Effect Of Hypoxia-inducible Factor-1alpha (HIF-1α) And Its Inhibitor On Neural Cell Apoptosis Of Hypoxia-ischemia Brain Damage In Neonatal Rats

Objective: Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the mostcommon neonatal diseases, and it is one of major reasons caused neonatal acute death andchronic nervous system damage, and there is no effective treatment at present. Researchhas shown that inhibition of HIF-1α (hypoxia-inducible factor-1alpha) expression aftercerebral ischemia in adult animal models produced neuroprotective effects[3,4]. Thisexperiment give inhibitor of HIF-1α, which is2-methoxyestradiol (2ME2), interventionthrough the establishment of neonatal rats with hypoxic-ischemic brain damage (HIBD)model, to observe brain tissue pathological change after newborn rats with HIBD, andmeasure expression of HIF-1α, Bax, Bcl-2at every time point and calculate ratio of Bax/Bcl-2, so as to explore the role of2ME2, HIF-1α on neonatal rats with HIBD, neuralprotective effect of2ME2and possible mechanisms, with a view to clinical treatment ofneonatal HIE with some guidelines.Methods:120seven-day-old Wistar rats (male or female) with birth weight of11-15gram were randomly divided into3groups: sham operation group (Sham group, n=8),hypoxia-ischemia model group (HIBD group, n=56),2ME2intervention group (2ME2group, n=56). The following two groups were divided into7groups based on differenttime points after HIBD (3h,6h,12h,24h,48h,72h, and7d groups), each subgroup has8rats. Rats in Sham group were given median neck incision and free left common carotidartery, without hypoxic-ischemic, rats in HIBD group and2ME2group were establishedHIBD model by Rice’s method:prepared by keeping rats into hypoxic environment of8%O2and92%N2for2hours after freed and ligated left common carotid artery. Eachsubgroup of2ME2group was given a one-time intraperitoneal injection of2ME2(15mg/kg, dissolved by DMSO, diluted by PBS)10minutes after HIBD. Sham group andHIBD group were given the same amount of DMSO and PBS. Newborn rats were openhead and taken out brain tissue at corresponding time point, paraffin sections then HEstained to observe cerebral pathological changes under the light microscope,immunohistochemical method to measure expression of HIF-1α, Bax, Bcl-2of the threegroups and TUNEL method to count brain cell apoptosis at different time points.Results:(1) HE staining: Brain tissue had clear hierarchy in Sham group, arrangementand morphology of neurons was normal; neural cell of cortex and hippocampus of leftbrain in HIBD group was swelling, disordered; neural cell morphological changes of2ME2group was slighter than that in HIBD group, and arrangement of cells still ruled.(2) Immunohistochemical staining: Positive staining of HIF-1α, Bax, Bcl-2were brownyellow particle deposition, positive cells were mainly found in the cerebral cortex andhippocampus; HIF-1α positive staining was in the nucleus and cytoplasm, Bax, Bcl-2positive staining was located mainly in the neuronal cytoplasm.①HIF-1α: Expression ofHIF-1α in Sham group was weak; HIF-1α significantly elevated at3h after model prepared,12h reached peak, then declined, but still higher than Sham group; the2ME2group had thesame trend with HIBD group, expression levels were reduced;②Bax: Expression of Baxin Sham group was weak; in HIBD group, it was obviously increased at3h after HIBD, to24h reached the peak, then reduced, but still higher than that of Sham group. Baxexpression trend in2ME2group first decreased then increased, it was minimum at24h,expression levels of each time point was lower than those in HIBD group.③Bcl-2: Bcl-2expression in Sham group was generally maintained at the same level at each time point, inHIBD group it started to increase at3h after HIBD, reached peak at24h, then decreased,but still higher than Sham group.2ME2group had same expression trend with HIBDgroup, but expression level elevated.(3) TUNEL method to measure brain cell apoptosis(%): Cell apoptosis in Sham group was less, HIBD group increased significantly; in2ME2group, apoptosis increased significantly compared with Sham group, decreased obviouslycompared with HIBD group, the differences among the three groups and between two-twocomparisons were statistically significant.Conclusion:(1) HIF-1α was harmful on hypoxic-ischemic brain damage in neonatalrats; when2ME2inhibited HIF-1α, expression of HIF-1α was significantly reduced, andexpression of Bax decreased, Bcl-2increased, caused ratio of Bax/Bcl-2decreased,eventually the brain cell apoptosis was significantly reduced, brain damage reduced.(2)2ME2playing a neuroprotective role may lie in its early inhibition of HIF-1α expressionduring hypoxia-ischemia and thereby reduced the expression of pro-apoptotic gene,reduced brain injury, which can provide new treatment method for the treatment ofneonatal HIBD.