Huang Fei, Li Shangrong, Gan Xiaoliang, Wang Ren, Chen Zhonggang
From the Department of Anaesthesiology, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China.
Eur J Anaesthesiol. 2014 Apr;31(4):219-24. doi: 10.1097/01.EJA.0000435059.98170.da.
Liver abnormalities are seen in a small proportion of patients following anaesthesia with sevoflurane.
To investigate whether the cytotoxicity of sevoflurane against rat liver cells was mediated by gap junction intercellular communications, and the effect of propofol on sevoflurane-induced cytotoxicity.
Experimental study.
The study was carried out in the central laboratory of The Third Affiliated Hospital, Sun Yat-sen University.
BRL-3A rat liver cells.
Immortal rat liver cells BRL-3A were grown at low and high density. Colony-forming assays were performed to determine clonogenic growth of these cells. To investigate the effect of oleamide and propofol on gap junction function, we measured fluorescence transmission between cells using parachute dye-coupling assays. Immunoblotting assays were performed to determine connexin32 and connexin43 expression.
Our colony formation assays revealed that, in low-density culture, sevoflurane caused no apparent inhibition of clonogenic growth of BRL-3A cells. In high-density culture, 2.2 to 4.4% sevoflurane markedly inhibited clonogenic growth of BRL-3A cells with 67.6 (0.34)% and 61.2 (0.17)% of the cells being viable, respectively (P = 0.003 vs. low-density culture), suggesting cell density dependency of sevoflurane-induced cytotoxicity. Our colony formation assays revealed that propofol markedly attenuated the suppression by sevoflurane of the clonogenic growth of BRL-3A cells (viability: propofol and sevoflurane, 91.5 (0.014)% vs. sevoflurane, 56.6 (0.019)%; P <0.01). Blocking gap junctions with 10 μmol l oleamide significantly attenuated 4.4% sevoflurane-induced suppression with a viability of 83.6 ± 0.138% (oleamide and sevoflurane vs. sevoflurane, P < 0.01). Immunoblotting assays further showed that propofol (3.2 μg ml) markedly reduced CX32 levels and significantly inhibited gap junctional intercellular communications as revealed by parachute dye-coupling assays. Values are mean (SD).
This study provides the first direct evidence that sevoflurane-induced cytotoxicity, which is mediated through gap junctions, is attenuated by propofol, possibly by its action on Cx32 homomeric or heteromeric complexes.
在一小部分接受七氟醚麻醉的患者中可观察到肝脏异常。
研究七氟醚对大鼠肝细胞的细胞毒性是否由缝隙连接细胞间通讯介导，以及丙泊酚对七氟醚诱导的细胞毒性的影响。
实验研究。
该研究在中山大学附属第三医院中心实验室进行。
BRL-3A大鼠肝细胞。
将永生大鼠肝细胞BRL-3A以低密度和高密度培养。进行集落形成试验以确定这些细胞的克隆生长情况。为研究油酰胺和丙泊酚对缝隙连接功能的影响，我们使用降落伞染料偶联试验测量细胞间的荧光传递。进行免疫印迹试验以确定连接蛋白32和连接蛋白43的表达。
我们的集落形成试验显示，在低密度培养中，七氟醚对BRL-3A细胞的克隆生长无明显抑制作用。在高密度培养中，2.2%至4.4%的七氟醚显著抑制BRL-3A细胞的克隆生长，细胞存活率分别为67.6(0.34)%和61.2(0.17)%(与低密度培养相比，P = 0.003)，提示七氟醚诱导的细胞毒性具有细胞密度依赖性。我们的集落形成试验显示，丙泊酚显著减轻了七氟醚对BRL-3A细胞克隆生长的抑制作用(存活率：丙泊酚和七氟醚组为91.5(0.014)%，七氟醚组为56.6(0.019)%;P <0.01)。用10μmol/l油酰胺阻断缝隙连接可显著减轻4.4%七氟醚诱导的抑制作用，存活率为83.6±0.138%(油酰胺和七氟醚组与七氟醚组相比，P <0.01)。免疫印迹试验进一步表明，丙泊酚(3.2μg/ml)显著降低CX32水平，并如降落伞染料偶联试验所示显著抑制缝隙连接细胞间通讯。数值为平均值(标准差)。
本研究提供了首个直接证据，表明七氟醚诱导的细胞毒性通过缝隙连接介导，丙泊酚可减轻这种毒性，可能是通过其对Cx32同聚体或异聚体复合物的作用。
