Sharifi Ali M, Eslami Habib, Larijani Bagher, Davoodi Jamshid
Department of Pharmacology and Cellular and Molecular Research Center, School of Medicine, Iran University of Medical Sciences, P.O. Box 14155-6183, Tehran, Iran.
Neurosci Lett. 2009 Aug 7;459(2):47-51. doi: 10.1016/j.neulet.2009.03.100. Epub 2009 Apr 5.
Hyperglycemia, which occurs under the diabetic condition, is widely recognized as the causal link between diabetes and its serious complications. Diabetic neuropathies, which are among the most frequent complications of diabetes, affect sensory, motor, and autonomic nerves. The exact molecular mechanisms of high glucose-induced toxicity on neuronal cells, is still unclear. We previously reported that high glucose can induce apoptosis in PC12 cells, as evidenced by DNA fragmentation and high Bax/Bcl-2 ratio. The present study examined the involvement of caspase-3, the executioner, and two initiators of apoptosis, caspase-8 and caspase-9, during high glucose-induced apoptosis in PC12 cells, a neuronal cell line. Cells were exposed to high glucose with or without z-VAD-fmk, a pan-caspase inhibitor. Cell viability was measured by MTT assay. Caspase activity was determined spectrophotometrically using enzyme specific substrates. To correlate and confirm the caspase activity with changes in protein expression, procaspase-8, -9, and -3 were evaluated by Western blot analysis. The DNA-fragmentation was determined by DNA ladder using gel electrophoresis. The PC12 cell viability on high glucose exposure was decreased compared to controls, which was reversed by z-VAD-fmk. The activities of caspase-8, -9, and -3 were significantly increased in treated cells compared to controls. Moreover, high glucose exposure induced a significant decrease in protein levels of procaspases, indicating conversion of pro-form into the mature caspases. Finally, DNA fragmentation (Ladder) was shown in treated cells by high glucose. Based on the current data, it could be concluded that high glucose-induced apoptosis in PC12 cells is mediated, in part, by activation of caspase-8, -9, and -3 dependent pathways.
糖尿病状态下出现的高血糖被广泛认为是糖尿病与其严重并发症之间的因果联系。糖尿病神经病变是糖尿病最常见的并发症之一，会影响感觉神经、运动神经和自主神经。高糖对神经元细胞产生毒性的确切分子机制仍不清楚。我们之前报道过高糖可诱导PC12细胞凋亡，DNA片段化和高Bax/Bcl-2比值可证明这一点。本研究检测了凋亡执行者caspase-3以及凋亡的两个起始者caspase-8和caspase-9在高糖诱导PC12细胞（一种神经元细胞系）凋亡过程中的作用。细胞分别暴露于添加或不添加泛半胱天冬酶抑制剂z-VAD-fmk的高糖环境中。通过MTT法测定细胞活力。使用酶特异性底物通过分光光度法测定半胱天冬酶活性。为了将半胱天冬酶活性与蛋白质表达变化相关联并进行确认，通过蛋白质免疫印迹分析评估前半胱天冬酶-8、-9和-3。通过凝胶电泳使用DNA梯状条带测定DNA片段化。与对照组相比，暴露于高糖环境中的PC12细胞活力降低，而z-VAD-fmk可使其逆转。与对照组相比，处理组细胞中caspase-8、-9和-3的活性显著增加。此外，高糖暴露导致前半胱天冬酶的蛋白质水平显著降低，表明前体形式转化为成熟的半胱天冬酶。最后，高糖处理的细胞中出现了DNA片段化（梯状条带）。根据目前的数据，可以得出结论，高糖诱导PC12细胞凋亡部分是由caspase-8、-9和-3依赖性途径的激活介导的。
