高糖促进成年大鼠心脏成纤维细胞periostin表达及其信号转导机制

High glucose increases periostin expression and the related signal pathway in adult rat cardiac fibroblasts

心肌纤维化是糖尿病心肌病时心室舒缩功能障碍的主要机制之一。心脏成纤维细胞分泌的一种细胞外基质蛋白periostin与心肌纤维化及间质重塑密切相关。本研究旨在观察高糖对心脏成纤维细胞periostin表达的影响及其相关信号转导机制。在离体培养的成年大鼠心脏成纤维细胞,采用低糖(5.5mmol/L)和高糖(25mmol/L)干预,RT-PCR检测periostinmRNA表达,免疫印迹法检测periostin蛋白表达,荧光法检测细胞内活性氧(reactive oxygen species,ROS)含量。结果显示,高糖刺激12h可使periostin mRNA表达增加117.26%,刺激24h可使其蛋白表达上调93.12%,高糖刺激30min可显著促进心脏成纤维细胞内ROS的产生,蛋白激酶C(protein kinase C,PKC)抑制剂白屈菜红碱可减少高糖诱导的ROS生成增加。高糖诱导的periostin蛋白表达增加可被白屈菜红碱或ROS清除剂N-乙酰-L-半胱氨酸(N-acetylcysteine,NAC)所抑制。高糖刺激30和60min可显著增加磷酸化c-Jun氨基末端激酶(c-jun N-terminal protein kinase,JNK)的含量,应用白屈菜红碱或NAC可抑制高糖诱导的磷酸化JNK含量增加。JNK抑制剂SP600125预孵育明显抑制了高糖诱导的periostin蛋白表达增加。上述结果提示,高糖刺激可促进periostin的mRNA和蛋白表达,激活PKC/ROS/JNK通路介导高糖诱导的periostin表达增加。

Cardiac fibrosis is a major mechanism contributing to myocardial systolic and diastolic dysfunction in diabetic cardiomyopathy. Periostin is a novel extracellular matrix protein, secreted from cardiac fibroblasts, and closely related with cardiac fibrosis and remodeling. The present study aimed to investigate the effect of high glucose on periostin expression and the related signal transduction pathway in cardiac fibroblasts. Adult rat cardiac fibroblasts were cultured and stimulated with high glucose （25 mmol/L）. The mRNA and protein expressions of periostin were detected by RT-PCR and Western blot, respectively. Intracellular reactive oxygen species （ROS） production was measured using 2, 7-dichlorofluorescein diacetate （DCF-DA）, an oxidant-sensitive fluorescent probe. Results showed that the mRNA expression of periostin in adult rat cardiac fibroblasts was increased by 117.26% when treated with high glucose for 12 h. Incubation with high glucose for 24 h enhanced periostin protein expression by up to 93.12%. High glucose induced the production of ROS in adult rat cardiac fibroblasts, which was reduced by chelerythrine （CLT）, a protein kinase C （PKC） inhibitor. High glucoseinduced periostin protein expression was decreased significantly when pretreated with CLT or N-acetylcysteine （NAC）, a ROSscavenger. The phosphorylation of c-jun N-terminal protein kinase （JNK） was increased markedly when stimulated with high glucose for 30 and 60 min, which was abolished when pretreated with CLT or NAC. SP600125, a specific JNK inhibitor, significantly decreased periostin expression induced by high glucose. In conclusion, high glucose stimulates periostin protein expression via a PKC/ROS/JNKdependent pathway in adult rat cardiac fibroblasts.