.01,P<0.05) and ZNF2group(P<0.01,P<0.05) and ZNF3group(P<0.05),but no reduced immobility time in ZNF1group (P>0.05)3) Results of western blotCompared of normal group, the protein level of NF-L and SYP were conspicuous down-regulationed in the model group (P<0.01,P<0.05), in comparison with the model group, the protein level of NF-L and SYP were noticeable up-regulated in F group (P<0.01,P<0.05) and in ZNF1,ZNF2and ZNF3(P<0.05).(3) Antidepressive mechanism of Zhuyang-ningshen Formula on hippocampal astrocyte plasticity1) Mechanism of antidepressant action results of Zhuyang-ningshen Formula on hippocampal astrocyte plasticityIn comparison with normal group, the protein level of GFAP and BDNF and GDNF were conspicuous down-regulated in the model group(P<0.01, P<0.01and P<0.01),compared with the model group, the expression level of GFAP and BDNF were clearly increased(P<0.05,P<0.01), the expression level of GDNF only had a little increasion, but no distinct difference(P>0.05),the expression level of GFAP and BDNF and GDNF were up-regulated in ZNF2and ZNF3(P<0.05or P<0.01),but in ZNF1only could up-regulated GFAP and GDNF(P<0.05or P<0.01),there was no noticeably went up in the expression of BDNF(P>0.05).2) Results of immunohistochemical expressionIn the hippocampal CA3region and CA1region, in comparison with normal group, positive regional total optical density value and positive region with a total area of GFAP in model group, decreased obviously(P<0.05or P<0.01),compared with the model group,there were clear up about positive regional total optical density value of GFAP in the F,ZNF1,ZNF2and ZNF3(P<0.05or P<0.01); In the hippocampal CA3region,ZNF1and ZNF2could up-regulated positive region with a total area of GFAP, so did ZNF2in the hippocampal CA1region (P<0.05or P<0.01); In the hippocampal DG region, positive regional total optical density value of GFAP were up-regulated in the group of F,ZNF1,ZNF2and ZNF3(P<0.05or P<0.01),but there was no significant risen about positive region with a total area of GFAP(P>0.05).3) Results of steorologyCompared with the normal group,the number of the GFAP positive cells (astrocyte)in hippocampus in Model group,was decreased,in comparison with model group,the number of the GFAP positive cells were increased obveriously in fluoxetine group, high dose group and middle dose group of Zhuyang-ningshen formula (P<0.05、P<0.01、P<0.05), but low dose group of Zhuyang-ningshen formula only had an uptrend of increasing the number of the GFAP positive cells (P>0.05).Conclusion:(1) Zhuyang-ningshen Formula have the effect of antagonism on the action of depression.(2) Antidepressive effect of Zhuyang-ningshen Formula may not affect monoamine neurotransmitter.(3) Zhuyang-ningshen Formula may up-regulate astrocyte plasticity, enhancement of neurotrophic factor on neuronal injury and repair, improve hippocampal plasticity, exert antidepressant effects.更多还原