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The cysteinyl leukotrienes (CysLTs) are inflammatory mediators closely associated with neuronal injury after brain ischemia through the activation of their receptors, CysLT1R and CysLT2R. Here we investigated the involvement of both receptors in oxygen-glucose deprivation/recovery (OGD/R)-induced ischemic neuronal injury and the effect of the novel CysLT2R antagonistin comparison with the CysLT1R antagonist montelukast. In primary neurons, neither the nonselective agonist leukotriene D4 (LTD4) nor the CysLT2R agonist N-methyl-leukotriene C4 (NMLTC4) induced neuronal injury, and HAMI 3379 did not affect OGD/R-induced neuronal injury. However, in addition to OGD/R, LTD4 and NMLTC4 induced cell injury and neuronal loss in mixed cultures of cortical cells, and neuronal loss and necrosis in neuron-microglial cocultures. Moreover, they induced phagocytosis and cytokine release (interleukin-1b and tumor necrosis factor-a) from primary microglia, and conditioned medium from the treated microglia induced neuronal necrosis. HAMI 3379 inhibited all of these responses, and its effects were the same as those of CysLT2R interference by CysLT2R short hairpin RNA, indicating CysLT2R dependence. In comparison, montelukast moderately inhibited OGD/R-induced primary neuronal injury and most OGD/R-and LTD4-induced (but not NMLTC4-induced) responses in mixed cultures, cocultures, and microglia. The effects of montelukast were both dependent and independent of CysLT1Rs because interference by CysLT1R small interfering RNA had limited effects on neuronal injury in neuron-microglial cocultures and on cytokine release from microglia. Our findings indicated that HAMI 3379 effectively blocked CysLT2R-mediated microglial activation, thereby indirectly attenuating ischemic neuronal injury. Therefore, CysLT2R antagonists may represent a new type of therapeutic agent in the treatment of ischemic stroke.

Background Transforming growth factor-β1 (TGF-β1) is an important regulator of cell migration and plays a role in the scarring response in injured brain. It is also reported that 5-lipoxygenase (5-LOX) and its products, cysteinyl leukotrienes (CysLTs, namely LTC 4 , LTD 4 and LTE 4 ), as well as cysteinyl leukotriene receptor 1 (CysLT 1 R) are closely associated with astrocyte proliferation and glial scar formation after brain injury. However, how these molecules act on astrocyte migration, an initial step of the scarring response, is unknown. To clarify this, we determined the roles of 5-LOX and CysLT 1 R in TGF-β1-induced astrocyte migration. Methods In primary cultures of rat astrocytes, the effects of TGF-β1 and CysLT receptor agonists on migration and proliferation were assayed, and the expression of 5-LOX, CysLT receptors and TGF-β1 was detected. 5-LOX activation was analyzed by measuring its products (CysLTs) and applying its inhibitor. The role of CysLT 1 R was investigated by applying CysLT receptor antagonists and CysLT 1 R knockdown by small interfering RNA (siRNA). TGF-β1 release was assayed as well. Results TGF-β1-induced astrocyte migration was potentiated by LTD 4 , but attenuated by the 5-LOX inhibitor zileuton and the CysLT 1 R antagonist montelukast. The non-selective agonist LTD 4 at 0.1 to 10 nM also induced a mild migration; however, the selective agonist N-methyl-LTC 4 and the selective antagonist Bay cysLT2 for CysLT 2 R had no effects. Moreover, CysLT 1 R siRNA inhibited TGF-β1- and LTD 4 -induced astrocyte migration by down-regulating the expression of this receptor. However, TGF-β1 and LTD 4 at various concentrations did not affect astrocyte proliferation 24 h after exposure. On the other hand, TGF-β1 increased 5-LOX expression and the production of CysLTs, and up-regulated CysLT 1 R (not CysLT 2 R), while LTD 4 and N-methyl-LTC 4 did not affect TGF-β1 expression and release. Conclusions TGF-β1-induced astrocyte migration is, at least in part, mediated by enhanced endogenous CysLTs through activating CysLT 1 R. These findings indicate that the interaction between the cytokine TGF-β1 and the pro-inflammatory mediators CysLTs in the regulation of astrocyte function is relevant to glial scar formation.

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