Fas/FasL Mediated PC12 Cell Damage Induced By Ropivacaine

Objective: Local anesthetics are widely used in clinical anesthesia,but many studies have shown that most local anesthetics have potential neurotoxicity,mainly characterized by abnormal sensory and motor function.Apoptosis is a way of cell death controlled by genes,it plays an important role in maintaining the stability of the internal environment and the growth and development of cells,as well as in many pathological processes.Studies have shown that,the neurocomplications caused by local anesthetics is related to its neurotoxicity,and apoptosis is the main factor of neurotoxicity.The classical process of apoptosis is activated mainly through two pathways,intrinsic pathway(also known as mitochondrial pathway)and extrinsic pathway(also known as death receptor pathway).Previous studies show that mitochondrial pathway participates in the regulation of neuronal apoptosis induced by local anesthetics.However,it is not clear whether the death receptor pathway is involved.This topic will explore whether the Fas/FasL death receptor pathway is involved in the apoptosis of neurons induced by local anesthetics,so as to provide new evidence for further explore the mechanisms of local anesthetics’ neurotoxicity.Methods:To establish an in vitro model of neurotoxicity of local anesthetics,we treated the rat adrenal pheochromocytoma PC12 cells with different concentrations of ropivacaine at different time,and then carry out the following experiments.(1)The cell viability of cells was detected by CCK-8 assay.(2)The cell morphological changes were observed under optical microscope.(3)The necrosis was detected by Hoechst 33342/PI staining.(4)The apoptosis rate was detected by Annexin V-FITC/PI staining using flow cytometry.(5)The expression of Fas,FasL,caspase-3 and caspase-8 mRNA by qPCR(6)The expression of Fas,FasL and cleaved caspase-3 protein by immunofluorescence and western blot.Results:(1)Ropivacaine decreased the viability of PC12 cells,and there was a difference between different concentrations and time(P < 0.05).(2)The cells treated with ropivacaine exhibited round and shrunken shapes with the disappearance of neuritis.(3)The necrotic PC12 cells increased after ropivacaine treatment,and there was a difference between the groups(P < 0.05).(4)The apoptosis rate of PC12 cells increased after ropivacaine treatment,and there was a difference between the groups(P < 0.05).(5)The expression of Fas,FasL,caspase-3 and caspase-8 mRNA of PC12 cells increased after ropivacaine treatment,and there was a difference between the groups(P < 0.05).(6)The expression of Fas,FasL and cleaved caspase-3 protein of PC12 cells increased after ropivacaine treatment,and there was a difference between the groups(P < 0.05).Conclusion:The toxicity of ropivacaine on PC12 cell is concentration and time dependent,and the mechanism of inducing apoptosis of PC12 cells may be related with the up-regulation of Fas/FasL.