葡萄糖浓度变化对乳鼠心肌细胞的影响及其机制的探讨

Influence of various glucose levels on the structure and function of cultured neonatal rats cardiomyocytes

目的观察不同浓度的葡萄糖培养对乳鼠心肌细胞形态及功能的影响，并初步探讨其机制。方法建立乳鼠心肌细胞培养模型，随机分成低糖（5．5mmol／L）组、高糖（25．5mmol／L）组、间歇性高糖（5．5mmol／L和25．5mmol／L交替培养）组以及高糖（25．5mmol／L）＋蛋白激酶C（PKC）抑制剂Ro-3l-8220（50nmol／L）组，培养5d后，观察心肌细胞搏动情况，测定心肌细胞直径，并应用Westernblot检测心肌细胞PKC-α、PKC-β2、p-PKC-α、p-PKC-β2、核因子（NF）-κB和c-fos的蛋白表达水平。结果不同浓度葡萄糖培养的心肌细胞在搏动频率、细胞直径以及PKC-α、PKC-β2、p-PKC-α、p-PKC-β2、NF—κB和c-fos的蛋白表达水平存在差异，其中高糖组心肌细胞搏动频率、细胞直径以及PKC-α、PKC-β2、p-PKC-α、p-PKC-β2、NF—κB和c-fos的蛋白表达水平显著高于低糖组[高糖组比低糖组：搏动频率（64．15±1．55）次／min比（57．22±1．91）次／min，细胞直径（20．1l±0．91）Ixm比（17．18±0．82）／xm，P均〈0．01]，间歇性高糖组的上述指标则显著高于高糖[间歇性高糖组比高糖组：搏动频率（66．60±2．02）次／min比（64．15±1．55）次／min，细胞直径（22．39±0．92）μm比（20．11±0．91）μm，P均〈0．01]和低糖组[间歇性高糖组比低糖组：搏动频率（66．60±2．02）次／min比（57．22±1．91）次／min，细胞直径（22．39±0．92）μm比（17．18±0．82）μm，P均〈0．01]，而PKC抑制剂Ro-31—8220则能逆转高糖所诱导的上述变化[高糖＋PKC抑制剂Ro-31-8220组比高糖组：搏动频率（62．17±1．58）次／min比（64．15±1．55）次／min，细胞直径（18．41±0．78）μm比（20．11±0．91）μm，P均〈0．01]。结论高糖或间歇性高糖的环境能够导致心肌细胞肥大，搏动频率增加，同时影响心肌细胞PKC-α、PKC-β2、NF—κB和c—fos的表达和活性，而PKC抑制剂Ro-31-8220能够逆转高糖所诱导的上述变化。因此高糖特别是间歇性高糖可能通过PKC／NF-κB／c-fos途径诱导了心肌细胞结构和功能的改变，导致了糖尿病心肌损害。

Objective To study the influence of various glucose levels on the structure and function of euhured neonatal rats cardiomyocytes. Method Cultured neonatal ventricular cardiomyocytes were treated with various glucose levels for 5 days： control （5.5 mmol/L） ;high （25.5mmol/L） ;intermittent high （5.5 mmol/L or 25.5 mmol/L in every 12 hours interval） ;high （25.5 mmol/L） ＋ PKC inhibitor Ro-31-8220 （50 nmol/L）. Then, the cell beating frequency was counted,the cardiomyocytes diameters were measured and the expressions of PKC-α, PKC-β2 , p-PKC-α, p-PKC-β2 , NF-κB and c-los were determined by Western blot. Results Compared with control group, cardiomyocytes beating frequency, diameters as well as the expressions of PKC-α, PKC-β2, p-PKC-α, p-PKC-β2, NF-κB and c-los were significantly increased in high glucose concentration （ all P 〈 0. 05 ） and intermittent high glucose treatment further amplified these changes （ all P 〈 0. 05 vs. high glucose and control groups）. High glucose induced changes could he significantly attenuated with PKC inhibitor Ro-31-8220. Conclusion High, especially intermittent high glucose could lead to diabetic cardiomyopathy by promoting cardiac hypertrophy, increasing beating frequency via activating PKC/NF-κB/c-fos pathways.