罗哌卡因对人软骨细胞C28/I2的损伤诱导作用观察及机制探讨

Effect of ropivacaine on human chondrocyte C28/I2 injury induction and its mechanism

目的体外实验观察罗哌卡因对人软骨细胞C28/I2的损伤诱导作用,并探讨相关机制。方法培养C28/I2细胞并分为对照组、实验1组、实验2组、实验3组。实验1、2、3组分别给予0.25、0.50、1.00 mmol/L的罗哌卡因处理48 h;对照组不添加药物。采用CCK-8法检测细胞活力,光学显微镜下观察细胞形态,检测线粒体活性氧(ROS)水平和线粒体膜电位,采用Western blotting法检测细胞中的自噬相关蛋白(LC3、p62)及铁死亡相关蛋白[谷胱甘肽过氧化物酶4(GPX4)、重组人铁蛋白重链1(FTH1)]。结果各实验组细胞存活率低于对照组,且实验1、2、3组细胞存活率依次下降(P均<0.05)。对照组细胞呈多边形、三角形等不规则形态,细胞体积大,具有人软骨细胞的结构及特征;各实验组随着罗哌卡因作用浓度增加,软骨细胞数量逐渐减少,细胞形态逐渐变得细长,胞质减少,部分细胞呈球状、皱缩,凋亡、坏死细胞数量增多。实验3组细胞线粒体ROS水平高于对照组,实验1、2、3组细胞线粒体ROS水平依次升高(P均<0.05)。对照组、实验1组、实验2组、实验3组LC3-Ⅱ/LC3-Ⅰ依次增加,线粒体膜电位及p62、FTH1、GPX4蛋白表达依次下降(P均<0.05)。结论罗哌卡因可诱导软骨细胞发生损伤,降低细胞活力,破坏细胞形态,增强氧化应激,导致线粒体功能障碍,且作用呈剂量依赖性;罗哌卡因对软骨细胞的损伤诱导作用可能与上调细胞自噬水平和诱导铁死亡有关。

Objective In vitro experiments were conducted to observe the injury-inducing effects of ropivacaine on human chondrocytes C28/I2 and to investigate the related mechanisms.Methods C28/I2 cells were cultured and divid⁃ed into the control group,experimental group 1,experimental group 2,and experimental group 3.Cells in the experimen⁃tal groups 1,2 and 3 were treated with 0.25,0.50 and 1.00 mmol/L of ropivacaine for 48 h,respectively;no drug was added to the cell in the control group.Cell viability was detected by CCK-8,cell morphology was observed under optical microscope,and mitochondrial reactive oxygen species(ROS)levels and mitochondrial membrane potential were detect⁃ed.Autophagy-related proteins(LC3,p62)and ferroptosis-related proteins[glutathione peroxidase 4(GPX4)and ferri⁃tin heavy polypeptide 1(FTH1)]were detected by Western blotting.Results The cell survival rates of the experimental groups were lower than that of the control group,and the cell survival rate of the experimental groups 1,2 and 3 decreased successively(all P<0.05).In the control group,the cells showed irregular shape,such as polygon and triangle,and the cells were large,with the structure and characteristics of human chondrocytes.With the increase of ropivacaine concentra⁃tions in all groups,the number of chondrocytes gradually decreased,cell morphology gradually became elongated,cyto⁃plasm decreased,some cells were spherical and shrunken,and the number of apoptotic and necrotic cells increased.The level of mitochondrial ROS in the experimental group 3 was higher than that in the control group,and the level of mitochondrial ROS in the experimental groups 1,2 and 3 increased gradually(all P<0.05).The ratios of LC3-Ⅱ/LC3-Ⅰin the control group,experimental group 1,experimental group 2 and experimental group 3 increased successively,while the mi⁃tochondrial membrane potential and protein expression levels of p62,FTH1 and GPX4 decreased successively(all P<0.05).Conclusions Ropivacaine can induce chondrocyte injury,reduce cell vitality,destroy cell morphology,en⁃hance oxidative stress,and lead to mitochondrial dysfunction in a dose-dependent manner.The effect of ropivacaine on chondrocytes may be related to the up-regulation of autophagy and the induction of ferroptosis.