Effects Of HAMI3379,a Selective Cysteinyl Leukotriene Receptor-2Antagonist,on Ischemic Brain Injury

Backgrounds:Inflammation is a critical pathological basis for brain injury caused by cerebral ischemia and reperfusion. Cysteinyl leukotrienes (CysLTs; including LTG4LTD4and LTE4) are potent inflammatory mediators, and play important roles in cerebral ischemia, traumatic brain injury and other diseases in the central nervous system. The actions of CysLTs are mediated by their specific receptors, the CysLT receptors (CysLT1R and CysLT2R), which have been cloned and characterized.After focal cerebral ischemia, CysLT1R expression is up-regulated. In the acute phase, the up-regulated CysLT1R is restricted to injured neurons in the ischemic core; while in the late phase, it is restricted to activated microglia (ischemic core) and hypertrophic astrocytes (boundary zone). Our group has reported the protective effects of the CysLT1R antagonists, pranlukast and montelukast, on acute and chronic brain injury after focal cerebral ischemia in rats and mice. The possible mechanisms may be that they inhibit BBB disruption and inflammation in the acute/subacute phases of focal cerebral ischemia, and astrocyte proliferation and the related glial scar formation in the chronic phase.The CysLT2R is expressed in the heart, umbilical vein endothelial cells, cremaster muscle vasculature, and myenteric neurons. In human brain, the CysLT2R is expressed in cerebral vascular smooth muscle and infiltrated granulocytes. The expression of CysLT2Rs is relatively higher than that of CysLT1Rs in the brain. In normal rat brain, CysLT2Rs are localized in astrocytes in the cortex and around the ventricles; however, after focal cerebral ischemia, the CysLT2R expression is up-regulated in injured neurons in the acute phase; and in proliferated astrocytes and microglia in the late phases. The spatiotemporal profiles of CysLT2R expression and distribution are similar to those of the CysLT1R. Moreover, the CysLT2R is associated with ischemic injury in PC12cells, and mediates the expression of the water channel aquaporin-4(AQP4) induced by ischemic injury or LTD4in astrocytes. These findings suggest that the CysLT2R is an important regulator in cerebral ischemia, so protective effects of CysLT2R antagonists on ischemic stroke or other brain diseases may be anticipated.HAMI3379is a selective CysLT2R antagonist recently reported. HAMI3379is devoid of CysLTR agonism, and shows>10,000-fold affinity for CysLT2R versus CysLT1R. Bay CysLT2, another selective CysLT2R antagonist, protects mice from myocardial infarction and inhibits LTD4-induced Evans blue dye leakage in the mouse ear vasculature; it protects astrocytes from ischemic injury as well. Recently, it has been reported that HAMI3379shows no direct effect on OGD/R-induced neuronal injury in neuron cultures, but inhibits reduction in cell viability and LDH release in the neuron-microglial cocultures and mixed cultures of cortical cells. In these cultures, HAMI3379attenuates ischemic neuronal injury mediated by inhibiting microglial activation. However, whether HAMI3379has an in vivo protective effect on focal cerebral ischemia in rats is unknown.It has been proved that the CysLT2R, not the CysLT1R, mediates ischemic neuronal injury through activating microglia. Since BV2microgial cells, a mouse microglioma cell line, have most characteristics similar to primary microgia, they have been widely used in the related research. Whether HAMI3379as well as pranlukast have similar protective effects on activation of BV2cells after exposure to OGD? What is the related mechanism? These problems remain to be further studied.Aims:Our goals were to clarify the protective effects of the CysLT2R antagonist HAMI3379on brain injury afer focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in rats, and its effects on BV2cell activation after ischemic injury induced by oxygen-glucose deprivation (OGD). In this study, the following investigations were performed:(1) At first, we determined the neuroprotective effect of HAMI3379via intracerebroventricular (i.c.v.) injection on acute brain injury after focal cerebral ischemia in rats. Its effect was compared with that of the selective CysLTiR antagonist pranlukas. Second, we administered HAMI3379via intraperitoneal injection (i.p), which is much closer to clinical medication, to futher determine the effects of HAMI3379on acute brain injury, sunacute injury and inflammation, and chronic injury after focal cerebral ischemia in rats.(2) Then, we investigated the effects of HAMI3379and pranlukast on the activation of BV2cells. The phagocytosis, the release and expression of cytokines, autophagy and CysLT1R-CysLT2R interaction were determined in BV2cells after exposure to OGD.Methods:Focal cerebral ischemia was induced by middle cerebral arterial occlusion (MC AO) in rats. Neurological deficit scores were observed and an inclined board test was performed to assess the behavioral dysfunction. Regional cerebral blood flow (rCBF) was monitored by laser Doppler flowmetry. The coronal slices were stained with TTC to determine infarct volume and brain edema. The neuron morphology was assessed in brain sections stained with toluidine blue or Cresyl violet. Degenerating neurons were determined by Fluoro-Jade B staining. Blood-brain barrier permeability, microglia, activated microglia, neutrophils and astrocyte were determined by immunohistochemistry. The survival neurons were determined by immunofluorescence. IFN-y, IL-4and IL-10in the serum and cerebrospinal fluid (CSF) were measured using CBA Flex set kits. The cytokines IL-1β, IL-6and TNF-a were measured using ELISA kits.In in vitro experiments, BV2cell viability was determined by MTT reduction assay, and BV2cell death by measuring LDH release after exposure to different durations of OGD followed by recovery (R); the optimal OGD/R condition was selected. The expression of LC3, CysLT1R and CysLT2R in BV2cells after OGD/R were detected by immunofluorescence. Phagocytosis was detected by fluorescence microscopy, and apoptosis was detected by flow cytometry with Annexin V/PI kits. The CysLT1R-CysLT2R interactions in BV2cells were detected by PLA kits. TNF-α and IL-6release in the culture media of BV2cells were detected with CBA kits. mRNA expression of all the cytokines in BV2cells were detected by real-time PCR.Results:Part Ⅰ. Protective effects of HAMI3379on focal cerebral ischemia in ratsIntracerebroventricular injection of HAMI3379(100ng) had no effect on the mean arterial blood pressure, arterial pH, PaO2, PaCO2, and blood glucose30min after reperfusion; it did not significantly affect the pattern of rCBF change as well. HAMI3379at10and100ng (but not1ng) attenuated the neurological deficits, and reduced infarct volume, brain edema, IgG exudation, neuronal degeneration and neuronal loss. This protective effect was similar to that of pranlukast (positive control). Intracerebroventricular injection of CysLT2R shRNA also reduced infarct volume, brain edema. Thus, HAMI3339is neuroprotective against acute brain injury after focal cerebral ischemia in rats, which is CysLT2R dependent.Intraperitoneal injection of HAMI3379had no effect on mortality rates and physiological parameters, such as mean arterial blood pressure and blood glucose, after MCAO in rats. HAMI3379attenuated acute brain injury24h after MCAO with an effective dose range of0.1-0.4mg/kg and a therapeutic window of1h. It attenuated neurological deficits, and reduced infarct volume, brain edema, neuron loss and degeneration24and72h after MCAO. Also, it inhibited release of the cytokines IL-1β IFN-γ and TNF-α in the serum and CSF24h after MCAO. Moreover, HAMI3379ameliorated the microglial activation and neutrophil accumulation in the ischemic regions, but did not affect astrocyte proliferation72h after MCAO. In comparison, the CysLT1R antagonist pranlukast did not affect microglial activation and IFN-γ release, but inhibited astrocyte proliferation and reduced serum IL-4. These results indicated that HAMI3379has a protective effect on acute and subacute brain ischemic injury, and attenuates microglia-related inflammation. After MCAO operation, HAMI3379(0.1mg/kg) was injected intraperitoneally for6consecutive days. HAMI3379improved neurological deficits and increased holding angles in the inclined board test during0-7days after ischemia. HAMI3379also ameliorated brain atrophy and lesion, increased neuronal density in the ischemic border zone, and inhibited microgila activation and glial scar formation (astrocyte proliferation)14days after MCAO. These results confirmed the long-term neuroprotective effect of HAMI3379on chronic brain injury after focal cerebral ischemia in rats.Part II. Effect of HAMI3379on BV2cell activation after exposure to OGDOGD1h followed by recovery (R)48h moderately activated BV2cells, namely induced changes in the morphology, enhanced the phagocytotic function, the production and release of cytokines, autophagy, and the upregulation and nuclear translocation of the CysLT1R and CysLT2R with spaciotermporal consistentance. HAMI3379reversed OGD/R-induced BV2cell activation, reduced production and release of cytokines, autophagy, and inhibited upregulation and nuclear translocation of the CysLT1R and CysLT2R. The CysLTiR antagonist pranlukast had similar effects to HAMI3379, but did not affect phagocytosis. PLA results indicated that there was an interaction between the CysLT1R and CysLT2R, which was enhanced by OGD1h/R48h. HAMI3379inhibited the interaction between the CysLT1R and CysLT2R, and also increased the expression of CysLT1R mRNA.Conclusions:(1) In rat model of focal cerebral ischemia, at first, we primarily have demonstrated that intracerebroventricular injection of HAMI3379protects against acute brain injury after focal cerebral ischemia in rats. Intraperitoneal injection of HAMI337protects against acute and subacute brain injury, and attenuates post-ischemic inflammation, especially the potent inhibitory effect on microglial activation. The effective dose of HAM3379is0.1-0.4mg/kg; and the therapeutic window is within1h after MCAO. Also, HAMI3379has a long-term neuroprotective effect on chronic brain injury, and promotes recovery of neural function. These effects are similar to those of the CysLTiR antagonist pranlukast, but have some distinct properties.(2) In the mouse microglial line, BV2cells, HAMI3379inhibits BV2cell activation; it reduces phagocytosis, production and release of cytokines release, and phagocytosis after exposure to OGD/R. Pranlukast has similar effects, but does not inhibit enhanced phagocytosis. In addition, HAMI3379inhibits OGD/R-enhanced CysLTiR-CysLT2R interaction and intracellar movement, and increases CysLT1R expression as well.(3) Our study, for the first time, comprehensively demonstrates the protective effects of CysLT2R antagonist(s) on ischemic brain injury, and further confirm that neuroprotection by HAMI3379may be mediated inhibiting microglial activation. Our findings provide experimental evindence for the potential application of CysLT2R antagonists in the treatment of ischemic stroke.