[Nphe1]-NC(1–13)-NH2, An Antagonist Of The Orphanin FQ/nociceptin Receptor, Improves Neuronal Survival After Traumatic Neuronal Injury In Vitro

Orphanin FQ receptor(orphanin receptor,ORL1),which was found in 1994,is aendogenous opioid peptide receptor and is widely distributed in central andperipheral organizations.It participate in many physiological and pathologicalprocess in our body. Nociceptin(orphanin-FQ,OFQ),first found in 1995,is the onlynociceptin receptor endogenous ligand in our body, and it plays an important role asORL1. N/OFQ contents increased greatly after traumatic brain injury in brain inearlier studies in vivo, and this may suggest that the OFQ/ORL1 system may play animportant role after traumatic brain injury.For deeply understanding the effect ofOFQ/ORL1 system after traumatic neuronal injury, our study tested if [Nphe1]-NC(1–13)-NH2, an antagonist of the N/OFQ receptor, could affect neuronalviability in cultured neurons from rat embryonic cortices after traumatic neuronalinjury. And we studied both the effects of different doses of Nphe and its role in thetime window preliminarily, and the possible mechanisms were discussed, too.Part I mechanically injured model of cultured cortical neuronsAbstract Objective For further experiment, we repeated a mechanically injured model of cortical neurons of rat in vitro, and the reliability and stability of it wasexamed. MethodMethods We identify cortical neurons by microscope andimmunohistochemistry methods after 1 week in vitro cultivation.A plastic tip wasused to make mechanical injury to the cultured neurons by crossing onto the culturedish.After that, morphological changes were observed. And in addition, cellularviability and lactate dehydrogenase (LDH) concentration were detected to evalue themechanical injury effects. Results The neuronal nucleus was observed with roundoutline, and it was large and clear.With the useage of microscope, the membrane ofnucleus and the nucleolus could easily found.Many big-figure neurons was observed,with bright cytoplasm and many dendrites and axons connected to each other ,whichform a complicated and special reticular pattern.We use NF200 to identify corticalneurons, and found that more than 90% was positive neurons.After process ofmechanical injury, necrosis was observed immediately. The cellular viabilitydecreased greatly, and the LDH concentration increased significantly aftermechanical injury of neurons in vitro.Conclusion In our study, cortical neurons were successfully cultured. We establisha mechanically injured model sucessfully, and it can simulate the pathologicalmechanism of traumatic brain injury.Part II ORL-1 expression and the neuroprotective effects of Nphe after themechanical injuryObjective In the present study, we checked the expression of ORL-1 aftertraumatic neuronal injury in cultured neurons from rat embryonic cortices andwhether [Nphe1]-NC(1–13)-NH2(Nphe), an antagonist of the N/OFQ receptor,could affect neuronal viability after traumatic neuronal injury was also tested.Methods The expression of ORL-1 was measured by Western Blot. The neuroprotective effects of Nphe on injured neurons were observed by MTT assay andLDH (lactate dehydrogenase) assay. ResultResults The ORL-1 level was not alteredfollowing injury as measured by Western Blot. Nphe increased the neuronal viabilityin a does-dependent manner after traumatic neuronal injury in vitro both by LDHassay and MTT assay. Conclusions The ORL-1 level was not altered followinginjury as measured by Western Blot. [Nphe1]-NC(1–13)-NH2, an antagonist of theN/OFQ receptor, could improve neuronal viability in cultured neurons from ratembryonic cortices after traumatic neuronal injury. And these data suggest thatN/OFQ and ORL1 might be implicated in injury-induced responses in the traumaticbrain injury.Part III The possible mechanisms of the neuroprotective effects of Nphe afterthe mechanical injuryObjective The experiment before has proved that Nphe improves neuronalviability in cultured neurons from rat embryonic cortices after traumatic neuronalinjury.To understand the mechanisms, Calcium level and expression of Caspase-3was detected in our study. Methods We checked calcium level and theexpression of Caspase-3 12 h after the mechanical injury process. Results Nphecould decrease Ca2+ concentration (P＜0.05) and the expression of Caspase-3(P＜0.05) after traumatic neuronal injury . Conclusions the Ca2+ concentration andCaspase-3 level in neurons decreased significantly after treated by Nphe. Themechanisms of the neuroprotective effects of Nphe after the mechanical injury mayinclude the decrease of Ca2+ concentration and the inhibition of apoptosis in neurons.