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

doi:10.13481/j.1671-587x.20160314

摘要/Abstract

摘要：

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^-1LMCTS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^-1LMCTS。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。

关键词: 壳聚糖, 心肌细胞, 缺氧, 复氧, 氯离子通道3

Abstract:

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 1 h,then they got reoxygenation at 37℃ for 24 h to 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 600 mg·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 600 mg·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-3 expression in cardiomyocytes of the rats associates with the cardiomyocyte H/R injuriy.LMCTS could decrease the ClC-3 expression level and protect the cardiomyocytes away from H/R injury by the down-regulation of ClC-3 expression level.

Key words: chitosan, cardiomyocytes, hypoxia, reoxygenation, chloride channel 3

中图分类号:

R329.28

万鑫, 何建平, 张欢, 林玲辉, 张甜, 费瑜. 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[J]. 吉林大学学报(医学版), 2016, 42(03): 491-495.

WAN Xin, HE Jianping, ZHANG Huan, LIN Linghui, ZHANG Tian, FEI Yu. Effects of low molecular weight chitosan on ClC-3 expression in cardiomyocytes of rats with hypoxia-reoxygenation injury[J]. Journal of Jilin University Medicine Edition, 2016, 42(03): 491-495.

参考文献

[1] 刘威,张成义,沈楠,等.五味子乙素对乳鼠缺氧/复氧损伤心肌细胞的保护作用及其机制[J].吉林大学学报:医学版,2014,40(5):977-980.

[2] Jentsch TJ.ClC chloride channels and transporters from genes to protein structure[J].Crit Rev Biochem Mol Biol,2008,43(1):3-36.

[3] Qian Y,Du YH,Tang YB,et al.ClC-3 chloride channel prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells through mitochondria dependent pathway[J].Apoptosis,2011,16(5):468-477.

[4] Zhu L,Yang H,Zuo W,et al.Differential expression and roles of volume-activated chloride channels in control of growth of normal and cancerous nasopharyngeal epithelial cells[J].Biochem Pharmacol,2012,83(3):324-334.

[5] 邱少波,梁燕,刘萍.冠心康对大鼠缺血再灌注损伤心肌氯通道CLC-2、CLC-3、CFTR表达的影响[J].中西医结合心脑血管病杂志,2013,11(12):1482-1485.

[6] 刘静娜,张家骊,夏文水.壳聚糖降低脂质过氧化的作用[J].食品与生物技术学报,2010,29(6):836-841.

[7] 陈伟,陈惠英,张彦.壳聚糖在医药领域的应用[J].医学信息,2007,20(3):507-508.

[8] 韩宝芹,王剑,蔡文娣,等.水溶性低分子壳聚糖对糖尿病大鼠血糖的影响[J].中国海洋大学学报:自然科学学报,2011,41(1/2):87-92.

[9] Chen Z,Hu Z,Lu ZQ,et al.Differential microRNA profiling in a cellular hypoxia reoxygenation model upon post-hypoxic propofol treatment reveals alterations in autophagy signaling network[J].Oxid Med Cell Longev,2013,2013(4):378484.

[10] 杜毓菁,徐一心,王紫璇,等.细胞缺氧/复氧模型的建立方法与应用[J].南昌大学学报:医学版,2015,55(2):88-91.

[11] 杨虎,张永亮,龚海英,等.蕨麻对急性低压缺氧大鼠脑组织保护作用及Caspase 3表达的影响[J].武警后勤学院学报:医学版,2014,23(11):893-900.

[12] 姚寅生,刘家传,杨艳艳,等.缺氧预处理对创伤性脑损伤大鼠Claudin-5表达及血-脑屏障通透性的影响[J].中国微侵袭神经外科杂志,2014,19(4):180-183.

[13] Guo R, Pan F,Tian Y,et al.Down-regulation of ClC-3 expression reduces epidermal stem cell migration by inhibiting volume-activated chloride currents[J].J Membr Biol,2016,1(1):1-12.

[14] Duan DD.The ClC-3 chloride channels in cardiovascular disease[J].Acta Pharmacol Sini,2011,32(6),:675-684.

[15] 任彦锋,张帆,王莉,等,球囊损伤腹主动脉大鼠血浆NO水平的变化及WSC对其影响[J].中国实验诊断学,2013(8):1369-1371.

[16] 闵清,白育庭,余薇,等.黄芪多糖对乳鼠心肌细胞缺氧/复氧损伤的保护作用[J].中国药理学通报,2010,26(12):1661-1664.

[17] 余薇,李璐,姚社,等.大蒜多糖对乳鼠心肌细胞缺氧/复氧损伤的保护作用[J].中国药理学通报,2010,26(5):697-699.

[18] 刘颖,纪超,吴伟康.附子多糖保护缺氧/复氧乳鼠心肌细胞及其抗内质网应激机制的研究[J].中国病理生理杂志,2012,28(3):459-463.

[19] 朱娜,要瑞莉,刘俊双,等.大鼠肢体缺血/再灌注后心肌细胞凋亡及氧化应激的调节作用[J].中国老年学杂志,2015,35(23):6666-6668.