Laboratory of Physiology, Medical School, University of Ioannina, 45110, Ioannina, Greece.
Biol Trace Elem Res. 2012 Dec;150(1-3):460-6. doi: 10.1007/s12011-012-9492-8. Epub 2012 Sep 16.
Zinc (Zn), a cell-protective metal against various toxic compounds, is the key agent for neutral endopeptidase (NEP) functional structure. NEP is a zinc metalloenzyme which degrades endogenous opioids and is expressed in human keratinocytes (HaCaT). Ropivacaine, a widely used opiate local anaesthetic, exerts cell toxic and apoptotic effects against HaCaT cells. The aim of the present study is to investigate whether zinc modulates the effects of ropivacaine on proliferation, viability, apoptosis and NEP expression in HaCaT cells. To investigate the role of ropivacaine in NEP function, HaCaT cells overexpressing NEP were generated via cell transfection with plasmids carrying NEP cDNA. Ropivacaine's anti-proliferative effect was tested by Neubauer's chamber cell counting, and induction of cell death was demonstrated by trypan blue exclusion assay. Apoptosis due to ropivacaine was tested via DNA fragmentation and poly-ADP-ribose-polymerase (PARP) cleavage. NEP and PARP expression was performed by western blot analysis. Results showed that zinc (15 μΜ) inhibited proliferation and cell death induction by ropivacaine (0.5, 1 and 2 mM) (p < 0.05) as well as apoptosis induced by the drug (0.5 and 1 mM) in HaCaT cells. Ropivacaine (1.0, 2.0 and 5.0 mM) downregulated NEP expression in the presence of zinc (15 μΜ) while NEP overexpression enhanced ropivacaine's apoptotic effect. In conclusion, the abilities of zinc to inhibit the toxic and apoptotic effects of ropivacaine, to maintain NEP downregulation induced by the drug and, consequently, to enhance its anaesthetic result suggest that zinc may have a significant role in pain management and tissue protection.
锌（Zn）是一种抵抗各种有毒化合物的细胞保护金属，是中性内肽酶（NEP）功能结构的关键试剂。NEP 是一种锌金属酶，可降解内源性阿片肽，并在人角质形成细胞（HaCaT）中表达。罗哌卡因是一种广泛使用的阿片类局部麻醉剂，对 HaCaT 细胞具有细胞毒性和凋亡作用。本研究旨在探讨锌是否调节罗哌卡因对 HaCaT 细胞增殖、活力、凋亡和 NEP 表达的影响。为了研究罗哌卡因在 NEP 功能中的作用，通过转染携带 NEP cDNA 的质粒，生成过表达 NEP 的 HaCaT 细胞。通过 Neubauer 室细胞计数法测试罗哌卡因的抗增殖作用，并通过台盼蓝排除试验证明细胞死亡的诱导。通过 DNA 片段化和多聚（ADP-核糖）聚合酶（PARP）裂解测试罗哌卡因诱导的细胞凋亡。通过 Western blot 分析检测 NEP 和 PARP 的表达。结果表明，锌（15 μM）抑制罗哌卡因（0.5、1 和 2 mM）引起的增殖和细胞死亡诱导（p < 0.05）以及药物诱导的细胞凋亡。罗哌卡因（1.0、2.0 和 5.0 mM）在锌（15 μM）存在下下调 NEP 表达，而 NEP 过表达增强了罗哌卡因的凋亡作用。总之，锌抑制罗哌卡因的毒性和凋亡作用、维持药物诱导的 NEP 下调以及增强其麻醉效果的能力表明，锌在疼痛管理和组织保护中可能具有重要作用。
