Protective Effect Of Hydrogen Sulfide On Bupivacaine-induced Cell Damage In N2a Cells

Local anesthetics are widely used in clinical anesthesia.However when high concentrations or long-term local anesthetics act on nerve tissue,it can cause neurotoxic damage and even lead to serious neurological complications.It is of great significance for clinical work to study the neurotoxicity mechanism and prevention of local anesthetics.Bupivacaine is a commonly used local anesthetic in clinical practice.Bupivacaine induces neurotoxicity through numerous mechanisms including apoptosis and autophagy.Autophagy is an intracellular clearance pathway that helps remove damaged organelles and misfolded or aggregated proteins.Hydrogen sulfide is the third gas signal molecule discovered after nitric oxide and carbon monoxide.Endogenous H₂S is involved in a series of physiological and pathological processes in the body.Many studies have found that H₂S has neuroprotective effects.However studies on whether hydrogen sulfide can alleviate local anesthetic-induced neurotoxicity have not been reported.In this study,we constructed a model of bupivacaine-induced N2a neurotoxicity in vitro,and observed the protective effect and possible mechanism of hydrogen sulfide on bupivacaine-induced cell damage in N2a cells.Part 1 Protective effect of H₂S on bupivacaine-induced cell damage in N2a cellsObjective To establish bupivacaine-induced N2a cells injury model in vitro,and to explore the protective effect of H₂S on bupivacaine-induced cell damage.Method N2a cells were treated with different concentrations of bupivacaine（0,100,300,600,900,1200μM）for 48 h,the cell viability was detected by CCK-8assay,and the morphological changes of cells were observed by microscope.N2a cells were treated with different concentrations of H₂S donor Na SH（0,100,200,400,800μM）for 48 h,and the cell viability was detected by CCK-8 assay to observe whether Na SH had an effect on cell survival.The experiment was divided into Control group,Bup group（300μM）and Bup+Na SH（100,200,400,800μM）group.The cell viability was detected by CCK-8 assay,and the morphological changes of cells were observed by microscope.The experiment was divided into Control group,Bup group（300μM）,Na SH group（400μM）and Bup+Na SH group.Hoechst 33342 staining was used to observe the morphological changes of apoptosis.The apoptosis rate was detected by flow cytometry.The expressions of apoptosis-related proteins Bax and Bcl-2 were detected by Western blot.Result 1.The results of CCK-8 assay showed that different concentrations of bupivacaine reduced the cell viability,and the difference was statistically significant（P<0.05）.Microscope observation found that different concentrations of bupivacaine caused different degrees of morphological damage to cells,and its cell damage was concentration-dependent.2.After different concentrations of Na SH acted on cells for 48 hours,the results of CCK-8 assay showed that different concentrations of Na SH had no significant effect on the cell viability（P>0.05）.3.The results of CCK-8 assay showed that the cell viability in the Bup group was significantly lower than that in the control group（P<0.05）.There was no significant difference in cell viability after 100μM Na SH pretreatment compared with the Bup group（P>0.05）,while 200,400,and 800μM Na SH significantly increased the cell viability（P<0.05）.Microscopy showed that the morphological damage of cells exposed to bupivacaine after Na SH treatment showed varying degrees of improvement.4.Hoechst 33342 staining showed that some cells in the Bup group had characteristic changes of apoptosis,and the number of apoptotic cells in the Bup+Na SH group was significantly less than that in the Bup group.Flow cytometry showed that the apoptosis rate of Bup group was significantly higher than that of Control group（P<0.05）,while the apoptosis rate of Na SH pretreated cells was significantly lower than that of Bup treatment group alone（P<0.05）.The results of Western blot analyses showed that the expression of the proapoptotic protein Bax in the Bup group was significantly increased（P<0.05）,and the expression of the antiapoptotic protein Bcl-2 was significantly decreased compared with the Control group（P<0.05）.The expression of Bax in the Bup+Na SH group was lower than that in Bup group（P<0.05）,the expression of Bcl-2 was higher than that in the Bup group（P<0.05）.Conclusion 1.The toxic damage of bupivacaine in N2a cells was dose dependent.2.100～800μM H₂S donor Na SH has no toxic effect on N2a cells.3.H₂S pretreatment improved bupivacaine-induced cytotoxicity and increased the cell viability.4.H₂S attenuated bupivacaine-induced cytotoxicity via inhibition of apoptosis in N2a cells.Part 2 H₂S attenuated bupivacaine-induced cell damage in N2a cells by enhancing autophagic fluxObjectiveTo explore whether the mechanism of H₂S attenuated bupivacaine-induced cell damage in N2a cells is related to the regulation of autophagy.Method N2a cells were treated with different concentrations of bupivacaine（0,100,300 and 600μM）for 48 h,and the expression of key protein molecules Beclin-1,LC3-I,LC3-II and p62 in the process of autophagy were detected by Western blot.The experiment was divided into Control group,Bup group（300μM）,Na SH group（400μM）and Bup+Na SH group.The expression of Beclin-1,LC3-I,LC3-II,and p62 were detected by Western blot.The level of LC3 and p62 was detected by immunofluorescence,and the autophagosomes were observed by transmission electron microscope.The experiment was divided into Control group,Bup group（300μM）,Bup+Na SH group（400μM）and Bup+Na SH+3-MA group.The expression of Beclin-1,LC3-I,LC3-II,and p62were detected by Western blot.LC3 and p62 expressions were detected by immunofluorescence,intracellular autophagosomes were observed by transmission electron microscope,morphological changes of apoptosis were observed by Hoechst 33342 staining,apoptosis rate was detected by flow cytometry,and Bax and Bcl-2 were detected by Western blot.Morphological changes of cells were observed by microscope,and cell viability was detected by CCK-8 assay.Result 1.After 100,300 and 600μM of bupivacaine treated cells for 48 h,the expression of Beclin-1 had no significant difference compared with the Control group（P>0.05）.100μM Bup groups made LC3-II/LC3-I ratio and p62 no difference compared with the Control group（P>0.05）.The ratio of LC3-II/LC3-I and the level of p62 were significantly increased in 300 and 600μM Bup groups compared with the Control group（P<0.05）.2.There was no significant difference in the protein expression of Beclin-1among the groups（P>0.05）.The ratio of LC3-II/LC3-I and the level of p62 in the Bup group were higher than those in the Control group（P<0.05）.The ratio of LC3-II/LC3-I in the Bup+Na SH group was higher than that in the Bup group（P<0.05）,while the expression of p62 in the Bup+Na SH group was lower than that in Bup group（P<0.05）.The fluorescence intensity of LC3 in Bup group was significantly stronger than that in the Control group（P<0.05）.The fluorescence intensity of LC3 in Bup+Na SH group was higher than that in Bup group（P<0.05）.The fluorescence intensity of p62 in Bup group was significantly stronger than that in Control group（P<0.05）.The fluorescence intensity of p62in Bup+Na SH group was lower than that in Bup group（P<0.05）.The autophagosomes in each treatment group were observed by transmission electron microscope.There were almost no autophagosome-like microstructures in the cells of the Control group and Na SH group,while the autophagosomes in the Bup group were significantly increased.Autophagosomes in the Bup+Na SH group were less than those in the Bup group.3.The results of Western blot showed that compared with the Bup+Na SH group,the expression of Beclin-1 and the ratio of LC3-II/LC3-I were decreased,and the level of p62 was increased in the Bup+Na SH+3-MA group（P<0.05）.The fluorescence intensity of LC3 in the Bup+Na SH group was higher than that in the Bup group（P<0.05）.The fluorescence intensity of LC3 in the Bup+Na SH+3-MA group was lower than that in the Bup+Na SH group（P<0.05）.The fluorescence intensity of p62 in the Bup+Na SH group was lower than that in the Bup group（P<0.05）.The fluorescence intensity of p62 in the Bup+Na SH+3-MA group was higher than that in Bup+Na SH group（P<0.05）.The autophagosomes in the Bup+Na SH group were less than that in the Bup group,while the autophagosomes in the Bup+Na SH+3-MA group were less than those in the Bup+Na SH group.4.Hoechst 33342 staining showed that the number of apoptotic cells in the Bup+Na SH group was significantly less than that in the Bup group,and the number of apoptotic cells in the Bup+Na SH+3-MA group was significantly higher than that in the Bup+Na SH group.Flow cytometry was used to detect the apoptosis rate of cells in different groups.The apoptosis rate of cells pretreated with Na SH was significantly lower than that in the Bup group（P<0.05）.The apoptosis rate of Bup+Na SH+3-MA group was significantly higher than that in the Bup+Na SH group（P<0.05）.The results of Western blot showed that compared with the Bup group,the expression of Bax was decreased and the level of Bcl-2 was increased in the Bup+Na SH group（P<0.05）.The expression of Bax in Bup+Na SH+3-MA group was higher than that in Bup+Na SH group（P<0.05）,and the expression of Bcl-2 was lower than that in Bup+Na SH group（P<0.05）.5.Microscope observation showed that the morphological damage in the Bup+Na SH group was significantly improved than that in the Bup group.The cells in the Bup+Na SH+3-MA group showed more cell damage than the Bup+Na SH group.The CCK-8 assay showed that the cell viability in the Bup+Na SH group was significantly higher than that in the Bup group（P<0.05）.The cell viability in Bup+Na SH+3-MA group was significantly lower than that in Bup+Na SH group（P<0.05）.Conclusion 1.Bupivacaine inhibited the autophagic flux in N2a cells.2.H₂S attenuated bupivacaine-induced cell damage in N2a cells by enhancing autophagic flux.Part 3 H₂S enhanced autophagy by inhibiting the PI3K/AKT/m TOR signaling pathwayObjective To explore the role of the PI3K/AKT/m TOR signaling pathway in H₂S-enhanced autophagy.Method The experiment was divided into Control group,Bup group（300μM）,Bup+Na SH group（400μM）and Bup+Na SH+740 Y-P group.After treating cells according to different groups for 48 h,Western blot was used to detect the protein expressions of PI3K,p-PI3K,AKT,p-AKT and m TOR,p-m TOR and autophagy-related proteins Beclin-1,LC3-I,LC3-II and p62 expression levels.Result 1.There was no significant difference in the ratios of p-PI3K/PI3K,p-AKT/AKT,and p-m TOR/m TOR between the Bup group and the Control group（P>0.05）.The ratios of p-PI3K/PI3K,p-AKT/AKT,and p-m TOR/m TOR in the Bup+Na SH group were significantly lower than those in the Control group（P<0.05）.Compared with the Bup group,the ratios of p-PI3K/PI3K,p-AKT/AKT,and p-m TOR/m TOR were decreased in the Bup+Na SH group（P<0.05）.After using the PI3K agonist 740 Y-P to activate the PI3K/AKT/m TOR pathway,the ratios of p-PI3K/PI3K,p-AKT/AKT,and p-m TOR/m TOR in the Bup+Na SH+740 Y-P group were significantly higher than those in the Bup+Na SH group（P<0.05）.2.Compared with the Bup+Na SH group,the expression of Beclin-1 and the ratio of LC3-II/LC3-I were significantly decreased,and the level of p62 was increased in the Bup+Na SH+740 Y-P group（P<0.05）.Conclusion H₂S enhanced autophagy by inhibiting the PI3K/AKT/m TOR signaling pathway.