| ObjectiveH9c2 rat myoblast cells were treated with hypoxia and reoxygenation as a simulation of myocardial ischemia-reperfusion injury model in vitro, which is respectively devided into three parts:Part One:effects of diversed lidocaine pretreatment on H9c2 cardiomyocytes against hypoxia/reoxygenation(H/R)-induced injury. Part Two:effect of lidocaine pretreatment on caspase-3 activity and calcium overload following injuries of H9c2 cardiomyocytes induced by hypoxia/ reoxygenation. Part Three:protective effects of lidocaine pretreatment on injury of H9c2 cardiomyocytes cultrued in high-glucose culture medium. Therefore, the objectives of this study are as follow:①To investigate the protective effects of different-concerntration lidocaine pretreatment on H9c2 cardiomyocytes against hypoxia/reoxygenation(H/R)-induced oxidative injury.②To investigate the protective effects of lidocaine pretreatment on H9c2 cardiomyocytes against H/R-induced apoptosis and its potential mechanisms. ③To investigate the protective effects of lidocaine pretreatment on hypoxia/ reoxygenation-induced injury of H9c2 cardiomyocytes cultured in high-glucose culture medium and roles of high-glucose culture in them.MethodsPart One:Cultured H9c2 cardiomyocytes were divided randomly into seven groups:(1)normal control group(Group NC); (2) H/R control group(Group HR):A 3-hour hypoxic period was followed by 2 hour of reoxygenation; (3) lidocaine pretreatment group(Group LP):according to the diversed concentration of lidocaine(1,2.5,5,10 and 20μmol/L), Group LP were redevided into five groups, including Group LP1, LP2, LP3, LP4 and LP5, respectivly. Cells were pretreated with lidocaine before H/R treatment. Colorimetric assay was used to detect cell viability and lactate dehydrogenase (LDH) release to evaluate cell injury. Superoxide dismutase (SOD) activity and malonaldehyde (MDA) content were measured by colorimetric assay to evaluate cell antioxidant ability.Part Two:Cultured H9c2 cardiomyocytes were divided into three groups:(1) NC group; (2) H/R group:A 3-hour hypoxic period was followed by 2 hours of reoxygenation; (3) LPC group:cells were pretreated with lidocaine(10μmol/L) before H/R treatment. Colorimetric assay was used to detect cell viability and lactate dehydrogenase (LDH) release to evaluate cell injury. Apoptotic rate of H9c2 cardiomyocytes were determined by flow cytometer. Caspase-3 activity was detected by immunocytochemistry and intracellular calcium was tested by fluorospectrophotometry.Part Three:Cultured H9c2 cardiomyocytes were divided randomly into six groups:normal glucose-cultured control group(Group NC), normal glucose-cultured HR group(Group NHR), normal glucose-cultured lidocaine pretreatment group(Group NLP) and high glucose-cultured control group(Group HC), high glucose-cultured HR group(Group HHR), high glucose-cultured lidocaine pretreatment group(Group HLP). Colorimetric assay was used to detect cell viability and lactate dehydrogenase (LDH) release to evaluate cell injury. Superoxide dismutase (SOD) activity and malonaldehyde (MDA) content were measured by colorimetric assay.ResultsPart One:The results showed that after treatment with H/R, cell viability was significantly weakened compared with NC group. LDH release was significantly higher than that in NC group. Lidocaine pretreatment markedly improved cell viability and reduced LDH release. Cells treated with H/R had a lower SOD activity and a higher MDA content compared with Group NC. Cells with lidocaine pretreatment had a higher SOD activity and a lower MDA content compared with HR group. Besides, the change of cell viability was positively correlated significantly to that of LDH release and MDA content, negatively to that of SOD activity.And cell viability was positively correlated significantly to the increasing concentration of lidocaine; LDH release and MDA content were negatively to it.Part Two:The results showed that after treatment with hypoxia and reoxgenation, cell viability was significantly reduced to 0.188±0.012 compared with NC group (0.325±0.009). LDH release and apoptotic rates were 80.18±4.70U/L and 20.12±2.19% respectively, significantly higher than that of NC group (17.13±2.40U/L and 0.57±0.30%). Lidocaine pretreatment markedly improved cell viability to 0.234±0.015 and reduced LDH activity and apoptotic rates to 38.51±2.45U/L and 8.03±1.50%. Caspase-3 activity and intracellular calcium were increased to 0.163±0.011 and 440.59±22.30nmol/L respectively, significantly higher than NC group after hypoxia/reoxgenation-induced injury. Cells with lidocaine pretreatment had a lower caspase-3 activity of 0.135±0.007 and intracellular calcium of 329.22±13.74nmol/L compared with H/R group. Part Three:①In normal glucose-cultured group:after treatment with hypoxia and reoxygenation, cell viability and SOD activity were significantly reduced compared with NC group. Reversely, LDH release and MDA content were significantly higher than that in Group NC. Lidocaine pretreatment markedly improved cell viability and reduced LDH release. Cells treated with lidocaine had a higher SOD activity and a lower MDA content compared with NHR group.②In high glucose-cultured group:after treatment with hypoxia and reoxygenation, cell viability and SOD activity were significantly reduced compared with HC group. Reversely, LDH release and MDA content were significantly higher than that in HC group. Lidocaine pretreatment markedly improved cell viability and reduced LDH release. Cells pretreated with lidocaine had a higher SOD activity and a lower MDA content compared with HHR group;③compared with normal glucose-cultured group, after co-cultured with high glucose, cell viability and SOD activity were lower and LDH release and MDA content were significantly higher in Group HC than that in Group NC;④The interactive effect of two factors between high glucose and experimental treatment (group) was significantly statistically significant.ConclusionsBased on the results observed, it is concluded that:①Lidocaine pretreament may protect cardiomyocytes from H/R-induced oxidative injury via inhibiting cell lipoperoxidation in a concerntration-dependent manner. And the better anti-oxidative-injury of it gets, the higher concentrations of lidocaine become.②Lidocaine pretreatment may protect cardiomyocytes from being injured by H/R. The underlying mechanisms may include repressing caspase-3 activity and reducing intracellular calcium overload.③Lidocaine(10μmol/L) may protect cardiomyocytes from being oxidatively injury induced by hypoxia and reoxygenation in the both kinds of conditions:normal glucose and high glucose, respectively; High glucose may result in stress injury in the normal condition, but markedly strengthen the anti-H/R ability of H9c2 cells and also have some cardioprective effects in the hypoxic condition. |
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