Protective Effect Of Monosialo Four Hexose Gangliosides' Pretreatment On Bupivacaine-induced N2a Cellular Neurotoxicity

PURPOSE This experiment is taking neuroblastoma N2a cells cultured in vitro of mouse as researching mode, discussing protective effect and possible mechanism of action of Monosialo Four hexose gangliosides' on bupivacaine-induced N2a cellular neurotoxicity after pretreatment. This experiment will provide a theoretical basis and experimental data for the clinicaltreatment of the potential neurotoxicity induced by bupivacaine, to find a more effective treatment.METHOD All experiment are taking neuroblastoma N2a cells cultured in vitro of mouse as researching modes.(?) experimental group:(1) in Experiment 1 : N2a cells were divided into the following groups: control group (Group C):N2a cells without any intervention and bupivacaine 0 ?mol / L;Bupivacaine group, N2a cells were cultured with different concentration of bupivacaine 300, 600, 900, 1200, 1500, 2000?mol/L (B1, B2, B3, B4, B5 group).(2) in Experiment 2 : cells were divided into the following groups: control group (group A): bupivacaine injury and GM-1 intervention; Bupivacaine group(group B): bupivacaine minimum effective damage concentration and 24h of GM-1 protection; different concentrations of GM-1 preconditioning group(group BG): in front of the concentration of bupivacaine minimum effective damage 24 hours in advance in culture solution containing different concentrations of GM-1 0.001,0.01,0. 1,1,10 mol / L (BG1, BG2, BG3, BG4 and BG5 group).(?)To observe the effects of different concentrations of bupivacaine on N2a cells,and to find the most .suitable model for the establishment of cell damage.(1) Evaluation index: apoptosis rate(2) Procedure: N2a cells acted with different concentrations (0, 600, 900,1500, 2000 mol/L) of Bup respectively, 24h, 12h, 6h and 36h, then to evaluate the cell survival rate with CCK-8 method. Each experiment has been repeated 3 times.(?) To clarify whether GM-1 has a protective effect on Bup induced N2a cellular injury.(1) Evaluation index: cytomorphology, cell apoptosis rate, nucleus shrinkage.(2) Procedure: after N2a cells have been pretreated with different concentrations (0, 0.001, 0.1, 10 mol/L) GM-1 for 24h add Bup (900 mol/L) to continue to have acted for 12 h, to establish good experimental model. The cell morphological changes were observed under optical microscope; Cell survival rate was evaluated by CCK8 method. The apoptosis rate was detected by flow cytometry annexin V / PI double staining method. Hoechst 33258 staining method was used to detect the pyknotic nuclei shrinkage.(?) To describe the possible protective mechanism of Bup on GM-1-induced N2a cell damage.(1) Evaluation index: expression levels of mRNA and protein of apoptosis factors—Caspase-3, Caspase-8 , Caspase-9.(2) Procedure: RT-PCR and Westernblot methods were used to determine the expression levels of mRNA and protein of genes of injured neurons of caspase-3 csapase-8,caspase-9 in different concentrations of GM-1. Each experiment has been repeated for 3 times.RESUIT (1) CCK-8 test results show that Bup can reduce the survival rate of N2a. The higher and the longer the time of the Bup concentration is, the stronger the neurotoxic effect is, with of double dosage and time dependence.The time of the lowest effective concentration of bupivacaine induced N2a neuronal damage is determined by 900 ?mol/L which has been cultured for 12h.(2) GM-1 has a significant protective effect on the nerve injury of Bup, and its protective effect is related to the dose. The maximum protection concentration of this experiment is 10 mu mol/L. Optical microscopy group BUP induced N2acell morphology change obviously, cell survival rate decreased significantly,apoptosis rate and cell nuclei pyknotic rates increased significantly. The results of the index showed that the control and GM-1 pretreatment group, the apoptosis rate and cell nucleus pyknotic rates are significantly lower than bupivacaine injury group; control group lower than that of GM-1 pretreatment group.(3) The neurotoxicity induced by bupivacaine is related to the apoptosis of mitochondrial pathway. Pretreatment with GM-1 can inhibit the neurotoxicity induced by bupivacaine. Control group and Bupivacaine group and GM-1 pretreatment protection group were observed in the cells of Caspase-3 and caspase-9 mRNA and protein expression, but the expression level of each group cells are correspondingly different; BUP group mRNA and protein expression was higher than that of the control group and GM-1 pre treatment protecting groups (P < 0. 05), The expression of mRNA and protein content is below and GM-1 pre treatment protecting groups (P < 0.05); compared with the control group,GM-1 pretreatment groups between mRNA and protein expression, the difference is statistically significant (P < 0.05). There was no significant change in the expression of caspase-8 in control group, Bup group and GM-1 pretreatment group (P>0.05).CONCLUSION: Bupivacaine will injure N2a cell. Dose and timing were positively correlated. GM-1 has significant protective effect on BUP nerve injury, which can be seen in the expression of Caspase-3 and caspase-9 of apoptotic factors when BUP induced nerve cell damage.