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右美托咪定对缺氧/复氧诱导的A549细胞凋亡及caspase-12表达的影响

罗梓垠, 项冰倩, 高慧, 邱晓晓, 郝卯林, 王万铁

温州医科大学缺血-再灌注损伤研究所，温州 325035；湖北省中西医结合医院病理科，武汉 430015

摘要

本研究旨在探讨右美托咪定(dexmedetomidine, DEX)对缺氧/复氧(hypoxia/reoxygenation, H/R)诱导的A549细胞凋亡以及caspase-12表达的影响。取对数生长期的A549细胞，随机分为对照组、DEX组、H/R组和DEX+H/R组。对照组和DEX组细胞常氧培养30 h，H/R组和DEX+H/R组细胞缺氧6 h、复氧24 h建立H/R模型，其中DEX组和DEX+H/R组给予1 nmol/L DEX干预。造模结束后用倒置显微镜观察细胞形态学的变化；原位末端标记(TUNEL)法检测A549细胞的凋亡指数(apoptotic index, AI)；CCK-8法检测A549细胞活力；caspase-3活性检测试剂盒检测各组caspase-3酶的活性；Western blot和RT-PCR分别检测A549细胞GRP78、caspase-12蛋白和mRNA的表达水平。结果显示，与对照组相比较，H/R组细胞发生融合现象，形态呈多边型改变，细胞活力明显降低(P < 0.01)，凋亡细胞数增加，AI值升高(P < 0.01)；caspase-3酶活性上升(P < 0.01)；GRP78、caspase-12蛋白和mRNA表达显著上升(P < 0.01)。经DEX干预后，与H/R组相比较，DEX+H/R组细胞损伤减轻，细胞活力上调(P < 0.01)；凋亡细胞数减少，AI值显著下降(P < 0.01)，caspase-3酶活性显著下降(P < 0.01)；caspase-12蛋白和mRNA表达下降(P < 0.01)。本研究结果表明，DEX对H/R损伤时的A549细胞具有一定的保护作用，其机制可能与其下调caspase-12的表达、抑制细胞凋亡有关。

关键词： 右美托咪定; 肺缺氧/复氧损伤 ; A549细胞 ; 细胞凋亡; caspase-12

分类号：R363

Effects of dexmedetomidine on hypoxia/reoxygenation injury-induced cell apoptosis and caspase-12 expression in A549 cells

LUO Zi-Yin, XIANG Bing-Qian, GAO Hui, QIU Xiao-Xiao, HAO Mao-Lin, WANG Wan-Tie

Ischemia/Reperfusion Injury Research Institute of Wenzhou Medical University, Wenzhou 325035, China； Pathology Department of Hubei Provincial Hospital of Integrated Chinese &； Western Medicine, Wuhan 430015, China

Abstract

To investigate the effects of dexmedetomidine (DEX) on hypoxia/reoxygenation (H/R) injury-induced cell apoptosis and caspase-12 expression, A549 cells were randomly divided into 4 groups: control group, DEX group, H/R group and DEX+H/R group. Cells of control and DEX groups were cultured in the normoxic incubator for 30 h. Cells of H/R and DEX+ H/R groups were incubated in the anoxic cultivation for 6 h, followed by normoxic culture for 24 h, and DEX (1 nmol/L) was added into the culture medium in DEX and DEX+H/R groups. Morphological changes were observed under the inverted microscope. Cell viability was detected by CCK-8. The apoptosis index (AI) of A549 cells was detected by TUNEL method. The activity of caspase-3 enzyme in cells was detected by using caspase-3 kit. The expressions of GRP78, caspase-12 protein and mRNA were determined by Western blot and RT-PCR respectively. Compared with control group, the morphological changes of the cultured cells were observed: some of the cell fusion occurred and the shape of the cells was multilateral; the cell viability was decreased significantly (P < 0.01), the number of apoptotic cells and the AI value, caspase-3 activity, and the expressions of GRP78, caspase-12 protein/mRNA were significantly increased (P < 0.01) in H/R group. While the administration of DEX alleviated the H/R injury-induced cell damage, obviously increased the cell viability (P < 0.01), significantly decreased the increment of apoptotic cells and the AI value induced by H/R injury (P < 0.01), and also dramatically decreased the H/R injury-induced high level of caspase-3 activity (P < 0.01) as well as high expression of caspase-12 protein and mRNA (P < 0.01). Taken together, the results suggest that DEX can effectively protect A549 cells from the H/R injury, which may be mediated by down-regulating the expression of caspase-12 and inhibiting cell apoptosis.

Key words: dexmedetomidine; lung hypoxia/reoxygenation injury ; A549 cells ; cell apoptosis; caspase-12

收稿日期：2016-11-28　　录用日期：2017-05-18

通讯作者：郝卯林,王万铁　　E-mail: wwt@wmu.edu.cn, hml@wmu.edu.cn

引用本文：

罗梓垠, 项冰倩, 高慧, 邱晓晓, 郝卯林, 王万铁. 右美托咪定对缺氧/复氧诱导的A549细胞凋亡及caspase-12表达的影响[J]. 生理学报 2017; 69 (4): 437-444.

LUO Zi-Yin, XIANG Bing-Qian, GAO Hui, QIU Xiao-Xiao, HAO Mao-Lin, WANG Wan-Tie. Effects of dexmedetomidine on hypoxia/reoxygenation injury-induced cell apoptosis and caspase-12 expression in A549 cells. Acta Physiol Sin 2017; 69 (4): 437-444 (in Chinese with English abstract).