| Part Ⅰ Protective effect of pretreatment with GM1 on bupivacaine-induced neurotoxicity in N2 a cellsSection 1: Establishment of N2 a cell injury model induced by bupivacaine Objective: To establish a bupivacaine induced N2 a cell injury model in vitro,and to observe the neurotoxic effect of bupivacaine on N2 a cells.Methods: N2 a cells were incubated with different concentrations of bupivacaine for different time periods,and the morphological changes of N2 a cells were observed by contrast inversion microscope.CCK-8 assay was used to detect the time-concentration effect of bupivacaine on N2 a cell viability.The apoptosis rate of N2 a cells in each group was detected by flow cytometry.Results: With the increasing concentration of bupivacaine and the prolongation of action time,the morphology of N2 a cells appear wrinkled,rounded,synaptic contraction or fracture.CCK-8 assay was used to detect N2 a cell viability.Meawhile,the cell viability decreased gradually with the increase of bupivacaine concentration and the prolongation of action time when compared with the control group.The cell viability in 400 μM,800 μM,1200 μM and 1600 μM bupivacaine groups decreased significantly after incubation for 12,24 and 36 hours,which were statistical significance when compared with the control group(P < 0.05).Flow cytometry was used to detect the apoptosis rate of N2 a cells.The apoptosis rate of N2 a cells increased with the increase of bupivacaine incubation concentration.The apoptosis rate of N2 a cells incubated with 800 μM bupivacaine for 24 h was significantly higher than that of the control group,the difference was statistically significant(P < 0.05).With the increase of bupivacaine incubation concentration,the apoptosis rate of N2 a cells increased significantly.Conclusion:The toxicity of bupivacaine on N2 cells was time-dependent and dose-dependent.In this study,N2 A cells were treated with 800 μM bupivacaine for 24 hours to establish an experimental model of bupivacaine neurotoxicity for subsequent studies.Section 2: To establish the protective model of GM1 against bupivacaineinduced N2 a cytotoxicityObjective: To explore the protective effect of GM1 on bupivacaine,and to screen the best protective concentration of GM1 against bupivacaine neurotoxicity.Methods: According to the results of the previous experiment,N2 a cells were pretreated with different concentrations of GM1,and then incubated with medium containing 800μM bupivacaine for 24 h.The morphological changes of cells were observed by contrast inversion microscope.N2 a cell viability was detected by CCK-8 assay.The apoptosis rate of N2 a cells in each group was detected by flow cytometry.Results: The morphological changes of N2 a cells in 0.1 μM and 1 μM GM1 pretreatment groups were not significant compared with those in bupivacaine group.With the increase of GM1 concentration,the morphological changes of N2 a cells in 10 μM and 20 μM GM1 pretreatment groups were significant compared with those in bupivacaine group,and the polymorphism of N2 a cells and their synapses were still can been observable.CCK-8 assay showed that the N2 a cell viability was significantly inhibited in bupivacaine group.After pretreatment with 0.1 μM and 1 μM GM1,the cell viability in bupivacaine group recovered compared with that in bupivacaine group,but the difference was not statistically significant.With the increase of GM1 concentration,the activity of N2 a cells in the 10μM GM1 pretreatment group was significantly improved compared with that in the bupivacaine group(P < 0.05),and the concentration of GM1 was further increased to 20 μM,but the activity of N2 a cells did not further increase with the increase of GM1 concentration.N2 a cells were pretreated with GM1 at a concentration of 10 μM for 24 h,and then incubated with 800 μM bupivacaine for 24 h.Cell apoptosis rate then was detected by flow cytometry.The results showed that the bupivacaine-induced apoptosis rate of N2 a cells pretreated with 10 μM GM1 was significantly lower than that in the bupivacaine group(P < 0.05).Conclusion:In a certain dose range,GM1 pretreatment can effectively reduce the apoptotic rate of N2 a cells and alleviate the neurotoxicity induced by bupivacaine in a certain dose range.Part Ⅱ: The mechanism of GM-1 pretreatment to alleviate bupivacaineinduced neurotoxicity in N2 a cellsSection 1 Bupivacaine on PERK/e IF2α/ATF4 signaling pathway of endoplasmic reticulum stressObjective: To investigate the role of endoplasmic reticulum stress PERK/e IF2α/ATF4 signaling pathway in bupivacaine-induced neurotoxicity.Methods: Neurotoxic injury model of bupivacaine was established according to experimental grouping,and the changes of ER signaling pathway proteins included PERK,p-PERK,p-e IF2α and ATF4 in each group were detected by Western blot.The apoptotic rate of each group was detected by flow cytometry Results: In the bupivacaine group,the protein levels of PERK was not significantly increased,but the levels of p-PERK,p-PERK/PERK,p-e IF2α and ATF4 were significantly increased when compared with the control group(P <0.05).When the cells were pretreated with endoplasmic reticulum inhibition inhibitor(4-PBA)and PERK specific inhibitor(GSK2656157),and then incubated with bupivacaine,the results showed that the levels of pPERK/PERK,p-e IF2α and ATF4 protein were significantly lower than those in BUP group(P < 0.05).The relative expression of ATF4 protein was determined by immunofluorescence,and the results were consistent with Western blot.Annexin V-FITC/PI double staining method was used to detect the apoptosis rate of each group.The apoptosis rate of bupivacaine group was significantly increased when compared with the control group(P < 0.05).However,when the endoplasmic reticulum stress was inhibited by 4-PBA or GSK pretreatment,and then the cells were incubated with bupivacaine,the apoptosis rate was decreased,there were statistical significance compared with the bupivacaine group(P < 0.05).Conclusion: Bupivacaine can increase the phosphorylation levels of endoplasmic reticulum transmembrane signaling protein PERK,and increase the expression levels of p-e IF2α and ATF signaling pathways,cause the increase of cell apoptosis rate.Section 2 GM-1 alleviate bupivacaine-induced neurotoxicity by affecting the PERK/e IF2α/ATF4 signaling pathwayObjective: To clarify whether the protective effect of GM1 on bupivacaine is related to endoplasmic reticulum stress,and to explore the role of PERK/e IF2α/ATF4/CHOP signaling pathway in mediating apoptosis pathway.Methods: According to experimental groups,the neurotoxic injury model of bupivacaine was established in N2 a cells.The expressions of ER signaling pathway proteins included PERK,p-PERK,e IF2α and ATF4 were detected by Western blot.Meanwhile,the changes of cell apoptosis pathway markers proteins included CHOP,caspase-3,Bax,and Bcl-2 were also detected.The level of pPRRK was detected by immunofluorescence.Results:The protein expressions of p-PERK/PERK,p-e IF2α and ATF4 in bupivacaine group were significantly up-regulated,and the differences were statistically significant when compared with those in control group and GM1group(P < 0.05).After pretreatment with GM1,the cells then were incubated with bupivacaine.The results showed that the levels of p-PERK /PERK,p-e IF2α and ATF4 proteins were significantly lower than those in the group treated with bupivacaine alone(P < 0.05).In addition,the expression levels of pro-apoptotic proteins CHOP,caspase-3,and Bax were significantly up-regulated in bupivacaine group,while the expression of anti-apoptotic protein Bcl-2 was decreased when compared with the control group(P < 0.05).While pretreatment with GM1,the results showed that the expression of pro-apoptotic proteins CHOP,caspase-3 and Bax decreased significantly compared with the bupivacaine group alone(P < 0.05),and the expression of Bcl-2 was significantly increased in the GM1 pretreatment group with statistical significance(P < 0.05).However,the anti-apoptotic effect of GM1 on bupivacaine was completely reversed when the ER stress response was activated by the ER activator TM.Conclusion: The protective mechanism of GM-1 against bupivacaine-induced cellular neurotoxicity may be related to its inhibition of ER stress response,The PERK/e IF2α/ATF4 signaling pathway mediated the endoplasmic reticulumapoptosis plays important role in this process. |
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