| With the wide application of local anesthesia, neurotoxicity and systemic toxicity reaction of local anesthetics have aroused more and more people’s attention. Especially peripheral nerve damage induced by local anesthetics, as will bring obvious adverse affect for the patient lives and production.In clinic, some patients have transient neurological syndrome manifested burning, radiating, convulsive and continuous pain after intravertebral anesthesia; and some patients even show cauda equina syndrome, one more serious disorder of nervous functions. Generally, nerve damage resulted by local anesthetics is related with the dose, concentration, and the time of the nerve exposure to the local anesthetics. The mechanisms of the nerve damage induced by local anesthetics remain unclear, it maybe related with many factors.Cell calcium overload is one of the important mechanisms of the local anesthetics nerve injury. Extracellular calcium ions can enter into cell through the voltage-dependent calcium ion channels or receptor-dependent calcium ion channels, activate cells calcium-dependent enzyme and apoptosis, and induce the nerve damage. At the same time, calcium ion entered into the cell can produce calcium induced calcium release(CICR), cause intracellular calcium overload, result in cell apoptosis and nerve damage. Another important mechanism of the local anesthetics nerve damage is the p38mitogen-activated protein kinase (p38MAPK). Extracellular calcium ions enter into cells, activate the calcium-dependent protease, further activate apoptosis signal-regulating kinase1(ASK1), induce p38MAPK activation, activate Ca2+/CaMK Ⅱ-ASK1-p38MAPK pathways, p38MAPK activation can induce Caspase-independent apoptosis.This study adopt SH-SY5Y cell model, detected the subtype of T-type calcium channel expression by western blotting and real-time PCR, and observed the changes of the T-type channel expression with lidocaine hydrochloride treatment for24h. Then we constructed pshRNA-CACNA1G-SH-SY5Y (RNAi2) with RNA interfere and measured the effects of lidocaine hydrochloride on the pshRNA-CACNA1G-SH-SY5Y (RNAi2) and the expression of p38MAPK to test the mechanism of p38MAPK related to the local anesthethics nerve injury.Chapter1Effects of lidocaine hydrochloride on the expression of Cav3.1in SH-SY5Y cellsExperiment1Expression of T-type calcium channel in SH-SY5Y cellsObjective To observe the mRNA and protein expression of the T-type calcium channel (Cav3.1, Cav3.2and Cav3.3) in SH-SY5Y cells and identify the subtype of the T-type calcium channel in SH-SY5Y cells.Method We cultured SH-SY5Y cells in vitro, measured the expression of protein and mRNA of Cav3.1, Cav3.2and Cav3.3with western blotting (WB) and real-time PCR. The films of WB were scanned, Quantity one, one picture analysis software, was adopted to analysis absorbance of the target band, the ratio of absorbance referred to the band of β-actin was regard as the expression of protein. Expression of mRNA was calculated with the formula of2-ΔΔCt, Ct was the threshold of cycle,Δ ΔCt=[Ct (target gene of experiment)-Ct (β-actin)]-[Ct (target gene of control)-Ct (β-actin)]. Data were record as mean±standard deviation (x±s), SPSS10.0software was used to analysis the data. Completely randomized single-factor analysis of variance was made by LSD (homogeneous variance) and Dunnet’s T3or Welch (heterogeneity of variance). P<0.05was regard as the significant difference.Results We found that all three subtype, Cav3.1, Cav3.2and Cav3.3mRNA or protein, were detected in SH-SY5Y cells. However, Cav3.1was the primary subtype, the expression of Cav3.1protein (F=777.113,P=0.000) and mRNA (F=194898.000,P=0.000) was more than Cav3.2and Cav3.3.Conclusion Cav3.1, Cav3.2and Cav3.3protein and mRNA were detected in SH-SY5Y cells, and Cav3.1was the primary subtype.Experiment2Effects of lidocaine hydrochloride on the expression of Cav3.1in SH-SY5Y cellsObjective Cav3.1was the main subtype in SH-SY5Y cells, the physiology characteristics of T-type calcium were reflected mainly by Cav3.1in SH-SY5Y cells. This study was to observe the changes of Cav3.1protein and mRNA in SH-SY5Y cells treated with lidocaine hydrochloride for24h.Methods SH-SY5Y cells cultured in vitro were divided into two group, one group SH-SY5Y cells were treated with lOmM lidocaine hydrochloride for24h and another were not treated with lidocaine hydrochloride. Cav3.1were detected with the western blotting and real-time PCR. The films of WB were scanned, Quantity one, one picture analysis software, was adopt to analysis absorbance of the target band, the ratio of absorbance referred to the band of β-actin was regard as the expression of protein. Expression of mRNA was calculated with the formula of2-ΔΔCt, Ct was the threshold of cycle, ΔΔCt=[Ct (target gene of experiment)-Ct (β-actin)]-[Ct (target gene of control)-Ct (β-actin)]. Data were record as mean±standard deviation (x±s), SPSS10.0software was used to analysis the data. Paired t-test was used to treated the data of Cav3.1mRNA and protein expression. P<0.05was regarded as the significance difference.Results Compared with Control group, SH-SY5Y cells treated with10mM lidocaine hydrochloride for24h can up-regulate Cav3.1protein (t=9.932, P=0.000) and mRNA expression (t=12.573, P=0.000)Conclusion SH-SY5Y cells treated with10mM lidocaine hydrochloride for24h can up-regulate Cav3.1protein and mRNA expression.Chapter2Construction and identification of pshRNA-CACNAIG-SH-SY5YObjective Up to now there were no specific antagonist of T-type calcium channel, which has become the main hinder of T-type calcium channel research.With the development of gene interference and genetic engineering, gene interference to silence the tagart gene has become one important method in science research. This study constructed the pshRNA-CACNA1G-SH-SY5Y cells with the RNAi to silence the Cav3.1gene, and test this cells with western blotting and real-time PCR.Method Three oligonucleotides of shRNA interferenc sequence tagarting to CACNA1G and one negative control oligonucleotide were designed. Those olgonucletides after annealed were jointed with vector enzyme cutting, and were transfered into Top10, The recombinant plasmid were measured by DNA sequencing after confirmed by enzyme digestion analysis. The recombinant plasmid then was transferred into SH-SY5Y cell after a series of experimental procedure. Cav3.1gene and protein expression of shRNA-CACNA1G-SH-SY5Y was identified by real-time PCR and western blotting. Data were record as mean±standard deviation(x±s), SPSS10.0software was used to analysis the data. Completely randomized single-factor analysis of variance was made by LSD (homogeneous variance) and Dunnett’s T3(heterogeneity of variance). P<0.05was regarded as the significance difference.Results The three oligonucleotides of shRNA interference sequence and negative control sequence were successfully insertted pSUPERretro-puro by the DNA sequencing. All three shRNA interference sequence can silence the Cav3.1gene and down-regulate the Cav3.1protein expression, especially the RNAi(2), with the highest efficiency of interference, however, negative control had no effects on the Cav3.1gene and protein.Conclusion We successfully constructed shRNA-CACNA1G-SH-SY5Y cells identified with western blotting and real-time PCR and provided one cell model with silenced Cav3.1gene for the further experiments.Chapter3The effects of lidocaine hydrochloride on pshRNA-CACNA1G-SH-SY5Y (RNAi2) cellsObjective We had constructed the pshRNA-CACNA1G-SH-SY5Y(RNAi2) cells with the gene interference to silence the Cav3.1gene expression, which provided the cell model to investigate the role of Cav3.1in the neurotoxicity induced by lidocaine. This study was designed to observe the effects of10mM lidocaien on the pshRNA-CACNA1G-SH-SY5Y (RNAi2) cells and to test the roles of Cav3.1in the neurotoxicity induced by lidocaine.Methods There were three control groups followed as:SH-SY5Y group, RNAi2group [pshRNA-CACNA1G-SH-SY5Y (RNAi2)] and NC group(pNC-puro-CACNA1G-SH-SY5Y). As well as, three experiment group were SH-SY5Y+Lido group, RNAi2+Lido group and NC+Lido group, which treated with10mM lidocaine for24hours. After drug treated for24hours, the cell shape were observed with microscope, cell viability were detected with MTT,cell apoptosis rate measured with flow cytometry, Cav3.1and Caspase-3were detected with western blotting. Data were record as mean±standard deviation (x±s), SPSS10.0software was used to analysis the data. Two factors factorial design variance analysis (LSD for the homogeneity of variance and Welch or Dunnett’s T3for the unequal variances and were adopted when separate effect analysis as necessary) was used to analysis the cell viability, cell apoptosis rate, Cav3.1and Caspase-3expression. P<0.05was regard as the significance difference.Results(1) Cell shapeCell in those control groups grew well in reticulation with a spindle shape cell and many protuberance extended from the cell body. However, cells in experiment groups (SH-SY5Y+Lido group and NC+Lido group) curled up and float, protuberance disappeared. Although cell in RNi2+Lido group also appeared above conditions, cell shape was intact and some protuberance can still be found.(2) Cell viabilityAfter treated with lidocaine for24hours, cell viability of cells in SH-SY5Y group, NC group, RNAi2group,SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group were (100.0±0.0)%,(98.3±5.9)%,(96.5±5.9)%,(50.0±2.4)%,(50.6±6.6)%,(65.2±3.0)%respectively. Factor analysis showed the main effects of lidocaine hydrochloride (F=798.768, P=0.000) and sequence insert (F=7.141,P=0.003) and the interaction effect (F=14.896,P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects (F=0.829, P=0.456) to the cells viability when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=22.267,P=0.000) among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group. When fixed no sequence insert factor, cell viability of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=2500.000, P=0.000).When fixed negative sequence insert factor, cell viability of the cells in NC group and NC+Lido group had significant difference (F=175.377, P=0.000). When fixed the interfere sequence insert factor, cell viability of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=142.746, P=0.000), tab3-1and fig3-2.(3) Apoptosis rateApoptosis rate of the cells in SH-SY5Y group, NC group, RNAi2group,SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group were (7.0±1.4)%,(6.7±1.0)%,(6.8±1.5)%,(61.5±2.7)%,(61.7±2.7)%,and (46.5±5.2)%respectively. Factor analysis showed the main effects of lidocaine hydrochloride (F=2871.057,P=0.000) and sequence insert(F=29.358,P=0.000) and the interaction effect (F=29.380,P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects (F=0.096, P=0.909) to the apoptosis rate when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=33.082,P=0.000) among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group. When fixed no sequence insert factor, apoptosis rate of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=1875.947, P=0.000). When fixed negative sequence insert factor, apoptosis rate of the cells in NC group and NC+Lido group had significant difference (F=2231.557,P=0.000). When fixed the interfere sequence insert factor, apoptosis rate of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=327.044,P=0.000), tab3-2and fig3-3.(4) Cav3.1expressionThere was no difference of the Cav3.1expression in cells that in SH-SY5Y group and NC group. Compared with the above two group, Cav3.1expression reduced in RNAi2group. After treated with10mM lidocaine hydrochloride, Cav3.1expression of the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group up regulated, but increase of Cav3.1expression of the cells in RNAi2+Lido group was less than the other two experiment group, see fig3-4. Factor analysis showed the main effects of lidocaine hydrochloride (F=93.860,P=0.005) and sequence insert (F=973.876,P=0.000) had significant difference. However, the interaction effect (F=2.042, P=0.147) of those two factors had no significant difference, see tab3-3.(5) Caspase-3expressionThe expression of cleavedcaspase-3(the activated caspase-3) or procaspase-3(the inactivated caspase-3) was no difference among the cells in the three control groups. Compared with the control groups, cleavedcaspase-3expression of the cells in experiment group increased and procaspase-3expression decreased. Factor analysis about the effects of lidocaine hydrochloride and sequence insert on the procaspase-3and cleaved caspase-3protein expression showed the main effects of lidocaine hydrochloride (F=1150.570,P=0.000and F=582.441,P=0.000) and sequence insert (F=91.081, P=0.000and F=32.372, P=0.000) and the interaction effect (F=92.318, P=0.000and F=33.529, P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects (F=0.304, P=0.742and F=0.932, P=0.416) to the procaspase-3and cleavedcaspase-3protein expression when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=227.787, P=0.000and F=57.604, P=0.000)among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group. When fixed no sequence insert factor, procaspase-3and cleavedcaspase-3protein expression of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=672.256, P=0.000and F=216.216, P=0.000). When fixed negative sequence insert factor, procaspase-3and cleavedcaspase-3protein expression of the cells in NC group and NC+Lido group had significant difference (F=671.226, P=0.000and F=415.059, P=0.000). When fixed the interfere sequence insert factor, procaspase-3and cleavedcaspase-3protein expression of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=672.256, P=0.000and F=216.216, P=0.000), tab3-4,3-5and fig3-5.(6)[Ca2+]free[Ca2+]free of the cells in SH-SY5Y group, NC group, RNAi2group,SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group were (417± 13)nM,(413±16)nM,(408±15)nM,(608±15) nM,(607±13) nM and (520±12) nM, respectively. Factor analysis showed the main effects of lidocaine hydrochloride (F=1217.976,P=0.000) and sequence insert (F=43.549,P=0.000) and the interaction effect (F=32.035, P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects (F=0.514,P=0.608) to the [Ca2+]free when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=81.695,P=0.000) among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group. When fixed no sequence insert factor,[Ca2+]free of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=538.156, P=0.000). When fixed negative sequence insert factor,[Ca2+]free of the cells in NC group and NC+Lido group had significant difference (F=503.833, P=0.000). When fixed the interfere sequence insert factor,[Ca2+]free of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=207.950, P=0.000), tab3-6and fig3-6.Conclusion Cav3.1expression increased after treated with lOmM lidocaine hydrochloride for24hours. Compared with the control group, lOmM lidocaine hydrochloride caused the cell shape change, cell viability decrease,[Ca2+]free increase, cell apoptosis rate increase and cleavedcaspase-3upregulate. Cav3.1gene silence of the cells in RNAi2+Lido group can protect the cells damage from the lidocaine hydrochloride.Chapter4SH-SY5Y cells injury induced by lidocaine hydrochloride via p38MAPK regulated by Cav3.1Objective To ivestigate the relation of the protection effects of Cav3.1gene silence with the p38MAPK in the damage induced by lidocaine hydrochloride, we detected the expression of the p38MAPK of the SH-SY5Y cells treated with lidocaine hydrochloride for24hours.Methods There were three control groups followed as:SH-SY5Y group, RNAi2group [pshRNA-CACNA1G-SH-SY5Y (RNAi2)] and NC group(pNC-puro-CACNA1G-SH-SY5Y). As well as, three experiment group were SH-SY5Y+Lido group, RNAi2+Lido group and NC+Lido group, which treated with10mM lidocaine for24hours. After drug treated for24hours, the cell shape were observed with microscope, cell viability were detected with MTT,cell apoptosis rate measured with flow cytometry, p38MAPK and p-p38MAPK were measured with western blotting. Data were record as mean±standard deviation(x±s), SPSS10.0software was used to analysis the data. Two factors factorial design variance analysis (LSD for the homogeneity of variance or Dunnet’s T3for the unequal variances and Paired Sample t test were adopted when separate effect analysis as necessary) was used to analysis the cell viability, cell apoptosis rate, p38MAPK and p-p38MAPK protein expression. P<0.05was regarded as the significance difference.Results Cell shape, cell viability and cell apoptosis rate were the same to the Part Ⅲ. There was no significant difference of the p38MAPK expression among the groups. Factor analysis about the effects of lidocaine hydrochloride and sequence insert on the p38MAPK protein expression showed the main effects of lidocaine hydrochloride (F=0.071, P=0.791) and sequence insert (F=0.061, P=0.941) and the interaction effect (F=0.363, P=0.699) of those two factors had no significant difference. Factor analysis about the effects of lidocaine hydrochloride and sequence insert on the p-p38MAPK protein expression showed the main effects of lidocaine hydrochloride (F=911.349,P=0.000) and sequence insert(F=53.082, P=0.000) and the interaction effect (F=49.737, P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects(F=0.145, P=0.866) to the p-p38MAPK protein expression when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=59.673, P=0.000) among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group.When fixed no sequence insert factor, p-p38MAPK protein expression of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=264.266, P=0.000). When fixed negative sequence insert factor, p-p38MAPK protein expression of the cells in NC group and NC+Lido group had significant difference (F=520.990, P=0.000). When fixed the interfere sequence insert factor, p-p38MAPK protein expression of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=278.400, P=0.000), fig4-1and tab4-1,4-2.Conclusion pshRNA-CACNA1G-SH-SY5Y (RNAi2) can reduce the cell injury induced by lidocaine hydrochloride. And this pretection maybe invovled with the p-p38MAPK downregulation. |
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