Liu An-Heng, Cao Ya-Nan, Liu Hong-Tao, Zhang Wei-Wei, Liu Yan, Shi Tang-Wang, Jia Guo-Liang, Wang Xiao-Ming
Department of Geriatrics, Xijing Hospital, Fourth Military Medical University, Xi'an, China.
Cell Physiol Biochem. 2008;22(1-4):177-86. doi: 10.1159/000149795. Epub 2008 Jul 25.
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a non-selective chloride channel blocker, has been shown to prevent cell apoptosis, however, the underlying mechanisms remain undefined, thus the present study was to explore whether phosphatidylinositol 3'-kinase (PI3K)/Akt and its downstream molecules are involved in the cytoprotective effect of DIDS.
Neonatal rat cardiomyocytes were exposed to staurosporine (STS) in the presence or absence of DIDS. Cell viability, apoptosis and expressions of Akt, phospho-Akt (p-Akt), eNOS, phospho-eNOS (p-eNOS), Bcl-2/Bax and nitric oxide (NO) production were determined, and Bax translocation was assessed by double immunofluorescence labeling and Western blotting.
DIDS markedly improved cell viability and exerted an anti-apoptotic effect on STS-exposed cardiomyocytes. DIDS resulted in a 2.1-fold increase of p-Akt over control levels, prevented the reduction in eNOS expression and phospho-eNOS levels induced by STS and significantly increased NO production (all P<0.01 vs. STS alone). Treatment with LY294002, a selective PI3K inhibitor, abolished DIDS-induced increases in p-Akt, eNOS, p-eNOS and NO production, and completely abrogated the DIDS-induced anti-apoptotic effect (P<0.01). Treatment with L-NAME, a non-selective NOS inhibitor similarly inhibited the increased NO but only partly abolished protective effects of DIDS (P<0.05). In addition, DIDS effectively inhibited STS-induced Bax translocation to mitochondria, which was also reversed by LY294002.
DIDS protects cardiomyocytes against STS-induced apoptosis via activating PI3K/Akt signaling pathway, including increasing eNOS phosphorylation and the subsequent NO production and inhibiting Bax translocation.
4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS）是一种非选择性氯离子通道阻滞剂，已被证明可预防细胞凋亡，然而，其潜在机制仍不明确，因此本研究旨在探讨磷脂酰肌醇3'-激酶（PI3K）/Akt及其下游分子是否参与DIDS的细胞保护作用。
将新生大鼠心肌细胞暴露于星形孢菌素（STS）中，同时存在或不存在DIDS。测定细胞活力、凋亡以及Akt、磷酸化Akt（p-Akt）、内皮型一氧化氮合酶（eNOS）、磷酸化eNOS（p-eNOS）、Bcl-2/Bax的表达和一氧化氮（NO）的产生，并通过双重免疫荧光标记和蛋白质印迹法评估Bax易位。
DIDS显著提高细胞活力，并对暴露于STS的心肌细胞发挥抗凋亡作用。DIDS导致p-Akt比对照水平增加2.1倍，防止了STS诱导的eNOS表达和磷酸化eNOS水平降低，并显著增加NO产生（与单独使用STS相比，所有P<0.01）。用选择性PI3K抑制剂LY294002处理可消除DIDS诱导的p-Akt、eNOS、p-eNOS和NO产生增加，并完全消除DIDS诱导的抗凋亡作用（P<0.01）。用非选择性NOS抑制剂L-NAME处理同样抑制了NO增加，但仅部分消除了DIDS的保护作用（P<0.05）。此外，DIDS有效抑制STS诱导的Bax易位至线粒体，LY294002也可逆转这一现象。
DIDS通过激活PI3K/Akt信号通路保护心肌细胞免受STS诱导的凋亡，包括增加eNOS磷酸化及随后的NO产生，并抑制Bax易位。
