Shao Beili, Bayraktutan Ulvi
Stroke, Division of Clinical Neuroscience, School of Medicine, University of Nottingham, UK.
Redox Biol. 2014 May 28;2:694-701. doi: 10.1016/j.redox.2014.05.005. eCollection 2014.
Blood-brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2 (•-) generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2 (•-) by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2 (•-) production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase.
血脑屏障破坏是缺血性中风后高血糖加重脑损伤的一个关键特征。尽管其潜在机制在很大程度上仍不清楚,但蛋白激酶C(PKC)的激活被认为起着关键作用。本研究检测了人脑微血管内皮细胞(HBMEC)的凋亡是否可能导致高血糖诱发的屏障损伤,并评估了PKC在这一现象中的具体作用。用高血糖(25 mM)或佛波酯(PMA,一种蛋白激酶C激活剂,100 nM)处理显著增加了NADPH氧化酶活性、O2(•-)生成、促凋亡蛋白Bax表达、TUNEL阳性染色以及caspase-3/7活性。通过其特异性抑制剂对NADPH氧化酶、PKC-α、PKC-β或PKC-βI进行药理抑制,以及用细胞可渗透的超氧化物歧化酶模拟物MnTBAP中和O2(•-),可使高血糖诱导的上述所有增加恢复正常。抑制这些PKC同工型也消除了高血糖对决定整体酶活性的NADPH氧化酶膜结合成分Nox2和p22-phox蛋白表达的刺激作用。通过使用特异性siRNA沉默PKC-βI基因消除了高血糖和PMA对内皮细胞NADPH氧化酶活性、O2(•-)产生和凋亡的影响,从而改善了由HBMEC、星形胶质细胞和周细胞组成的人脑屏障体外模型的完整性和功能。高血糖介导的HBMEC凋亡导致脑屏障功能障碍,并受PKC-βI和NADPH氧化酶的顺序激活调节。