Melatonin Attenuates Ropivacaine-induced Apoptosis By Inhibiting Excessive Mitophagy

Background:Melatonin,as an endogenous circadian indoleamine,is secreted from the pineal gland to regulate the circadian rhythm of the organism.Melatonin has been proven to have a wide range of biological functions,including anti-tumor,anti-inflammatory,anti-oxidant and neuroprotective effects.The neuroprotective effect of melatonin makes it to be a potential therapeutic for many nerve damages.But its effect on ropivacaine-induced neurotoxicity remains unclear.Objective:The aims of our research is to explore the impact and mechanism of melatonin on ropivacaine-induced neurotoxicity.Methods:Rat adrenal pheochromocytoma PC 12 cells and mouse hippocampal neuron HT22 cells were selected.After treatment with different concentrations of melatonin for 24h,cell viability was detected by the CCK-8 assay to explore the toxin of melatonin on PC 12 and HT22 cells.Based on our previous study,1mM ropivacaine was added to construct a model of local anesthetic induced neurotoxicity in vitro.Cell was pretreated with melatonin for 2h,then ropivacaine was added to co-culture for 24h,48h,and 72h.Then,the cell viability was detected by CCK-8 assay;the morphology was observed under an optical microscope;the protein expression of Bax,Bcl-2 and activated Caspase-3 were detected by Western-blot;cell apoptosis was measured by Hoechst 33258/PI staining and Caspase-3 detection kit;and the detection of mitochondrial reactive oxygen species and mitochondrial membrane potential were to evaluate the mitochondrial function.In addition,the levels of mitophagy were detected by Western-blot and immunofluorescence.Finally,in order to explore the relationship between the inhibition of melatonin on ropivacaine-induced apoptosis with mitophagy,cells were pretreated with rapamycin and 3-MA,and the levels of mitophagy and apoptosis were detected.Results:(1)The results from CCK-8 assay showed that melatonin was no toxic to PC12 cells and HT22 cells,melatonin pretreatment could significantly inhibit the decline of cell viability induced by ropivacaine in a concentration-dependent manner(p<0.05).and melatonin improved the cell morphology damaged from ropivacaine(3)Compared with the control group,ropivacaine promoted the protein expression of Bax,activated Caspase-3,and inhibited Bcl-2 levels.It increased the number of apoptotic and necrotic cells,and up-regulated the activity of Caspase-3 so that it induced apoptosis.But melatonin pretreatment inhibited the apoptosis from ropivacaine.(4)Compared with the control group,ropivacaine increased the mitochondrial reactive oxygen species and reduced the mitochondrial membrane potential.But melatonin pretreatment improved ropivacaine-induced mitochondrial dysfunction.(5)Compared with the control group,ropivacaine increased LC3II/LC3I ratio,promoted the protein expression of Beclinl,PINK1,and Parkin,and down-regulated p62,Tomm20,COXIV levels.It also increased the co-localization between autophagosome and mitochondria.But melatonin pretreatment inhibited ropivacaine-induced mitophagy.(6)Compared with the melatonin+ropivacaine group,rapamycin relieved the inhibition of melatonin on ropivacaine-induced mitophagy,thereby reducing the protective effect of melatonin on apoptosis;on the contrary,3-MA enhanced the effect of melatonin anti-mitophagy function,while increasing the cytoprotective effects of melatonin.Conclusion:Melatonin pretreatment attenuated ropivacaine-induced neurotoxicity,including improving cell viability and morphology,inhibiting cell apoptosis,and rescuing impaired mitochondria,which may be achieved by inhibiting excessive mitophagy.