| Objective Cerebrovascular diseases are still the major causes of leading death and disability. Moreover ,the morbility of the cerebrovascular diseases raises stepwise, about 80 percent of the cerebrovascular diseases are caused by cerebral ischemia. Endothelial nitric oxide synthase (eNOS) plays an important role in the regulation of the cerebral blood circulation. Up-regulating expression and/or activity of eNOS can vasodilate cerebral vessels and increase cerebral blood flow, especially in ischemic penumbra, and protect neurons from ischemic injury. Many studies have reported that puerarin ,a traditional Chinese herb, is very effective to treat or prevent from the injury caused by cerebral ischemia,especially in the early stage. However, the neuroprotective effect mechanism of puerarin is still unclear. Whether the neuroprotective effect of puerarin in the early stage of cerebral ischemia is associated with the up-reguration expression of eNOS in brain tissue? This issue has not been reported. We will investigate it, which may provide experiment evidence for clinical prevention and treatment of cerebral ischemia. Methods Animal Model: we made a rat model of the middle cerebral artery occlusion (MCAO) by introducing a monofilament in left common carotid artery and advancing it. Animal Groups: Rats were randomly divided into three groups: Sham- operate group (S,n=5);cerebral ischemia group (M,n=20); puerarin pretreatment group (P,n=20).M and P groups were further divided into four groups: cerebral ischemia 0.5h,1h,2h and 4h group (P0.5h,P1h,P2h,P4h and M 0.5h,M 1h,M2h,M4h,n=5).Puerarin was administered with intraperitoneal injection (100mg/kg, i.p.) in P groups 10 minute before MCAO.The vehicle was administered by equal volume in M groups and S group at the same time. Extracting The Sample And Observation:The rats were killed respectively at the corresponding time after cerebral ischemia. Neurological deficit scores were applied to evaluate the functional changes of the central nervous system before the rats were killed in S,M2h,M4h,P2h,and P4h groups.Hematoxylin and eosine (HE) dyeing and Neuron Nissl Body staining were used to observe the brain tissue pathologic histological changes of different groups,and the distribution and expression of eNOS protein in the brain tissue was evaluated by immunohistochemistry and western blot.Results Neurological deficit scores in all sub-groups of P groups showed significantly decrease compared with the corresponding sub-groups of M groups (P<0.05). The extents of the neuron damage of P groups were more slight than M groups in the brain tissue of HE staining, and the dissolved Nissl Bodies were decreased in the neurons of P groups in the brain tissue of Neuron Nissl Body staining. All sub-groups of P groups showed evidently an increase of the eNOS protein expression of brain tissue compared with the corresponding sub-groups of M groups by immunohistochemistry and western blot,and the eNOS protein expression of brain tissue in M1h and M2h groups was strong than S group(P<0.05 or P<0.01).However, comparison of the eNOS protein expression of brain tissue revealed no significant difference between M0.5h group, the super early stage of cerebral ischemia, and S group (P>0.05).Conclusions The eNOS protein expression of rat brain tissue is increased after MCAO. However, this increasing is not obvious in the super early stage . The pretreatment of puerarin can significantly up-regulate the protein expression of eNOS in brain tissue in the super early stage of cerebral ischemia, and it can also prolong the time of eNOS protein expression and effectively reduce the damage of brain tissue after MCAO. The mechanism that the pretreatment of puerarin protects brain tissue from the early stage of cerebral ischemia may be associated with up-regulating the protein expression of eNOS in brain tissue. |
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