从5-HT3受体介导的Ca2+/CaMKII/ERK1/2信号通路探讨小半夏汤防治化疗性恶心呕吐机制

Emesis is a common and severe side effect of anticancer drugs such as cisplatin, which is usually named chemotherapy-induced nausea and vomiting (CINV). CINV is a major reason of non-complicance with cancer treatment. CINV caused by cytotoxic chemotherapeutic drugs is associated with an increase of 5-HT level centrally and peripherally. Cytotoxic drugs stimulate 5-HT release from the enterochromaffin (EC) cells in the small intestine. The released 5-HT activates 5-HT3 receptor on the end of vagal afferent nerves and induces depolarization of the vagal afferent nerves. The impulse of the vagal afferent nerves stimulates the vomiting center located in the brainstem and finally emesis occurs. It is confirmed that 5-HT and 5-HT3 receptor play a key role underlying the phathological mechanism of CINV. There are evidence indicates the pharmacological pivotal roles of 5-HT3 receptor in the control of CINV. Actually, the 5-HT3 receptor antagonists such as ondansetron, palonosetron, are the most efficient anti-emetic drugs in current clinic..Xiaobanxia Decoction (XD) is a traditional anti-emetic remedy, which is recorded in JinGuiYaoLue, a medicinal classic written by Zhang Zhongjing nearly two thousand years ago during Chinese Dong Han dynasty. XD is composed of two crude herbs, Banxia (Pinelliae tuber, Pinellia ternate (Thunb) Breit.) and Shengjiang (ginger, Zingiber officinale Roscoe). Clinical evidence has proved its potentials of anti-emetic effects, including control of CINV. Our previous studies showed that XD has a potent anti-emetic effect in cisplatin-induced rat pica and mink vomiting models, one of the key machenism underlying its anti-emetic effects is decreasing 5-HT level and downregulating 5-HT3 receptor in small intestine and brainstem..It was reported that 5-HT3 receptor-mediated emesis occurs by the activation of Ca2+/CaMKII-dependent ERK1/2 signaling pathway. According to our previous studies combined with the current literature reportings, we predict that regulating 5-HT3 receptor-mediated Ca2+/CaMKII-dependent ERK1/2 signaling pathway could be a pivotal machenism underlying XD’s anti-emetic effect..In the present applying project, we plan to explore the anti-emetic mechanism of XD focusing on 5-HT3 receptor-mediated Ca2+/CaMKII-dependent ERK1/2 transdution signals. In our potocol, intracellular Ca2+ levels in rat brainstem slices incubated with Ca2+ indicator fluo-3/AM will be observed using Laser Scanning Confocal Microscope. Male adult Wistar rat brain slices will be used to to investigate the effect of XD and its active ingredients 6-gingerol, 6-shogaol and alkaloids of Pinellia ternata on 2-Me-5-HT (a selective 5-HT3 receptor agonist) -elicited Ca2+ increase in the AP and NTS regions of the brainstem emetic nuclei. Pica models will be induced in male adult rats by intraperitoneal injection of cisplatin and 2-Me-5-HT, respectively. XD will be observed how to regulate 5-HT3 receptor-mediated Ca2+/CaMKII-dependent ERK1/2 transduction signals in above pica models. In vivo and in vitro methods will be used, including immunohistochemistry, immunoprecipitation, immunocytochemistry and Western blot on tissues of both small intestine and brainstem of rats and isolated EC cells from rat small intestine..The purpuse of the present project will be evaluating XD how to regulate 5-HT3 receptor-mediated Ca2+/CaMKII-dependent ERK1/2 signaling pathway underlying its anti-emetic mechanism. The research results will be helpful to improve the control of CINV in clinic. The project will be an good example of studying classical prescription using modern methods, and will facilitate to modernize traditional Chinese herbs.

恶心呕吐是困扰肿瘤化疗的难题。小半夏汤为止呕之祖方，防治化疗性呕吐疗效确切。我们前期研究表明，5-HT3R是小半夏汤止吐的关键靶点。5-HT激动5-HT3R发生呕吐，需要5-HT3R介导的胞内信号系统的转导和放大。已知5-HT3R介导呕吐与Ca2+/CaMKII/ERK1/2信号通路的活化有关。我们认为：对5-HT3R介导的Ca2+/CaMKII/ERK1/2胞内信号通路的调控，可能是小半夏汤止吐的重要机制。本项目拟在前期工作基础上，以5-HT3R介导呕吐的胞内信号转导系统为切入点，体内外实验相结合，从整体、离体、细胞等水平，采用传统药理学与现代分子生物学相结合的实验技术，从中枢和外周，从行为学、形态、功能等方面，观察小半夏汤及其主要有效成分对5-HT3R介导呕吐的胞内信号系统的影响，深入阐明小半夏汤止呕机制。本项目对于中西医结合肿瘤防治、对于中医经方的现代研究和中药现代化均具有重要意义。

目前对于化疗性恶心呕吐（chemotherapy-induced nausea and vomiting, CINV）没有理想防治方法，从疗效确切的传统中药中寻找新型止吐药物具有重要意义。本项目深入探讨了小半夏汤防治CINV的作用机制，达到了预期研究目标。.1．采用顺铂和5-HT3R选择性激动剂1-PBG分别建立大鼠异食癖恶心呕吐模型，观察了小半夏汤对5-HT3R介导的胞内信号通路的影响。结果表明，小半夏汤可显著抑制模型大鼠摄食高岭土，说明对激动5-HT3R所致呕吐具有确切止吐作用。顺铂和1-PBG均可增加小肠和脑组织中CaM和5-HT3R的含量，并增强两个蛋白的共定位作用，说明5-HT与5-HT3R的结合可以促进CaM的表达，而小半夏汤对CaM和5-HT3R荧光表达增强具有明显抑制作用，并抑制小肠和脑组织中CaMKII、pCaMKII、ERK1/2和pERK1/2蛋白表达升高。说明抑制5-HT3R介导的CaM/CaMKII/ ERK1/2信号通路的活化是小半夏汤防治CINV的重要机制。.2．研究发现，小半夏汤下调模型大鼠胃肠组织NLRP3炎症小体相关蛋白表达，阻断NLRP3炎症小体的组装，抑制NLRP3激活，最终抑制炎症因子分泌。说明小半夏汤通过抑制NLRP3炎症小体活化发挥防治CINV作用。.3．对化疗大鼠回肠组织进行了转录组学高通量测序，小半夏汤显著抑制顺铂上调的基因343个，显著抑制顺铂下调的基因75个。通过GO功能注释及KEGG富集分析，发现顺铂诱导大鼠异食癖与炎症介质和炎症信号通路密切相关。随机选取20个炎症差异基因进行了qRT-PCR验证，小半夏汤可抑制顺铂导致的炎症基因过表达和炎症信号通路异常激活。采用顺铂诱导的大鼠小肠上皮细胞（IEC-6）损伤模型，观察到小半夏汤中主要成分6-姜酚和6-姜烯酚对顺铂诱导细胞存活率降低、ROS产生及细胞坏死均有显著改善作用。.4．16S rDNA基因测序结果表明，小半夏汤及6-姜酚可显著改善化疗大鼠肠道菌群。.5．6-姜酚的止吐作用与抑制中枢和外周5-HT合成、促进5-HT代谢和转运从而降低5-HT水平，同时下调5-HT3R表达有关。.已发表学术论文14篇，培养研究生8名，其中已毕业3名。.本项目针对CINV病理机制，从多个角度对小半夏汤防治CINV作用机制进行了深入探讨，对于阐明该方止吐的现代科学内涵具有重要意义。