STIM1/TRPV1/calpain通路在顺铂所致耳毒性中的作用及葛根素的保护机制研究

Cisplatin is a highly effective anti-cancer drug that is widely used in clinic now.However, serious ototoxicity of cisplatin become an important factor affecting its use. At present, the mechanism of cisplatin ototoxicity is still unclear. Our previous study found that cisplatin could activate transient receptor potential vanilloid 1 (TRPV1) in cochlear cell, TRPV1 caused calcium influx and overload, which causing cochlear cell apoptosis finally. Store-operated calcium channels (SOCC) are important channels of the inflow of extracellular calcium, stromal interacting molecule (STIMI), Orail and transient receptor potentials (TRPs) are the keys to constitute the SOCC proteins, and they can modulate with each other. We found that after giving cisplatin, the expression of STIM1, Orail and TRPV1 is increased with a dose-effect relationship. At the same time, the expression of calcium protease enhanced obviously, which was positive correlated with cell apoptosis rate. On this basis, we deduce that STIM1 / TRPV1 / calpain pathway may be involved in cisplatin-induced ototoxicity. Our latest studies suggest that puerarin as Chinese native medicine preparation, can effectively protective cisplatin ototoxicity, but its protective mechanism is to be explored. This project intends to investigate the role of STIM1 / TRPV1 / calpain pathway in cisplatin ototoxicity and the protective mechanism of puerarin by using molecular biological, micromorphological and other techniques, so as to provide new route for clinical prevention and treatment of drug deafness.

顺铂是一线抗癌药，其耳毒性机制仍不明晰。前期研究中，我们发现顺铂可激活耳蜗细胞上瞬时感受器电位香草酸受体1（TRPV1），导致钙内流和钙超载，从而引发细胞凋亡。钙池操纵性钙通道（SOCC）是胞外钙内流的重要通道，基质交感分子1（STIM1）、Orail和TRPs 是构成SOCC的关键蛋白，三者间相互调控。我们发现给予顺铂后，STIM1、Orail和TRPV1的表达呈剂量依赖性升高，同时钙蛋白酶（calpain）的表达也明显增强，且与细胞凋亡率呈正相关。据此，我们推论STIM1/TRPV1/calpain通路可能参与了顺铂耳毒性过程。我们最新研究提示，葛根素作为中药制剂，可有效防护顺铂耳毒性，但其防护机制有待阐明。综上，本课题拟利用分子生物学、显微形态学及其它相关技术，研究STIM1/TRPV1/calpain通路在顺铂所致耳毒性中的作用及葛根素的防护机制，为临床防治药物性聋提供新途径。

顺铂是一线抗癌药，其耳毒性机制仍不明晰。前期研究中，我们发现顺铂可激活耳蜗细胞上瞬时感受器电位香草酸受体1（TRPV1），导致钙内流和超载，从而引发细胞凋亡。钙池操纵性钙通道（SOCC）是胞外钙内流的重要通道，基质交感分子1（STIM1）、Orail和TRPs 是构成SOCC的关键蛋白，三者间相互调控。我们发现给予顺铂后，STIM1、Orail和TRPV1的表达呈剂量依赖性升高，同时钙蛋白酶的表达也明显增强，且与细胞凋亡率呈正相关。我们最新研究提示，葛根素作为中药制剂，可有效防护顺铂耳毒性，但其防护机制有待阐明。据此，我们提出STIM1/TRPV1/calpain通路可能参与了顺铂耳毒性过程，且葛根素通过以上通路减轻顺铂所致耳蜗毒性的科学假说。我们的研究内容包括1）明确STIM1在正常及顺铂致毒小鼠听觉传导细胞（外毛细胞和螺旋神经节细胞）的表达及定位，2）明确STIM1在顺铂耳毒性中的作用，探讨STIM1的激活在顺铂耳毒性损伤中的机制，3）从临床病例和动物实验两个方面，探讨葛根素对顺铂耳毒性的防护作用。通过应用小鼠在体和离体STIM1沉默模型和顺铂耳毒性模型，我们研究发现：1）在正常小鼠听觉传导细胞（外毛细胞和螺旋神经节细胞）中STIM1和Orai1有表达，且顺铂致毒后两者水平均显著增强，2）钙超载是顺铂耳毒性的关键机制，且STIM1/TRPV1/calpain通路的激活直接导致听觉细胞钙超载，小鼠听觉丧失，3）葛根素通过抑制STIM1/TRPV1/calpain通路的激活，降低耳蜗细胞的钙超载和氧化应激水平，进而防护顺铂耳毒性。我们的研究结果提示，顺铂可能通过STIM1/TRPV1/calpain通路导致钙超载，激活下游各种凋亡相关因子，最终损伤听功能；而葛根素对顺铂小鼠听功能保护作用可能是通过减少ROS的生成，进而抑制STIM1/TRPV1/calpain通路所致钙超载来实现的。本研究阐明了由STIM1诱发的顺铂耳毒性的发病机制，初步探讨葛根素与顺铂的联合应用的可能性，为临床化疗患者防护顺铂耳毒性提供新的潜在靶点和治疗方案。