抗抑郁药物的神经可塑性促进作用及机制研究
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摘要
目的：抑郁症是一种慢性易复发的疾病,世界上约五分之一的人口受到抑郁症的困扰。抑郁症的发病机理尚不清楚,大量文献报道抑郁症与脑源性神经营养子和神经可塑性之间存在一定的联系,然而,其具体的关系尚缺乏系统的研究。单次注射氯胺酮可以在30min内起到抗抑郁效果,并且可以持续1个星期,虽证实可能与脑源性神经营养因子(BDNF)和神经可塑性相关,但氯胺酮的速效及长效的机制尚不清楚。进一步阐明抑郁症与BDNF和神经可塑性的系统关系,研究氯胺酮在治疗抑郁时的速效和长效与BDNF和神经可塑性的具体关系和作用机制,有利于进一步深入了解抑郁症的病理生理学机制,为抗抑郁药物的研发提供理论基础。常春藤皂苷元具有一定的抗抑郁生物活性,然而,其活性不强,对其结构改造以提升其抗抑郁活性的研究较少。
本研究旨在检测在抑郁的发病和治疗过程中,BDNF和神经可塑性的作用；研究糖皮质激素皮质酮对氯胺酮在BDNF表达中的促进作用的影响,并探讨氯胺酮的速效和长效机制与神经可塑性及BDNF的关系；研究氯胺酮增强小鼠海马神经元神经可塑性可能的信号转导通路；对具有抗抑郁活性的常春藤皂苷元进行结构改造,并检测其抗抑郁效果及机制。
方法：1.以C57BL/6小鼠作为研究对象,采用长期皮质酮暴露的方法造成焦虑/抑郁模型,经过30d的饮水给药(皮质酮,35mg·L-1)以后,通过高架迷宫实验和悬尾实验,检测小鼠的行为学变化。当确认小鼠已经达到焦虑/抑郁样状态以后,进入治疗、康复阶段,每天腹腔注射氟西汀(18mg·kg-1)。20d以后,检测小鼠的行为学变化。在整个实验过程中,分别在第10d,20d,35d,45d,60d取材,进行高尔基染色和免疫荧光染色。计数海马CA1,CA3区椎体神经元树突棘密度的变化和DG区颗粒神经元树突棘密度的变化,检测海马CA1区,CA3区和DG区(包括新生神经元和成熟神经元)神经元内BDNF的表达变化。
2.以C57BL/6小鼠为研究对象,研究单次注射氯胺酮(3.0mg·kg-1)对小鼠BDNF的表达和神经可塑性的影响。采用长期皮质酮暴露(饮水中给予皮质酮35mg·L-1)造成焦虑/抑郁模型,单次给予腹腔注射氯胺酮以后,使用Western blotting法检测氯胺酮对BDNF的促进作用在抑郁鼠和正常鼠的不同,并运用糖皮质激素受体抑制剂RU486进行进一步确认。在此基础上,一方面研究短期给予皮质酮对氯胺酮在BDNF表达中的促进作用中的影响,并探讨氯胺酮的速效(30min)和长效(7d)的病理生理学基础：采用皮质酮暴露的方法,给皮质酮3天以后,单次腹腔注射氯胺酮(3.0mg·kg-1)后分别在第30min和第7d取材,检测BDNF的表达和突触素(Synaptophysin)的变化。一方面研究氯胺酮提高小鼠海马神经元突触可塑性的机制：以C57BL/6小鼠为研究对象,在单次给予氯胺酮后的15min,45min,4h和24h时间点取材,应用Western blotting法检测Racl信号通路中的RaclGTPase, Tiaml, RacGAPl,和P-cofilin的变化情况,拟探讨氯胺酮提高小鼠海马神经元突触可塑性与Racl信号通路的关系。
3.设计并合成常春藤皂苷元酰胺衍生物N-(3-二甲氨基丙基)-常春藤皂苷元-17-甲酰胺(HGA)。以常春藤皂苷元为原料,经DCC/NHS将常春藤皂苷元28位的羧基活化,加入二甲氨基丙胺反应得HGA。经质谱,核磁氢谱和碳谱对其结构进行确认。然后,经高效液相色谱(HPLC)检测其纯度。在此基础上,在细胞水平,以原代海马神经元为研究对象,用皮质酮(10μmol·L-1)损伤造模,探讨HGA对原代海马神经元的保护作用；在整体动物水平,应用急性给药的方法,研究HGA急性给药对C57BL/6小鼠行为学的影响。并且,从BDNF和神经可塑性的角度研究其抗抑郁作用的机制。
结果：1.神经可塑性和BDNF在抑郁症发病及治疗中的变化
(1)慢性皮质酮暴露及长期的氟西汀治疗对小鼠行为学的影响
给予皮质酮暴露30d以后,高架迷宫实验结果显示,小鼠进入开臂的次数著减少(t=2.202,P=0.043),悬尾实验结果显示小鼠的不动时间显著延长(t=2.094,P=0.047)：经过20d的氟西汀治疗以后,小鼠在高架迷宫实验中进入开臂的次数显著增多(P=-0.007),悬尾实验中小鼠的不动时间显著(P=0.000)减少。
(2)慢性皮质酮暴露及长期氟西汀治疗对小鼠神经可塑性的影响
与相应的对照组相比,10d和20d的皮质酮处理对海马各区神经元树突棘密度没有产生显著的影响,经过35d的皮质酮的处理以后,与相应的溶剂对照组(VEH)的神经元的树突棘的密度相比,CA1区(t=2.626,P=0.030)和CA3区(t=2.982,P=0.018)神经元树突棘的密度显著降低。
经过45d的皮质酮处理以后的,海马的所有亚区的神经元树突棘的密度均明显少于相应的溶剂对照组的神经元树突棘的密度(CA1：P=0.004；CA3：P=0.000；DG：P=0.002)。经过60d的皮质酮处理以后海马各区神经元树突棘的密度均明显的少于相应的溶剂对照组的树突棘的密度(CA1：P=0.001；CA3：P=0.000；DG：P=0.004)。
经过10d的氟西汀处理以后,与相应的CORT组相比,经氟西汀处理的CORT+FLX组的CA1区(P=0.000)和CA3区(P=0.004)的神经元树突棘的密度显著提高。经过25d的氟西汀处理以后,与相应的CORT组相比,CORT+FLX组的海马所有亚区的神经元树突棘的密度均显著升高(CA1：P=0.000；CA3：P=0.000；DG：P=0.010)。
(3)慢性皮质酮暴露及长期氟西汀治疗对小鼠海马区神经元胞体内BDNF水平的影响
经过10d的皮质酮处理后,与相应的对照组相比,DG区的新生神经元(t=4.416,P=0.002)与成熟神经元(t=3.371,P=0.010)胞体中的BDNF的水平显著升高。经过20d的皮质酮处理后,与相应的对照组相比,DG区成熟神经元(t=3.848,P=0.005),DG区新生神经元(t=3.991,P=0.004)和CA3(t=3.495,P=0.008)区神经元胞体中的BDNF的水平显著升高。经过35d的皮质酮处理后,与相应的对照组相比,DG区成熟神经元(t=2.501,P=0.037),CA3区(t=3.670,P=0.006),CA1区(t=3.549,P=-0.008)神经元中的BDNF水平显著提高。
与相应的VEH组相比,45天的皮质酮处理后,CA1区神经元(P=0.001)和DG区新生神经元（P=0.001)的BDNF的水平显著减少。经过60d的皮质酮处理后,与相应的VEH组相比,海马CA1区、CA3区神经元和DG区新生神经元中的BDNF的水平均显著升高(CA1：P=0.031；CA3：P=0.008；DG-N：P=0.012)。
经过10d(36d-45d)的氟西汀处理后,与相应的CORT组相比,DG区成熟神经元(P=0.000)和新生神经元(P=0.003)胞体内的BDNF的水平显著减少。与相应的各CORT组相比,经过25d的氟西汀处理后,海马CA3区神经元、DG区成熟神经元和新生神经元中的BDNF的水平均显著降低(CA3：P=0.025；DG-M：P=0.001；DG-N：P=0.001)。
2.氯胺酮抗抑郁作用与神经可塑性和BDNF的关系及其机制研究
(1)氯胺酮对BDNF的促进作用及糖皮质激素皮质酮的影响
与给予单次氯胺酮注射的正常鼠相比,给抑郁鼠单次注射氯胺酮,给药后30min时,抑郁鼠海马BDNF的表达显著升高(F=12.493,P=0.008),并且糖皮质激素受体抑制剂Ru486可以显著抑制这种升高(F=15.499,P=0.004)。但是短期(3d)的皮质酮处理却不能达到相似的效果,氯胺酮可显著促进海马区BDNF的表达(F=927.149,P=0.000)；短期(3d)的糖皮质激素受体抑制剂处理不会对氯胺酮的BDNF促进作用产生影响(F=0.165,P=0.690)。单剂量给予氯胺酮以后30min时,小鼠海马突触素(Synaptophysin)表达水平较对照组没有发生显著变化(F=0.897,P=0.358)。糖皮质激素受体抑制剂Ru486对突触素的表达也没有显著影响(F=1.524,P=0.235)。单剂量给予氯胺酮以后7d时,与对照组相比,小鼠海马BDNF水平没有出现显著改变(F=1.539,P=0.233)。并且短期的糖皮质激素受体抑制剂Ru486处理也没有对氯胺酮的BDNF促进作用产生显著影响(F=2.275,P=0.151)。单剂量给予氯胺酮以后7d时,给予了氯胺酮的各组小鼠海马突触素(Synaptophysin)表达水平显著提高(F=466.104,P=0.000)。并且短期的Ru486处理(3d)不影响突触素的表达(F=4.308,P=0.054)。
(2)氯胺酮对Rac1信号通路的影响
与对照组相比,在给予单剂量氯胺酮后,Rac1-GTPase在15min(t=4.023,P=0.016)和45min(t=3.939,P=0.017)两个时间点均显示升高,在给药后的4h时,恢复到正常水平(t=0.570,P=0.599)；在给予单剂量氯胺酮后15min时间点,与对照组相比,Tiam1水平显著升高(F=953.626,P=0.000),并且在给予单剂量氯胺酮后45min时,Tiam1仍显著升高(F=70.369,P=0.000),在单剂量注射氯胺酮以后4h时间点Tiam1的表达恢复到基础水平(F=1.259,P=0.278)；在给予单剂量氯胺酮后15min时,与对照组相比,RacGAP1表达水平显著降低(F=20.750,P=0.000),并且持续到给予单剂量氯胺酮后45mmin时间点,RacGAP1仍显著降低(F=181.808,P=0.000)。给予单剂量氯胺酮以后4h时间点,RacGAP1的表达恢复到基础水平(F=0.343,P=0.566)；在给予单剂量氯胺酮的15min,与对照组相比,Cofilin的磷酸化水平显著升高(F=617.735,P=0.000)；单剂量给予氯胺酮后的45min(F=259.295,P=0.000)和4h(F=148.986,P=0.000),Cofilin的磷酸化水平均持续显著升高。在单剂量给予氯胺酮后的24h的时间点,Cofilin的磷酸化水平回到基础水平(F=2.453,P=0.137)。
3.常春藤皂苷元结构改造与生物活性及其机制研究
(1)通过普通化学合成的方法,经柱色谱分离,得到了白色粉末状化合物N-(3-二甲氨基丙基)-常春藤皂苷元-17-甲酰胺(HGA)。质谱图显示分子离子峰是558.0([M+H]+)而HGA的相对分子质量为556.86；核磁氢谱的数据：1H-NMR(CD3OD):δ5.26(t,J=3.4Hz,1H,C=C-H),3.99(dd,J1=14Hz,J2=7.2Hz,1H),3.48-3.52(m,1H),3.41(d,J=10.8,1H),3.05-3.11(m,1H),2.91(t,J=7.2Hz,2H),2.71(s,6H,NMe2),1.91(s,1H),1.05-1.43(m,22H),0.78-0.95(m,18H),0.686(3H,s,CH3),0.596(3H,s,CH3),与HGA的结构相吻合；核磁碳谱数据：6180.02(C=0),143.67(C=C),122.65(C=C),72.30,65.76,60.07,55.38,46.20,46.10,42.22,41.82,41.51,40.99,39.20,37.97,36.43,35.90,33.58,33.20,33.02,31.81,30.14,27.06,25.93,25.03,24.87,23.06,22.57,22.44,19.38,17.62,16.56,14.78,12.98,11.24,与HGA的结构相吻合。综合以上的分析,合成所得化合物为目标化合物HGA。利用高效液相色谱(HPLC),以甲醇-水-冰醋酸-乙二胺(87：13：0.04：0.02)为流动相,210nm为检测波长,发现,HGA出峰时间为59.09min,归一化法的计算结果显示HGA的纯度为94.08%(HPLC)。
(2)常春藤皂苷元酰胺衍生物HGA的抗抑郁生物活性及机制研究
HGA神经保护作用实验中,MTT结果显示,化合物HGA可以在0.1μmol·L-1(P=0.042)。而相同实验条件下1μmoL·L-1的HG(P=0.866)不能对神经元产生显著的保护作用。急性给药的小鼠悬尾实验的结果显示,HGA在10mg·kg-1时可以显著(P=0.010)缩短C57BL/6小鼠的不动时间。在此基础上,从BDNF和神经可塑性的角度对其机制进行研究,Western blotting的结果显示,HGA可以显著逆转皮质酮所导致的BDNF(P=0.000)和突触素(Synaptophysin, P=0.000)蛋白的表达水平的下降。
结论：1.在抑郁的发生发展过程中BDNF的表达水平先升高,再降低,最后又升高,呈波浪形变化。并且在给予氟西汀治疗以后,BDNF的表达水平与抑郁但未接受治疗的小鼠相比,显著下降,说明BDNF与小鼠的抑郁状态密切相关,但关系复杂。在抑郁的发生阶段,BDNF应激性的升高可能对神经可塑性起到一定的保护作用。海马神经元树突棘密度则随着小鼠的抑郁状态的发生、发展而不断降低,当给与氟西汀治疗以后,小鼠海马神经元树突棘密度迅速升高而且与小鼠的行为学状态呈正相关,说明神经可塑性的变化很可能是抑郁的病理生理学基础。
2.氯胺酮在抑郁鼠身上对BDNF表达的促进作用更加显著,而这种显著的促进作用可以被糖皮质激素受体抑制剂Ru486所阻断。但短期(3d)的皮质酮处理不能显著影响氯胺酮对BDNF的促进作用。单剂量注射氯胺酮的速效抗抑郁作用可能与促进BDNF的表达相关,其长效可能与其改善了神经可塑性有关。单剂量氯胺酮可能是通过激活Racl信号通路,促进Cofilin的磷酸化,抑制Cofilin的剪切作用,最终改善神经可塑性,起到长效的抗抑郁作用。
3.经设计和普通化学合成,成功得到了N-(3-二甲氨基丙基)-常春藤皂苷元-17-甲酰胺(HGA),HGA可以在0.1μmo1·L-1的浓度下对原代海马神经元产生显著的保护作用；HGA可以在10mg·kg-1的给药剂量下,小鼠在悬尾实验中起到显著的抗抑郁作用,HGA的抗抑郁作用可能跟HGA可以促进BDNF和突触素的表达有关。
Objective:
Depression is a chronic, recurring and potentially life-threatening illness that affects up to20%of the population of the word. However the patho physio logy of depression is still not clear. Many studies revealed that BDNF and the neuronal plasticity were correlated with depression, however the specific and the correlation between them are still unknown. Single injection of ketamine could treat depression in30min and the effect could last for7days. However the underlying mechanisms are still unknown. To clarify the correlation among depression, BDNF and plasticity would let us know some more about the depression, and would support us new avenue to explore the novel antidepressants. Previous studies revealed that hederagenin showed effect on the treatment of depression, however, the biological activity is not very strong.
To clarify the systematic correlations among BDNF, neuronal plasticity and depression. And then, one side, reconstruct the hederagenie to HGA, and test the biological activity on the treatment of depression, then studied the possible mechanism of HGA on the treatment of depression. Another side, make a study on the mechanism underlying the fast and long lasting antidepressant effect of ketamine, specially to reveal the correlation between the effect of ketamine and the variance of the BDNF and the synaptophysin.
Methods:
1. C57BL/6mice were exposured to chronic corticosterone to make the anxiety/depression-like model, elevated plus maze and tail suspension test were used to test the behavioral state of the mice after30days exposure of corticosterone. After the confirmation of the behavioral state of the mice. Mice were give fluoxetine(18mg·kg-1, ip) to reverse the anxiety/depression-like state. After20days treatment of fluoxetine, EPM and TST were used to test the behavioral state of the mice. During this period, mice were sacrificed at day10,20,35,45,60. The brains were fixed with4%paraformaldehyde solution and then the Golgi-cox-staining and immunofluorescence were carried out. For Golgi-cox-staining dendritic spines were scored from10-12neurons from each mouse in each subregion of hippocampus. For CA1and CA3, pyramidal neurons were selected for spine density evaluation, and for DG, granule neurons were selected for spine density evaluation. The spines were counted from the last branch point to the terminal tip of the dendrite. The dendritic spine density was counted at1000×(at least20μm in length) from different neurons in hippocampal subregions CA1, CA3and DG. For Immunofluorescence staining, images were acquired with a60×objective lens of a confocal microscope (FV10i, Olympus) in11subareas for each slice, and6-12cell bodies in each subarea were selected randomly for the measurement. The BDNF levels were expressed as a percentage of the vehicle group. An average value was obtained for each mouse for statistical analysis.
2. The effect of ketamine on the expression of BDNF and the neuronal plasticity was studied. Chronic corticosterone exposure was recruited to make the depressive mice model, and then single injection of ketamine was carried out to study the different effect of ketamie on BDNF on depressive mice and normal mice by western blotting. On the basis of this, one side, whether the short-term exposure of corticosterone could affect the influence of ketamine on the expression of BDNF was studied, the other side, the pathophysiology underlying the fast effect and long-lasting effect of ketamine on the treatment of depression was studied. C57BL/6mice were exposured to corticosterone for3days then were sacrificed30min or7days after the single injection of ketamine (3mg·kg-1). The variation of BDNF and synaptophysin was detected by western blotting.
Finally, the mechanism underlying the promotion of the neuronal plasticity of ketamine was studied. Racl-cofilin signalling pathway was studied after the single injection of ketamine. The variations of the related proteins. Total Racl, RaclGTPase, Tiaml, RacGAPl, cofilin and P-cofilin were studied by western blotting.
3. The restructuring derivative of HG (HGA) was designed and synthesized. First the carboxyl group was activated by DCC/NHS, then the DMAPA (dimethylamino-1-propylamine) was added, and the aim product HGA was produced. Using mass-spectrography and NMR. After the thorough confirmation of HGA, HPLC was carried out to check the purity of HGA.
Primary cultured hippocampal neurons from one day old SD rats were taken as the research object. The cell model was made by corticosterone in the primary cultured neurons. The protective effect of HGA on the injury induced by corticosterone was analyzed by MTT. The effect of HGA on the treatment of depression was analyzed by TST through acute drug delivery on C57BL/6mice.
Results:
1. The veriation of BDNF and neuronal plasticity during the procedures of the development and treatment of depression.
(1) The effect of the chronic corticosterone exposure and long term treatment of fluoxetine on the behavior of the mice.
After30days exposure of corticosterone, in the EPM, the number of entries into the open arms was significantly decreased after chronic corticosterone exposure in the corticosterone group compared with the vehicle group (t=2.202, P=0.043); in the TST, chronic corticosterone treatment induced a significant increase in the duration that the mice remained immobile compared to the vehicle group(t=2.094, P0.047). After25days treatment of fluoxetine (FLX), in the EPM, the number of entries into the open arms were significantly increased after20days treatment with fluoxetine compared with the corticosterone (CORT) group (P=0.007); while in the TST, the immobility duration decreased significantly compared with the corticosterone group (P=0.000).
(2) The chronic effect of corticosterone and long term treatment of fluoxetine on the spine density of the neurons in the hippocampus.
The Spine density was unchanged, following10and20days of corticosterone exposure in the hippocampal subregions CA1, CA3and DG, compared with corresponding vehicle group. After35days of corticosterone exposure, the spine density in CA1(t=2.626, P=0.030) and CA3(t=2.982, P=0.018) were significantly decrease compared with the vehicle group.45days of corticosterone exposure, significantly decreased the spine density in all subregions (CA1:P=0.004; CA3:P=0.000; DG:P=0.002) of the hippocampus compared with the vehicle group. Following60days corticosterone exposure, the spine density was also decreased in all of the neurons in the three subregions (CA1:P=0.001; CA3:P=0.000; DG:P=0.004), compared with the corresponding vehicle group. After10days treatment of fluoxetine, the spine density in CA1(P=0.000) and CA3(P=0.004) was significantly increased, compared with the corresponding CORT group. Following25days of fluoxetine treatment, spine density was significantly increased in all three subregions CA1(P=0.000), CA3(P=0.000) and DG (P=0.010), compared with the corresponding CORT group.
(3) The effects of chronic corticosterone exposure and the long term treatment of fluoxetine on the quantity of BDNF in neuronal cell bodies in the hippocampus
After10days exposure of the corticosterone, the expression of BDNF in mature neuronal cell bodies (t=3.371, P=0.010) and newborn neuronal cell bodies (t=4.416, P=0.002) in DG were significantly increased, compared with the corresponding VEH group. The expression of BDNF in neuronal cell bodies in CA3(t=3.495, P=0.008) mature neuronal cell bodies (t=3.848, P=0.005) and newborn neuronal cell bodies (t=3.991, P=0.004) in DG were significantly increased, compared with the corresponding VEH group. After35days of corticosterone exposure, the quantity of BDNF were increased significantly in mature neuronal cell bodies in the DG (t=2.501, P=0.037), CA3(t=3.670, P=0.006) and CA1(t=3.549, P=0.008) compared with the VEH group. After45days of corticosterone treatment, the quantity of BDNF were significantly decreased in the neuronal cell bodies in CA1(P=0.001) and the newborn neuronal cell bodies in DG (P=0.001) compared with the corresponding vehicle group. Following60days of corticosterone exposure, the quantity of BDNF in CA1(P=0.031),CA3(P=0.008) and the newbornal cell bodies in DG (P=0.012) were all increased.
After10days fluoxetine treatment, the BDNF levels was significantly decreased in mature neuronal cell bodies (P=0.000) and new born neuronal cell bodies (P=0.003) in DG compared with corticosterone group. After25days of fluoxetine treatment, the levels of BDNF in the neuronal cell bodies of CA3, and the mature as well as the newborn neuronal cellbodies in DG were significantly decreased compared with the corticosterone group (CA3:P=0.025; DG-M:P=0.001; DG-N: P=0.001).
2. The role of BDNF and neural plasticity in ketamine treatment in depression, and the mechanisms of strengthened neuronal plasticity induced by ketamine.
(1) The role of ketamine on BDNF levels and the role of glucocorticoid corticosterone in the course of ketamine-induced BDNF expression.
Compared with single injection of ketamine in normal mice, after30min, the administration of a single ketamine injection in depressed mice can make the expression of BDNF in hippocampus increased significantly (F=12.493, P=0.008). And the glucocorticoid receptor inhibitor Ru486can significantly inhibit this increased (F=15.499, P=0.004). But three days of corticosterone treatment can't achieve a similar effect. Meanwhile, synaptophysin expression has no change after30min following a single ketamine in hippocampus (F=0.897, P=0.358). After7days, following a single injection of ketamine treatment, hippocampal BDNF expression was no longer affected in each group, it showed no significant change (F=1.539, P=0.233). Interestingly, synaptophysin expression was significantly increased after7days, following a single injection of ketamine treatment (F=466.104, P=0.000), and the Ru486pretreatment has no effect on the increasing expression of synaptophysin after7days following a single injection of ketamine treatment (F= 4.308, P=0.054).
(2) The effect of ketamine on Racl signaling pathway
Following a single intraperitoneal injection of ketamine, the western blotting results revealed that, compared with the vehicle group, the Racl-GTPase protein level increased significantly at15min (t=4.023, P=0.016) and45min (t=3.939, P=0.017) in hippocampus, and recovered to normal level at4h (t=0.570, P=0.599). Following a single intraperitoneal injection of ketamine, the western blotting results revealed that, compared with the vehicle group, the Tiaml protein level increased significantly at15min (F=953.626, P=0.000) and45min (F=70.369, P=0.000) in hippocampus, and recovered to normal level at4h (F=1.259, P=0.278). Following a single intraperitoneal injection of ketamine, the western blotting results revealed that, compared with the vehicle group, the RacGAPl protein level significantly reduced in ketamine treated mice at15min (F=20.750, P=0.000) and45min (F=181.808, P=0.000) time points. Following a single intraperitoneal injection of ketamine, the western blotting results revealed that, compared with the vehicle group, inactive p-cofilin was significantly increased at15min (F=494.566, P=0.000),45min (F=207.595, P=0.000), and4h(F=119.280, P=0.000) time points, and return to basic levels at24h (F=2.453, P=0.137), following ketamine treatment.
3. The study on the synthesis, structural characterization, the biological activity validation as well as the underlying mechanism of the HGA
(1) The synthesis and the characterization of HGA By ordinary chemical synthesis and column chromatography separation, a white powdery compound was obtained. The Mass spectrum reveals the molecular ion peak is558.0([M+H]+), while the molecular weight of HGA is556.86. The1hNMR (CDC13) revealed:1H-NMR(CD3OD):δ5.26(t, J=3.4Hz,1H, C=C-H),3.99(dd, J1=14Hz, J2=7.2Hz,1H),3.48-3.52(m,1H),3.41(d, J=10.8,1H),3.05-3.11(m,1H),2.91(t,J=7.2Hz,2H),2.71(s,6H, NMe2),1.91(s,1H),1.05-1.43(m,22H),0.78-0.95(m,18H),0.686(3H, s, CH3),0.596(3H, s, CH3), and the datas are agreed with the structure of HGA. Data from13CNMR show that:δ180.02(C=O), 143.67(C=C),122.65(C=C),72.30,65.76,60.07,55.38,46.20,46.10,42.22,41.82,41.51,40.99,39.20,37.97,36.43,35.90,33.58,33.20,33.02,31.81,30.14,27.06,25.93,25.03,24.87,23.06,22.57,22.44,19.38,17.62,16.56,14.78,12.98,11.24. Based on the above analysis, the purity of HGA was checked by high performance liquid chromatography (HPLC) with methanol-water-acetic acid-the ethylenediamine (87:13:0.04:0.02) as the mobile phase,210nm as the detection wavelength. HPLC revealed that the peak time of HGA is about59.09min, and the normalization method results show that the HGA's purity is94.08%(HPLC).
(2) The antidepressant activity and the underlying mechanism of HGA
In the neuroprotective experiments of HGA, MTT results showed that the compound HGA (P=0.042) produced significant protective effect in a concentration of0.1μmol·L-1on the injury caused by corticosterone (10μmol·L-1) in primary cultured neurons, however, under the same experimental conditions, HG (P=0.866) did not have a significant protective effect on neurons at a concention of1μmol·L-1. The western blotting results show that the HGA can reverse the decline of expression of BDNF (P=0.000) and synaptophysin (P=0.000) caused by corticosterone exposure. And the results of the acute delivery of HGA (10mg·kg-1) in mouse tail suspension test revealed that HGA could significantly (P=0.010) decrease the immobility duration.
Conclusions:
1. During the occurrence and development of depression our data revealed an increase-decrease-increase fluctuation in the levels of BDNF. After treatment with antidepressants, fluoxetine reduced the stress-induced up-regulation of BDNF. Our data indicated that BDNF correlate with depression in a complicated increase-decrease-increase style.
Dendritic spine density of hippocampal neurons decreased during the development of depression in mice, and increased after giving fluoxetine treatment. The impairment of dendritic spines in the hippocampus may underlie the development of depression. These results indicate that changes in spine density in the hippocampus maybe a pathophysiological mechanism underlying depression.
2. Ketamine could promote the BDNF expression significantly in depressed mice model compared with the normal mice, and such significant promoting effect can be blocked by the glucocorticoid receptor inhibitor Ru486. However, the short-term (3days) corticosterone treatment did not affect the ketamine's promotion in BDNF expression. The expression of BDNF was significantly increased after30min following a single injection of ketamine, but the neuronal plasticity was not affected. After7days following a single injection of ketamine, BDNF expression returned to the basic level, while the expression of synaptophysin was significantly increased. The single dose ketamine improved neuronal plasticity may recruit Racl-GTPase signaling pathway, and ultimately increase the neuronal plasticity, to achieve the antidepressant effect.
3. The hederagenin amide derivative HGA was designed and successfully synthesized. HGA can show protective effect on injuries induced by corticosterone in the primary cultured hippocampal neurons at a concention of0.1μmol·L-1. HGA could significantly decrease the immobility duration in tail suspension test by acute delivery. HGA can promote the expression of BDNF and synaptophysin which was reduced by corticosterone (10μmol·L-1).
本研究旨在检测在抑郁的发病和治疗过程中,BDNF和神经可塑性的作用；研究糖皮质激素皮质酮对氯胺酮在BDNF表达中的促进作用的影响,并探讨氯胺酮的速效和长效机制与神经可塑性及BDNF的关系；研究氯胺酮增强小鼠海马神经元神经可塑性可能的信号转导通路；对具有抗抑郁活性的常春藤皂苷元进行结构改造,并检测其抗抑郁效果及机制。
方法：1.以C57BL/6小鼠作为研究对象,采用长期皮质酮暴露的方法造成焦虑/抑郁模型,经过30d的饮水给药(皮质酮,35mg·L-1)以后,通过高架迷宫实验和悬尾实验,检测小鼠的行为学变化。当确认小鼠已经达到焦虑/抑郁样状态以后,进入治疗、康复阶段,每天腹腔注射氟西汀(18mg·kg-1)。20d以后,检测小鼠的行为学变化。在整个实验过程中,分别在第10d,20d,35d,45d,60d取材,进行高尔基染色和免疫荧光染色。计数海马CA1,CA3区椎体神经元树突棘密度的变化和DG区颗粒神经元树突棘密度的变化,检测海马CA1区,CA3区和DG区(包括新生神经元和成熟神经元)神经元内BDNF的表达变化。
2.以C57BL/6小鼠为研究对象,研究单次注射氯胺酮(3.0mg·kg-1)对小鼠BDNF的表达和神经可塑性的影响。采用长期皮质酮暴露(饮水中给予皮质酮35mg·L-1)造成焦虑/抑郁模型,单次给予腹腔注射氯胺酮以后,使用Western blotting法检测氯胺酮对BDNF的促进作用在抑郁鼠和正常鼠的不同,并运用糖皮质激素受体抑制剂RU486进行进一步确认。在此基础上,一方面研究短期给予皮质酮对氯胺酮在BDNF表达中的促进作用中的影响,并探讨氯胺酮的速效(30min)和长效(7d)的病理生理学基础：采用皮质酮暴露的方法,给皮质酮3天以后,单次腹腔注射氯胺酮(3.0mg·kg-1)后分别在第30min和第7d取材,检测BDNF的表达和突触素(Synaptophysin)的变化。一方面研究氯胺酮提高小鼠海马神经元突触可塑性的机制：以C57BL/6小鼠为研究对象,在单次给予氯胺酮后的15min,45min,4h和24h时间点取材,应用Western blotting法检测Racl信号通路中的RaclGTPase, Tiaml, RacGAPl,和P-cofilin的变化情况,拟探讨氯胺酮提高小鼠海马神经元突触可塑性与Racl信号通路的关系。
3.设计并合成常春藤皂苷元酰胺衍生物N-(3-二甲氨基丙基)-常春藤皂苷元-17-甲酰胺(HGA)。以常春藤皂苷元为原料,经DCC/NHS将常春藤皂苷元28位的羧基活化,加入二甲氨基丙胺反应得HGA。经质谱,核磁氢谱和碳谱对其结构进行确认。然后,经高效液相色谱(HPLC)检测其纯度。在此基础上,在细胞水平,以原代海马神经元为研究对象,用皮质酮(10μmol·L-1)损伤造模,探讨HGA对原代海马神经元的保护作用；在整体动物水平,应用急性给药的方法,研究HGA急性给药对C57BL/6小鼠行为学的影响。并且,从BDNF和神经可塑性的角度研究其抗抑郁作用的机制。
结果：1.神经可塑性和BDNF在抑郁症发病及治疗中的变化
(1)慢性皮质酮暴露及长期的氟西汀治疗对小鼠行为学的影响
给予皮质酮暴露30d以后,高架迷宫实验结果显示,小鼠进入开臂的次数著减少(t=2.202,P=0.043),悬尾实验结果显示小鼠的不动时间显著延长(t=2.094,P=0.047)：经过20d的氟西汀治疗以后,小鼠在高架迷宫实验中进入开臂的次数显著增多(P=-0.007),悬尾实验中小鼠的不动时间显著(P=0.000)减少。
(2)慢性皮质酮暴露及长期氟西汀治疗对小鼠神经可塑性的影响
与相应的对照组相比,10d和20d的皮质酮处理对海马各区神经元树突棘密度没有产生显著的影响,经过35d的皮质酮的处理以后,与相应的溶剂对照组(VEH)的神经元的树突棘的密度相比,CA1区(t=2.626,P=0.030)和CA3区(t=2.982,P=0.018)神经元树突棘的密度显著降低。
经过45d的皮质酮处理以后的,海马的所有亚区的神经元树突棘的密度均明显少于相应的溶剂对照组的神经元树突棘的密度(CA1：P=0.004；CA3：P=0.000；DG：P=0.002)。经过60d的皮质酮处理以后海马各区神经元树突棘的密度均明显的少于相应的溶剂对照组的树突棘的密度(CA1：P=0.001；CA3：P=0.000；DG：P=0.004)。
经过10d的氟西汀处理以后,与相应的CORT组相比,经氟西汀处理的CORT+FLX组的CA1区(P=0.000)和CA3区(P=0.004)的神经元树突棘的密度显著提高。经过25d的氟西汀处理以后,与相应的CORT组相比,CORT+FLX组的海马所有亚区的神经元树突棘的密度均显著升高(CA1：P=0.000；CA3：P=0.000；DG：P=0.010)。
(3)慢性皮质酮暴露及长期氟西汀治疗对小鼠海马区神经元胞体内BDNF水平的影响
经过10d的皮质酮处理后,与相应的对照组相比,DG区的新生神经元(t=4.416,P=0.002)与成熟神经元(t=3.371,P=0.010)胞体中的BDNF的水平显著升高。经过20d的皮质酮处理后,与相应的对照组相比,DG区成熟神经元(t=3.848,P=0.005),DG区新生神经元(t=3.991,P=0.004)和CA3(t=3.495,P=0.008)区神经元胞体中的BDNF的水平显著升高。经过35d的皮质酮处理后,与相应的对照组相比,DG区成熟神经元(t=2.501,P=0.037),CA3区(t=3.670,P=0.006),CA1区(t=3.549,P=-0.008)神经元中的BDNF水平显著提高。
与相应的VEH组相比,45天的皮质酮处理后,CA1区神经元(P=0.001)和DG区新生神经元（P=0.001)的BDNF的水平显著减少。经过60d的皮质酮处理后,与相应的VEH组相比,海马CA1区、CA3区神经元和DG区新生神经元中的BDNF的水平均显著升高(CA1：P=0.031；CA3：P=0.008；DG-N：P=0.012)。
经过10d(36d-45d)的氟西汀处理后,与相应的CORT组相比,DG区成熟神经元(P=0.000)和新生神经元(P=0.003)胞体内的BDNF的水平显著减少。与相应的各CORT组相比,经过25d的氟西汀处理后,海马CA3区神经元、DG区成熟神经元和新生神经元中的BDNF的水平均显著降低(CA3：P=0.025；DG-M：P=0.001；DG-N：P=0.001)。
2.氯胺酮抗抑郁作用与神经可塑性和BDNF的关系及其机制研究
(1)氯胺酮对BDNF的促进作用及糖皮质激素皮质酮的影响
与给予单次氯胺酮注射的正常鼠相比,给抑郁鼠单次注射氯胺酮,给药后30min时,抑郁鼠海马BDNF的表达显著升高(F=12.493,P=0.008),并且糖皮质激素受体抑制剂Ru486可以显著抑制这种升高(F=15.499,P=0.004)。但是短期(3d)的皮质酮处理却不能达到相似的效果,氯胺酮可显著促进海马区BDNF的表达(F=927.149,P=0.000)；短期(3d)的糖皮质激素受体抑制剂处理不会对氯胺酮的BDNF促进作用产生影响(F=0.165,P=0.690)。单剂量给予氯胺酮以后30min时,小鼠海马突触素(Synaptophysin)表达水平较对照组没有发生显著变化(F=0.897,P=0.358)。糖皮质激素受体抑制剂Ru486对突触素的表达也没有显著影响(F=1.524,P=0.235)。单剂量给予氯胺酮以后7d时,与对照组相比,小鼠海马BDNF水平没有出现显著改变(F=1.539,P=0.233)。并且短期的糖皮质激素受体抑制剂Ru486处理也没有对氯胺酮的BDNF促进作用产生显著影响(F=2.275,P=0.151)。单剂量给予氯胺酮以后7d时,给予了氯胺酮的各组小鼠海马突触素(Synaptophysin)表达水平显著提高(F=466.104,P=0.000)。并且短期的Ru486处理(3d)不影响突触素的表达(F=4.308,P=0.054)。
(2)氯胺酮对Rac1信号通路的影响
与对照组相比,在给予单剂量氯胺酮后,Rac1-GTPase在15min(t=4.023,P=0.016)和45min(t=3.939,P=0.017)两个时间点均显示升高,在给药后的4h时,恢复到正常水平(t=0.570,P=0.599)；在给予单剂量氯胺酮后15min时间点,与对照组相比,Tiam1水平显著升高(F=953.626,P=0.000),并且在给予单剂量氯胺酮后45min时,Tiam1仍显著升高(F=70.369,P=0.000),在单剂量注射氯胺酮以后4h时间点Tiam1的表达恢复到基础水平(F=1.259,P=0.278)；在给予单剂量氯胺酮后15min时,与对照组相比,RacGAP1表达水平显著降低(F=20.750,P=0.000),并且持续到给予单剂量氯胺酮后45mmin时间点,RacGAP1仍显著降低(F=181.808,P=0.000)。给予单剂量氯胺酮以后4h时间点,RacGAP1的表达恢复到基础水平(F=0.343,P=0.566)；在给予单剂量氯胺酮的15min,与对照组相比,Cofilin的磷酸化水平显著升高(F=617.735,P=0.000)；单剂量给予氯胺酮后的45min(F=259.295,P=0.000)和4h(F=148.986,P=0.000),Cofilin的磷酸化水平均持续显著升高。在单剂量给予氯胺酮后的24h的时间点,Cofilin的磷酸化水平回到基础水平(F=2.453,P=0.137)。
3.常春藤皂苷元结构改造与生物活性及其机制研究
(1)通过普通化学合成的方法,经柱色谱分离,得到了白色粉末状化合物N-(3-二甲氨基丙基)-常春藤皂苷元-17-甲酰胺(HGA)。质谱图显示分子离子峰是558.0([M+H]+)而HGA的相对分子质量为556.86；核磁氢谱的数据：1H-NMR(CD3OD):δ5.26(t,J=3.4Hz,1H,C=C-H),3.99(dd,J1=14Hz,J2=7.2Hz,1H),3.48-3.52(m,1H),3.41(d,J=10.8,1H),3.05-3.11(m,1H),2.91(t,J=7.2Hz,2H),2.71(s,6H,NMe2),1.91(s,1H),1.05-1.43(m,22H),0.78-0.95(m,18H),0.686(3H,s,CH3),0.596(3H,s,CH3),与HGA的结构相吻合；核磁碳谱数据：6180.02(C=0),143.67(C=C),122.65(C=C),72.30,65.76,60.07,55.38,46.20,46.10,42.22,41.82,41.51,40.99,39.20,37.97,36.43,35.90,33.58,33.20,33.02,31.81,30.14,27.06,25.93,25.03,24.87,23.06,22.57,22.44,19.38,17.62,16.56,14.78,12.98,11.24,与HGA的结构相吻合。综合以上的分析,合成所得化合物为目标化合物HGA。利用高效液相色谱(HPLC),以甲醇-水-冰醋酸-乙二胺(87：13：0.04：0.02)为流动相,210nm为检测波长,发现,HGA出峰时间为59.09min,归一化法的计算结果显示HGA的纯度为94.08%(HPLC)。
(2)常春藤皂苷元酰胺衍生物HGA的抗抑郁生物活性及机制研究
HGA神经保护作用实验中,MTT结果显示,化合物HGA可以在0.1μmol·L-1(P=0.042)。而相同实验条件下1μmoL·L-1的HG(P=0.866)不能对神经元产生显著的保护作用。急性给药的小鼠悬尾实验的结果显示,HGA在10mg·kg-1时可以显著(P=0.010)缩短C57BL/6小鼠的不动时间。在此基础上,从BDNF和神经可塑性的角度对其机制进行研究,Western blotting的结果显示,HGA可以显著逆转皮质酮所导致的BDNF(P=0.000)和突触素(Synaptophysin, P=0.000)蛋白的表达水平的下降。
结论：1.在抑郁的发生发展过程中BDNF的表达水平先升高,再降低,最后又升高,呈波浪形变化。并且在给予氟西汀治疗以后,BDNF的表达水平与抑郁但未接受治疗的小鼠相比,显著下降,说明BDNF与小鼠的抑郁状态密切相关,但关系复杂。在抑郁的发生阶段,BDNF应激性的升高可能对神经可塑性起到一定的保护作用。海马神经元树突棘密度则随着小鼠的抑郁状态的发生、发展而不断降低,当给与氟西汀治疗以后,小鼠海马神经元树突棘密度迅速升高而且与小鼠的行为学状态呈正相关,说明神经可塑性的变化很可能是抑郁的病理生理学基础。
2.氯胺酮在抑郁鼠身上对BDNF表达的促进作用更加显著,而这种显著的促进作用可以被糖皮质激素受体抑制剂Ru486所阻断。但短期(3d)的皮质酮处理不能显著影响氯胺酮对BDNF的促进作用。单剂量注射氯胺酮的速效抗抑郁作用可能与促进BDNF的表达相关,其长效可能与其改善了神经可塑性有关。单剂量氯胺酮可能是通过激活Racl信号通路,促进Cofilin的磷酸化,抑制Cofilin的剪切作用,最终改善神经可塑性,起到长效的抗抑郁作用。
3.经设计和普通化学合成,成功得到了N-(3-二甲氨基丙基)-常春藤皂苷元-17-甲酰胺(HGA),HGA可以在0.1μmo1·L-1的浓度下对原代海马神经元产生显著的保护作用；HGA可以在10mg·kg-1的给药剂量下,小鼠在悬尾实验中起到显著的抗抑郁作用,HGA的抗抑郁作用可能跟HGA可以促进BDNF和突触素的表达有关。
Objective:
Depression is a chronic, recurring and potentially life-threatening illness that affects up to20%of the population of the word. However the patho physio logy of depression is still not clear. Many studies revealed that BDNF and the neuronal plasticity were correlated with depression, however the specific and the correlation between them are still unknown. Single injection of ketamine could treat depression in30min and the effect could last for7days. However the underlying mechanisms are still unknown. To clarify the correlation among depression, BDNF and plasticity would let us know some more about the depression, and would support us new avenue to explore the novel antidepressants. Previous studies revealed that hederagenin showed effect on the treatment of depression, however, the biological activity is not very strong.
To clarify the systematic correlations among BDNF, neuronal plasticity and depression. And then, one side, reconstruct the hederagenie to HGA, and test the biological activity on the treatment of depression, then studied the possible mechanism of HGA on the treatment of depression. Another side, make a study on the mechanism underlying the fast and long lasting antidepressant effect of ketamine, specially to reveal the correlation between the effect of ketamine and the variance of the BDNF and the synaptophysin.
Methods:
1. C57BL/6mice were exposured to chronic corticosterone to make the anxiety/depression-like model, elevated plus maze and tail suspension test were used to test the behavioral state of the mice after30days exposure of corticosterone. After the confirmation of the behavioral state of the mice. Mice were give fluoxetine(18mg·kg-1, ip) to reverse the anxiety/depression-like state. After20days treatment of fluoxetine, EPM and TST were used to test the behavioral state of the mice. During this period, mice were sacrificed at day10,20,35,45,60. The brains were fixed with4%paraformaldehyde solution and then the Golgi-cox-staining and immunofluorescence were carried out. For Golgi-cox-staining dendritic spines were scored from10-12neurons from each mouse in each subregion of hippocampus. For CA1and CA3, pyramidal neurons were selected for spine density evaluation, and for DG, granule neurons were selected for spine density evaluation. The spines were counted from the last branch point to the terminal tip of the dendrite. The dendritic spine density was counted at1000×(at least20μm in length) from different neurons in hippocampal subregions CA1, CA3and DG. For Immunofluorescence staining, images were acquired with a60×objective lens of a confocal microscope (FV10i, Olympus) in11subareas for each slice, and6-12cell bodies in each subarea were selected randomly for the measurement. The BDNF levels were expressed as a percentage of the vehicle group. An average value was obtained for each mouse for statistical analysis.
2. The effect of ketamine on the expression of BDNF and the neuronal plasticity was studied. Chronic corticosterone exposure was recruited to make the depressive mice model, and then single injection of ketamine was carried out to study the different effect of ketamie on BDNF on depressive mice and normal mice by western blotting. On the basis of this, one side, whether the short-term exposure of corticosterone could affect the influence of ketamine on the expression of BDNF was studied, the other side, the pathophysiology underlying the fast effect and long-lasting effect of ketamine on the treatment of depression was studied. C57BL/6mice were exposured to corticosterone for3days then were sacrificed30min or7days after the single injection of ketamine (3mg·kg-1). The variation of BDNF and synaptophysin was detected by western blotting.
Finally, the mechanism underlying the promotion of the neuronal plasticity of ketamine was studied. Racl-cofilin signalling pathway was studied after the single injection of ketamine. The variations of the related proteins. Total Racl, RaclGTPase, Tiaml, RacGAPl, cofilin and P-cofilin were studied by western blotting.
3. The restructuring derivative of HG (HGA) was designed and synthesized. First the carboxyl group was activated by DCC/NHS, then the DMAPA (dimethylamino-1-propylamine) was added, and the aim product HGA was produced. Using mass-spectrography and NMR. After the thorough confirmation of HGA, HPLC was carried out to check the purity of HGA.
Primary cultured hippocampal neurons from one day old SD rats were taken as the research object. The cell model was made by corticosterone in the primary cultured neurons. The protective effect of HGA on the injury induced by corticosterone was analyzed by MTT. The effect of HGA on the treatment of depression was analyzed by TST through acute drug delivery on C57BL/6mice.
Results:
1. The veriation of BDNF and neuronal plasticity during the procedures of the development and treatment of depression.
(1) The effect of the chronic corticosterone exposure and long term treatment of fluoxetine on the behavior of the mice.
After30days exposure of corticosterone, in the EPM, the number of entries into the open arms was significantly decreased after chronic corticosterone exposure in the corticosterone group compared with the vehicle group (t=2.202, P=0.043); in the TST, chronic corticosterone treatment induced a significant increase in the duration that the mice remained immobile compared to the vehicle group(t=2.094, P0.047). After25days treatment of fluoxetine (FLX), in the EPM, the number of entries into the open arms were significantly increased after20days treatment with fluoxetine compared with the corticosterone (CORT) group (P=0.007); while in the TST, the immobility duration decreased significantly compared with the corticosterone group (P=0.000).
(2) The chronic effect of corticosterone and long term treatment of fluoxetine on the spine density of the neurons in the hippocampus.
The Spine density was unchanged, following10and20days of corticosterone exposure in the hippocampal subregions CA1, CA3and DG, compared with corresponding vehicle group. After35days of corticosterone exposure, the spine density in CA1(t=2.626, P=0.030) and CA3(t=2.982, P=0.018) were significantly decrease compared with the vehicle group.45days of corticosterone exposure, significantly decreased the spine density in all subregions (CA1:P=0.004; CA3:P=0.000; DG:P=0.002) of the hippocampus compared with the vehicle group. Following60days corticosterone exposure, the spine density was also decreased in all of the neurons in the three subregions (CA1:P=0.001; CA3:P=0.000; DG:P=0.004), compared with the corresponding vehicle group. After10days treatment of fluoxetine, the spine density in CA1(P=0.000) and CA3(P=0.004) was significantly increased, compared with the corresponding CORT group. Following25days of fluoxetine treatment, spine density was significantly increased in all three subregions CA1(P=0.000), CA3(P=0.000) and DG (P=0.010), compared with the corresponding CORT group.
(3) The effects of chronic corticosterone exposure and the long term treatment of fluoxetine on the quantity of BDNF in neuronal cell bodies in the hippocampus
After10days exposure of the corticosterone, the expression of BDNF in mature neuronal cell bodies (t=3.371, P=0.010) and newborn neuronal cell bodies (t=4.416, P=0.002) in DG were significantly increased, compared with the corresponding VEH group. The expression of BDNF in neuronal cell bodies in CA3(t=3.495, P=0.008) mature neuronal cell bodies (t=3.848, P=0.005) and newborn neuronal cell bodies (t=3.991, P=0.004) in DG were significantly increased, compared with the corresponding VEH group. After35days of corticosterone exposure, the quantity of BDNF were increased significantly in mature neuronal cell bodies in the DG (t=2.501, P=0.037), CA3(t=3.670, P=0.006) and CA1(t=3.549, P=0.008) compared with the VEH group. After45days of corticosterone treatment, the quantity of BDNF were significantly decreased in the neuronal cell bodies in CA1(P=0.001) and the newborn neuronal cell bodies in DG (P=0.001) compared with the corresponding vehicle group. Following60days of corticosterone exposure, the quantity of BDNF in CA1(P=0.031),CA3(P=0.008) and the newbornal cell bodies in DG (P=0.012) were all increased.
After10days fluoxetine treatment, the BDNF levels was significantly decreased in mature neuronal cell bodies (P=0.000) and new born neuronal cell bodies (P=0.003) in DG compared with corticosterone group. After25days of fluoxetine treatment, the levels of BDNF in the neuronal cell bodies of CA3, and the mature as well as the newborn neuronal cellbodies in DG were significantly decreased compared with the corticosterone group (CA3:P=0.025; DG-M:P=0.001; DG-N: P=0.001).
2. The role of BDNF and neural plasticity in ketamine treatment in depression, and the mechanisms of strengthened neuronal plasticity induced by ketamine.
(1) The role of ketamine on BDNF levels and the role of glucocorticoid corticosterone in the course of ketamine-induced BDNF expression.
Compared with single injection of ketamine in normal mice, after30min, the administration of a single ketamine injection in depressed mice can make the expression of BDNF in hippocampus increased significantly (F=12.493, P=0.008). And the glucocorticoid receptor inhibitor Ru486can significantly inhibit this increased (F=15.499, P=0.004). But three days of corticosterone treatment can't achieve a similar effect. Meanwhile, synaptophysin expression has no change after30min following a single ketamine in hippocampus (F=0.897, P=0.358). After7days, following a single injection of ketamine treatment, hippocampal BDNF expression was no longer affected in each group, it showed no significant change (F=1.539, P=0.233). Interestingly, synaptophysin expression was significantly increased after7days, following a single injection of ketamine treatment (F=466.104, P=0.000), and the Ru486pretreatment has no effect on the increasing expression of synaptophysin after7days following a single injection of ketamine treatment (F= 4.308, P=0.054).
(2) The effect of ketamine on Racl signaling pathway
Following a single intraperitoneal injection of ketamine, the western blotting results revealed that, compared with the vehicle group, the Racl-GTPase protein level increased significantly at15min (t=4.023, P=0.016) and45min (t=3.939, P=0.017) in hippocampus, and recovered to normal level at4h (t=0.570, P=0.599). Following a single intraperitoneal injection of ketamine, the western blotting results revealed that, compared with the vehicle group, the Tiaml protein level increased significantly at15min (F=953.626, P=0.000) and45min (F=70.369, P=0.000) in hippocampus, and recovered to normal level at4h (F=1.259, P=0.278). Following a single intraperitoneal injection of ketamine, the western blotting results revealed that, compared with the vehicle group, the RacGAPl protein level significantly reduced in ketamine treated mice at15min (F=20.750, P=0.000) and45min (F=181.808, P=0.000) time points. Following a single intraperitoneal injection of ketamine, the western blotting results revealed that, compared with the vehicle group, inactive p-cofilin was significantly increased at15min (F=494.566, P=0.000),45min (F=207.595, P=0.000), and4h(F=119.280, P=0.000) time points, and return to basic levels at24h (F=2.453, P=0.137), following ketamine treatment.
3. The study on the synthesis, structural characterization, the biological activity validation as well as the underlying mechanism of the HGA
(1) The synthesis and the characterization of HGA By ordinary chemical synthesis and column chromatography separation, a white powdery compound was obtained. The Mass spectrum reveals the molecular ion peak is558.0([M+H]+), while the molecular weight of HGA is556.86. The1hNMR (CDC13) revealed:1H-NMR(CD3OD):δ5.26(t, J=3.4Hz,1H, C=C-H),3.99(dd, J1=14Hz, J2=7.2Hz,1H),3.48-3.52(m,1H),3.41(d, J=10.8,1H),3.05-3.11(m,1H),2.91(t,J=7.2Hz,2H),2.71(s,6H, NMe2),1.91(s,1H),1.05-1.43(m,22H),0.78-0.95(m,18H),0.686(3H, s, CH3),0.596(3H, s, CH3), and the datas are agreed with the structure of HGA. Data from13CNMR show that:δ180.02(C=O), 143.67(C=C),122.65(C=C),72.30,65.76,60.07,55.38,46.20,46.10,42.22,41.82,41.51,40.99,39.20,37.97,36.43,35.90,33.58,33.20,33.02,31.81,30.14,27.06,25.93,25.03,24.87,23.06,22.57,22.44,19.38,17.62,16.56,14.78,12.98,11.24. Based on the above analysis, the purity of HGA was checked by high performance liquid chromatography (HPLC) with methanol-water-acetic acid-the ethylenediamine (87:13:0.04:0.02) as the mobile phase,210nm as the detection wavelength. HPLC revealed that the peak time of HGA is about59.09min, and the normalization method results show that the HGA's purity is94.08%(HPLC).
(2) The antidepressant activity and the underlying mechanism of HGA
In the neuroprotective experiments of HGA, MTT results showed that the compound HGA (P=0.042) produced significant protective effect in a concentration of0.1μmol·L-1on the injury caused by corticosterone (10μmol·L-1) in primary cultured neurons, however, under the same experimental conditions, HG (P=0.866) did not have a significant protective effect on neurons at a concention of1μmol·L-1. The western blotting results show that the HGA can reverse the decline of expression of BDNF (P=0.000) and synaptophysin (P=0.000) caused by corticosterone exposure. And the results of the acute delivery of HGA (10mg·kg-1) in mouse tail suspension test revealed that HGA could significantly (P=0.010) decrease the immobility duration.
Conclusions:
1. During the occurrence and development of depression our data revealed an increase-decrease-increase fluctuation in the levels of BDNF. After treatment with antidepressants, fluoxetine reduced the stress-induced up-regulation of BDNF. Our data indicated that BDNF correlate with depression in a complicated increase-decrease-increase style.
Dendritic spine density of hippocampal neurons decreased during the development of depression in mice, and increased after giving fluoxetine treatment. The impairment of dendritic spines in the hippocampus may underlie the development of depression. These results indicate that changes in spine density in the hippocampus maybe a pathophysiological mechanism underlying depression.
2. Ketamine could promote the BDNF expression significantly in depressed mice model compared with the normal mice, and such significant promoting effect can be blocked by the glucocorticoid receptor inhibitor Ru486. However, the short-term (3days) corticosterone treatment did not affect the ketamine's promotion in BDNF expression. The expression of BDNF was significantly increased after30min following a single injection of ketamine, but the neuronal plasticity was not affected. After7days following a single injection of ketamine, BDNF expression returned to the basic level, while the expression of synaptophysin was significantly increased. The single dose ketamine improved neuronal plasticity may recruit Racl-GTPase signaling pathway, and ultimately increase the neuronal plasticity, to achieve the antidepressant effect.
3. The hederagenin amide derivative HGA was designed and successfully synthesized. HGA can show protective effect on injuries induced by corticosterone in the primary cultured hippocampal neurons at a concention of0.1μmol·L-1. HGA could significantly decrease the immobility duration in tail suspension test by acute delivery. HGA can promote the expression of BDNF and synaptophysin which was reduced by corticosterone (10μmol·L-1).
引文
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