下丘脑室旁核Nesfatin-1通过胰岛素信号通路调控慢性应激大鼠的抑郁相关行为

Nesfatin-1 is an anorectic molecule localized widely in hypothalamus and has been identified to play a role in the regulation of food intake and glucose metabolism. Recently, it has also been found that serum nesfatin-1 concentration was significantly increased in major depression disorder (MDD) patients. Moreover, restraint stress could induce c-fos expressions in nesfatin-1-immunoreactive neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus, the nucleus of solitary tract (NTS), locus coeruleus (LC) and dorsal raphe nucleus (DR) in the brain stem. Intracerebroventricular administration of nesfatin-1 could induce c-fos expressions in the CRH-immunoreactive neurons in PVN. These results indicated that nesfatin-1 might be associated with the pathogenetics of depression. To test our hypothesis that nesfatin-1 could regulate the depression-like behavior through impairing hypothalamic insulin signaling pathway, the depression rat models would be established in the present study by using chronic unpredictable mild stress (CUMS), the depression-like behaviors would be detected using behavior tasks including sucrose preference test (SPT), forcing swimming test (FST), and conditional preference response (CPP), and the activity of hypothalamic-pituitary-adrenal (HPA) axis would be tested also. The rats would be given a catheter through right jugular vein to observe the dynamic changes of serum concentrations of nesfatin-1, corticosterone, insulin, and glucose with the frequency once a week during CUMS procedure. Immunohistochemistry and in situ hybridization technique would be adopted to detect the expression of nesfatin-1 in rat hypothalamus, and the mRNA expression of CRH would be measured using real-time quantitative PCR(Q-PCR). The association of serum concentration and hypothalamic expression of nesfatin-1 with depression-like behavior should be analyzed. Moreover, the depression-like behavior should be tested in rats with nesfatin-1 injected or conditionally knocked-out in the PVN. Furthermore, the expression of key moleculars in the insulin signaling pathway including insulin receptor, phosphatidylinositol-3 kinase (PI3K), Akt and insulin receptor substrate (IRS) in hypothalamus would be detected, and the relationship between their changes and nesfatin-1 should be analyzed subsequently. To further investigate whether insulin signaling pathway plays an important role in the effect of nesfatin-1 on the depression-like behavior and the activity of HPA axis, insulin sensitizer rosiglitazone or inhibitors of insulin signaling pathway would be administrated in vivo and in vitro. The present study should be helpful to reveal the nesfatin-1-operative mechanism that triggers depression-like behavior, which should throw light in understanding the neurobiological mechanism of depression and developing new kinds of antidepressant targeting at nesfatin-1.

Nesfatin-1是最近发现的摄食抑制因子，广泛存在于下丘脑及其它中枢和外周组织，研究表明抑郁症患者血浆Nesfatin-1浓度显著增高。本项目提出“应激导致的下丘脑Nesfatin-1水平增高可通过影响胰岛素信号通路活性介导抑郁相关行为的发生”理论假设，拟建立慢性不可预见温和应激大鼠模型，检测抑郁相关行为及Nesfatin-1的动态变化，探讨下丘脑及血浆Nesfatin-1水平、胰岛素信号通路关键分子与抑郁相关行为的关系；采用下丘脑室旁核干预Nesfatin-1表达、胰岛素增敏剂或胰岛素通路关键分子PI3K的特异性抑制剂等，观察对Nesfatin-1介导的抑郁相关行为和HPA轴活性的影响，明确胰岛素信号通路在Nesfatin-1调控抑郁相关行为中的作用。旨在揭示Nesfatin-1与抑郁症的因果关系并阐明Nesfatin-1调控抑郁相关行为的潜在机制，为开发新型抗抑郁药物提供实验依据。

抑郁症是一种严重影响人类健康的身心疾病，本课题组长期关注其发生的下丘脑调节机制。本研究采用急性应激、慢性不可预见温和应激、nesfatin-1腹腔注射及高脂饮食诱导的整体动物模型、体外培养的SH-SY5Y细胞模型，结合旷场试验、糖水偏爱实验、强迫游泳实验、Morris水迷宫实验等神经行为学实验方法，重点关注了摄食抑制因子nesfatin-1与抑郁症之间的因果关系，并在此基础上探讨其可能机制。结果表明：急性应激，而非慢性应激，可导致大鼠HPA轴过度激活相关的外周血及下丘脑nesfatin-1的上调性改变；体外实验结果表明，皮质酮可诱导SH-SY5Y细胞nesfatin-1的mRNA和蛋白表达增强，抗抑郁药盐酸氟西汀可逆转这一改变；Nesfatin-1（10、20、40g/kg）单次腹腔注射可诱导大鼠绝望行为及HPA轴过度激活，连续3周腹腔注射nesfatin-1（10、20、40g/kg）可诱导大鼠抑郁相关行为及HPA轴过度激活，其机制与免疫激活及与此相关的下丘脑突触可塑性改变有关；体外实验结果表明，nesfatin-1可上调SH-SY5Y细胞突触相关蛋白synapsinⅠ及p-ERK的蛋白表达，并且这一过程由CRHR1介导。高脂饮食连续喂养4周不仅可诱导大鼠糖脂代谢紊乱，还可导致大鼠学习记忆能力下降及抑郁相关行为，nesfatin-1可能通过参与调控海马及前额叶皮层copine6及Wnt信号转导通路关键蛋白的表达从而参与这一过程；Quercetin灌胃给药可改善高脂饮食诱导的大鼠高脂血症、学习记忆能力下降及抑郁相关行为，其机制可能涉及到对nesfatin-1丰度、海马和前额叶皮层copine6蛋白及Wnt信号转导通路关键蛋白的调节。. 综上所述，本研究结果提示nesfatin-1的丰度上调是导致抑郁症发生的可能原因之一，靶向于nesfatin-1的治疗可能为抑郁症治疗药物开发提供新的思路。