| In recent years,with the wide application of ultrasound-guided nerve block,the clinical use of local anesthetics has been increasing.However,studies have shown that local anesthetics produce direct toxic effects on nerve cells,which may lead to related neurological complications and even devastating effects.Therefore,this study selected edaravone,a neuroprotective drug,to explore its antioxidant and anti-apoptotic effects in bupivacaine toxicity model and its relationship with Nrf2/HO-1 signaling pathway,so as to provide theoretical and experimental basis for the protective mechanism of bupivacaine toxicity.Part Ⅰ Protective effect of Edaravone pretreatment on toxicity induced by bupivacaine in N2 a cellsSection 1: Establishment of an N2 a cell injury model induced by bupivacaine in vitroObjective To observe the toxicity to N2 a cells injury induced by bupivacaine at various concentration.Methods Incubate N2 a cells with bupivacaine at various concentration levels for 24 hours,and observe cell morphological changes with inverted phase contrast microscope;use the CCK-8 method and the flow cytometer to check the effect on N2 a cell viability and changes in apoptosis rate.Results With the increasing concentration of bupivacaine,morphology tests show that N2 a cells appear wrinkled,rounded,synoptically contracted and shedded etc.Using the CCK-8 method,the cell viability in the 0.4m M,0.8m M,1.2m M and 1.6m M bupivacaine concentration groups progressively decreases 24 hours after incubation compared with the control group(P<0.05).The apoptosis rate,detected by a flow cytometry,increases significantly 24 hours after incubation in the 0.8m M,1.2m M and 1.6m M bupivacaine concentration groups(P < 0.05).Compared with the control group,after N2 a cells with 0.8m M bupivacaine being incubated for 24 hours,the N2 a cell viability decreased to(57.0±5.2)%,and the apoptosis rate increased significantly to(20.1±2.2)%.Conclusion Bupivacaine is toxic to N2 a cells,and its toxicity is dosedependent.Section 2: Protective effect of Edaravone on toxicity of N2 a cells injury induced by bupivacaineObjective The purpose of this study was to clarify whether Eda has antioxidative stress and anti-apoptotic effects on the toxicity of bupivacaine.Methods In this experiment,we used different concentrations(0.01,0.1,1,10 μM)of Eda to pretreat N2 a cells for 24 hours,and added the medium containing 0.8 m M bupivacaine(BUP)to incubate the cells for 24 hours.The effects of different concentrations of Eda pretreatment on N2 a cell viability and cell morphology were detected by CCK-8 method and inverted phase contrast microscope.1μM Eda was selected for subsequent modeling.The cells were divided into four groups,Control group,Eda group,BUP group,and BUP+ Eda group.Changes of mitochondrial membrane potential were detected by JC-1fluorescence probe;the apoptosis rate and reactive oxygen species(ROS)level of cells in each group were detected by flow cytometry;The changes of apoptosis pathway marker proteins Bcl-2,Bax and Casepase-3 were detected by Western blot;the content of oxidative stress index malondialdehyde(MDA)and the activity of antioxidant index superoxide dismutase(SOD)were detected by microplate reader colorimetry.Results We used the CCK-8 method to detect N2 a cell viability,and it shows that the cell viability in the bupivacaine groups were significantly inhibited.In the Eda pretreatment groups with concentration of 0.1,1,and 10 μM,the N2 a cell viability significantly improved compared with the bupivacaine groups(P<0.05).Compared with the Bupivacaine groups,the morphology of N2 a cells in the Eda pretreatment groups at concentrations of 0.01 and 0.1μM under light microscope did not change significantly.In the Eda pretreatment groups with concentrations of 1 and 10μM,the cell morphology of N2 a has been significant improved comparing to the bupivacaine groups.The Eda pretreatment group with1μM concentration was selected for subsequent modeling.The results showed that compared with the bupivacaine group,when N2 a cells were pretreated with Eda at a concentration of 1 μM for 24 h,the mitochondrial membrane potential was significantly increased,the apoptosis rate was significantly decreased,and the expression of apoptosis-related protein Bcl-2 was increased.and the expressions of Bax and Casepase-3 were significantly decreased.Meanwhile,after pretreatment of Eda,the level of ROS was significantly decreased,the content of MDA was significantly decreased,and the activity of SOD enzyme was increased(P<0.05).Conclusion Edaravone pretreatment has a protective effect on BUPinduced toxicity in N2 a cells,which may be related to edaravone antagonizing BUP-induced oxidative stress and apoptosis on N2 a cells.Part Ⅱ The mechanism of edaravone pretreatment to alleviate bupivacaine-induced toxicitySection 1: Effect of edaravone on Nrf2 and HO-1 expression in N2 a cellsObjective To investigate the effect of different concentrations of Eda on Nrf2/HO-1 signaling pathway in N2 a cells.Methods N2 a cells were incubated with Eda at different concentrations(0.01,0.1,1,10μM)for 24 h according to experimental groups.The changes of Nrf2 and HO-1 proteins in and outside the nucleus were detected by Western blot.Meanwhile Nrf2 nuclear migration and HO-1 protein level were detected by immunofluorescence.Results Compared with the control group,Eda coule activate Nrf2,and Western blot showed that 1 and 10μM Eda up-regulated Nrf2 expression in nucleus,accompanied by the up-regulated HO-1 protein expression in a dosedependent manner(P<0.05).Immunofluorescence results also showed that Eda promoted Nrf2 to enter the nucleus and increased HO-1 expression.Conclusion Eda promoted Nrf2 entry into N2 a cells and increased HO-1protein expression.Section 2: Eda alleviates bupivacaine toxicity through Nrf2/HO-1 signaling pathwayObjective To clarify whether the protective effect of Eda on bupivacaine toxicity is related to Nrf2/HO-1 signaling pathway,and to explore its relationship with antioxidant stress.Methods To study the expression of Nrf2 and HO-1 proteins in each group,the logarithmic phase cells were divided into four groups: Control group,Eda group,BUP group and BUP+Eda group.The changes of Nrf2 and HO-1 proteins in and outside the nucleus were detected by Western blot.Nrf2 nuclear inward migration and HO-1 protein level were detected by immunofluorescence method.To study the relationship between the antioxidant capacity of Eda and Nrf2/HO-1 signaling pathway,cells were divided into 4 groups: Control group,BUP group,BUP+Eda group,ML385+BUP+Eda group.The changes of Nrf2,HO-1,Bcl-2,Bax and Casepase-3 proteins were detected by Western blot.Cell viability of each group was detected by CCK-8 method.The ROS level and apoptosis rate of each group were detected with flow cytometry.The content of oxidative stress index MDA and the activity of antioxidant index SOD were detected by colorimetry.Results Western Blot and immunofluorescence showed that compared with the control group,Nrf2 expression in nucleus and HO-1 protein expression increased in BUP group.Compared with BUP group,Eda promoted Nrf2 to enter the nucleus and increased HO-1 expression in bupivacaine toxicity model(P<0.05).Compared with BUP group,the expression levels of nucleus Nrf2,HO-1and Bcl-2 were significantly increased in BUP+Eda group,while the expression levels of Bax and Caspase 3 were significantly decreased.The cell activity significantly increased,the oxidative stress index of ROS and MDA contents decreased,SOD activity increased,apoptosis rate decreased(P<0.05).After using Nrf2 inhibitor ML385,compared with BUP+Eda group,the expression levels of nucleus Nrf2,HO-1 and Bcl-2 were significantly decreased in ML385+BUP+Eda group,while the expression levels of Caspase 3 and Bax were significantly increased.The cell activity decreased significantly,the oxidative stress index of ROS and MDA contents increased,the activity of SOD decreased,and the apoptosis rate increased(P<0.05).Conclusion Eda pretreatment could reduce the toxic effect of bupivacaine in N2 a cells,and its protective mechanism may be related to the activation of Nrf2/HO-1 signaling pathway,inhibition of oxidative stress and improvement of apoptosis rate. |
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