The Neuroprotective Effects And Mechanisms Of Hydroxyethylpeurarin On Cerebral Ischemia/Reperfusion Injury In Rats

Objective: Cerebrovascular diseases have the characteristics of high morbility, high rate of mutilation and high mortality, therefore they can do great harm to human health. The patho-physiological mechanisms of cerebrovascular diseases haven't been fully made out till now. Ideal drugs which can successfully prevent and treat cerebrovascular diseases are very few according to recent reports. Our present study was performed both in vivo and in vitro to investigate the patho-physiological mechanisms of cerebral ischemia/reperfusion injury, focusing on whether a new member of isoflavine, hydroxyethylpuerarin (HEP) has neuroprotective effects on brain injury after cerebral ischemia/reperfusion based on our previous study and implore its probable mechanisms as well. This study may have an important implication in hydroxyethylpuerarin growing to a new creative drug which has independence intellectual property rights for the future treatment of patients suffered with cerebrovascular diseases.Methods: There are two parts of our study. For in vivo experiments, typical rat thread model performed by middle cerebral artery occlusion (MCAO) was used to induce the reversible focal cerebral ischemia/reperfusion injury under different glycemic conditions. Rats under normoglycemic condition were divided into sham-operate group; focal cerebral ischemia/reperfusion injury group which were subject to one hour of ischemia followed by 48 hours of reperfusion; drug treatment groups(focal cerebral ischemia/reperfusion injury and pretreatment with different doses of drugs). The behavioral tests were used to evaluate the damage to central nervous system. 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining method was used to assess the percentage of brain infarct area. H&E staining and flow cytometry methods were used to observe the pathologic histological changes and the apoptotic occurrence of hippocampus CA₁ region. Colorimetric method was used to determine the changes of cerebral energy metabolism. Nitroreductase method was used to determine the NO and NOS activity in brain homogenate. Radioimmunoassay method was used for determination of endothelin-1(ET-1) and interleukin-6 (IL-6) in brain tissue and blood. Rats under hyperglycemic condition prepared by pre-injection glucose were divided into sham-operate group; focal cerebral ischemia/reperfusion injury group which were subject to one hour of ischemia followed by three hours of reperfusion; drug treatment groups (focal cerebral ischemia/reperfusion injury and pretreatment with different drugs). TTC staining method was used to assess the percentage of brain infarct area and brain edema in the brain slices. Immunoblot methods were used for the protein expressions of Manganese-superoxide dismutase (Mn-SOD), Complexin I , Complexin II and glutamate transporter GLAST. For in vitro experiments, cultured neonate rat cortical astrocytes were used. Na₂S₂O₄ and sugar free Earle's solution were used to perform the chemical oxygen glucose deprivation (OGD) model, which followed by changing the culture into complete medium to mimic the ischemia/reperfusion injury. 3-(4,5-dimethylthialzol-2-yl) -2-5 -diphenyl- tetrazolium bromide (MTT) method and lactate dehydrogenase (LDH) leakage method were used for evaluations of cell viability and cell lysis respectively. Nitroreductase method was used to determine cellular NO level and NOS activity. Colorimetric method was used to determine the changes of celluar energy metabolism, malondialdehyde (MDA) level and superoxide dismutase (SOD) activities.Results: For in vivo experiments, compared with sham-operate group, rats under normoglycemic condition subject with one hour of ischemia followed by 48 hours of reperfusion exhibited severe neural injury as shown in significant increase of neurological scores, significant increase of infarct area ratio, significant cell loss and apoptosis in CA₁ region of hippocampus disorder of cerebral energy metabolism, increase of NO level and NOS activity, increase of levels of ET-1 and IL-6 in either brain tissue or blood. Compared with sham-operate group, rats under hyperglycemic condition subject with one hour of ischemia followed by three hours of reperfusion also exhibited severe neural injury as shown in significant increase of infarct area and edema ratio, significant down-regulation of protein expressions of Mn-SOD, Complexin I and Complexin II, significant up-regulation of glutamate transporter GLAST protein expression. For in vitro experiments, compared with sham-group, cultured neonate rat cortical astrocytes subject with one hour of OGD followed by 24 hours of reperfusion exhibited severe injury as shown in significant decrease of cell viability and increase of cell lysis, disorder of cellular energy metabolism, increase of cellular NO level and NOS activity, increase of malondialdehyde (MDA) level and decreases of SOD activities. Pretreatment with different doses of hydroxyethylpuerarin can reverse or attenuate those changes of cerebral ischemia/reperfusion injury to some extent.Gonclusions1. Pre-treatment of hydroxyethylpuerarin exhibits neuroprotective effects oncerebral ischemia/reperfusion injury in rats under normoglycemic conditions, the mechanism of its effects is possibly through improving cerebral energy metabolism, attenuating the neurotoxicity of NO, decreasing injury of blood vessel endothelium and inhibiting the production of inflammatory cytokines.2. Pre-treatment of hydroxyethylpuerarin exhibits neuroprotective effects on cerebral ischemia/reperfusion injury in rats under hyperglycemic conditions, the mechanism of its effects is possibly through increasing the expression of anti-oxidative enzyme, up-regulating the protein expression of Complexins and down-regulating the potein expression of glutamate transpoter, GLAST which contributes to attenuate glutamate-mediated excitotoxity after cerebral ischemia/reperfuion.3. Pre-treatment of hydroxyethylpuerarin exhibits neuroprotective effects on OGD/reoxygenation injury in rats' cortical astrocytes, the mechanism of its effects is possibly through improving cellular energy metabolism, increasing cellular anti-oxidative ability and attenuating the neurotoxicity of NO.