摘要
3,6′-二芥子酰基蔗糖(3,6′-disinapoyl sucrose, DISS)是一种从中药远志中提取的新寡糖酯类化合物，课题组前期研究发现，该成分具有明确的抗抑郁作用，能明显改善抑郁模型小鼠行为学指标，可增强5-HT系统功能，并能够保护受损的神经细胞，对慢性应激大鼠HPA轴激素水平具有调节作用，增加抑郁动物海马中磷酸化CREB表达，并提高CREB下游靶基因BDNF的mRNA及蛋白的表达。
本课题主要以神经元再生与保护为切入点，聚焦于神经营养通路中的关键靶点CREB，针对其调节的上下游通路（细胞生存与凋亡、细胞氧化应激），围绕DISS参与的促神经元再生和神经营养保护作用通道进行研究，旨在深入探讨DISS通过促进神经元再生和神经保护发挥抗抑郁的细胞分子机制，希望发现其可能作用的具体靶点或参与调节的信号传递途径，对同类新型抗抑郁剂的研发及抑郁症发病机理的认识做出有益探索。
DISS对CREB磷酸化通路调节下游凋亡靶基因，参与神经细胞生存与凋亡的影响
采用体外培养人神经母细胞瘤株(SH-SY5Y细胞)，用MTT法筛选Glu致细胞损伤的最佳浓度，在建立损伤细胞模型的基础上，用MTT法测定DISS对细胞存活率的影响及乳酸脱氢酶(LDH)含量；流式细胞仪PI染色测定细胞凋亡比率；Real-time PCR技术测定促凋亡基因bax，抗凋亡基因bcl-2 mRNA的相对表达。研究发现，与Glu损伤后的模型组相比，DISS可以提高细胞存活率，减少LDH的释放，降低细胞凋亡比率，下调bax mRNA表达，升高bcl-2 mRNA表达；提示DISS可能参与细胞凋亡基因的调节，抑制细胞凋亡促进神经细胞再生。
DISS对CREB磷酸化下游通路调节，参与神经细胞氧化应激的影响
筛选H_2O_2最佳损伤浓度，确定最佳损伤浓度为300μM，干预0.5h后加入终浓度为0.6，6，60μMDISS继续培养12h结束培养，测定细胞存活率，超声裂解细胞测定细胞内SOD，MDA，CAT，GSH-PX的含量。研究表明，与H_2O_2组比较，终浓度为0.6，6，60μM的DISS能减轻H_2O_2引起的SH-SY5Y神经细胞的损伤，且呈现剂量依赖趋势，能明显提高细胞的存活率，升高SOD，CAT，GSH-PX的含量，降低MDA含量。该结果提示提高机体抗氧化能力和清除自由基能力参与细胞氧化应激的调节，可能是DISS促神经再生和保护的另一途径作用。
DISS对CREB磷酸化上游信号通路关键靶点的影响
采用Western-blot检测技术，分别对CREB上游三条通路中的关键蛋白进行拮抗后Glu损伤，给予DISS保护，检测末端蛋白CREB和pCEEB拮抗前后表达差异。H89为PKA拮抗剂，拮抗后末端蛋白表达变化不明显，推测DISS神经保护作用与AC/cAMP/PKA通路关联不明显。U0126为MEK拮抗剂，拮抗后CREB和pCREB表达变化明显，据此推测DISS作用与MEK/ERK/RSK通路有关。KN93为CaMK拮抗剂，拮抗后CREB和pCREB表达有明显差异，推测DISS作用与Ca~(2+)/CaM/CaMK通路有关。
综上所述，DISS可能通过(MAPK)通路和钙调蛋白激酶(CaMK)通路激活CREB，磷酸化后的CREB可以进一步促进抗凋亡基因bcl-2的表达，抑制凋亡基因bax的表达，促进神经细胞再生并抑制其凋亡；同时，DISS促进CREB磷酸化后的启动了下游氧化应激的通路，通过上述转导通路系统，DISS最终影响神经细胞的神经可塑性、神经分化、神经营养、细胞生存与凋亡、细胞氧化应激功能等，发挥抗抑郁作用。
3,6′-disinapoyl sucrose (DISS) is the active oligosaccharide ester component found in the root ofPolygala tenuifolia Willd (Radix Polygala). Recorded as“YuanZhi”in the Pharmacopoeia of the People’sRepublic of China, the root has been used in traditional medicine as, among other things, an expectorant, tonic,tranquillizer and antipsychotic agent. Our previous studies indicated that DISS has notable antidepressanteffects in pharmacological depression models, an action closely related to the potentiation of central 5-HT andNE systems. Our recent study also found that DISS increased expression in the hippocampus of threenoradrenergic-regulated plasticity genes (laminin, CAM-L1 and CREB) and one neurotrophic factor (BDNF).
This research is aimed to investigate the neuroprotective and nerve regeneration effect and mechanism ofDISS by CREB related upstream and downstream signal pathway and to find the detailed targets and passwayactiving by DISS, which may be useful to the antidepressant new drug development.
The effect of DISS on the CREB downstream passway and its antiapoptosis activity
SH-SY5Y neuronal cells were pretreated with glutamate (8mM) for 30min followed by co-treatment withDISS for 12 h. Cell viability was determined by (3,4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide(MTT) assay, and apoptosis was confirmed by cell morphology and flow cytometry assay, evaluated withpropidium iodide dye. Treatment with DISS (0.6, 6, and 60μM) increased cell viability dose-dependently,inhibited LDH release and attenuated apoptosis. The mechanisms by which DISS protected neuron cells fromglutamate induced excitotoxicity included the downregulation of proapoptotic gene bax and the upregulationof anti-apoptotic genes bcl-2.
The effect of DISS on the CREB downstream passway and its antioxidant activity
SH-SY5Y neuronal cells were pretreated with H_2O_2 (300μM) for 30min followed by co-treatment withDISS for 12 h. Cell viability was determined by (3,4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide(MTT) assay, and antioxidant effect was confirmed by SOD,CAT,GSH-PX and MDA level. Treatment withDISS (0.6, 6, and 60μM) increased cell viability dose-dependently, inhibited H_2O_2 induced injurey, increasedthe SOD, CAT, GSH-PX level and decreased the MDA level. The mechanisms by which DISS protectedneuron cells from H_2O_2 induced cell toxicity included its antioxidant activity.
The effect of DISS on the CREB upstream passway and its detail targets
The DISS-induced phosphorylation of CREB was completely blocked by U0126 [a mitogen-activatedprotein (MEK) kinase inhibitor] and KN93 (a CaMK inhibitor), but not by inhibitors of protein kinase A, H89kinase. These results suggest that antidepressants acutely increase CREB activity in CaMK and ERKdependent manners, which might contribute to DISS increased pCREB expression in SY5Y cells.
In summary, basing on the results, the present study speculated that the ERK-CREB and CaM-CREBsignal system may be involved in the mechanism that DISS exerts antidepressant actions, and the neuronalmechanism of depression. Moreover the present findings also indicated that DISS exerts the neuroprotectiveeffects by actived CREB phosphorylation and further adjusted its downstream gene (proapoptotic gene bax andanti-apoptotic genes bcl-2.) and antioxidant system proteins, which participant the neural plasticity,differentiation, neurotrophy, apoptotic and oxidative stress function.
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