Song Zhan, Fan Ting-Jun
a Laboratory for Corneal Tissue Engineering , College of Marine Life Sciences, Ocean University of China , Qingdao , Shandong province , P. R. China.
Cutan Ocul Toxicol. 2018 Dec;37(4):350-358. doi: 10.1080/15569527.2018.1468342. Epub 2018 May 9.
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 in vitro model to study the effects of tetracaine on HCS cells.
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.
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).
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.
丁卡因是一种广泛用于眼科诊断和眼科手术的局部麻醉剂，在临床剂量下可能会导致一些不良反应和并发症。为了评估丁卡因的细胞毒性和分子毒性机制，我们使用人角膜基质（HCS）细胞作为体外模型来研究丁卡因对HCS细胞的影响。
通过检测丁卡因浓度为10 g/L至0.078125 g/L处理HCS细胞时细胞生长、形态、活力和细胞周期进程的变化，研究丁卡因对HCS细胞的细胞毒性。为了证明丁卡因对HCS细胞的细胞毒性与诱导凋亡有关这一假设，我们进一步检测了丁卡因处理后HCS细胞的多种变化，包括质膜（PM）通透性、磷脂酰丝氨酸（PS）取向、基因组DNA完整性和细胞超微结构。此外，通过检测各种半胱天冬酶的激活、线粒体跨膜电位（MTP）的变化、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节蛋白的量，探索丁卡因诱导的促凋亡信号通路。
浓度高于0.15625 g/L的丁卡因对HCS细胞具有剂量和时间依赖性细胞毒性，导致细胞生长抑制、增殖延迟、形态异常和活力降低。同时，我们发现用丁卡因处理的HCS细胞具有与凋亡相关的典型特征，包括PM通透性增加、PS外化、DNA片段化和凋亡小体形成。丁卡因不仅导致半胱天冬酶-3、半胱天冬酶-8和半胱天冬酶-9激活以及MTP破坏，还下调Bcl-2和Bcl-xL并上调Bad和Bax，同时上调细胞质细胞色素c（Cyt. c）和凋亡诱导因子（AIF）。
这些结果表明，丁卡因诱导的凋亡可能通过Fas死亡受体触发，并由线粒体依赖性途径中的Bcl-2家族蛋白介导。我们的研究结果确定了丁卡因的细胞毒性和分子机制，可为该药物的安全性以及眼科临床的前瞻性治疗干预提供参考价值。
