Minocycline Modulates Inflammation And Repair After Focal Cerebral Ischemia Through Inhibiting 5-lipoxygenase Activation

A line of experimental and clinical evidence indicates that the inflammatory responses after ischemic stroke aggravate brain injury.As one of the pro-inflammatory molecules,5-lipoxygenase(5-LOX)is the rate-limiting enzyme in the metabolism of arachidonic acid to produce leukotrienes,including leukotriene B₄(LTB₄)and cysteinyl leukotrienes(CysLTs,namely LTC₄,LTD₄ and LTE₄).As potent inflammatory mediators, leukotrienes can induce leukocyte chemotaxis,smooth muscle contraction,microvascular leakage and other pathophysiological changes.Initially,5-LOX had been investigated in the peripheral diseases such as asthma and allergic rhinitis.Recently,it has been reported that 5-LOX is also expressed in the brain, and involved in various central nervous diseases,such as cerebral ischemia,trauma, tumor and epilepsy.After cerebral ischemia,5-LOX expression is increased and the contents of its metabolites,leukotrienes,are elevated in the ischemic brain.Moreover, 5-LOX inhibitors[such as AA861,MK-886,caffeic acid,and nordihydroguaiaretic acid (NDGA)]and the antagonists of leukotrienes(such as pranlukast and montelukast)exert protective effects against ischemic brain injury,indicating the involvement of 5-LOX in cerebral ischemia.One property in the late phase of cerebral ischemia is astrocyte proliferation and glial scar formation,which may be act as a physical/chemical barrier to reinnervation. Prevention of this reactive astrogliosis may be beneficial for repair and recovery.In our recent studies,we found that 5-LOX expression was increased and primarily localized in the proliferated astrocytes in the boundary zone after brain ischemia,indicating that 5-LOX was involved in glial scar formation.5-LOX inhibitor caffeic acid and CysLT-1 receptor antagonist pranlukast inhibited the astrocyte proliferation,glial scar formation, and exerted long-term protective effects.Furthermore,the importance of 5-LOX in stroke has been proven by the reports that the gene encoding 5-LOX activating protein confers risk of stroke.These findings indicate that 5-LOX is involved in ischemic brain injuries in the early and late phases,and inhibition of 5-LOX may have therapeutic benefits for cerebral ischemia.However,properties of 5-LOX involved in the inflammation after cerebral ischemia are not clearly clarified.It is also unknown whether the CysLT₁ receptor mediates 5-LOX actions on ischemic brain injury.On the other hand,minocycline,a second generation semi-synthetic tetracycline antibiotic,has been reported to possess neuroprotective effects on cerebral ischemic injury and other brain injuries,which is independent of its antibacterial activity.The neuroprotective effect of minocycline relates to its anti-inflammatory and anti-apoptotic activities,such as inhibiting the activation and proliferation of microglia,the expressions of inducible nitric oxide synthetase(iNOS),IL-1βconverting enzyme(ICE)and cyclooxygenase-2(COX-2),and the caspase-dependent and -independent cell apoptotic pathways.Recently,we have reported that minocycline protects PC12 cells from in vitro ischemic-like injury or NMDA-induced excitotoxicity;it can inhibit 5-LOX translocation to the nuclear membranes(a phenomenon of 5-LOX activation).These findings indicate that the in vitro protective effects of minocycline on ischemic or excitotoxic injuries may be partly mediated by inhibiting 5-LOX activation.In the present study,we determined whether minocycline exerts an in vivo anti-inflammatory effect mediated by inhibiting. 5-LOX activation after focal cerebral ischemia in rats.Taking together,following questions need to be clarified in in vivo studies:(1)what are the properties of 5-LOX-related inflammatory responses after cerebral ischemia?(2) whether the role of 5-LOX in regulating brain inflammation is mediated via CysLT₁ receptor?(3)whether minocycline inhibits inflammation,improves repair and accelerates function recovery via inhibiting 5-LOX expression and activation? In the present study, we designed and performed series of experiments in rats with focal cerebral ischemia-reperfusion to answer these questions.Partâ… 5-Lipoxygenase/cysteinyl leukotriene receptor-1 pathway mediates brain intlammation after focal cerebral isehemia in ratsAim:To determine the properties of 5-LOX-related post-ischemic inflammation, and the mediation of CysLT₁ receptor for the inflammation in rats with focal cerebral ischemia.Methods:Focal cerebral ischemia was induced by 30 min of middle cerebral artery occlusion and followed by reperfusion for 72 h.The ischemic injuries,endogenous IgG exudation,and accumulation of neutrophils and macrophage/microglia,and ICAM-1 and P-selectin expression were determined 72 h after reperfusion.5-LOX metabolites (leukotriene B₄ and cysteinyl leukotrienes)were measured by ELISA 3 h after reperfusion.Results:The 5-LOX inhibitor,NDGA(10 mg/kg,ip,for 3 days),and the CysLT-1 receptor antagonist,pranlukast(0.1 mg/kg,ip,for 3 days),attenuated ischemic injuries,IgG exudation,and accumulation of neutrophils,and ICAM-1 expression 72 h after reperfusion.NDGA also inhibited P-selectin expression 72 h after reperfusion,and the production of leukotrienes 3.h after reperfusion.However,both of them did not inhibit accumulation of macrophage/microglia.Conclusion:Focal cerebral ischemia induces accumulation of inflammatory cells,and increases blood-brain barrier permeability and adhension molecule expression.5-LOX inhibitor and CysLT₁ receptor antagonist partly attenuate the inflammatory responses,suggesting 5-LOX may be involved in post-ischemic inflammation partly mediated by CysLT₁ receptor.Partâ…¡Minocycline inhibits 5-lipoxygenase activation and brain inflammation after focal cerebral ischemia in ratsAim:To determine whether the effect of minocycline on post-ischemic brain inflammation is mediated by inhibition of 5-LOX expression and enzymatic activation in rats with focal cerebral ischemia.Methods:Focal cerebral ischemia was induced by 30 min of middle cerebral artery occlusion and followed by reperfusion for 72 h. Minocycline(22.5 and 45 mg/kg)was ip injected for 3 days after ischemia.The ischemic injuries,endogenous IgG exudation,and accumulation of neutrophils and macrophage/microglia determined 72 h after reperfusion;the expression of 5-LOX mRNA and protein were detected by RT-PCR and immunohistochemical analysis. 5-LOX metabolites(leukotriene B₄ and cysteinyl leukotrienes)were measured by ELISA 3 h after reperfusion.Results:Minocycline(22.5 and 45 mg/kg)attenuated ischemic injuries,lgG exudation,and accumulation of neutrophils and macrophage/microglia 72 h after reperfusion.5-LOX expression was increased.The increased 5-LOX was primarily distributed in the neurons in the ischemic core and in the astrocytes in the boundary zone of the ischemic core 72 h after reperfusion.Minocycline inhibited the increased 5-LOX expression 72 h after reperfusion,and the production of leukotrienes 3 h after reperfusion. Conclusion:Minocycline inhibits post-ischemic brain inflammation partly mediated by inhibiting 5-LOX expression and its enzymatic activation.partâ…¢Minocycline inhibits 5-lipoxygenase expression and accelerates function recovery in chronic phase of focal cerebral ischemia in ratsAim:To explore the long-term protective effects of minocycline on chronic post-ischemic brain injury,and the relation with inhibition of 5-LOX expression in rats with focal cerebral ischemia.Methods:Focal cerebral ischemia was induced by 60 rain of middle cerebral artery occlusion and followed by reperfusion for 36 days.Minocycline (45 mg/kg)was ip injected for 6 days after ischemia.The sensorimotor function outcome was evaluated by limb placing test,foot fault test,and adhensive tape test 1,3,7,14,21, and 28 days after ischemia.Cognitive function was determined by eight arms maze from 30 to 35 days after ischemia.At 36 days after reperfusion,pathohistological changes were evaluated by detecting infarct volume and survival neuron density,and gliosis and 5-LOX expression were determined by immunohistochemical staining.Results: Minocycline(45 mg/kg for 6 days)accelerated the recovery of the sensorimotor and cognitive functions,while did not affect the infarct volume.At 36 days after reperfusion, 5-LOX expression was increased.The increased 5-LOX was primarily distributed in the proliferated astrocytes in the boundary zone around the ischemic core,and in the macrophages/microglia in the ischemic core.Minocycline significantly inhibited gliosis and 5-LOX expression.Conclusion:Minocycline accelerates functional recovery,which is partly mediated by inhibition of 5-LOX expression in the chronic phase of focal cerebral ischemia in rats. Conclusions1.5-LOX inhibitor NDGA and CysLT-1 receptor antagonist pranlukast inhibit post-ischemic brain inflammation 72 h after focal cerebral ischemia.These findings indicate that 5-LOX is involved in regulating brain inflammation partly mediated by CysLT₁ receptor.2.5-LOX expression and activation are increased 72 h after focal cerebral ischemia in rats;minocycline inhibits 5-LOX expression and enzymatic activation,and attenuates post-ischemic brain inflammation as well.These findings indicate that minocycline inhibites post-ischemic brain inflammation partly mediated by inhibiting 5-LOX expression and activation.3.Minocycline accelerates the recovery of sensorimotor and cognitive function, inhibits astrocytes proliferation in the boundary zone and microglia/macrophage accumulation in the ischemic core,and inhibits glial scar formation in the chronic phase of focal cerebral ischemia.It also inhibits the increased 5-LOX expression in the proliferated astrocytes.These findings indicate that minocycline exerts the long-term protective effects mediated via inhibiting 5-LOX expression.4.The present study provides new experimental evidence of the properties and mechanism of the central actions of minocycline for its use in the treatment of central nervous system diseases like ischemic stroke.