低分子壳聚糖对缺氧/复氧大鼠心肌细胞中ClC-3表达的影响

Effects of low molecular weight chitosan on ClC-3 expression in cardiomyocytes of rats with hypoxia-reoxygenation injury

目的:研究低分子壳聚糖(LMCTS)对缺氧/复氧(H/R)损伤大鼠心肌细胞中氯离子通道3(ClC-3)表达的影响,探讨LMCTS对大鼠心肌细胞H/R损伤的保护作用及其与ClC-3通道的关系。方法:原代培养的大鼠心肌细胞分为空白对照组,模型组和200、400及600mg·L-1 LMCTS组。除空白对照组细胞不做任何处理、正常培养外,其余各组细胞均进行低压缺氧处理1h,之后在37℃条件下复氧24h,构建心肌细胞H/R损伤模型,其中模型组不加药物,正常培养;而不同浓度LMCTS组细胞在构建模型前分别给予200、400和600mg·L-1 LMCTS。采用RT-PCR法检测大鼠心肌细胞中ClC-3mRNA的表达水平;Western blotting法和免疫荧光法检测大鼠心肌细胞中ClC-3蛋白的相对表达水平。结果:RT-PCR法和Western blotting法检测,与空白对照组比较,模型组大鼠心肌细胞中ClC-3mRNA和蛋白表达水平明显升高(P<0.01);与模型组比较,不同浓度LMCTS组大鼠心肌细胞中ClC-3mRNA和蛋白表达水平明显降低(P<0.05),其中以600mg·L-1LMCTS组心肌细胞中ClC-3mRNA和蛋白表达水平最低。免疫荧光检测,与空白对照组比较,模型组大鼠心肌细胞荧光强度明显增强;与模型组比较,不同浓度LMCTS组大鼠心肌细胞荧光强度明显降低。结论:大鼠心肌细胞中ClC-3表达水平与心肌细胞H/R损伤有关联,LMCTS可降低ClC-3表达水平,其可能通过降低ClC-3表达水平对大鼠心肌细胞H/R损伤发挥保护作用。

Objective：To investigate the effects of low molecular weight chitosan（LMCTS）on the expression of chcoride channel 3（CIC-3）in cardiomyocytes of the rats with hypoxia-reoxygenation（H/R）injury,and to probe the protective effect of LMCTS on the myocardial H/R injury and its correlation with the expression of ClC-3.Methods：The cardiomyocytes of rats were cultured and divided into blank control group,model group,and 200,400 and 600 mg·L-1 LMCTS groups.The cells in blank control group were cultured regularly without any treatment;and the cells in other groups were treated under hypobaric and hypoxic condition for 1h,then they got reoxygenation at 37℃for 24 hto establish the cardiomyocyte H/R injury models.The cells in model group were cultured regularly without drugs and the cells in three different concentrations of LMCTS groups were given 200,400 and 600mg·L-1 LMCTS before establishing the H/R injury models.The ClC-3 mRNA expression level in cardiomyocytes of the rats was detected by RT-PCR method;the relative expression level ClC-3 protein in cardiomyocytes of the rats was detected by Western blotting method and immunofluorescence method.Results：The Western blotting and RT-PCR results indicated that the expression levels of ClC-3 mRNA and protein in cardiomyocytes of the rats in model group were higher than those in blank control group（P〈0.01）;but the expression levels in different concentrations of LMCTS groups were lower than those in model group（P〈0.05）,especially in 600mg·L-1 LMCTS group.The immunofluorescence experiment results showed that compared with blank control group,the fluorescence intensities in cardiomyocytes of the rats in model group were increased,but the fluorescence intensities in different concentrations of LMCTS groups were lower than that in model group.Conclusion：The ClC-3expression in cardiomyocytes of the rats associates with the cardiomyocyte H/R injuriy.LMCTS could decrease the ClC-3expression level and protect the cardiomyocytes away from H/R injury by the down-regulation of ClC-3expression level.