Research On ROS Burst And Its Targets In Bupivacaine-induced SH-SY5Y Cell Injury

In recent years, the ratio of anesthesiologists applying nerve block, intrathecal anesthesia, and postoperative analgesia increased year by year. But the neurological complications occurred in some patients after anesthesia. Pristine studies demonstrated that neurotoxicity of local anesthetic was positively correlated with dosage, concentration and action time. In vivo and in vitro experiments had showed that local anesthetic could make the nervous tissue engorgement, degeneration, demyelination, vacuolization and chromosome solution. However, the mechanism of neurotoxicity induced by local anesthetic was still unclear.Bupivacaine is one of the frequently used local anesthetic, and the reports about the toxicity reaction of nerves and cardiovascular system induced by bupivacaine increased gradually, which attract people's utmost attention. The studies have shown that bupivacaine may inhibit the complexⅠof the mitochondrial respiratory chain, uncouple oxidative phosphorylation, decrease the production of ATP, increase the mitochondrial permeability and collapse the mitochondrial membrane potential, leading to apoptosis. In addition, a study with a Schwann cell line demonstrated that bupivacaine could induce apoptosis mediated by an increase of intracellular reactive oxygen species (ROS) and activation of caspase-3 (intracellular kinase related to apoptosis). However, the mechanism underlying the production and release of ROS, increase of mitochondrial permeability and collapse of the mitochondrial membrane potential induced by bupivacaine is still unclear.AMP-activated protein kinase (AMPK) acts as a kind of serin/threonine protien kinase and is composed of a protein complex containing one catalyticα-subunit and two regulatory subunits (βandγ). AMPK that exists in eucaryotic cell generally regulates the energy metabolism and is activated following a rise in the AMP to ATP ratio. The activated AMPK kinase system can inhibit the constructive metabolism to reduce the expenditure of ATP, and promote atabolism to increase the production of ATP when the intracellular ATP decreases. Studies have established that the sustained AMPK activation can result in a marked increase of intracellular ROS that contributes to apoptosis. These evidences have formed the basis for our presumption that bupivacaine provokes AMPK activation by interfering oxidative phosphorylation, inhibiting the complex I of the mitochondrial respiratory chain and decreasing the production of ATP, and triggers the burst increase of intracellular ROS subsequently, leading to apoptosis.Cell apoptosis pathways consist of two signal transduction pathways. One is the death receptor pathway. The death receptor of cell surface binds the death ligand by the ectodomain, introduces the death signal into cell, and activates caspase-8 and caspase-3, leading to apoptosis. The other is mitochondria pathway. Stimulating factors, such as oxygen free radical and calcium overload, increase the permeability of mitochondria, decrease mitochondria membrane potential, release cytochrome C and apoptosis-inducing factor (AIF), and subsequently activate caspase-9 and caspase-3, leading to apoptosis. Cell apoptosis is related to the opening of mitochondrial permeability transition pore (mPTP). The mPTP is composed of voltage dependent anion channel (VDAC), adenine nueleotide translocator (ANT) and cyclophilin D (Cyp-D). The influx of chloridion (Cl-) into mitochondria can occur through the open VDAC and can cause mitochondrial membrane damage and collapse the membrane potential, leading to release of pro-apoptotic molecule such as cytochrome C and AIF. Accordingly, the increase in intracellular ROS induced by bupivacaine prompts the opening of VDAC, causing the influx of Cl- into mitochondria, increase of mitochondrial osmotic pressure, swell of matrix, decrease of mitochondrial membrane potential and augmentation of permeability, and release of pro-apoptotic factors, which leads to cell apoptosis.P38 mitogen-activated protein kinase (MAPK) is an important member of MAPK family. The p38 MAPK signaling pathway is activated by different stimuli associated with stress, such as H2O2, TNF-a, lipopolysaccharide and hyperosmotic fluid. Previous studies demonstrated that the p38 MAPK signaling pathway controls much gene expression activity of transcription factor, affects the production of many cytokines, and regulates the synthesis of NO and cytoskeleton protein. p38 MAPK participates in immunological regulation, inflammatory reaction and cell apoptosis under the circumstance of stress. Studies found that peripheral nerve toxicity of lidocaine is closely related to the activation of p38 MAPK. Lirk found that the p38 MAPK inhibitor 4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1H-imidazole (SB203580) can inhibit neurocyte axon degeneration. Researches showed that p38 MAPK may participate in the activation of pain signal factor and takes an important role in the formation and development of algesthesia sensitization of central nerve. Accordingly, it is presumed that p38 MAPK may be closely related to never injury induced by bupivacaine.In this study, the recombinant plasmids pEGFP-N1-AMPKα2 and pGPU6/GFP/Neo-shRNA AMPKa2 were transfected into SH-SY5Y cells in order to regulate the expression of AMPKa2 in SH-SY5Y cells. It is the aim to investigate whether bupivacaine induces the considerable increase of intracellular ROS and cell apoptosis by AMPK. On the other hand, the phospho-p38 MAPK (p-p38 MAPK) and mitochondria Cl- concentration were measured in bupivacaine-induced cell injury in order to identify the target of ROS with the interference of p38 MAPK inhibitor SB203580 and Cl- channel inhibitor 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid disodium (DIDS).Chapter 1 Construction and identification of pEGFP-N1-AMPKα2 expression vector targeting to human AMPKa2 geneObjective To construct pEGFP-N1-AMPKα2 expression vector and observe its up-regulation effect on human AMPKα2 gene expression in the SH-SY5Y cell line.Methods The special primers for AMPKα2 were designed according to human AMPKα2 gene. The human neuroblastoma cell line (the SH-SY5Y cell line) was cultured and the total RNA of cells was extracted. Gene AMPKα2 fragment were amplified, retrieved from agarose gel electrophoresis, purified and cloned into the pEGFP-N1 vector. The connective product was transfected into competence escherichia coli DH5a. The recombinant vector was extracted after amplification. The recombinant vector was confirmed by DNA sequencing and enzyme digestion analysis. The recombinant vector was transfected by lipofectamine into the SH-SY5Y cells. After the screening by G418, the expression levels of AMPKa2 mRNA and protein were detected by RT-PCR and Western blot.Measurement data were presented as mean±standard deviation. SPSS 13.0 software was used for statistical treatment. Optical density value was analyzed by one-way ANOVA and comparisons among groups were made by LSD (homogeneous variance) or Dunnett's T3 (heterogeneity of variance). P<0.05 was considered statistically significant.Results The total and integrity RNA of SH-SY5Y cell was obtained. The target gene was amplified at 1659 bp by RT-PCR. The target band retrieved from agarose gel was connected with pEGFP-Nl vector successfully. The recombinant expression vector pEGFP-N1-AMPKα2 was successfully constructed, which was confirmed by the DNA sequencing and the enzyme digestion analysis. The vector pEGFP-N1-AMPKα2 can significantly up-regulate the mRNA and protein expression of AMPKα2 effectively after transfection into the SH-SY5Y cell line, compared with the untransfected group (P<0.01).Conclusion pEGFP-N1-AMPKα2 expression vector was successfully constructed. The expression of AMPKα2 gene was up-regulated effectively in SH-SY5Y cells transfacted with pEGFP-N1-AMPKα2, which laid a basis for its application in the research of cell injury induced by bupivacaine.Chapter 2 Construction and identification of shRNA vector targeting to human AMPKα2 geneObjective To construct pGPU6/GFP/Neo-shRNA expression vector targeting to human AMPKαgene and to observe its silencing effect in the SH-SY5Y cell line.Methods The four oligonucleotides of shRNA interference sequence targeting to AMPKα2 were designed by Ambion on-line CAD software and a negative control oligonucleotide was designed. Double strands of shRNA were cloned into the pGPU6/GFP/Neo vector after positive-sense and antisense strands of shRNA were synthesized by chemical synthesis. The connective product was transfected into competence escherichia coli DH5a. The recombinant plasmids were screened from kanamycin plates and subsequently the kanamycin-resistant colonies were amplified and the recombinant plasmids were extracted. The recombinant vectors were measured by DNA sequencing after confirmed by enzyme digestion analysis. The recombinant plasmids were transfected by lipofectamine mediated transfection into the SH-SY5Y cell line. After the transfection and screening by G418, the expression levels of AMPKα2 mRNA and protein were detected by RT-PCR and Western blot.Measurement data were presented as mean±standard deviation. SPSS 13.0 software was used for statistical treatment. Optical density value was analyzed by one-way ANOVA and comparisons among groups were made by LSD (homogeneous variance) or Dunnett's T3 (heterogeneity of variance). P<0.05 was considered statistically significant.Results The four shRNA expression vectors, pGPU6/GFP/Neo-shRNA AMPKα2(1), pGPU6/GFP/Neo-shRNA AMPKα2(2), pGPU6/GFP/Neo-shRNA AMPKa2(3), pGPU6/GFP/Neo-shRNA AMPKα2(4) and a negative control vector pGPU6/GFP/Neo NC were successfully constructed, which was confirmed by the DNA sequencing and the enzyme digestion analysis. In the pGPU6/GFP/Neo-shRNA AMPKα2(3) group, the mRNA and protein expressions were significantly lower than those of the untransfected group and the negative control group after transfection with the four recombinant plasmids and the negative control plasmid into SH-SY5Y cells (P<0.01). pGPU6/GFP/Neo-shRNA AMPKα2(3) can inhibit the expression of AMPKα2 gene in SH-SY5Y cells effectively and the inhibition rate was 63%.Conclusion The four shRNA expression vectors targeting to AMPKα2 gene and a negative control vector were successfully constructed. The expression of AMPKα2 gene was inhibited effectively in SH-SY5Y cells transfacted with pGPU6/GFP/Neo-shRNA AMPKα2(3), which laid a basis for its application in the research of cell injury mediated by AMPK. Chapter 3 Effect of AMPK on ROS increase and apoptosis induced by bupivacaine in the SH-SY5Y cell lineObjective To investigate whether the activation of AMPK mediates the considerable increase of ROS and cell apoptosis induced by bupivacaine in the SH-SY5Y cell line.Methods Cells were divided into the pEGFP-N1-AMPKα2 group (AMPKα2 group), the pEGFP-N1 group (blank group), the pGPU6/GFP/Neo-shRNA AMPKα2 group (siAMPKα2 group), the pGPU6/GFP/Neo NC group (NC group) and the untransfected group (control group). AMPKα2 and phospho-AMPKa2 (p-AMPKα2) proteins were measured by western blot. Each group was treated with culture fluid containing 1 mmol/l bupivacaine. At 1,2,3,4 and 5 h after treatment with bupivacaine, intracellular ROS was measured by flow cytometry (FCM). At 24 h after bupivacaine treatment, cells of each group were collected, cell viability was measured by MTT, and apoptosis rate was determined by Hoechst33258 staining and FCM. Meanwhile, the same indicators were detected in the cells of each group untreated with bupivacaine at the same time.Measurement data were presented as mean±standard deviation. SPSS.13.0 software was used for statistical treatment. After plasmids transfection, AMPKa2 protein expression was analyzed by one-way ANOVA, and multiple comparisons tests were performed by LSD, and p-AMPKα2 protein expression was analyzed by corrected F analysis (Welch analysis), and multiple comparisons tests were performed by Dunnett's T3. After bupivacaine treatment, factorial design ANOVA was used to analyze the data from cells-survival rate assay, ROS measurement, p-AMPKa2 protein expression and apoptosis rate. Multiple comparisons tests were performed by LSD (homogeneous variance) or Dunnett's T3 (heterogeneity of variance). A probability value of P<0.05 was considered to be statistically significant.Results After transfection with plasmids, the AMPKα2 protein expression level of AMPKα2 group was significantly higher than that of the control group (P<0.01), and the AMPKα2 protein expression level of siAMPKα2 group was significantly lower than that of the control group (P<0.01). After bupivacaine treatment, the p-AMPKα2 protein expression level of AMPKα2 group was significantly higher than that of the control group (P<0.01), and the p-AMPKα2 protein expression level of siAMPKα2 group was lower than that of the control group (P<0.05). The intracellular ROS levels of each group increased and peaked at 3 h after lmmol/l bupivacaine treatment, and then gradually attenuated at 4 and 5 h (P<0.01). In addition, the ROS levels of AMPKα2 group dramatically increased as compared with those of other groups at 1-5 h, and the ROS levels of the siAMPKα2 group were lower than those of the blank, NC and control groups at 2,3 and 4 h, and the difference had statistical significance (P<0.01). The results of MTT assay showed that the survival rate of the AMPKα2 group was 38.92±4.76% and lower than that of the control group (52.46±2.93%) after bupivacaine treatment (P<0.01). The survival rate of the siAMPKα2 group was 65.80±4.10% and higher than that of the control group (P<0.01). FCM showed that the apoptosis rate of the AMPKα2 group was 40.22±3.11% and higher than that of the control group (31.76±3.73%) (P<0.01). The apoptosis rate of the siAMPKα2 group was 23.34±3.68% and lower than that of the control group (P<0.01). Hoechst33258 staining revealed that cells of AMPKα2 group presented as coalesced, condensed, and segmented nuclei with a brighter blue fluorescence, and the apoptosis rate of the AMPKα2 group was 42.82±1.96% and higher than that of the control group (32.57±3.18%) (P<0.01). The apoptosis rate of the siAMPKα2 group was 22.21±2.76% and lower than that of the control group and the difference had statistical significance (P<0.01). Conclusion Down-regulation of AMPKa2 expression attenuated ROS production and cell apoptosis induced by bupivacaine, and up-regulation of AMPKa2 expression promoted ROS production and cell apoptosis induced by bupivacaine. It is probably mediated by the activation of AMPK that bupivacaine induce the ROS burst production and cell apoptosis in SH-SY5Y cells.Chapter 4 The effects of bupivacaine on mitochondria and p38 MAPK in SH-SY5Y cellsObjective To explore the downstream targets of ROS after bupivacaine-induced increase in intracellular ROS by examining the effect of bupivacaine on mitochondria and p38 MAPK.Methods SH-SY5Y cells randomly divided into four groups:DIDS group, SB203580 group, DIDS+SB203580 group and non-pretreated group. Cells of DIDS group, SB203580 group and DIDS+SB203580 group were pretreated respectively with 50μmol/l DIDS,10μmol/l SB203580, and 50μmol/l DIDS plus 10μmol/l SB203580 30 min prior to the treatment with the culture medium containing 1 mmol/l bupivacaine. Cells of non-pretreated group treated with the culture medium. containing 1 mmol/l bupivacaine. At 3 h after bupivacaine treatment, intracellular ROS level was measured by FCM, the mitochondrial membrane potential was measured by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole-carbocyanide iodine (JC-1), the p38 and p-p38 MAPK protein was examined by western blots, and mitochondrial Cl- concentration was measured by laser scanning confocal microscope. At 24 h after bupivacaine treatment, the cell viability was examined by MTT assay, and cell apoptosis was examined by Hoechst33258 staining and FCM. Meanwhile, the same indicators were detected in the cells of each group untreated with bupivacaine at the same time. Measurement data were presented as mean±standard deviation. SPSS 13.0 software was used for statistical treatment. Factorial design ANOVA was used to analyze the data from ROS measurement, Cl-fluorescence intensity, mitochondrial membrane potential assay, cells survival rate assay, cell apoptosis rate assay, p38 and p-p38 MAPK protein expression. Multiple comparisons tests were performed by LSD (homogeneous variance) or Dunnett's T3 (heterogeneity of variance). A probability value of P<0.05 was considered to be statistically significant.Results After bupivacaine treatment, the ROS levels of DIDS group and DIDS +SB203580 group were lower than that of non-pretreated group (P<0.01). The results of fluorescence intensity measured by FCM showed that the ratios of mitochondrial membrane JC-1 polymer/monomer in DIDS group and DIDS+ SB203580 group were 0.74±0.04 and 0.76±0.05 respectively, which were higher than that of non-pretreated group (0.49±0.01). The difference had statistical significance (P<0.01). Mitochondrial Cl- concentration measured by laser scanning confocal microscope showed that the Cl- concentration of DIDS group and DIDS+ SB203580 group were lower than that of non-pretreated group (P<0.01). Western blot showed that the p-p38 MAPK protein expression of DIDS group and DIDS+ SB203580 group were lower than that of non-pretreated group (P<0.01). MTT assay revealed that the survival rates of DIDS and SB203580 groups were higher than that of non-pretreated group (P<0.01), but lower than that of DIDS+SB203580 group (P<0.05). Cells apoptosis rates of group DIDS, DIDS+SB203580, SB203580 and non-pretreated were 31.88±4.41%,26.51±3.56%,33.30±2.01% and 39.00±3.18%, which were measured by FCM after bupivacaine treatment. The apoptosis rate of SB203580 group was lower than that of non-pretreated group (P<0.01), but there were no distinct difference in ROS levels and the ratios of JC-1 polymer/monomer fluorescence intensity between the SB203580 group and non-pretreated group (P>0.05). Hoechst33258 staining assay revealed that the apoptosis rates of group DIDS, DIDS+SB203580, SB203580 and non-pretreated were 33.23±3.00%, 28.80±4.69%,34.74±4.24% and 43.05±5.26%.Conclusion These findings indicate that the mitochondrial VDAC channel and p38 MAPK pathway are implicated in bupicavaine-induced apoptosis. Bupivacaine-induced intracellular ROS increase has effects on the opening of mitochondrial VDAC channel and results in an influx of Cl- into mitochondria, which seems to be responsible for the mitochondrial depolarization, the increase in the permeability of the mitochondria and the p38 MAPK activation.