罗哌卡因通过抑制STAT3的磷酸化诱导PC12细胞线粒体自噬

Ropivacaine induces mitophagy of PC12 cells via inhibiting phosphoryla⁃tion of STAT3

目的:体外实验探究罗哌卡因对大鼠嗜铬细胞瘤PC12细胞线粒体自噬的作用及其对信号转导及转录激活因子3(STAT3)磷酸化的影响。方法:PC12细胞被随机分为对照组和罗哌卡因处理(0.5、1、2、4和8 mmol/L)组,CCK-8实验测定细胞活力,光学显微镜下观察细胞形态,通过线粒体活性氧和线粒体膜电位来评估罗哌卡因对细胞线粒体功能的影响,通过免疫荧光和Western blot实验比较不同处理组中细胞的线粒体自噬水平以及STAT3和p-STAT3的蛋白水平。结果:与对照组比较,罗哌卡因处理24 h后PC12细胞的活力显著下降(P<0.01),呈剂量依赖性;同时光学显微镜下可以观察到细胞形态的改变:细胞变圆,突起减少,坏死凋亡细胞数量增加。与对照组相比,罗哌卡因组PC12细胞中的线粒体活性氧显著增加,线粒体膜电位显著下降,呈剂量依赖性。进一步比较不同处理组中细胞线粒体自噬水平,结果表明罗哌卡因促进了PC12细胞线粒体自噬,包括增加线粒体与自噬体间的共定位,调节线粒体自噬相关蛋白的表达。Western blot和免疫荧光的结果显示,与对照组比较,罗哌卡因组中p-STAT3的蛋白水平显著降低(P<0.01),呈剂量依赖性,以2 mmol/L罗哌卡因组差异最显著。结论:罗哌卡因通过促进PC12细胞的线粒体自噬来引发细胞损伤,其机制可能与抑制细胞中STAT3的磷酸化有关。

AIM:To investigate the role of ropivacaine in the mitophagy level and signal transducer and acti-vator of transcription 3(STAT3)phosphorylation in rat pheochromocytoma PC12 cells in vitro.METHODS:The PC12 cells were randomly divided into control group and ropivacaine(0.5,1,2,4 and 8 mmol/L)groups.The cell viability was measured by CCK-8 assay,the morphological changes of the cells were observed under optical microscope,and the mitochondrial function was evaluated by detection of mitochondrial reactive oxygen species and mitochondrial membrane potential.Finally,immunofluorescence and Western blot were used to detect the mitophagy level and the protein levels of STAT3 and p-STAT3 in the PC12 cells.RESULTS:Compared with control group,the PC12 cell viability was significant-ly decreased after ropivacaine treatment for 24 h in a dose-dependent manner(P<0.01).The morphological changes of the cells were observed after treatment with ropivacaine,including that the cells became round,the protrusions were de-creased,and the number of necrotic and apoptotic cells was increased.The levels of mitochondrial reactive oxygen species were increased and the mitochondrial membrane potential was decreased in a dose-dependent manner in ropivacaine group.Ropivacaine also promoted the mitophagy of PC12 cells,including promotion of the fusion of mitochondria and autophagosomes,and regulation of the mitophagy-related protein expression.Finally,compared with control group,the pro-tein levels of p-STAT3 in ropivacaine group was significantly reduced in a dose-dependent manner(P<0.01),especially in 2 mmol/L group.CONCLUSION:Ropivacaine induces PC12 cell damage via promoting excessive mitophagy,which may be related to inhibiting the phosphorylation of STAT3.