title: Ropivacaine- and bupivacaine-induced death of rabbit annulus fibrosus cells in vitro: involvement of the mitochondrial apoptotic pathway.
abstract: OBJECTIVE
  The purposes of this study were to assess whether local anesthetics (LAs), such as ropivacaine and bupivacaine, could induce apoptosis of rabbit annulus fibrosus (AF) cells in vitro and further to explore the possible underlying mechanism.
  
  
  METHODS
  Rabbit AF cells at second passage were treated with saline solution and various concentrations of LAs. Apoptosis of AF cells were examined by cell counting kit-8 (CCK-8), Annexin V assays, Hoechst 33342 staining, and Caspase-3, -9 activity assays. The expression of apoptosis-related markers was detected by real-time PCR (RT-PCR) and Western Blot. The JC-1 staining was used to evaluate the change of mitochondrial membrane potential (MMP). Moreover, the levels of reactive oxygen species (ROS) were determined with fluorescent probe DCFH-DA.
  
  
  RESULTS
  The results of flow cytometry indicated that LAs could induce apoptosis of rabbit AF cells in a dose-dependent manner. Apoptosis was confirmed by cell morphology, condensed nuclei and activation of Caspase-3 and -9. In addition, the molecular data showed that LAs could significantly up-regulate the expression of Bax, accompanied by a significant down-regulation of Bcl-2 expression. Furthermore, we also observed that LAs resulted in alteration of MMP and accumulation of intracellular ROS in AF cells. Blockade of ROS production by N-acetyl-l-cysteine (NAC) inhibited LAs-induced apoptosis.
  
  
  CONCLUSIONS
  These findings suggest that LAs in clinically relevant concentrations could induce apoptosis of rabbit AF cells in vitro, and the mitochondrial pathway was, at least in part, involved in the LAs-mediated apoptosis. Further investigations focusing on the potential cytotoxicity of LAs on IVD cells are needed.
authors.affiliation: Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Electronic address: xianyi_cai@hust.edu.cn.
authors.forename: X-Y
authors.initials: XY
authors.lastname: Cai
authors.affiliation: Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Electronic address: shining1010@sina.cn.
authors.forename: Y
authors.initials: Y
authors.lastname: Xia
authors.affiliation: Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Electronic address: shuhua_yang@yeah.net.
authors.forename: S-H
authors.initials: SH
authors.lastname: Yang
authors.affiliation: Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Electronic address: liuxianzhe@gmail.com.
authors.forename: X-Z
authors.initials: XZ
authors.lastname: Liu
authors.affiliation: Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Electronic address: zwspro@126.com.
authors.forename: Z-W
authors.initials: ZW
authors.lastname: Shao
authors.affiliation: Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Electronic address: sdbzyxylyl@126.com.
authors.forename: Y-L
authors.initials: YL
authors.lastname: Liu
authors.affiliation: Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Electronic address: Yangwenaya@hotmail.com.
authors.forename: W
authors.initials: W
authors.lastname: Yang
authors.affiliation: Department of Orthopaedic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. Electronic address: tony_xiong@hotmail.com.
authors.forename: L-M
authors.initials: LM
authors.lastname: Xiong
mesh_terms: D000577:Amides / Q000494:pharmacology*; D000779:Anesthetics, Local / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002045:Bupivacaine / Q000494:pharmacology*; D053148:Caspase 3 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D053453:Caspase 9 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects*; D015536:Down-Regulation / Q000187:drug effects; D066298:In Vitro Techniques; D007403:Intervertebral Disc / Q000166:cytology / Q000187:drug effects*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D000077212:Ropivacaine
chemical_list: D000577:Amides; D000779:Anesthetics, Local; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D000077212:Ropivacaine; D053148:Caspase 3; D053453:Caspase 9; D002045:Bupivacaine
keywords: Apoptosis; Bupivacaine; Intervertebral disc; Mitochondria; Ropivacaine