| Local anesthetics are commonly used for local anesthesia and pain therapy, but they have the potential neurotoxicity. With the wide application in nerve block anesthesia, reports increase gradually hat local anesthetics cause neurotoxicity, and clinical workers pay high attention to this phonemenon. Many research found that this neurotoxicity caused by local anesthetics cause was associated with cell apoptosis, but the exact mechanism is still not fully understood.Many factors can induce nerve cell apoptosis, such as DNA damage agent, peroxide, the adjustment of the phosphorylated proteins factor, steady state of calcium ions, nitric oxide, neurotrophic factor etc. Research found that the mitochondrial membrane function change, inner membrane potential disappeared and protease activation thing in the mitochondria released, and arouse all kinds of apoptosis related metabolic changes, then the apoptotic cells were induced to produce characteristic form change and DNA degradation.Burst of reactive oxygen species (ROS) is one of the main factors causing the nerve cells acute injury. ROS accumulation can affect oxidoreduction balance and damage the mitochondrial membrane in nerve cells. In addition, ROS can directly affect protein, lipid, nucleic acid and make them lose function. moreover, ROS can also be a messenger, further cause nerve damage. Study has showed that bupivacaine can induce intracellular ROS generation and cell apoptosis in Schwann cells. But whether that neurotoxicity induced by ropivacaine is associated with ROS generation and mediation is still not identified.In previous studies, Our team found bupivacaine may interfere with oxidative phosphorylation and inhibit the complex â… of the mitochondrial respiratory chain, decrease the production of ATP, lead to single phosphoric acid active adenosine protein kinase (AMP-activated protein kinase, AMPK) continuous activation, and cause the mitochondria create and release more ROS in the cells, then cause cell damage and apoptosis through the mechanisms of ROS-induced-ROS release, further study showed bupivacaine may induce nerve cell apoptosis by via p38MAPK and mitochondrial Cl-channel pathway.Ropivacaine, one of common amide local anesthetics, is similar with bupivacaine in physical and chemical properties, but theirs roles in cells are not exactly the same. Ropivacaine, a long-term and new type of amide local anesthetics, is less fat soluble than bupivacaine, and has rare heart toxicity. moreover, it is a suitable drug for fetal. Therefore, Ropivacaine is an important drug and gets a wide application, especially for the postoperative analgesia and obstetric anesthesia. But it still has the potential neurotoxicity. Thus, trying to understand neurotoxicity mechanisms of ropivacaine has an important significance in avoiding and reducing neurotoxicity complications induced by it. Whether the neurotoxicity induced by ropivacaine is the same as bupivacaine, Whether ropivacaine may also induce neurotoxicity through AMPK activation and ROS generation? There are not related studies that have been reported. If ropivacaine can cause ROS increased in nerve cells, are their functions similar between ropivacaine with bupivacaine? What are the main targets of ROS to induce cell apoptosis?These questions need a further research.ROS is an important factor inducing DNA damage. The mechanism of repairmen is complex after DNA damage, and it attracts more and more attention for DNA damage and repairmen directly related to a cellular structure and function recovery. It is still not clear that the repair mechanism in nerve cells after oxidative stress and DNA damage caused by local anesthetics. Previous study has found that8-oxygen guanine (8-oxoguanine,8-oxoG) is an oxidized product of DNA guanine (dG)in cells attacted by ROS. There are two pathways of8-oxoG mutation repair, one pathway is that OGG1(8-oxoG-DNA glycosylase) cut8-oxoG directly; Another pathway is initiated with the cutting of A (Adenine) paired with8-oxoG by MSH2/MYH repair enzymes, and ended with8-oxoG cutting by OGG1. The antioxidant defense system and DNA repair system can effectively resist the DNA damage caused by reactive free radicals. But if the damage factors overwhelm the body’s defense system, the DNA repair system can not repair all the accumulated mutations, the cellular apoptosis and the a variety of diseases may then occur.We assume that8o-xoG mutation caused by ROS is one of the probable mechanisms of never injury induced by ropivacaine, then DNA repair enzymes including hMYH,hOGG1and hMSH2. HOGG1may be activated and play an important role in repairment in order to resist the apoptosis of never cells.To sum up, we apply biology and molecular techniques to tell the difference of ROS lever and cell apoptosis caused by ropivacaine and bupivacaine in SH-SY5Y cells, construt overexpression AMPK cells and lower expression AMPK cells, then make a further research on whether AMPK is related with ROS production induced by ropivacaine. moreover, we use chlorine ion blockers DIDS and MAPK antagonists SB203580to pretreatm SH-SY5Y cells and observe if ropivacaine has an effect on p38MAPK and mitochondria, and determine if ropivacaine induces cell apoptosis via p38MAPK signaling pathways and chlorine ion channels. In addition, we will make a further study on discussing the functions of the DNA repair enzymes system after nerve damage and determine the mechanism of DNA repair system after nerve injury (including8-oxoG production and three DNA repair enzymes called hOGG1, hMYH and hMSH2enzyme), and provide theoretic basis on preventing neurotoxicity induced by ropivacaine and developing effective drug treatment for neurotoxicity. Objective To determine the relationship of ROS increase and cell apoptosis induced by ropivacaine and bupivacaine in the SH-SY5Y cell line.Methods SH-SY5Y cells treated with culture fluid containing3mmol/l ropivacai-ne or bupivacaine for1,2,3,4,5and6h, and intracellular ROS was detected by flow cytometry (FCM); following treatment with3mmol/l ropivacaine or bupivacaine for4h, cell viability and apoptosis rate were assessed.Measurement data were presented as mean±standard deviation, and were statistically analysed by SPSS17.0software. Repeated measurement double factor analysis of variance was used to analyse the intracellular ROS in cells treated with different drugs at different time. Completely random single factor analysis of variance was used to analyse cell viability and apoptosis rate, the comparisons between groups were performed by LSD. A probability value of P<0.05was considered to be statistically significant.Results The intracellular ROS levels of each group increased and peaked at4h(ROS levels of ropivacaine were146.07±4.68, ROS levels of bupivacaine were268.06±12.73) after3mmol/l ropivacaine or3mmol/l bupivacaine treatment, then gradually attenuated at5and6h (P<0.01). Compared with groups untreated with ropivacaine or bupivacaine treatment, the ROS levels of treated group markly increased at1-6h, and the difference had statistical significance (P<0.01).Meanwhile, the intracellular ROS levels of groups treated with ropivacaine were lower than those of the bupivacaine groups at1-6h, and the difference had statistical significance (P<0.01).Cell viability of groups treated with ropivacaine or bupivacaine for4h was lower than that of untreated groups, and the difference had statistical significance (P<0.01).while cell viability of ropivacaine group(60.91%±1.91%) higher than that of bupivacaine group(35.91%±1.66%), and the difference had statistical significance (P<0.01).The apoptosis rate of untreated groups was4.44%±0.63%, ropivacaine and bupivacaine groups were respectively36.34%±2.74%and64.19%±2.99%. Compared with untreated groups, the apoptosis rate ropivacaine and bupivacaine groups markedly increased and the difference had statistical significance (P<0.01). while cell apoptosis rate of ropivacaine group obviously lower than that of bupivacaine group, and the difference had statistical significance (P<0.01).Conclusion Either ropivacaine or bupivacaine promoted intracellular ROS production and cell apoptosis in SH-SY5Y cells. The intracellular ROS levers ropivacaine induced were lower than bupivacaine did in SH-SY5Y cells, moreover, cell apoptosis induced by ropivacaine was lower than bupivacaine.Objective To determine if activation of AMP-activated protein kinase (AMPK) is involved in ropivacaine-induced reactive oxygen species (ROS) production and apoptosis in human neuroblastoma cell line SH-SY5YMethods Plasmids pGPU6/GFP/Neo-shRNA AMPKα2and pEGFP-N1-AMPKa2were transfected into the SH-SY5Y cell line. Cells were divided into the pEGFP-N1-AMPKa2group (AMPKa2group), the pEGFP-N1group (blank group), the pGPU6/GFP/Neo-shRNA AMPKa2group (siAMPKα2group), the pGPU6/GFP/Neo NC group (NC group) and the untransfected group (control group). The expression of AMPKα2and phospho-AMPKα2(p-AMPKa2) were measured by western blot analysis. Each group was treated with culture fluid containing3mmol/l ropivacaine for4h. Following treatment with ropivacaine, intracellular ROS was detected by flow cytometry (FCM), cell viability was quantitatively determined by MTT colorimetry assay, and apoptosis was assessed by FCM and Hoechst33258staining. moreover, the same indicators were detected in the cells of each group untreated with ropivacaine at the same time.Measurement data were presented as mean±standard deviation, and were analysed by SPSS17.0software for statistical treatment. The expression of AMPKa2 protein in the cells with plasmids transfection was analyzed by one-way ANOVA, and multiple comparisons tests were performed by LSD, and p-AMPKa2protein expression was analysed by corrected F analysis (Welch analysis), and multiple comparisons tests between groups were performed by Dunnett’s T3. After ropivacaine treatment, factorial design ANOVA was used to analyse the data from cell viability, ROS lever, p-AMPKa2protein 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<0.05was considered to be statistically significant.Results Within the cells with plasmids transfection, the AMPKa2protein expression level of AMPKa2group was significantly higher than that of the control group (P<0.01), and the AMPKa2protein expression level of siAMPKa2group was significantly lower than that of the control group (P<0.01). After ropivacaine treatment, the p-AMPKa2protein expression level of AMPKa2group was significantly higher than that of the control group (P<0.01), and the p-AMPKa2protein expression level of siAMPKa2group was lower than that of the control group (P<0.01). The intracellular ROS levels of each group increased after ropivacaine treatment, moreover, the ROS levels of AMPKa2group were higher as compared with those of other groups, and the difference had statistical significance(P<0.01) but the ROS levels of the siAMPKa2group were lower than those of the blank, NC and control groups, and the difference had statistical significance (P<0.01). After ropivacaine treatment, MTT colorimetry assay showed that cell viability of the AMPKa2group was43.74%±2.27%,which was lower than that of the control group (53.81%±4.31%)(P<0.01). The cell viability of the siAMPKa2group was67.22%±2.51%and higher than that of the control group (P<0.01). FCM showed that the apoptosis rate of the AMPKa2group was46.65%±4.44%and higher than that of the control group (33.34%±2.80%)(P<0.01). The apoptosis rate of the siAMPKa2group was21.77%±2.83%and lower than that of the control group (P<0.01). Hoechst33258staining revealed that more cells of in AMPKa2group presented as coalesced, condensed, and segmented nuclei with a brighter blue fluorescence, and the apoptosis rate of the AMPKa2group was46.51%±4.94%and higher than that of the control group (33.84%±2.82%)(P<0.01). The apoptosis rate of the siAMPKa2group was22.70%±2.68%and lower than that of the control group, and the difference had statistical significance (P<0.01).Conclusion Up-regulation of AMPKa2expression promoted intracellular ROS production and cell apoptosis induced by ropivacaine, while down-regulation of AMPKa2expression attenuated intracellular ROS production and cell apoptosis induced by ropivacaine, AMPK probably mediates ROS production and cell apoptosis induced by ropivacaine in SH-SY5Y cells.Objective To investigate the effects of ROS burst induced by ropivacaine on mitochondrial chloride channel and p38MAPK. And to reveal the ropivacaine-induced cellular apoptosis pathways related with mitochondrial chloride channel and p38MAPK.Methods SH-SY5Y cells randomly divided into four groups:DIDS group, SB203580group, DIDS+SB203580group and non-pretreated group. Cells of DIDS group, SB203580group and DIDS+SB203580group were pretreated respectively with50μmol/l DIDS,10μmol/l SB203580, and50μmol/l DIDS plus10μmol/l SB20358030min prior to the treatment with the culture medium containing3mmol/l ropivacaine. Cells of non-pretreated group treated with the culture medium containing3mmol/l ropivacaine. At4h after ropivacaine treatment, intracellular ROS level was measured by FCM, the mitochondrial membrane potential was measured by5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazole-carbocyanide iodine (JC-1), the expression of p38and p-p38MAPK protein was examined by western blot analysis, and mitochondrial Cl-concentration was measured by laser scanning confocal microscope. At4h after ropivacaine treatment, the cell viability was examined by MTT assay, and cell apoptosis was examined by Hoechst33258staining and FCM. Meanwhile, the same indicators were detected in the cells of each group untreated with ropivacaine at the same time.Results After ropivacaine treatment, the ROS levels of DIDS group and DIDS+SB203580group 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-1polymer/monomer in DIDS group and DIDS+SB203580group were0.81±0.02and0.83±0.03respectively, which were higher than that of non-pretreated group (0.52±0.02). The difference had statistical significance (P<0.01). Mitochondrial Cl-concentration measured by laser scanning confocal microscope showed that the difference of Cl-are not statistical significance. Western blot showed that the p-p38MAPK protein expression of DIDS group and DIDS+SB203580group were lower than that of non-pretreated group (P<0.01). MTT assay revealed that the cell viability of DIDS and SB203580groups were higher than that of non-pretreated group (P<0.01), but lower than that of DIDS+SB203580group (P<0.01). Cell apoptosis rates of group DIDS, DIDS+SB203580, SB203580and non-pretreated were27.70%±3.71%,17.21%±4.20%,28.13%±3.59%and42.76%±4.77%, which were measured by FCM after bupivacaine treatment. The apoptosis rate of SB203580group 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-1polymer/monomer fluorescence intensity between the SB203580group and non-pretreated group (P>0.05). Hoechst33258staining assay revealed that the apoptosis rates of group DIDS, DIDS+SB203580, SB203580and non-pretreated were43.05%±5.26%,28.01%±3.08%,16.77%±4.41%,27.15%±2.54%å'Œand42.98%±5.69%。Conclusion Ropicavaine probably induce SH-SY5Y cells apoptosis via the mitochondrial VDAC channel and p38MAPK pathway. Ropicavaine induced intracellular ROS increase and made mitochondrial VDAC channel open, the mitochondrial membrane potential decreased, and the p38MAPK activation. While mitochondrial Cl-concentration seems to be not related with mitochondrial membrane potential and cell apoptosis induced by ropicavaine. Objective To determine the relationship of cell apoptosis and8-oxoG production induced by ropivacaine treatment in the SH-SY5Y cell line, and the relationship of8-oxoG production and DNA repair enzymes. investigate the mechanisms.To reveal whether8o-xoG mutation is the mechanism of cell injury caused by ropivacaine, and DNA repair enzymes including hMYH,hOGG1and hMSH2. HOGG1are activated and involved in DNA repairmen during cell injury caused by ropivacaine.Methods SH-SY5Y cells treated with culture fluid containing ropivacaine for4h, and move culture fluid containing ropivacaine and replace culture fluid without ropivacaine, take samples following replacement for0,3,6,12,24h, with fluorescent antibody mark8-oxoG, laser confocal microscope observe intracellular8-oxoG lever at different time point before and after ropivacaine treatment, The expression of hOGG1, hMYH and hMSH2were measured by western blot analysis at different time points before and after ropivacaine treatment. and apoptosis was assessed by FCM at different time points.Measurement data were presented as mean±standard deviation, and were statistically analysed by SPSS17.0software. Completely random single factor analysis of variance was used to analyse8-oxoG lever, the expression of DNA repair enzymes and cell apoptosis rate at different time points, the comparisons between groups were performed by LSD or Dunnett’s T3. A probability value of P<0.05was considered to be statistically significant.Results The variation tendency of8-oxoG at different time points were in coincidence with that of3DNA repair enzymes and apoptosis rates. In comparison of control group, the intracellular8-oxoG level increased significantly in cells after treated with ropivacaine. The peak level reached at0h after culture media was changed. And then the level of8-oxoG decreased time-dependently. DNA repair enzymes, hOGG1, hMYH and hMSH2were only weakly detected in cells before ropivacaine treatment. But the levels of three DNA repair enzymes increased markedly after treated with ropivacaine. The peak levels of the enzymes were also detected at0h after ropivacaine treatment and then decreased time-dependently. Similarly, cellular apoptosis rates increased at different time points after ropivacaine treatment. Apoptosis peaked at0h after ropivacaine treatment, and time-dependent decrease of cellular apoptosis was then detected subsequently.Conclusion8o-xoG mutation may be one of the probable mechanisms of cell injury caused by ropivacaine, and DNA repair enzymes including hMYH,hOGG1and hMSH2. HOGG1play an important role in cell DNA injury caused by ropivacaine. |
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