Protective Effects And Mechanisms Of Lipoxin A4 Methyl Ester On Transient Cerebral Ischemia And Reperfusion Injury

PartⅠEffect of lipoxin A4 methyl ester on cerebral ischemia reperfusion injury in ratsObjective To establish intraluminal suture right middle cerebral artery occlusion (MCAO) model and investigate effects of lipoxin A4 methyl ester (LXA4 ME) on cerebral ischemia reperfusion injury in rats. Methods Fourty-eight adult male Sprague-Dawley rats were divided randomly into four groups:sham-operated group (sham group), ischemia/reperfusion group (I/R group), low dose of LXA4 ME group (0.03 nM LXA4ME, L group) and high dose of LXA4 ME group (0.3 nM LXA4 ME, H group). Rats were subjected to middle cerebral artery occlusion for 2 h. The neurological deficits, infarct volume and morphologic injury were assessed at 24 h after reperfusion. The myeloperxidase activity and the lipid peroxidation levels were evaluated. The concentration of IL-1βand TNF-αwere studied by ELISA assay. Results Compared with sham group, LXA4 ME, especially at high dose, reduced infarct volume, improved neurological recovery, decreased numbers of neutrophil and suppressed malondialdehyde levels at 24 h after reperfusion. Also concentration of IL-1βand TNF-a in LXA4 ME groups was lower significantly. Conclusion Intraluminal suture MCAO model in rats provided reproducible MCA territory infarctions and allowed reperfusion by retracting the suture. LXA4 ME may be a promising neuroprotective approach to against focal cerebral ischemia reperfusion injury.PartⅡLipoxin A4 methyl ester protects brain and reduces inflammation in a rat model of focal cerebral ischemia reperfusionObjective To investigate effects of LXA4 ME on the brain against focal ischemia reperfusion injury and its mechanism. Methods Male Sprague-Dawley rats were randomly divided into three groups:sham-operated group (sham group), ischemia reperfusion group (I/R group), lipoxin A4 methyl ester group (LXA4 ME group, intracerebroventricular injection of 0.3 nM lipoxin A4 ME), and subjected to a transient right-side middle cerebral artery occlusion for 2 h. The neurological deficits, infarct volume, morphologic injury, apoptosis neuronal, activated glia, MPO activity and the lipid peroxidation levels were evaluated at 24 h after reperfusion. The expression of cytokines TNF-a, IL-1β, IL-10, and TGF-β1 in brain tissue were detected at 0,6,12,24 h after reperfusion by ELISA assay. Activation of NF-κB was analyzed by Western blotting and EMSA. Results Intracerebroventricular administration of LXA4 ME ameliorated neurological dysfunctions, reduced infarction volume, attenuated neuronal apoptosis. Treatment with LXA4 ME suppressed neutrophils infiltration and lipid peroxidation levels, inhibited the activation of microglia and astrocytes, down-regulated the expression of pro-inflammatory cytokines TNF-a and IL-1β; and up-regulated the expression of anti-inflammatory cytokines IL-10 and TGF-β1 in the ischemic brain. In addition, activation of NF-κB was inhibited by LXA4 ME significantly. Conclusions These results demonstrate that treatment of LXA4 ME significantly reduced cerebral ischemia reperfusion injury in rats, and that these effects might be associated with its anti-inflammatory, which is related to inhibition of the activity of NF-κB.PartⅢEffects of lipoxin A4 methyl ester on JAK2/STAT3 signal pathway following transient focal cerebral ischemia reperfusionObjective To examine whether LXA4 ME affect JAK2/STAT3 pathway following transient focal cerebral ischemia reperfusion injury in rats. Methods Adult male Sprague-Dawley rats were randomly divided into sham-operated group (sham group), cerebral ischemia reperfusion group (I/R group), DMSO + ischemia reperfusion group (intracerebroventricular injection of 3% DMSO 5μl, DMSO group), AG490+ischemia reperfusion group (intracerebroventricular injection of AG490 100 nM, AG490 group), LXA4 ME + ischemia reperfusion group (intracerebroventricular injection of LXA4 ME 0.3 nM, LXA4 ME group). Rats were subjected to middle cerebral artery occlusion for 2 h. The neurological deficits were evaluated at 24 h after reperfusion. The concentration of IL-6 in brain tissue were measured at 6,24 h after reperfusion by ELISA assay. The expression of STAT3 and SOCS3 mRNA were analysed by real-time quantitative PCR. The protein level of p-JAK2, p-STAT3, SOCS3 were tested by western blotting. And the cellular localization of p-JAK2, p-STAT3, SOCS3 were evaluated by fluorescent immunohitochemistry. Results Compared with sham group, transient middle cerebral artery occlusion in rats led to significantly increase the concentration of IL-6, the mRNA level of STAT3 and SOCS3, phosphorylation of p-JAK2, p-STAT3 and their immunoreactive glia in the ipsilateral cortex at 24 h reperfusion. Intracerebroventricular injection of AG490 prevented the cerebral ischemia induced JAK2 and STAT3 phosphorylation and significantly decreased JAK2, STAT3 immunoreactive glia located in ischemic cortex. LXA4 ME reduced the concentration of IL-6, increased the expression of SOCS3 and inhibited JAK2 and STAT3 phosphorylation, attenuated JAK2 and STAT3 immunoreactive glia, ameliorated neurological dysfunctions. Conclusions LXA4 ME could inhibit JAK2/STAT3 activation, maybe via up-regulation of SOCS3 and inhibit expression of IL-6 in vivo, to ameliorate the cerebral ischemic injury.