TRPV1通道蛋白在局麻药神经毒性损伤中的作用及机制

It's reported that local anesthetics (LA) can induce calcium overload, activate p38 MAPK and lead to apoptosis. It's convinced that LA can activate TRPV1 channel protein and result in the increase of intracellular calcium ion.TRPV1 is involved in nerves injury and release of excitatory amino acids induced by LA. Accordingly, it's presumed that LA induce calcium ion inflow by activating TRPV1 channel protein. On the one hand the intracellular calcium stores release generous calcium and result in calcium overload by calcium induced calcium release mechanism (CICR), on the other hand calcium/calmodulin depedent kinase Ⅱ (CaMKⅡ) is activated and p38 MAPK is activated sequentially, leading to apoptosis. This study will intend to use SH-SY5Y cell line, gene transfection and RNAi technique to up-regulate and silence TRPV1 gene, detect intracellular calcium ion concentration, cell viability, cell apoptosis, the expression of CaMKⅡ and p38 MAPK protein and investigate whether LA targets CICR mechanism by TRPV1 channel protein and the relationship of TRPV1 and cell injury and apoptosis. Eventually, TRPV1 shRNA lentivirus vector will be constructed and the response of the rat model with TRPV1 low expression to LA injury will be observed in order to offer the basis of neurovirulent prevention and cure of LA.

研究报道局麻药(LA)可引起细胞内钙超载，激活p38 MAPK，导致细胞凋亡。有研究证实LA可激活TRPV1通道蛋白，导致细胞内钙离子浓度增高，参与了LA诱发的神经损伤和兴奋性氨基酸释放。因此，推测LA通过激活TRPV1通道蛋白，引起钙离子内流，一方面通过钙诱导的钙释放机制（CICR）使细胞内钙库释放大量钙离子，造成钙超载；另一方面激活钙/钙调蛋白依赖性激酶Ⅱ（CaMKⅡ），导致p38 MAPK激活，触发细胞凋亡。本研究拟采用SH-SY5Y细胞株，运用基因转染、RNAi技术上调和沉默TRPV1基因，检测LA损伤时细胞内钙离子浓度、细胞活力、细胞凋亡、CaMKⅡ和p38 MAPK蛋白表达等指标，研究LA是否通过TRPV1通道蛋白触发CICR机制及其与细胞损伤和凋亡的关系。最后构建TRPV1 shRNA 慢病毒载体，观测TRPV1蛋白低表达大鼠模型对LA损伤反应，为LA神经毒性的防治提供依据。

研究报道局麻药(LA)可引起细胞内钙超载，激活p38 MAPK，导致细胞凋亡。有研究证实LA可激活TRPV1通道蛋白，导致细胞内钙离子浓度增高，参与了LA诱发的神经损伤和兴奋性氨基酸释放。因此，推测LA通过激活TRPV1通道蛋白，引起钙离子内流，一方面通过钙诱导的钙释放机制（CICR）使细胞内钙库释放大量钙离子，造成钙超载；另一方面激活钙/钙调蛋白依赖性激酶Ⅱ（CaMKⅡ），导致p38 MAPK激活，触发细胞凋亡。本研究采用U87-MG细胞株，运用基因转染、RNAi技术上调和沉默TRPV1基因，检测LA损伤时细胞内钙离子浓度、细胞活力、细胞凋亡、CaMKⅡ和p38 MAPK蛋白表达等指标，结果表明利多卡因可通过TRPV1通道蛋白使细胞钙超载并导致细胞损伤和凋亡。TRPV1蛋白低表达可降低利多卡因对大鼠的神经功能损伤，这些结果为LA神经毒性的防治提供依据。