Neuroprotective Effects Of TRPC3 Channel Against Neonatal Hypoxic-ischemic Brain Damage Via ERK Signaling Pathway

Background Neonatal hypoxic-ischemic brain damage?HIBD?refers to perinatal asphyxia-or hypoxia-induced fetal or neonatal brain injury,which is one of leading causes of death and disability among neonates.However,there is no very effective therapeutic approach in clinical practice.Hypoxia/ischemia-induced neuronal necrosis and apoptosis are the major cause of brain injury.Thus,reducing neuronal necrosis and apoptosis and promoting neuronal survival could be one of the most important strategies to prevent and treat the nervous system injury caused by hypoxic-ischemic insult.The transient receptor potential canonical 3?TRPC3?channel is a subtype of non-specific cationic channels widely expressed in the fetal and perinatal central nervous system,which participate in various physiological and pathological processes,including cardiovascular and cerebrovascular diseases.Studies have shown that TRPC3 channel is an important regulator of intracellular calcium homeostasis,which regulates the activity of multiple intracellular calcium-sensitive signal transduction pathways including extracellular signal-regulated kinase?ERK?pathway,to modulate neuronal death or survival.Extracellular signal-regulated kinase?ERK?,a member of the mitogen activated protein kinases?MAPKs?family,is aserine/threonine-protein kinase.The activation of ERK is the key to transduction of extracellular signals from the cell membrane to the nucleus,which is high-profile because of its roles in cell proliferation,differentiation,survival and apoptosis.Emerging evidenc has shown that ERK pathway mediates pro-survival signals following HI insult,so the activation of ERK plays a protective role whereas its inhibition aggravates brain damage.Among various modulatory factors that may influence the ERK cascade activity,elevated free intracellular calcium concentrations is one of the most significant signaling events.It has been reported that TRPC channels,as an important modulator of intracellular calcium concentration,are involved in the activation of ERK pathway induced by various nocuous or irritant factors to affect a balance between cell apoptosis and survival.However,much less is known about the potential effects of TRPC3 channel on neonatal hypoxic-ischemic brain damage?HIBD?and the correlation with ERK cascade.In this study,we focused on TRPC3 channel,Ca²⁺ and ERK signal pathway to explore the effects and its underlying mechanism of TRPC3 on neonatal HIBD.Part one Protective effects of TRPC3 channel on OGD/R-induced neuronal injury ObjectiveTo investigate the neuroprotective effect of TRPC3 channel on OGD/R-induced neuronal injury in vitro.Methods1.Model: Oxygen glucose deprivation 1.5 h followed by reoxygenation 24 h in primary cortical neurons to mimic HI insult in vitro.2.Groups?1?For selective blockade of TRPC3 by Pyr3: Control group,Vehicle group,Pyr3?1,5,10,20 and 30?mol/L?groups,OGD/R group,OGD/R + Vehicle group,and OGD/R + Pyr3?1,5,10,20 and 30?mol/L?groups.?2?For si RNA interfering: Control group,OGD/R group,OGD/R + si-TRPC3 group,and OGD/R + si-Scramble group.3.Detection Indicator?1?MTT assay used to test cell viability;?2?LDH leakage used to assess cell injury;?3?Hochest33342 staining used to assay neuronal apoptosis;?4?Laser confocal used to detect the intracellular calcium ion concentration.ResultsCompared with OGD/R group,there were less cell viability,higher LDH leakage and higher neuronal apoptosis in OGD/R+Pyr3 groups and OGD/R+si-TRPC3 group.Blockade of TRPC3 channel reduced intracellular calcium concentration in OGD/R neurons.ConclusionSpecific blockade of TRPC3 or down-regulation of TRPC3 expression aggravated OGD/R-induced neuronal injury in vitro via.The underlying mechanisms may be partly related to disturbance of intracellular calcium homeostasis by blocking TRPC3.Part two Neuroprotective effects of TRPC3 channel on hypoxic-ischemic brain damage in neonatal rats ObjectiveTo investigate the neuroprotective effects of TRPC3 channel on hypoxic-ischemic brain damage in neonatal rats in vivo.Methods1.Model: HIBD model was induced by unilateral ligation of the right carotid artery followed by hypoxia?92% N2 + 8% O2?for 2 h in postnatal day 7 rat pups.2.GroupsThe SD rat pups were randomly assigned to the following groups:Sham group,Vehicle group,Pyr3?5,10,20 and 40mg/kg?groups,HI group,HI + Vehicle group,HI + Pyr3?5 and/or 20mg/kg?groups3.Detection Indicator?1?Brain water content used to estimate brain edema;?2?TTC stainingused to measure brain infarct volume;?3?HE staining used to observe morphologic changes in the brains?4?Neurobehavioral test batteries?neurologic deficit scores,balance beam test,and Morries water maze experiments?;?5?Ipsilateral/contralateral hemispheric weight ratio;?6?TUNEL staining used toassess neuronal apoptosis;?7?Western blotting used to detected the protein expressions.ResultsCompared with HI group,acute and long-term neurobehavior and brain injury of HI+Pyr3-20 mg group after HI insult were worse.Neurological deficit scores,balance beam test and water maze showed that specific blockade of TRPC3 by Pyr3 in HI animals aggravated the neurobehavioral deficits in coordination,counterbalance and spatial learning and memory ability.TTC staining,brain water content and ipsilateral/contralateral hemispheric weight ratio measurement revealed that Pyr3 administration increased brain edema and infarct volume as well as exacerbated brain atrophy following HI.TUNEL staining showed that the number of apoptotic neurons was by blocking TRPC3.Western blotting analysis demonstrated that p-ERK1/2 transiently increased in the ipsilateral hemisphere after HI.Furthermore,blocking TRPC3 reduced p-ERK1/2 expression,which accompanied by a marked up-regulation of proapoptotic protein,BAX,and a down-regulation of antiapoptotic protein,BCL-2.Conclusion1.TRPC3 channel is involved in neuroprotective effects in neonatal rats with hypoxic-ischemic brain damage.2.Blocking TRPC3 channel may aggravate neonatal hypoxic-ischemic brain damage,and its mechanism may be partially related that it regulates activation of ERK signaling pathway via intracellular calcium concentration alteration.