Kimura Hitoshi, Gules Ilker, Meguro Toshinari, Zhang John H
Department of Neurosurgery, University of Mississippi Medical Center, Jackson, MS 39216, USA.
Brain Res. 2003 Nov 14;990(1-2):148-56. doi: 10.1016/s0006-8993(03)03450-4.
Several studies reported that the levels of proinflammatory cytokines such as TNF-alpha, IL-1beta, IL-6, and IL-8 are elevated in the cerebrospinal fluid (CSF) of patients after subarachnoid hemorrhage (SAH). Cytokines in CSF may contribute to the development of vasospasm and cerebral ischemia. In the present study, we investigated the possible cytotoxic effects of these cytokines on cultured cerebral microvascular endothelial cells.
The effects of TNF-alpha, IL-1beta, IL-6, and IL-8 were tested using cell viability assay, DNA fragmentation analysis (DNA laddering), Western blot analysis (Anti-poly-(ADP-ribose) polymerase [PARP] antibody), and caspase-3 activity.
TNF-alpha and IL-1beta, but not IL-6 or IL-8, caused cell detachment in a dose-dependent manner (p<0.05). TNF-alpha (200 pg/ml) and IL-1beta (150 pg/ml) produced DNA ladders at 24-72 h. TNF-alpha but not IL-1beta cleaved the PARP from 116- to 85-kDa fragments and enhanced caspase-3 activity at 24-72 h after incubation with endothelial cells. Caspase-3 inhibitor at 10 micromol/l significantly prevented TNF-alpha-induced cell detachment (p<0.05).
TNF-alpha induces apoptosis in cultured cerebral endothelial cells through the cleavage of caspase-3. IL-1beta decreases the adherent cells, produces DNA ladders, but fails to cleave PARP or increase caspase-3 activity. IL-1beta may induce apoptosis in cerebral endothelial cells through different pathway from that of TNF-alpha.
多项研究报道，蛛网膜下腔出血（SAH）患者脑脊液（CSF）中促炎细胞因子如肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和白细胞介素-8（IL-8）水平升高。脑脊液中的细胞因子可能导致血管痉挛和脑缺血的发生。在本研究中，我们调查了这些细胞因子对培养的脑微血管内皮细胞可能的细胞毒性作用。
使用细胞活力测定、DNA片段化分析（DNA梯状条带）、蛋白质免疫印迹分析（抗聚（ADP-核糖）聚合酶[PARP]抗体）和半胱天冬酶-3活性检测TNF-α、IL-1β、IL-6和IL-8的作用。
TNF-α和IL-1β，而非IL-6或IL-8，以剂量依赖性方式导致细胞脱离（p<0.05）。TNF-α（200 pg/ml）和IL-1β（150 pg/ml）在24 - 72小时产生DNA梯状条带。TNF-α而非IL-1β将PARP从116 kDa切割为85 kDa片段，并在与内皮细胞孵育24 - 72小时后增强半胱天冬酶-3活性。10 μmol/l的半胱天冬酶-3抑制剂显著阻止TNF-α诱导的细胞脱离（p<0.05）。
TNF-α通过切割半胱天冬酶-3诱导培养的脑内皮细胞凋亡。IL-1β减少贴壁细胞，产生DNA梯状条带，但未能切割PARP或增加半胱天冬酶-3活性。IL-1β可能通过与TNF-α不同的途径诱导脑内皮细胞凋亡。
