Lidocaine induces ROCK-dependent membrane blebbing and subsequent cell death in rabbit articular chondrocytes.
Lidocaine induces ROCK-dependent membrane blebbing and subsequent cell death in rabbit articular chondrocytes.
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利多卡因在兔关节软骨细胞中诱导 ROCK 依赖性膜起泡和随后的细胞死亡。
DOI：
10.1002/jor.23092
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发表时间：
2016
期刊：
影响因子：
2.8
通讯作者：
Matsusue Y.
中科院分区：
文献类型：
--
作者：
Maeda T;Toyoda F;Imai S;Tanigawa H;Kumagai K;Matsuura H;Matsusue Y.
局部麻醉剂在骨科诊所和手术中用于关节内疼痛控制。虽然以前的研究表明局部麻醉剂对软骨细胞有毒性，但其潜在的细胞机制仍不清楚。本研究研究了与利多卡因诱导的关节软骨细胞毒性相关的急性细胞反应。将兔关节软骨细胞暴露于利多卡因，用活细胞显微镜观察其形态学变化。使用基于荧光的LIVE/DEAD测定法评价软骨细胞的活力。用利多卡因（3-30 mM）对软骨细胞进行急性处理，以时间和浓度依赖性方式诱导细胞表面的球形突起（所谓的“膜起泡”）。通过升高细胞外pH值，利多卡因效应的浓度-反应关系发生了显著变化，这与参与滤过泡形成的非离子化利多卡因的预期一致。ROCK（Rho激酶）抑制剂Y-27632和法舒地尔完全阻止了利多卡因诱导的膜起泡，表明ROCK激活是水泡形成所必需的。10 mM利多卡因未改变Caspase-3水平（p =0.325），Caspase抑制剂z-VAD-favorite不影响利多卡因诱导的水泡（p= 0.964）。GTP-RhoA水平显著增加（p< 0.001），但Rho抑制剂-1未能抑制膜起泡（p= 0.875）。利多卡因（30 mM）降低了分离的软骨细胞（p< 0.001）和原位软骨细胞（p <0.001）的细胞活力。通过用ROCK抑制剂或肌球蛋白-II抑制剂blebbistatin预处理细胞来减弱软骨毒性（p< 0.001）。这些结果表明，利多卡因诱导ROCK依赖性膜起泡，从而对软骨细胞产生细胞毒性作用。© 2015骨科研究学会。出版社：Wiley Periodicals，Inc. J Orthop Res 34：754-762，2016.
Local anesthetics are administered intraarticularly for pain control in orthopedic clinics and surgeries. Although previous studies have shown that local anesthetics can be toxic to chondrocytes, the underlying cellular mechanisms remain unclear. The present study investigates acute cellular responses associated with lidocaine‐induced toxicity to articular chondrocytes. Rabbit articular chondrocytes were exposed to lidocaine and their morphological changes were monitored with live cell microscopy. The viability of chondrocytes was evaluated using a fluorescence based LIVE/DEAD assay. Acute treatment of chondrocytes with lidocaine (3–30 mM) induced spherical protrusions on the cell surface (so called “membrane blebbing”) in a time‐ and concentration‐dependent manner. The concentration‐response relationship for the lidocaine effect was shifted leftward by elevating extracellular pH, as expected for the non‐ionized lidocaine being involved in the bleb formation. ROCK (Rho‐kinase) inhibitors Y‐27632 and fasudil completely prevented the lidocaine‐induced membrane blebbing, suggesting that ROCK activation is required for bleb formation. Caspase‐3 levels were unchanged by 10 mM lidocaine (p =0.325) and a caspase inhibitor z‐VAD‐fmk did not affect the lidocaine‐induced blebbing (p= 0.964). GTP‐RhoA levels were significantly increased (p< 0.001), but Rho inhibitor‐1 failed to suppress the membrane blebbing (p= 0.875). Lidocaine (30 mM) reduced the cell viability of isolated chondrocytes (p< 0.001) and in situ chondrocytes (p <0.001). The chondrotoxicity was attenuated by pretreatment of cells with ROCK inhibitors or a myosin‐II inhibitor blebbistatin (p< 0.001). These findings suggest that lidocaine induces ROCK‐dependent membrane blebbing and thereby produces a cytotoxic effect on chondrocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:754–762, 2016.