肌苷介导MAPK/ERK信号通路参与青幼期抑郁大鼠发病机制研究

Children and adolescents depression seriously endanger their physical and mental health and lives of juveniles, while its etiology and pathogenesis is still not clear. Our previous studies first found that the plasma levels of inosine in patients with children and adolescents depression are significantly more than health controls. And, after intraperitoneal injection of the inosine in the depressed animal model, we found their depressive-like behavior have been relieved. Recent studies reported that MAPK / ERK signaling pathway is significantly associated with the pathogenesis of adult depression, but there is no report for children and adolescents depression.Thus, based on the rats model in childhood depression and the MAPK/ERK signal pathway, the aim of this study was to investigate the pathogenesis of depression in children and adolescents, and to explore their potential therapeutic target. We plan to carry out the following work: ① we will confirm the changes of MAPK/ERK pathway in childhood rats from both of the protein and gene level, and look for the key protein and key brain areas of this pathway; ②After intraperitoneal injection of inosine in childhood depressed rats, we will determine the inosine content in their brain and evaluate animals’ behavioral changes, as well as detect the expression of key protein of MAPK/ERK pathway; ③the key protein antagonists of MAPK / ERK pathway will be injected into the key brain area and we will further investigate the inhibitory effect of inosine on MAPK/ERK pathway in childhood depressed rats according to their behavioral analysis.

儿童青少年抑郁严重危害未成年人身心健康和生命安全，且病因和发病机制迄今不清。课题组前期研究首次发现，儿童青少年抑郁患者血浆肌苷水平显著降低，且动物模型腹腔注射肌苷后可缓解抑郁样行为。最近研究报道，MAPK/ERK信号通路与成人抑郁发病密切相关，但国内外尚无该通路参与儿童青少年抑郁相关报道。因此，本项目以青幼期抑郁大鼠模型为载体,以肌苷介导MAPK/ERK信号通路为研究主线，探索儿童青少年抑郁发病机制及干预靶点。拟开展以下工作：①从蛋白和基因层面证实MAPK/ERK通路在青幼期抑郁大鼠中的改变，寻找该通路的关键蛋白和表达的关键脑区；②在青幼期抑郁大鼠腹腔注射肌苷，测定脑区肌苷含量和MAPK/ERK通路的关键蛋白表达的变化，并评价行为学改变；③进一步在关键蛋白表达脑区注射MAPK/ERK通路的关键蛋白拮抗剂后，观察其行为学改变，进一步验证肌苷通过MAPK/ERK通路调控青幼期大鼠的抑郁样行为。

儿童青少年抑郁严重危害未成年人身心健康和生命安全，但目前发病机制尚不清楚。课题组前期研究发现：儿童青少年抑郁患者血浆肌苷水平显著降低，且未造模的青幼期SD大鼠腹腔注射肌苷后可缓解抑郁样行为。同时结合国内外研究报道，MAPK/ERK信号通路可能是肌苷的作用靶点，并参与儿童青少年抑郁症的发病机制。因此，课题组以青幼期啮齿类动物（大鼠、小鼠）抑郁模型为载体，以肌苷介导MAPK/ERK信号通路为研究主线，探索儿童青少年抑郁发病机制及干预靶点，开展了以下工作：①通过Western blot技术验证腹腔注射肌苷后青幼期大鼠的海马和前额叶脑组织中ERK和CREB磷酸化蛋白水平（P-ERK和P-CREB），结果显示P-ERK和P-CREB明显增加，提示肌苷可能通过激活青幼期大鼠海马和前额叶的ERK-CREB信号通路来起到抗抑郁作用；②课题组构建青幼期慢性不可预见性温和刺激（CUMS）小鼠模型，对青幼期C57小鼠随机分为三组（CUMS+肌苷组，CUMS+生理盐水组，对照组），CUMS+肌苷组和CUMS+生理盐水组进行为期4周的慢性不可预见性温和刺激及肌苷或生理盐水腹腔注射，通过糖水偏好实验、旷场试验、高架十字迷宫实验进行了行为学测试，结果显示肌苷可以缓解青幼期CUMS抑郁小鼠模型抑郁样行为，其中CUMS+生理盐水组与CUMS+肌苷组的差异蛋白富集到MAPK信号通路，并进行MAPK信号通路关键蛋白验证；③基于前期对信号通路机制探索的实验结果，并查阅抗抑郁药可能相关的机制研究，课题组积极开展肌苷对抑郁小鼠肠道微生物的影响，通过对小鼠粪便进行16S rRNA检测，结果显示肌苷可以改善CUMS刺激引起的小鼠肠道微生物的改变，提示肌苷可能通过“微生物-肠-脑”轴调控青幼期小鼠抑郁样行为。本项目研究围绕肌苷作为儿童青少年抑郁症的潜在治疗靶点开展了动物行为学、关键脑区的蛋白分子层面及结合“微生物-肠-脑”轴等多个方面研究，证实了通过腹腔注射肌苷可能通过ERK-CREB信号通路改善青幼期啮齿类动物抑郁样行为，并可以逆转CUMS刺激引起小鼠肠道微生物的改变，提示肌苷可能通过“微生物-肠-脑”轴缓解小鼠抑郁样行为。