Inflammatory Mechanisms In Reperfusion Injury After Focal Cerebral Ischemia And The Protective Effects Of Hydroxyethylpuerarin

BACKGROUD AND OBJECTIVES Ischemic cerebral vascular disease(ICAD) is a neurological disorder caused by insufficient blood supply to a particular brain area and has become the third common cause of death around the world. Recent study has revealed that inflammatory mechanisms play an important role in the secondary injury after acute cerebral ischemia, which was characterized by a progressive increase in neutrophils adhesion and infiltration. Inflammatory cytokines such as tumor necrosis factor α (TNF- α ) and interleukin-1 β (IL-1 β) as well as adhesion molecules which are produced at the early stage of ischemia promote both the recruitment of inflammatory cells, their adherence to brain endothelial cells and the resultant activation of inflammatory processes. Nuclear factor-kappaB(NF-κB), a proinflammatory transcription factor, which is closely associated with ischemia-reperfusion injury, was also reported to be activated in the ischemic brain tissue. Activation of the NF-κB proteins plays a pivotal role in inflammation through the regulation of genes encoding pro-inflammatory cytokines, adhesion molecules, oxidative-stress related enzymes and cell surface receptors.Recent study has revealed that puerarin could exhibit its neuroprotective effect by inhibiting the inflammatory reactions in the brain tissue. But the lipid solubility of puerarin is comparatively low, which limits its effectiveness to some extent. Hydroxyethylpuerarin, which was modified on the structure basis of puerarin, has higher lipid solubility and BBB permeability as compared to puerarin. Our previous study have shown that hydroxyethylpuerarin could exhibit its protective effects against brain ischemia-reperfusioninjury by reducing damage of oxygen free radicals, increasing the activity of antioxidase and inhibiting cell apoptosis in ischemic area , it could also protect the astrocytes and brain microvascular endothelial cells from the injury of hydrogen peroxide^C^) in vitro. In our present study, the ischemia-reperfusion injury model was established by middle cerebral artery occlusion(MCAO) to investigate the role of inflammation in cerebral ischemia reperfusion injury and the potential neuroprotective mechanism of hydroxyethylpuerarin.METHODS Adult male Wistar rats weighing 270g~320g were used in the experiments. Animals were randomly divided into six groups: sham operation group(NS 5ml/kg, iv), ischemia-reperfusion(I/R) injured group(NS 5ml/kg, iv), high dose of hydroxyethylpuerarin group(40mg/kg, iv), middle dose of hydroxyethylpuerarin group(20 mg/kg, iv), low dose of hydroxyethylpuerarin group(10mg/kg iv), puerarin group(18 mg/kg, iv). Middle cerebral artery (MCA) occlusion was prepared by putting a nylon suture( cj> 0.2mm) in the MCA. Restoration of MCA blood flow in animals subjected to two hours of MCA occlusion was achieved by withdrawing the suture to the external carotid artery (EC A). Drugs were administered for two times(30minutes before occlusion and immediately after reperfusion). Twenty-four hours after reperfusion, rats were sacrificed and the ischemic brain tissues were immediately removed. Part samples were isolated for routine pathological examination and counting the number of viable pyramidal cells in hippocampal CA1 region, the rest were frozen in liquid nitrogen for the following study. Myeloperoxidase(MPO) activity was evaluated to determine the infiltration of neutrophils in brain tissue, protein expression of intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), P-selectin and E-selectin were evaluated by western blot analysis, IL-1 {3 and TNF- a content were observed with radioimmunoassay(RIA), mRNA expression of ICAM-1,VCAM-1, P-selectin, E-selectin, IL-1 (3 and TNF- a were examined with reverse transcriptase-polymerase chain reaction (RT-PCR), the nuclear translocation of NF-kB p65 subunit and the protein expression of IkB a were observed by western blot, the DNA binding activity of nuclear factor -kappaB(NF-KB ) was observed by electrophoretic mobility shift assay (EMSA). RESULTS Compared with sham operation group, rats in I/R group exhibited obviousneurological deficit and high mortality rate. Morphological examination of hippocampus CA1 region showed obvious ischemic damage. Hydroxyethylpuerarin 40 and 20mg/kg could improve the neurological deficit of ischemic rats and alleviate the pathological damage in hippocampal CA1 region. Hydroxyethylpuerarin lOmg/kg and puerarin 18mg/kg only had the tendency to improve the neurological deficit of ischemic rats and alleviate the pathological damage in hippocampal CA1 region. The MPO activity was obviously increased following 2 hours of ischemia and 24 h reperfusion as compared with sham group, hydroxyethylpuerarin and puerarin treatment could obviously decrease the MPO activity and alleviate the neutrophil infiltration in ischemic brain tissue. The mRNA and protein expression of ICAM-1, VCAM-1, P-selectin, E-selectin, IL-1 P and TNF- a were obviously increased in I/R injured group. Hydroxyethylpuerarin treatment could suppress the expression of ICAM-1, VCAM-1, P-selectin, E-selectin, IL-1 3 and TNF-a . Western blot analysis and EMS A study of NF-kB suggested that hydroxyethylpuerarin also inhibited the nuclear translocation and DNA binding activity of NF-kB. IkB a protein was dramatically reduced in MCAO rats as indicated by western blot, whereas no significant reduction of IkB a in the sham group was observed. The degradation of IkB a protein was markedly reduced by hydroxyethylpuerarin treatment.CONCLUSION Our results demonstrate that neutrophils, adhesion molecules, cytokines and NF-kB may play an important role in the pathogensis of cerebral ischemia reperfusion injury. Hydroxyethylpuerarin can protect the brain from ischemia-reperfusion injury in rats by improving neurological deficit and alleviating the pathological damage in hippocampus. Hydroxyethylpuerarin can inhibit neutrophil-mediated inflammatory response after brain ischemia reperfusion in rats by down-regulating the expression of adhesion molecules and cytokines and inhibiting the translocation and activation of NF-kB. These data suggest that hydroxyethylpuerarin may exhibit its protective effect against cerebral ischemia-reperfusion injury in rats by alleviating the excessive inflammatory reaction in ischemic brain tissue.