抑郁症 (Depression) 是一种较为常见的精神和情感障碍性疾病，其临床症状主要表现在心情低落、悲观、思维迟缓、绝望、意志活动减退、认知功能损害和一些其他躯体症状等，主要包括睡眠障碍、食欲减退、体重下降、乏力等，并且其发病率仍有逐年上升的趋势。抑郁症的治疗不仅是精神科而且也是其它学科的研究热点。

       抑郁症的发病机制涉及多个方便。目前临床上以抗抑郁药物作为治疗抑郁症状的主要手段，传统的抗抑郁药物主要是在“单胺类神经递质”假说的基础上发展而来的。对抗抑郁药物的研究虽然进展很大，但理想的药物仍然不足，尚不能满足临床治疗的需要。近年来新研制的抗抑郁药物虽然降低了毒性，并且减少了其不良反应，但这些新型药物的长期使用仍可能引起一些潜在的不良反应。因而尚需对其发病机制进行深入探讨，以指导新型抗抑郁药的研究。

       乌灵参 (Xylariasp) 是具有利尿、治失眠、补心神等药用价值的一种真菌的菌核。乌灵菌粉是乌灵参菌丝体经深层发酵技术培养制成粉末，本品为浅棕色至棕色粉末。其胶囊制剂在临床主要用于镇静安神和抗失眠的治疗。乌灵菌粉的抗抑郁、抗焦虑效果在临床上已经得到证实，显示中医药在这方面的独特优势。为了进一步确立这一优势，仍需在其抗抑郁作用机制方面进行深入探讨。本研究采用习得性无助和社会竞争失败两种小鼠抑郁症动物模型，观察乌灵菌粉的抗抑郁作用，并通过全基因表达谱芯片技术对差异基因的表达进行了分析，在分子水平上对乌灵菌粉的抗抑郁症作用机制进行研究，从而为乌灵菌粉的临床应用提供了理论基础。

第一部分 乌灵菌粉抗抑郁症药效学研究

1. 在乌灵菌粉对习得性无助抑郁症模型小鼠的作用研究中，模型组小鼠在逃避反应评价实验中的逃避失败次数明显增多 (P<0.01)，并且其逃避潜伏期也明显长于对照组动物 (P<0.01)。经乌灵菌粉中剂量和高剂量治疗后小鼠的逃避失败次数显著减少 (P<0.05)，其逃避潜伏期也显著缩短 (P<0.05)，并且其抗抑郁作用具有较好的剂量依赖关系。在新奇抑制摄食实验中，模型组小鼠的摄食潜伏期显著延长 (P<0.05)，低剂量的乌灵菌粉则显著缩短了其摄食潜伏期 (P<0.05)。在强迫游泳实验中，模型组小鼠的的累计不动时间明显长于对照组动物 (P<0.01)，中剂量和高剂量的乌灵菌粉治疗后，可显著缩短其累计不动时间 (P<0.01)，并且其抗抑郁作用具有较好的剂量依赖关系。

2. 在乌灵菌粉对社会竞争失败抑郁症模型小鼠的作用研究中，模型组小鼠在社交回避反应评价实验中的社交区停留时间比例明显低于对照组动物 (P<0.01)，而低剂量的乌灵菌粉 (P<0.05) 和中、高剂量的乌灵菌粉 (P<0.01) 治疗后可以显著提高其社交区停留时间比例。同时，模型组小鼠的角落区停留时间比例与对照组动物相比明显增大 (P<0.01)，低、中、高剂量的乌灵菌粉治疗后均显著降低了其角落区停留时间比例 (P<0.01)。

第二部分 乌灵菌粉抗抑郁作用的机制研究

1. 在习得性无助小鼠抑郁症模型中，采用全基因表达谱芯片对中脑组织进行了分析。结果显示与线粒体自噬功能密切相关的基因TSPO (18kDa转运蛋白) 的表达水平显著下调。

2. 以TSPO-VDAC1调控的线粒体自噬相关通路作为研究对象探讨乌灵菌粉在习得性无助小鼠抑郁症模型中的抗抑郁作用机制。与正常对照组相比，模型组小鼠中脑蛋白TSPO、VDAC1和PINK1的表达水平显著下降 (P<0.05)，而乌灵菌粉治疗两周后其表达水平显著增加 (P<0.05)；模型组小鼠中脑蛋白Parkin的表达水平明显高于正常对照组动物的表达水平 (P<0.01)，而低、中和高剂量的乌灵菌粉治疗两周后均可显著降低其表达水平 (P<0.01)；模型组小鼠中脑线粒体自噬标志蛋白Beclin1的表达水平明显低于对照组 (P<0.01)，乌灵菌粉治疗后其表达水平明显增加 (低剂量和中剂量P<0.05，高剂量P<0.01)；与神经元轴浆运输相关的蛋白KIFC2在模型组小鼠中脑的表达水平显著下降 (P<0.05)，而高剂量的乌灵菌粉显著增加了其表达水平 (P<0.05)。

3. 习得性无助抑郁症模型小鼠突触蛋白Synaptogyrin3免疫组化染色结果显示：模型组小鼠腹侧纹状体区Synaptogyrin-3的阳性神经纤维的累积光密度值 (IOD) 显著降低 (P<0.01)，而低剂量和中剂量乌灵菌粉治疗组Synaptogyrin-3的阳性神经纤维的IOD值显著增加 (P<0.01)，该研究结果提示乌灵菌粉对突触发生具有显著地改善作用。

4. 上述结果提示，在习得性无助小鼠抑郁症模型中，乌灵菌粉通过调控TSPO介导的线粒体自噬信号通路发挥抗抑郁作用。

    Depression (also known as depressive disorders) is a common mental disorder, with essential features are depressed mood, thought slowness, thinking volitional activity drops give priority to, cognitive dysfunction and physical impairment, including sleep disorders, fatigue, loss of appetite, weight loss and so on. The prevalence exhibited a gradually rising tendency. The treatment of depression has recently gained considerable interest not only for psychiatry, but also for other disciplines.

    The pathogenesis of depression remains incompletely understood. Antidepressants are the main drugs for the treatment of depressive symptoms. The traditional antidepressants were mainly developed from “monoamine hypothesis”. Although great progress has been made in the development of antidepressants, there is still lack of ideal therapeutic drugs. Aside lower toxicity and few side effects, the long-term use may still cause potential adverse reactions with the novel antidepressants. Thus, further investigation into the pathogenesis of depressive disorder remains to be done, and the development of the next generation of novel antidepressants is urgent.

    Xylariasp is the fungus sclerotia which grow in termite nests, and its medicinal properties include diuresis, filling the mind, to treat insomnia, vomiting blood and postpartum bleeding treatment. Wuling powder is a light brown to brown powder, which is made by cultivating Xylariasp mycelium using submerged fermentation technology. The capsule preparation of Wuling powder is mainly used to soothe nerves and anti-insomnia in clinic. The antidepressant and anti-anxiety effect of Wuling powder has been confirmed in clinic, which indicated the unique advantage of Chinese medicine. In order to further establish this advantage, it’s still need to in-depth research the mechanism of action. This research adopted two animal models of depression in mice, learned helplessness and social defeat stress, to observe the antidepressant effect of Wuling powder. We explored the underlying mechanism of Wuling powder by analyzing the difference of gene expression using the gene expression profile chip technology, and provided theoretical basis for the clinical application of Wuling powder.

Part I: Studies on the antidepressant effect of Wuling powder

1. In the learned helplessness animal model, mice were subjected to the shuttle box text to assess the helpless behavior. Results showed that the number of escape failures was significantly increased in model group (P<0.01). Meanwhile, the average escape latency to crossing shuttle box of model group was observably longer than that of the control group (P<0.01). While treatment with Wuling powder significantly decreased both the number of escape failures (P<0.05) and the average escape latency (P<0.05) in a dose-dependent pattern. In the novelty suppressed feeding test, the latency to chew the pellet is defined as feeding latency. Results showed that mice in model group spent significantly more time to initiate feeding (P<0.05). Treatment with Wuling powder shortened the latency to feed (P<0.05). In the forced swimming test, model mice displayed an obvious increase in immobility time (P<0.01). On the contrary, the immobility time was shortened in Wuling powder administrated mice (P<0.01) with a dose-dependent pattern.

2. In the social interaction test in social defeat stress animal model, the ratio of time model mice spent in the interaction zone in the presence of a target CD-1 compared with the absence of a target CD-1 was obviously decreased (P<0.01). After treatment with Wuling powder, the ratio was ameliorated observably (P<0.01). In addition, the ratio of time model mice spent in the corner zone in the presence of a target CD-1 compared with the absence of a target CD-1 was significantly increased (P<0.01), and treatment with Wuling powder dramaticlly decreased this ratio (P<0.01).

Part II: Studies on the antidepressant mechanisms of Wuling powder

1. In the learned helplessness animal model, the results of whole genomic expression profile analysis showed that 18kDa translocator protein (TSPO) differentially expressed by inescapable e-shock exposure, which plays an important role in regulating mitochondrial function.

2. We investigated the TSPO/VDAC1-mediated mitochondrial autophagy related pathway to study the antidepressant mechanisms of Wuling powder in the learned helplessness animal model. The down-regulation of TSPO、VDAC1 and PINK1 were observed in LH model mice (P<0.05), while Wuling powder enhanced their expression level dramatically (P<0.05). The expression of Parkin was upregulated in LH model mice (P<0.01), Wuling powder markedly reverse this change induced by inescapable e-shock exposure (P<0.01). Meanwhile, results showed that model mice experienced reduced level of Beclin1 expression (P<0.01), and Wuling powder was capable of alleviating this impact (Wuling-L/M P<0.05; Wuling-H P<0.01). KIFC2, a kinesin family member C2, may play a role in retrograde axonal transport. In this study, we observed a decrease in the expression level of KIFC2 (P<0.05), and Wuling powder increased its expression level (P<0.05).

3. Immunohistochemistry analysis indicated that the IOD of ventral striatum in LH model group decreased obviously P<0.01), and treatment of Wuling powder , which indicated the improvement of by Wuling powder.

4. Our results suggested that Wuling powder exhibited an obvious antidepressant effect, which could be due to the improvement of TSPO-mediated mitophagy signaling pathway.