| The reported of nerve toxicity reaction increased gradually, which caused the clinical attaches great importance to. However, currently the exact mechanism of neurotoxicity of Local anesthetics (LA) cause has not been fully elucidated. Lvobupivacaine (LB) is a new, long-term amide LA, whose physical and chemical properties, anesthesia efficiency are similar to bupivacaine, but the toxicity of cardiovascular system and central nervous system (CNS) are less than than bupivacaine, has a wide application prospect, but the specific mechanism of neurotoxicity of LB is unclear. Severe neurotoxicity of LA characterized by cell necrosis, however, more and more evidences indicates that apoptosis has become the main mechanism of neural toxicity.Neuroblastoma is a kind of tumor derived from the neural crest of newborn, which is one of the most common solid tumor in children. But compared with the other human cancer, neuroblastoma is one of tumor which has the highest spontaneous decay rate. SH-SY5Y cells is a clone of SK-N.S H in human neuroblastoma cell line, which is a very useful model to study of nerve cell toxicity. Mitogen-activated protein kinases (MAPKs) family is made up of kinase of MAPK kinase kinase (mitogen activated protein kinase kinase kinase, MAPKKKs, MKKK), MAPK kinase (mitogen activated protein kinase kinase, MAPKKs, MKK) and its downstream kinases MAPKs. MAPKs family capable react well to a variety of cell growth factor and promote mitosis substances, conduct the extracellular signal from the cell surface to the nucleus, participate in stroma cells, growth factors, inflammatory response, and other important signal pathways, mediates growth, development, division, differentiation, apoptosis and cellular interactions between a variety of cellular processes. Jun N-terminal kinase (JNK) signaling pathway is one of important pathways of MAPKs, a large number of studies have shown that the activation of JNK pathway play an important role in the neuronal apoptosis process. The incentive of apoptosis induce MAPKKKs activate, and then activate MKK, the activation of MKK phosphorylates the downstream of the JNK, promote the expression of target genes, thereby induced apoptosis. The activation of MKK are required in JNK pathway activation, but how the incentive of apoptosis prompt MKK medium is not entirely clear. It has reported the activation of JNK pathway is involved in the neurotoxic mechanism of ropivacaine and bupivacaine, and bupivacaine can stimulate nerve cells ROS (Reactive oxygen species, ROS) outbreak, induce apoptosis, and the generation of ROS can activates JNK pathway, therefore we assume LB neurotoxic mechanism related to ROS and JNK pathway.Adenosine Monophosphate Activated Protein Kinase (AMPK) is a kind of silk/threonine protein kinase, activation of AMPK can promote catabolism, inhibiting anabolic pathways, in order to increase the production of Adenosine Triphosphate (ATP). Some scholars suggest that continued activation of AMPK in the cells can lead to the production of large numbers of ROS, which can lead to cell damage and apoptosis. But it is not clear whether LB induced neurotoxicity associated with the activation of AMPK activate ROS generation.We have found that AMPK could be related to neurotoxicity of LA, such as bupivacaine and ropivacaine, triggers intracellular ROS production and release in previous research, however, it is still unknown whether the increased of ROS induced apoptosis through activate downstream JNK pathway in nerve cells. At present domestic clinical research of LB has focused on the pharmacodynamic and pharmacokinetic studies, little research about its neurotoxic mechanism has been reported. Thereby we speculate that LB activated AMPK pathway, led to an increased ROS production activate MKKs, then activated downstream of the JNK pathway, lead to cell apoptosis.Above all, we treated SH SY5Y cells with LB for24h, then detect cell survival, the average fluorescence intensity of ROS and apoptosis rate, JNK protein and P-JNK protein; for further study, we applied the ROS inhibitors, NAC, and through the transfection of small interfering RNAs (siRNA), MKK4siRNA and MKK7siRNA, to determine the targets of ROS activates JNK, in order to determine how LB activate ROS increase. To prove how LB increase ROS, we transfect SH SY5Y cells with recombinant plasmid pEGFP-N1-AMPK a2which has been built and identification by our research group in the laboratory, To verify the above conjecture, research the intracellular ROS, cell viability and cell apoptosis rate and downstream of phosphorylation JNK protein changes in the high expression of AMPK a2and SH-SY5Y cells which stimulated by LB, and compared to the group has not been transfected and the empty plasmid group.Our test is intended to explore what is the mechanism and principle of LB cause nerve toxicity, provide theoretical basis for the safe medication of clinical anesthesia. Chapter1LB induce ROS generation and apoptosis in SH SY5Y cellObjective To explore the relationship of ROS level and apoptosis induced by LB in SH SY5Y cellsMethod We culture SH SY5Y cell in vitro, cells were divided into12groups, C group as the control group (without medication), L0.5～L L2.5group (treated by0.5,1,0.5,2,2.5mM LB culture medium respectively for24h) and N group(NAC pretreatment for1h), N0.5～N2.5group (after NAC pretreatment for1h, treated by0.5,1,1.5,2,2.5mM LB culture medium respectively for24h). Detect the cell vitality with CCK-8method, fluorescent detection by Hochest33258, detect intracellular average ROS fluorescence intensity and cell apoptosis rate by flow cytometry (FCM).Statistical The measurement data were record mean±standard deviation, SPSS13.0statistics software was used to analysis the date. Two factors factorial analysis of variance was used to analysis the cell vitality, cellular ROS level, cell apoptosis rate; the comparison between groups was analysis by single factor analysis of variance (one-way ANOVA) in single effect analysis, multiple comparisons was analysis using LSD method for the homogeneity of variance and Welch or Dunnett’s T3for the unequal variances were adopted when separate effect analysis as necessary. P<0.05for the difference was statistically significant.Results1. Cell viability We observed that a dose dependent decrease in cell survival using CCK-8after LB treated SH-SY5Y cell for24h, results show that difference statistical among L0.5～L2.5groups (F=907.093, P=0.000), and the cell survival rate of L2.5group was the lowest as (19.50±0.58)%; cell survival rate of N-N2.5group increased after NAC pretreated (F=14.346, P=0.000). 2. Hoechst33258dyeing SH SY5Y cells was observed highly agglutinated and showed strong blue fluorescence after treated with different concentrations of LB, the number of cells present strong blue fluorescence nucleus was most especially in the L2.0group, thus numbers of cells show strong blue fluorescence was reduced when SH-SY5Y cells preprocessed by NAC.3. The apoptosis rate There has a statistically significant among the groups treated by different concentration of LB (F=205.123, P=205.123), and apoptosis rate of L2.0group is highest as (24.07±.12)%. Apoptosis rate were reduced after ROS inhibitors of NAC pretreatment (F=25.047, P=0.004).4. The average fluorescence intensity of ROS There has a statistically significant among the groups treated by different concentration of LB (F=64.098, P=64.098), including that the average fluorescence intensity of ROS of L2.0is highest as2113.33±188.81; after5mM NAC pretreatment forl h, the mean fluorescence intensity of ROS reduced (F=100.423, P=0.023), including that the fluorescence intensity of N2.0is the highest, whose reduction is the minimum after NAC pretreatment (F=35.960, P=0.004)Conclusion LB can cause dose dependent cell survival rate decreased in SH SY5Y cells presented with LB and ROS dependent apoptosis, NAC can suppress cell necrosis and apoptosis induced by LB, cells apoptosis rate was the highest when LB is2mM.Chapter2LB induced SH SY5Y cell apoptosis through ROS/MKK7/JNK pathwayExperiment1The effects of MKK4siRNA and MKK7siRNA on cellular ROS and apoptosis caused by LB Objective To test the link between MKK and ROS in the process of SH SY5Y cells apoptosisMethod Screened the highest transfection efficiency of MKK4siRNA and MKK7siRNA through Q-PCR and detected expression of MKK4mRNA and MKK7mRNA after0,0.5,1.51,2,2.5mM LB treated24h respectivily.Then transfected MKK4siRNA or MKK7siRNA into the cell, concentration of LB was choose as2mM because of its the highest cell apoptosis rate (it have confirmed in the first chapter). The cell were divided into below:the control group (without transfection and drug treatment); NAC group (treated by5mM NAC alone for1h); MKK4siRNA group (transfected by MKK4siRNA); LB group (treated by2mM LB for24h); LB+NAC group (pretreated by5mM NAC and then treated by2mm LB for24h); LB+MKK4siRNA group (transfected with MKK4siRNA, and then treated by2mM LB for24h); LB+NAC+MKK4siRNA group (transfected by MKK4siRNA, pretreated by5mM NAC and then treated by2mm LB for24h). The cell which transfected with MKK7siRNA are divided into groups as MKK4siRNA. FCM detect the apoptosis rate and the mean fluorescence intensity of ROS.Statistical The measurement data were record mean±standard deviation, SPSS13.0statistics software was used to analysis the date. MKK4mRNA and MKK7mRNA level, cellular ROS level, cell apoptosis rate were analysis by single factor analysis of variance (one-way ANOVA); multiple comparisons was analysis using LSD method for the homogeneity of variance and Welch or Dunnett’s T3for the unequal variances were adopted when separate effect analysis as necessary. P<0.05for the difference was statistically significant.Results 1. Screening the MKK4siRNA and MKK7siRNA We screened the exact silence effects of the MKK4siRNA and MKK7siRNA respectively, with the MKK4mRNA expression of siRNA-MKK4-3group is0.06±0.10, MKK7mRNA expression of siRNA-MKK7-1group is0.63±0.01. The following experiments choice of siRNA were siRNA-MKK4-3and siRNA-MKK7-1.2. The expression of MKK4mRNA and MKK7mRNA The expression of MKK4mRNA showed no statistical difference among groups after treated by different concentrations of LB (F=0.661, P=0.661), however the expression of MKK7mRNA were statistically difference among each group (F=1799.050, P=0.000), MKK7mRNA expression quantity was highest as205.63±5.19when LB=2mM.3. The apoptosis rate Compared with control group, the apoptosis rate of MKK4siRNA group showed no statistical difference (P=0.601), there had no statistical difference between the LB group and the LB+MKK4siRNA group (P=1.000); compared with control group, there was statistically difference in MKK7siRNA group (P=0.005), and there was statistically difference between the LB group and LB+MKK7siRNA group (P=0.003).4. The average fluorescence intensity of ROS Compared with the control group, the MKK4siRNA group showed no significant difference (P=0.095), and there has no statistical difference between the LB group and the LB+MKK4siRNA group (P=0.171); compared with the control group, MKK7siRNA group has no significant difference (P=1.000), there was no statistical difference between the LB group and the LB+MKK7siRNA group (P=1.000), however, there was statistically difference between LB and LB+NAC group (P=0.015), as well as the LB group and the LB+NAC+MKK7siRNA group (P=0.024). Conclusion LB can activate ROS/MKK7pathways leading to SH SY5Y cells apoptosisExperiment2The effect of MKK4siRNA and MKK7siRNA on the JNK and P-JNK protein expression induced by LBObjective Detection the link of ROS, MKK and JNK in the process of SH-SY5Y cells apoptosis induced by LBMethod JNK and P-JNK protein expression were detected by western blotting after cells were treated by difference concentrations of LB. The cells after transfection of MKK4siRNA were divided into below:the control group (without transfection and drug treatment); NAC group (treated by5mM NAC alone for1h); MKK4siRNA group (transfected by MKK4siRNA); LB group (treated by2mM LB for24h); LB+NAC group (pretreated by5mM NAC and then treated by2mm LB for24h); LB+MKK4siRNA group (transfected with MKK4siRNA, and then treated by2mM LB for24h); LB+NAC+MKK4siRNA group (transfected by MKK4siRNA, pretreated by5mM NAC and then treated by2mm LB for24h). The cell which transfected with MKK7siRNA are divided into groups as MKK4siRNA. Western blotting detected P-JNK protein expression of each group.Statistical The measurement data were record mean±standard deviation, SPSS13.0statistics software was used to analysis the date. JNK and P-JNK protein expression were analysised by single factor analysis of variance (one-way ANOVA); multiple comparisons was analysis using LSD method, the homogeneity of variance and Welch or Dunnett’s T3for the unequal variances were adopted when separate effect analysis as necessary. P<0.05for the difference was statistically significant.Results 1. The expression of JNK and P-JNK protein The cells treated with different concentrations of LB for24h, the expression of JNK showed significant difference (F=685.027, P=685.027); compared with the control group, the JNK protein expression decreased when LB=0.5,1,1.5,2.52mM, P value are0.000; compared with the group of LB=2mM group, JNK protein expression is higher when LB=0.5,1,1.5mM, P value are0.000; the JNK protein expression is lowest when LB=2.5mM. The P-JNK protein expression of the cells treated with different concentrations of LB24h shows siginificant difference (F=1449.76, P=0.000); compared with LB group=0, compared with the LB=0group, the P-JNK protein expression increased when LB=0.5,1,1.5,2.52mM, P value are0.000; compared with the group of LB=2mM, P-JNK protein expression is lowerr when LB=0.5,1,1.5mM, P value are0.000; the P-JNK protein expression is low when LB=2.5mM. Above all, the level of JNK phosphorylation level is highest when LB=2mM.2.The expression of P-JNK after MKK4siRNA transfection After transfection of MKK4siRNA, multiple comparison showed, there is no statistical difference between the control group and MKK4siRNA group (P=1.000), LB group and the LB+MKK4siRNA groups (P=0.548), the protein expression levels of P-JNK of LB+NAC group is lower than the LB group.3.The expression of P-JNK after MKK7siRNA After transfection of MKK7siRNA, western blotting results showed statistically difference among groups (F=2209.632, P=2209.632), multiple comparison shows, there had significant statistical difference between the control group and MKK7siRNA group (P=0.000), LB+MKK7siRNA group was lower than LB group (P=0.000), in addition, the P-JNK expression of LB+NAC+MKK7siRNA group was lower than LB+NAC group (P=0.000) and LB+MKK7siRNA group (P=0.000). Conclusion LB can activate ROS/MKK7/JNK pathway resulting in SH SY5Y cell apoptosisChapter3AMPKa2can promote LB induced ROS increased and apoptosis in SH SY5Y cellsObject Discussed whether overexpression of Adenosine Monophosphate Activated Protein Kinase (AMPKa2) mediated LB induced SH-SY5Y cells apoptosis through ROS/MKK7/JNK pathway.Methods We transfected plasmid pEGFP-N1-AMPK a2and plasmid pEGFP-N1into SH-SY5Y cell lines respectively, cells are divided into groups as belows: the control group (no plasmid transfection); pEGFP-N1group (transfection of pEGFP-N1); PEGFP-N1-AMPK alpha2groups (transfection of plasmid pEGFP-N1-AMPK a2); The LB group (no plasmid transfection and then treated by2mM LB for24h); LB+pEGFP-N1group (transfection of plasmid pEGFP-N1and then2mM LB processed after24h); LB+pEGFP-N1-AMPK a2group (transfection of plasmid pEGFP-N1-AMPK a2and then2MMLB processed after24h). We detected the intracellular ROS levels and cell apoptosis rate using FCM, determined the cell vitality using CCK-8method, western blot method to detect identification of P-JNK expression level.Statistical The measurement data were record mean±standard deviation, SPSS13.0statistics software was used to analysis the date. AMPKa2, P-JNK protein expression, CCK-8method to detect the cell vitality, ROS, apoptosis rate were analysis by two factors factorial design analysis of variance, multiple comparisons was analysis using LSD method for the homogeneity of variance and Welch or Dunnett’s T3for the unequal variances were adopted when separate effect analysis as necessary. P<0.05for the difference was statistically significant. Results After transfected recombinant plasmid pEGFP-N1-AMPK a2, the AMPK a2expression was increased in SH SY5Y cells; after processed with2mM LB, compared with empty plasmid pEGFP-N1cells group and the group which was not transfected with plasmid, the intracellular ROS content, P-JNK protein levels and cell apoptosis rate increased, the cell viability reduced(P=0.000); there were no significant differences in the cell vitality, intracellular ROS content and apoptosis rate, P-JNK protein levels between the empty plasmid pEGFP-N1cell group and the group which was not transfected with plasmid (P>0.05).Conclusion Overexpression of AMPKa2can promote LB induced SH SY5Y cells apoptosis through ROS/MKK7/JNK pathway. |
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