硫氧还蛋白系统对糖尿病大鼠心肌I_(to)钾电流的影响

Influence of thioredoxin system on myocardial Ito in rats with diabetic mellitus

目的：研究硫氧还蛋白（Trx）系统对糖尿病（DM）大鼠左室心肌细胞瞬时外向钾电流（Ito）的影响。方法采用链脲菌素（STZ）诱导的DM大鼠模型作为DM组（n=25），未经STZ诱导的大鼠作为对照组（n=20），应用全细胞膜片钳方法记录DM组与对照组大鼠心室肌Ito电流；用分光光度计测量氧化还原活性分子含量；采用5,5-二巯基双-2-硝基苯甲酸（DTNB）法评估蛋白质巯基的氧化程度；应用胰岛素对DM大鼠心肌细胞进行体外孵育，观察孵育前后心肌细胞Ito的变化。用TrxR抑制剂金诺芬或13-顺式视黄酸对经胰岛素孵育的大鼠心肌细胞进行处理，观察处理前后心肌细胞Ito的变化。结果 DM组存活22只，对照组存活20只。与对照组相比，DM组大鼠心肌硫氧还蛋白还原酶（TrxR）、谷胱甘肽还原酶（GR）明显降低[TrxR：（70.3±4.9）mU/mgvs.（147.9±18.2）mU/mg；GR：（34.4±2.4）mU/mg vs.（53.1±1.9）mU/mg，P＜0.05]，而硫氧还蛋白（Trx）、谷氧还蛋白（Grx）明显升高[Trx：（15.6±1.8）mU/mgvs.（9.2±1.9）mU/mg；Grx：（36.5±6.1）mU/mgvs.（14.7±1.0）mU/mg，P＜0.05]；同时，DM组大鼠左心室心肌自由巯基（P-SH）水平较对照组明显减少[（6.1±0.3）nmol/mgvs.（10.2±0.8）nmol/mg，P＜0.05]；DM组大鼠心肌细胞Ito密度较对照组显著降低[（16.8±2.4）pApFvs.（30.6±2.8）pApF，P＜0.05]；但胰岛素处理（4～5）h后Ito密度显著增加（31.2±2.1pApF，P＜0.05）；TrxR抑制剂金诺芬或13-顺式视黄酸均可以显著降低用胰岛素处理的DM大鼠心肌细胞Ito密度（P＜0.05）。结论DM大鼠心肌Trx系统功能下降，可造成心肌细胞Ito密度显著下降。

Objective To study the influence of thioredoxin system （Trx） on transient outward potassium current （Ito） of left ventricular myocardial cells in rats with diabetic mellitus （DM）.Methods DM model was established in rats by using STZ （DM group,n=25）, and other rats without using STZ were included in control group （n=20）. CardiomyocyteIto was recorded by applying whole-cell patch clamp method, and content of redox-active molecules was detected by using spectrophotometer. The oxidation degree of protein thiols was reviewed by using DTNB method. The myocardial cells from DM rats were cultured in vitro with insulin for observing the changes of Ito before and after culturing, and then the cells were treated with auranofin or 13-cis-retinoic acid （Trx inhibitors） for observing the change ofIto before and after treating.Results There were 22 rats survived in DM group and 20 in control group. Compared with control group, the levels of Trx reductase （TrxR） and glutathione reductase （GR） decreased significantly in DM group [TrxR： （70.3±4.9） mU/mgvs. （147.9±18.2） mU/mg; GR： （34.4±2.4） mU/mg vs. （53.1±1.9） mU/mg,P〈0.05], and levels of Trx and glutaredoxin （Grx） increased significantly [Trx： （15.6± 1.8） mU/mgvs. （9.2±1.9） mU/mg; Grx： （36.5±6.1） mU/mgvs. （14.7±1.0） mU/mg,P〈0.05]. At the same time, left ventricular myocardial free thiols decreased significantly in DM group compared with control group [（6.1±0.3） nmol/mgvs. （10.2±0.8） nmol/mg, P〈0.05]. The density ofIto decreased significantly in DM group compared with control group [（16.8±2.4） pApFvs. （30.6±2.8） pApF,P〈0.05], which increased significantly after insulin treating for 4-5 h （31.2±2.1pApF,P〈0.05）. Auranofin or 13-cis-retinoic acid reduced significantly the density ofIto treated with insulin （P〈0.05）.Conclusion The decrease of Trx function can induce the significant decrease of density ofIto.