Effects And Mechanisms Of Purmorphamine On Hypoxic-Ischemic Brain Damage In Neonatal Mice

Background:Hypoxic-ischemic brain damage(HIBD)in neonatal infants is a common disease of neonatal mortality and long-term neurological deficits which is caused by neonatal Hypoxic-ischemic encephalopathy(HIE).Perinatal asphyxia is the leading cause of HIBD.With the rapid development in perinatal care,the survival rate of neonatal infants suffering from HIBD has been significantly improved.However,many survivors of preterm labor still have long-term functional deficits,such as persistent motor deficits,sensory or cognitive abnormalities.HIBD is also a major cause of adult disability.So,effective pharmacological intervention is urgently required to prevent the brain damage and associated poor long-term outcome.Earlly prophylactic or neuroprotective interventions have the potential to save newborns from protecting their brains.Unfortunately,pharmacological options are scarce.The pathological features of HIBD include neuronal degeneration and necrosis,inflammation,edema,and hyperemia.The HIBD hindbrain reperfusion will produce a series of pathophysiological changes,such as a substantial increase in oxygen free radicals,leading to cell membrane breakdown,destruction of the blood-brain barrier,and then resulting in Increased brain edema.What’s more,the influx of Ca2+ prevents the mitochondrial oxidative phosphorylation process,and the accumulation of cytoplasmic calcium can also lead to the production of free radicals.The free radicals can also alter the function of the cell membrane sodium pump,leading to cell excitotoxicity.Excitotoxicity has led to energy consumption and Ca2 + accumulation,resulting in further brain damage.The interaction between excitotoxicity,oxidative stress,and inflammation contributes to the development of neuronal death patterns in neonates with HIBD,therefore it is needed to start studying for this point,pick a more effective treatment to prevent the brain damage and associated poor long-term outcome.The Sonic hedgehog(Shh)signaling pathway plays a key role in embryonic development and adult stem cell function.After Shh is secreted,it binds to its cognate receptor,Patched(Ptch),which is on the target cells surface.Then Ptch is activated and releasing Smoothened(Smo),which initiates the downstream signaling that controls the transcription factor Gli-1.Thereby Gli-1 transfers into the nucleus and regulates downstream target gene transcription.A report demonstrated that Shh plays a protective role in acute brain injury,and spinal cord injury could be improved by the application of recombinant Shh protein or intravenous hedgehog agonist,Ag11.1 in adult rats.Studies have shown that injection of exogenous Shh protein could exert angiogenesis and protect the brain from ischemic injury.Cerebrolysin treatment promoted neurogenesis and oligodendrocyte production and improved neurological function after ischemic brain injury,and these results are associated with up-regulation of the Shh pathway.PUR as an anagonist of Shh co-receptor Smoothened receptor have neuroprotective effects in acute experimental ischemic stroke.However,its role in neonatal mouse HIBD remains unknown.Whether Shh signaling pathway plays a role in HIBD and what role it plays has not been reported yet.In the present study,we investigated the influences of a short-term PUR administration on acute brain injury,and on long-term neurobehavioral dysfunction in neonatal HI mouse.Objective:The purpose of this study is to study the effects and mechanisms of purmorphamine on HIBD in neonatal mouse.Methods:We use Rice-Vannucci method to establish HIBD model on postnatal day(PND)7 to simulate human perinatal HIE.At first,C57BL/6J pregnant rats were bought and fed,and their PND 7 male pups were selected randomly for the experiment.Their right common carotid artery of pups was isolated and permanently double-ligated after anesthetized.After surgery,the pups were recovered for 1 h and then were placed in a hypoxia chamber(humidified 8%O2+92%N2)for 1.5 h to inducee HIBD,which similar to the human newborn HIBD changes obviously in neonatal ischemia-sensitive areas(ligation side of the cerebral cortex,hippocampus,striatum and thalamus)showed significant ischemic changes,the organization showed significant edema.Sham controls were just performed with anesthesia and exposing the right carotid artery.The PND 7 pups were randomly divided into five groups:Sham+saline group(Sham),HI + saline group(HI),HI + PUR group,HI+PUR+ Cyclopamine(The inhibitor of Shh pathway)group(HI+PUR+Cyc),and HI+ Cyc group.An equal amount of saline,PUR(10 ml/kg)and Cyc(0.5 ml/kg)were injected intraperitoneally at 24 h,48 h and 72 h after establishment of HIBD model respectively.For acute damage analyses,the mice were sacrificed at 72 hours after HIBD model(PND 10).For long-term analyses,the open field test was used to observe the locomotor activity of the mice at 14 days after HI insult(PND 21),and the Morris water maze was observed for cognitive function at 28 days after the HI insult(PND 35).The neuroprotective effect of PUR on HIBD and its molecular mechanism were studied systematically.The effects of PUR and Shh signaling pathway on apoptosis and oxidative stress were detected by immunohistochemistry,immunofluorescence and Western blot.Their benefit effects on apoptotic indices were studied by RT-PCR at the gene level as well.Results:1.The results showed that after treatment with PUR greatly improved the condition of HIBD cerebral edema,reduced brain edema volume,and neuronal cell death.2.PUR decreased TUNEL-positive cells and apoptotic markers.After PUR treatment,the Bax/Bcl-2 ratio was decreased and the caspase-3 activation was suppressed.3.PUR promoted phosphorylation of Akt 72h after HI insult and upregulated the expression of brain-derived neurotrophic factor(BDNF)and Phospho-cAMP response element-binding protein(p-CREB).4.PUR can reduce the content of ROS caused by HIBD and increase the expression of Nrf-2.5.PUR had long-term effects of protecting on the loss of ipsilateral brain and improving neurobehavioral outcomes 14 days and 28 days post-HI insult.6.PUR treatment after HI exposure up-regulated the expression of Shh pathway mediators(Shh,Patched and Gli-1).Conclusions:PUR have neuroprotective effect against HIBD in neonatal mice HIBD models via activating Shh signaling pathway.PUR has protective effects in HI-induced acute brain injury,as well as chronic memory deficits and impaired synaptic function.PUR ameliorated brain atrophy and locomotor activity.The beneficial effects of PUR in HI injury were associated with suppressed ted oxidative stress and up-regulated Nrf-2 expression.PUR treatment after HI exposure up-regulated the expression of synaptophysin and postsynaptic density protein 95 in ipsilateral cortex and promoted the synaptic remodling.Short-term application of PUR or Shh pathway agonists has great potential for the treatment of Hi-induced damage in newborn mice.PUR is a promising candidate for HI therapy,which specifically and sensitively targets the activation of the Shh signaling pathway and is expected to become an adjuvant for the treatment of neonatal hypoxic-ischemic brain damage,as well as for the clinical management of neonatal ischemia Hypoxic diseases provide new ideas.