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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 one of the main mechanisms of ventricular systolic and diastolic dysfunction in diabetic cardiomyopathy. Periostin, an extracellular matrix protein secreted by cardiac fibroblasts, is closely related to myocardial fibrosis and interstitial remodeling. The purpose of this study was to investigate the effects of high glucose on periostin expression in cardiac fibroblasts and its related signal transduction mechanisms. Periostin mRNA expression was detected by RT-PCR, periostin protein expression was detected by immunoblotting, and the cells were detected by fluorescence assay Reactive oxygen species (ROS) content. The results showed that periostin mRNA expression increased by 117.26% after 12 h of high glucose stimulation, and increased protein expression by 93.12% after stimulated for 24 h. ROS increased significantly in cardiac fibroblasts stimulated by high glucose for 30 min. Protein kinase C , PKC inhibitor chelerythrine can reduce high glucose-induced increase in ROS production. High glucose-induced increases in periostin protein expression can be inhibited by chelerythrine or the ROS scavenger N-acetylcysteine (NAC). High glucose stimulated 30 and 60 min significantly increased the phosphorylation of c-Jun N-terminal protein kinase (JNK) content, the application of chelerythrine or NAC can inhibit high glucose-induced phosphorylation of JNK content increase. Preincubation with JNK inhibitor SP600125 significantly inhibited high glucose-induced increase of periostin protein expression. The above results suggest that high glucose stimulation can promote periostin mRNA and protein expression, and activate PKC / ROS / JNK pathway to induce high glucose-induced increase of periostin expression.