br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion The brain is endowed with the 5-LOX enzymatic system (Lammers et al., 1996, Lindgren et al., 1984), but its physiological and pathological roles are still not clear. Inflammation is a characteristic of many neurodegenerative diseases such as Alzheimer's disease and PD. The level of inflammation in the brain is determined by the degree of activation of glial cells. Increasing evidence from human and mouse models suggests that inflammation is an important player in the pathogenesis of neurodegenerative disease (Wyss-Coray, 2006). Eicosanoids, including prostaglandins and leukotrienes derived from arachidonic IFN-alpha 1, human recombinant protein mg by the action of cyclooxygenase (COX-1 and COX-2) and 5-lipoxygenase (5-LOX) enzymes, exert important proinflammatory functions and other biological activities (Funk, 2001). 5-LOX is widely expressed in the brain and 5-LOX inhibition provides neuroprotection in cases of stroke and traumatic injury (Jatana et al., 2006, Zhang et al., 2006). Furthermore, recent studies indicate that 5-LOX plays a key role in AD (Chu et al., 2012, Chu and Pratico, 2012, Chu and Pratico, 2013, Manev et al., 2011). In addition, treatment with a COX-2/5-LOX inhibitor protects neuron against 3-NP-induced neurotoxicity (Kumar et al., 2011). Arachidonic acid and other polyunsaturated fatty acids are stereoselectively oxygenated at carbon 5 by the non-heme iron-containing enzyme 5-LOX (Ford-Hutchinson et al., 1994). The intervention of a second protein, 5-LOX-activating protein (FLAP), is required for 5-LOX to become catalytically active (Abramovitz et al., 1993). MK-886 binds to FLAP with high-affinity and prevents the activation of 5-LOX. Although previous studies showed that MK-886 induces apoptosis in murine pro-B lymphocytic cell lines (FL5.12) in a FLAP-unrelated manner (Datta et al., 1998, Wu et al., 2003), it was found here that MK-886 significantly ameliorates MPP+-induced neurotoxicity in differentiated SH-SY5Y cells without affecting the SH-SY5Y cell viability by itself. FL5.12 cells contained a substantial amount of FLAP protein and mRNA but had no measurable 5-lipoxygenase protein or 5-, 12-, or 15-lipoxygenase activity, which is quite different from neurons. It has been reported that both oxygen/glucose deprivation (OGD) and excitotoxic stimulations activate 5-LOX and elicit neuronal injury in rat primary cortical neuron culture (Ge et al., 2006). In addition, the gp120 protein of HIV also stimulates 5-LOX expression and causes cell death in a human neuronal cell line (Maccarrone et al., 1998). Therefore, the activation of the 5-LOX pathway may be involved in neurodegeneration. A recent study has indicated that the 5-LOX and 15-LOX isozymes contribute differentially to striatal vulnerability in response to MPTP challenge (Chou et al., 2013). Our animal study indicated that the expression of 5-LOX and FLAP in the striatum can be enhanced by MPTP. Furthermore, 5-LOX and GFAP (astrocyte marker) were colocalised, as shown by confocal microscopy. Recent in vitro studies have suggested that the activation of microglia and the subsequent activation of astrocytes via mediators released by injured dopaminergic neurons are involved in PD pathogenesis (Tufekci et al., 2012). Our results implied that 5-LOX and the activation of astrocytes are involved in the pathophysiology of the animal model of PD. In the in vitro study with rat mesencephalic neuron–glia cultures, we found that MK-886 also effectively protects dopaminergic neurons against MPP+-induced neuronal death. The 5-LOX product of 5-HETE is converted to LTA4. LTA4 is subsequently enzymatically converted into other leukotrienes (LTB4, LTC4, LTD4 and LTE4). Leukotrienes are biologically active molecules that may convey messages by interacting with specific membrane G-protein coupled receptors (Yokomizo et al., 1997) and likely regulate transcription by binding an intranuclear orphan receptor (Devchand et al., 1996). We found that LTB4, but not LTD4 or 5-HETE, enhanced the MPP+-induced cytotoxicity in the rat midbrain culture. Furthermore, MPP+ treatment increased the level of LTB4 in the supernatant of cultures in a time-dependent manner. In contrast, in vivo animal experiments in mice showed that MK-886 reduced the death of dopaminergic neurons in the substantia nigra and increased the dopamine, DOPAC and HVA concentrations in the striatum. In addition, the level of LTB4 in the striatum and substantia nigra was also increased following MPTP challenge. Previous studies have indicated that LTB4, a potent chemotactic agent, amplifies the inflammatory response and enhances the expression of IL-6, IL-12 and TNFα in the central nervous system (Phillis et al., 2006, Tassoni et al., 2008). It has also been reported that leukotrienes increase the proliferation of cultured astrocytes via the CysLT1 receptor and the CysLT1 receptor antagonist pranlukast inhibits glial scar formation in the chronic phase of cerebral ischemia (Yu et al., 2005). The increased leukotrienes in the acute phase may promote astrocyte proliferation in the boundary zone to ultimately form a glial scar, and the proliferated glia may produce LTB4 to induce further reactions (Zhou et al., 2006). LTB4 may thus be involved in MPP+-induced neuronal death. However, the signalling pathway needs further investigation.