Role And Mechanism Of Thyroxine On Brain Tissue Hypoxia-inducible Factor-1α Expression In Neonatal Rats With Hypoxia-ischemia Brain Damage

Background and ObjectiveNeonatal Hypoxic-Ischemic Encephalopathy (HIE) is caused by hypoxia anddecline or transitory lack of cerebral blood flow resulted from perinatal asphyxia andpresents a series of abnormal clinical symptoms and signs of central nervous.HIE isone of the most important causes of acute perinatal mortality and neurologicalsequelae.Therefore,the prevention and treatment strategy and intervention mechanismhave been the common concerns for clinicians and researchers.During neonatalhypoxic–ischemic brain injury, activation of transcription of a series of genes isinduced to produce protective and harmful reactions.The former includeerythropoiesis,anti-apoptosis,angiogenesis,and so on.The latter include apoptosis andnecrosis.A key factor mediating these gene transcriptions is hypoxia-induciblefactor-1α(HIF-1α).Under normal physiological conditions,HIF-1α participates in early brain development and proliferation of neuronal precursor cells.In brain tissueof neonatal rats after HIBD,HIF-1α protein is stabilized and heterodimerizes withHIF-1β to form HIF-1,subsequently regulating the expression of target genes,such asvascular endothelial growth factor,glucose transporter protein-1and erythropoietin,and playing a role in neuro protection.Therefore,looking for drugs,which canupregulate the expression of endogenous HIF-1alpha,is of great significance toexplore the treatment for HIBD.The function of thyroidea declined in patients withmoderate and serious neonatal HIE,and it is positively related with the severity of thedisease.If the function of thyroidea does not go back to its normal level until therecuperating phase,the replacement therapy with thyroxine tablers can prompt therecovering of the disease and improve the prognosis.The researches of the influenceof thyroxine（T4）on HIF-1α are mainly about in-vitro cell culture experiments,whichmainly involve the activation of Phosphoinositid3-kinase/Akt pathway.There is noreport on wether T4influences the expression of HIF-1α in brain tissue after HIBDand whether the PI3K/Akt signaling pathway is involved.The research objects of thisstudy were newborn rats with HIBD.The aim of this study is to explore the role andpossible mechanism of thyroxine on regulating brain tissue hypoxia-inducible factor1α expression after hypoxia-ischemia brain damage,which will provide a newtheoretical basis for the clinical application of T4on HIE.Materials and MethodsSixty-four healthy7-day-old clean grade Sprague-Dawley (SD) rats,male orfemale,weighing12~16g,were divided into4groups randomly:the sham operationgroup,model control group,solvent-treated group(M) and T4-treated group(T4).Thereare16pups in each group.Briefly,each pup was anesthetized with halothane.With thepup supine, the left common carotid artery (CCA) was exposed and permanentlyligated with a7-0silk suture through a midline cervical incision.After ligation of theCCA,the pups were returned to the mother rat for1h to recover from anesthesia.Aduration of2h of hypoxia (8%O2/92%N2) was used to produce hypoxia-ischemia brain injury.Sham controls received halothane anesthesia and exposure of the CCAwithout hypoxia or ligation of the CCA.The rats of T4-treated group wereadministrated with intraperitoneal injection of2ug/100g T4,once a day,for5daysafter HIBD.The rats of solvent-treated group were administrated with intraperitonealinjection of alkaline ethanol of the equal volume.After the last intraperitoneal injection,eight rats’ brains from each group werecollected for experiments.Briefly,each pup was anesthetized with halothane. Thoraciccavity is opened to expose the heart,and then impregnate the left ventricle with4%paraformaldehyd and then the brain is fixed in the same fixed fluid.Afterconventional dehydration and paraffin embedding,continuous coronary sections of3um are made to detect the expression of HIF-1α and p-Akt protein byimmunohistochemical staining.After the last intraperitoneal injection,another eight rats’ brains from each groupwere collected for experiments.Briefly,each pup was sacrificed after anesthetized withhalothane and the left brain was collected on ice plate to prepare tissue used fordetecting the level of HIF-1a mRNA by RT-PCR.Results1HIF-1alpha and p-Akt protein were both mainly distributed in the cortical andhippocampal neurons.The subcellular localization of HIF-1alpha are mainlyconcentrated in the nucleus,also in cytoplasm,and The subcellular localization of p-Akt protein is in the cytoplasm and cytomembrane.2The levels of p-Akt protein,HIF-1α protein and HIF-1αmRNA in model controlgroup are respectively50.168±4.259,72.795±6.121and0.448±0.035;The levels ofp-Akt protein,HIF-1α protein and HIF-1αmRNA in sham operation group arerespectively8.080±0.369,38.581±2.846and0.174±0.015;the levels of p-Aktprotein,HIF-1α protein and HIF-1αmRNA in solvent-treated group are respectively52.199±3.850,78.481±5.080and0.439±0.035.The levels of above factors in modelcontrol group are higher than sham operation group,the difference was statistically significant (p<0.05);while the difference compared with solvent-treated group wasnot statistically significant (p>0.05).3The levels of p-Akt protein,HIF-1α protein and HIF-1α mRNA in T4-treated groupwere respectively82.765±6.271,117.350±9.374and0.618±0.042,and are higher thanthose in model control group(p<0.05).4The levels of HIF-1α protein and p-Akt protein in model control group andT4-treated group were positively correlated, r（M）=0.635（p=0.048）,r（T4）=0.694（p=0.026）.Conclusions1The expression of HIF-1alpha mRNA and protein increased in newborn ratsafter HIBD.This process may be implemented through the PI3K/Akt signalingpathway.2Ectogenic T4upregulated the expression of HIF-1α mRNA and protein inneonatal rats with HIBD.Phosphatidylinositol-3-kinase/protein kinase B（PI3K/Akt）signaling pathway may be involved in T4up-regulating HIF-1α mRNA and proteinexpression.