Tetracaine induces apoptosis through a mitochondrion-dependent pathway in human corneal stromal cells <i>in vitro</i>

<b>Purpose: </b>Tetracaine is a local anesthetic widely used in ocular diagnosis and ophthalmic surgery and may lead to some adverse effects and complications at a clinical dose. To assess the cytotoxicity and molecular toxicity mechanisms of tetracaine, we used human corneal stromal (HCS) cells as an <i>in vitro</i> model to study the effects of tetracaine on HCS cells. <b>Materials and methods: </b>The cytotoxicity of tetracaine on HCS cells was investigated by examining the changes of cell growth, morphology, viability and cell cycle progressing when HCS cells were treated with tetracaine at concentrations from 10 g/L to 0.078125 g/L. To prove the hypothesis that the cytotoxicity of tetracaine on HCS cells was related with apoptosis induction, we further detected multiple changes in HCS cells, including plasma membrane (PM) permeability, phosphatidylserine (PS) orientation, genomic DNA integrality, and cell ultrastrcuture after treated with tetracaine. Furthermore, the pro-apoptotic signalling pathway induced by tetracaine was explored through detecting the activation of various caspases, the changes of mitochondrial transmembrane potential (MTP), the expression level of Bcl-2 family proteins and the amount of mitochondria-released apoptosis regulating proteins in cytoplasm. <b>Results: </b>Tetracaine at concentrations above 0.15625 g/L had a dose- and time-dependent cytotoxicity to HCS cells, which resulted cell growth inhibition, proliferation retardation, morphological abnormalities and decreased viability. Meanwhile, we found that the HCS cells treated with tetracaine had typical features associated with apoptosis, including an increase in PM permeability, PS externalization, DNA fragmentation and apoptotic body formation. Tetracaine not only resulted in caspase-3, caspase-8 and caspase-9 activation and disruption of MTP but also downregulated Bcl-2 and Bcl-xL and upregulated Bad and Bax, along with the upregulation of cytoplasmic cytochrome c (Cyt. c) and apoptosis-inducing factor (AIF). <b>Conclusions: </b>These results suggested that tetracaine-induced apoptosis might be triggered through Fas death receptors and mediated by Bcl-2 family proteins in the mitochondria-dependent pathway. Our findings identified the cytotoxicity and molecular mechanisms of tetracaine, which could provide a reference value for the safety of this medication and prospective therapeutic interventions in eye clinic.**目的：** 丁卡因（Tetracaine）是一种广泛应用于眼科诊断与眼科手术的局部麻醉剂，在临床剂量下可能引发部分不良反应与并发症。为探究丁卡因的细胞毒性及其分子毒性机制，本研究以人角膜基质（HCS）细胞作为体外（in vitro）模型，研究丁卡因对人角膜基质细胞的影响。 **材料与方法：** 本研究通过检测丁卡因在10 g/L至0.078125 g/L浓度梯度处理下，人角膜基质细胞的生长、形态、活力及细胞周期进程变化，探究丁卡因对其的细胞毒性。为验证“丁卡因对人角膜基质细胞的细胞毒性与凋亡诱导相关”这一假说，本研究进一步检测了丁卡因处理后人角膜基质细胞的多项变化，包括细胞膜（PM）通透性、磷脂酰丝氨酸（PS）外翻状态、基因组DNA完整性及细胞超微结构。此外，本研究通过检测各类半胱天冬酶（caspases）的激活情况、线粒体跨膜电位（MTP）变化、Bcl-2家族蛋白的表达水平，以及细胞质中线粒体释放的凋亡调控蛋白含量，探究丁卡因诱导的促凋亡信号通路。 **结果：** 浓度高于0.15625 g/L的丁卡因对人角膜基质细胞具有浓度与时间依赖性的细胞毒性，可导致细胞生长受抑、增殖迟缓、形态异常及活力下降。同时，经丁卡因处理的人角膜基质细胞呈现出细胞凋亡的典型特征，包括细胞膜通透性升高、磷脂酰丝氨酸外翻、DNA片段化及凋亡小体形成。丁卡因不仅可激活半胱天冬酶-3、-8、-9并破坏线粒体跨膜电位，还可下调Bcl-2与Bcl-xL的表达，上调Bad与Bax的表达，同时使细胞质内细胞色素c（Cyt. c）及凋亡诱导因子（AIF）的水平升高。 **结论：** 本研究结果显示，丁卡因诱导的细胞凋亡可能通过Fas死亡受体启动，并由线粒体依赖性通路中的Bcl-2家族蛋白介导。本研究明确了丁卡因的细胞毒性及其分子机制，可为该药物的临床用药安全性及眼科临床潜在治疗干预手段提供参考依据。