The Role Of12/15-LOX Pathway In Cerebral Ischemia And The Effect Of Oxymatrine Treatment On12/15-LOX Pathway

Part I The role of12/15-LOX in cerebral ischemiaObjective: Cerebral ischemia is one of the leading causes of death andadult disability wordwide. The secondary injury after cerebral ischemiaaggravated the progress of stroke and influenced the recovery of strokepatients. Despite the significant efficiency of neuroprotective reagents in alarge growing of experimental stroke models, none of these drugs was provedavailable in stroke patients. With the advanced understanding ofpathophysiology of cerebral ischemia in recent years, it has been found thatinflammation plays an important role in the progress of ischemic injury afterstroke. The production of inflammatory mediators induced by the recruitmentof blood-derived leukocytes and the activation of resident inmmune cells inischemic brain will damage the blood-brain barrier and promoted neuronalnecrosis after stroke. Because of the susceptibility of neurons to variousstimuli, local brain injury will cause permanent neuronal dysfunction afterstroke. Thereafter, inflammaroty response provides a new therapeutic strategyfor stroke therapy. Pro-inflammatory cytokines including interleukin-1β (IL-1β) and tumornecrosis factor α (TNFα) regulates the expression of arachidonicacid cascades enzymes through NF-κB and AP-2signaling pathways. In thebrain, arachidonic acid and its metabolites influence gene transcription, signaltransduction, neuronal activity, aptosis and other process.12/15-LOX is one ofthe major enzymes that catalyze the metabolism of arachidonic acid. Ourprevious studies have demonstrated that the expression of12/15-LOX wasup-regulated after stroke. Deficiency of12/15-LOX improved blood-brainbarrier permeability and functional recovery after stroke. In this study, weinvestigated the potential effect of12/15-LOX in cerebral ischemia by using the specific inhibitor, bacalain.Methods: Male and healthy Sprague-Dawley rats were subjected tomodified permanent middle cerebral artery occlusion (MCAO). Baicalein wasadministered at a dose of30mg/ml by jugular vein injection immediately afterMCAO. Rats were randomly assigned to four groups: Sham operated group(Sham), MCAO, vehicle (MCAO+DMSO), and baicalein group (MCAO+baicalein). At24h after ischemia neurological deficit was evaluated, brainwater content was measured by wet-dry method, infarct size were analyzedwith2,3,5-triphenyltetrazolium chloride (TTC) staining.Immunohistochemistry, reverse transcription–polymerase chain reaction(RT-PCR) and western blot were used to analyze the expression of12/15-LOX.Results:1The expression of12/15-LOX protein and gene as well as the12/15-LOX positive cells was significantly increased at24h after cerebralischemia.2The inhibitor of12/15-LOX, baicalein, efficiently suppressed theexpression of12/15-LOX in ischemic brain.3Rats in MCAO group, vehicle group and baicalein group all showedsome degree of neurological deficit at24h after stroke. The12/15-LOXinhibitor, baicalein, significantly improved functional recovery after stroke.4The brain water content of ischemic hemisphere was increased at24hafter stroke. Compared with vehicle treatment, baicalein significantlydecreased brain water content after stroke.5Compared with vehicle treatment, baicalein significantly decreasedinfarct size at24h after stroke.6Vehicle did not have effect on behavior test, brain edema and infarctsize.Conclusion: Inhibition of12/15-LOX significantly improvedneurological deficit, decreased brain water content and infarct size at24hafter stroke.12/15-LOX contributes to the brain injury after stroke. Part II The influence of12/15-LOX in the expression p38MAPK andcPLA2in cerebral ischemiaObjective: It has been demonstrated that activation of p38mitogen-activated protein kinase (p38MAPK) is one of the most importantmechanism for12/15-LOX pathway. Release of AA induced by cytosolicphospholipase A2(cPLA2) is the rate-limiting step in the12/15-LOX pathway.Several reports have shown that12/15-LOX derivatives from AA can directlyactivate p38MAPK and cPLA2, induce more release of AA. The activatedp38MAPK is also linked to activation and phosphorylation of cPLA2and AArelease. However, little is known about the relationship between12/15-LOXexpression and the activity of p38MAPK and cPLA2in ischemic stroke. Inthis study, we investigated whether inhibition of12/15-LOX influences thesefactors’ expression after stroke.Methods: Male and healthy Sprague-Dawley rats were subjected tomodified permanent middle cerebral artery occlusion (MCAO). Baicalein wasadministered at a dose of30mg/ml by jugular vein injection immediately afterMCAO. Rats were randomly assigned to four groups: Sham operated group(Sham), MCAO, vehicle (MCAO+DMSO), and baicalein group (MCAO+baicalein). At24h after ischemia the phosphorylated p38mitogen-activatedprotein kinase (p-p38MAPK) and cytosolic phospholipase A2(cPLA2)positive cells were detected using immunohistochemistry. And the expressionof p-p38MAPK and cPLA2protein was measured using western blot. RT-PCRwas used to analyze the gene expression of cPLA2.Results:1The number of p-p38MAPK and cPLA2positive cells was significantlyincreased after cerebral ischemia, and the expression of p-p38MAPK andcPLA2showed increase in western blot and RT-PCR.2Baicalein significantly decreased the expression of p-p38MAPK and p-p38MAPK positive cells in ischemic brain at24h after MCAO.3Baicalein significantly decreased the expression of cPLA2and cPLA2positive cells in ischemic brain at24h after MCAO.Conclusion: Bacalein protected the brain against cerebral ischemicdamage by inhibiting the activation of p38MAPK and down-regulating theexpression of cPLA2. Inhibition of AA metabolism contributes to theneuroprotection induced by baicalein after stroke. Part III The role of oxymatrine in the expression of12/15-LOX pathwayin cerebral ischemiaObjective: Oxymatrine is an alkaloid extracted from the root of Sophoraflavescent Arr (Kushen). Our previous studies have demonstrated thatoxymatrine protects the brain against ischemic injury by inhibitinginflammatory responses after stroke. In this study, we investigated whetheroxymatrine treatment has influence in the expression and activity ofarachidonic acid cascade enzymes after stroke, and demonstrated thatinhibition of arachidonic acid metabolism is an efficient therapeutic target forstroke.Methods: Male and healthy Sprague-Dawley rats were subjected topermanent MCAO. Oxymatrine was administered at a dose of120mg/ml byperitoneal injection immediately after MCAO. Rats were randomly assignedto three groups: Sham operated group (Sham), MCAO (MCAO+vehicle), andoxymatrine group (MCAO+oxymatrine). At24h after ischemia, neurologicaldeficit was evaluated, brain water content was measured by wet-dry method,and infarct size was analyzed with TTC staining. The expression of12/15-LOX, p-p38MAPK, and cPLA2was respectively detected usingimmunohistochemistry, western blot and RT-PCR at24h after ischemia. Results:1Oxymatrine significantly decreased infarct size at24h after stroke.2Oxymatrine significantly decreased ipsilesional but not contralesionalbrain water content at24h afte stroke.3Compared with MCAO group, the number of12/15-LOX positive cellsas well as the protein and gene expression of12/15-LOX in ischemic brainwas significantly decreased by oxymatrine at24h after stroke.4Compared with MCAO group, the number of p-p38MAPK positivecells as well as the protein expression in ischemic brain was significantlydecreased by oxymatrine at24h after stroke.5Compared with MCAO group, the number of cPLA2positive cells aswell as the protein and gene expression of cPLA2in ischemic brain wassignificantly decreased by oxymatrine at24h after stroke.Conclusion: Oxymatrine protected the brain against cerebral ischemicdamage by inhibiting the activation and expression of arachidonic acidcascade enzymes after stroke. Inhibition of arachidonic acid metabolism istherapeutic target for anti-inflammatory threatment after stroke.