| Hypoxia-inducible factor-1(HIF-1) is a transcription factor thatfunctions as a master regulator of cellular and systemic oxygen homeostasis. Itconsists of two subunits: HIF-1α and HIF-1β. The regulaton of HIF-1activityconcerns mostly the HIF-1α subunit. As an oxygen regulated protein, HIF-1αcan response to changes in oxygen levels. Under hypoxic conditions, theHIF-1α is activated and may contribute not only to the neuroprotectionby regulating the expression of genes that facilitate cellular adaptation to lowoxygen condition but also to the neurotoxicity by regulating the expression ofgenes that induce cell apoptosis or death. It has been reported recently thatreactive oxygen species (ROS) are involved in the regulation ofHIF-1α stabilization in various cerebral ischemic injury. But the reports aboutROS regulation are different.In present study the brain histomorphology, the activity of superoxidedismutase (SOD), the content of malondialdehyde (MDA), the expression ofHIF-1α, manganese superoxide dismutase (Mn-SOD) and inducible nitricoxide synthase (iNOS) in brain were taken to study the expression of HIF-1αand the effects of ROS on HIF-1α stabilization after focal cerebral ischemiain rats.Part one The expression of HIF-1α after the permanent focal cerebralischemia in ratsObjective:To study the expression of HIF-1α after the permanent focalcerebral ischemia in rats.Methods: The male Soragye-Dawley rats were randomly divided intosham group, ischemia2h,4h,8h,12h and24h group. A model of permanent focal cerebral ischemia was induced by inserting a filament to occlude theorigin of the middle cerebral artery (MCA). The rats in sham group underwentthe same surgical procedure without inserting filament. The histologicalchanges of brain tissue were examined under light microscope by HE stainingand the expression of HIF-1α in cerebral cortex were observed byimmunohistochemical method.Results:1Histologic analysis revealed that cerebral tissue structure in ischemicgroup showed apparent ischemic injury both in cortex and striatumcharacterized by the neuronal degeneration. The severe injury was observed at4h,8h, and12h after MCAO. No pathological changes were observed in shamgroup.2The immunochemical positive expression for HIF-1α appeared incytoplasm. Few expression of HIF-1α showed in sham group, while theexpression in ischemic groups were increased after MCAO. Quantitativeanalysis showed that the expression of HIF-1α was diphasic change, peaked at8h, decreased at12h and up-regulated at24h again after tMCAO.Conclusion: The biphasic change of HIF-1α expression was observedafter the focal permanent cerebral ischemia, which inferred that HIF-1α waslikely to have the neuroprotection at early phase and the neurotoxicity at latephase.Part two The expression and the regulatory mechanism of HIF-1αafter transient focal cerebral ischemia in ratsObjective: To study the expression of HIF-1α and the effect of ROS onHIF-1α stabilization after the transient focal cerebral ischemia in rats.Methods: The male Soragye-Dawley rats were randomly divided intosham group, ischemic-reperfusion2h,4h,8h,12h and24h group. A model oftransient focal cerebral ischemia was induced by inserting a filament occludethe origin of the MCA (tMCA). Reperfusion was achieved by removal of the filament. The rats in sham group underwent the same surgical procedurewithout inserting filament. The histological changes of brain tissue, theactivity of SOD and the content of MDA in serum, the expression of HIF-1α,Mn-SOD and iNOS in cerebral cortex after tMCAO were determined,respectively.Results:1No pathological changes were observed in sham group. The cerebraltissue structure in ischemic group showed ischemic injury both in cortex andstriatum characterized by the neuronal degeneration until24h after tMCAO.2The expression of HIF-1α: The immunochemical positive expressionfor HIF-1α in ischemic-reperfusion groups were increased after tMCAO. Afew positive staining were observed in sham group. The quantitative analysisshowed that the expressions of HIF-1α in cerebral ishcmic-reperfusion groupsincreased rapidly, peaked at4h, and declined till24h after tMCAO. Thecomparative levels of HIF-1α expression in cerebral ishcmic-reperfusiongroups were significantly higher than that in sham group (P<0.01).3The expression of iNOS: A weak positive expression for iNOS wasobserved in sham group. The expression of iNOS in ischemic-reperfusiongroups increased after MCAO. The quantitative analysis showed that thepositive expression of iNOS at4h after tMCAO was the highest, thendecreased. The comparative levels of iNOS expression at2h,4h,8h and12hafter tMCAO were higher compared with sham group (P<0.05, P<0.01). Theexpression of iNOS at24h increased too, but there was no significantdifference compared with sham group (P<0.05).4The expression of Mn-SOD: The strongest positive staining forMn-SOD was observed in sham group. The positive staining inischemic-reperfusion groups decreased until24h after tMCAO. There wassignificant difference compared with sham group (P<0.05, P<0.01).5The activity of T-SOD in serum was (257±24) U·ml-1at4h,(239±33)U·ml-1at8h,(198±37) U·ml-1at12h and (154±48) U·ml-1at24h after tMCAO,much lower than (283±14) U·ml-1in sham group (P<0.05, P<0.01). The activity of T-SOD in IR2h was (274±19) U·ml-1lower than that in sham group,but there was no significant difference compared with sham group (P<0.05).6MDA content in sham group was9.68±1.00umol·L-1. After tMCAO,the MDA content in serum increased continuously. They were9.77±0.54umol·L-1at2h,10.83±0.64umol·L-1at4h,12.01±1.42umol·L-1at8h,14.79±2.03umol·L-1at12h and16.57±2.42umol·L-1at24h, respectively. Thecontent of MDA in ischemic-reperfusion groups significantly higher than thatin sham group (P<0.05, P<0.01), except that in2h group.Conclusion: The up-regulation of HIF-1αexpression was likely to showa neurotoxicity after cerebral ischemic-reperfusion. It was inferred that ROSmight be involved in the regulation of HIF-1α stabilization after cerebralischemic-reperfusion, and its regulation was related to the activity of theanti-oxidase. |
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