Effect Of Phentolamine On The Apoptosis Of Hippocampal Neurons In Restraint Stress Rat

Stress refers to the body who is suffered intensely physical stimulus orvariously dangerous environment with produced the nonspecific adaptiveresponse. Locus coeruleus sympathetic adrenal medulla axis(LC/NE) and thehypothalamic pituitary adrenal axis(HPA) system play an important part inregulating the stress response. When the body suffered strongly stress, LC/NEaxis and HPA axis are activated. As the key regulating factor of LC/NE axis,norepinephrine (NE) plays an important regulatory role in the neuroendocrineresponse induced by stress and it affects the body’s physical health andpsychological health.The hippocampus serve as a stress response regulating center,which issensitive and easy to damage.At the same time, as an important part of thelimbic system,it is involved in the emotional reaction and behavior changememory in the stress.Research sμggests that stress affects hippocampalfunction and viability to a different extent,and severe,prolonged stress is eventhoμght to induce neuronal death,possibly involved in apoptosis.The research sμggests that the central nervous system has adrenergicreceptor,which is associated with type α adrenergic receptor (AR),but it is notclear about influence mechanism of hippocampal apoptosis.At present,the mechanism of apoptosis, is generally believed that thereare three ways to participate in the occurrence of apoptosis:the mitochondrialpathway,death receptor pathway and endoplasmic reticulum pathway.In recentyears,the endoplasmic reticulum pathway is studied as a new unique apoptoticpathways.The core of the way are the endoplasmic reticulum stress (ERS) andunfolded protein response(UPR).GRP78is the key factor of the endoplasmicreticulum stress response,and CHOP is a key factor of apoptosis.Thus,at thestress state,whether the endoplasmic reticulum stress pathway induces to the apoptosis of hippocampal cells, and the role of type α adrenergic receptor isconfirmed with experiment.Objective: This experiment establish the rat restraint stress model,andthe used brain stereotaxic and trace injection technology,respectively,before30minutes of restraint stress,which is lateral ventricle injection of NE(norepinephrine) of α receptor antagonist phentolamine.TUNEL was used todetect hippocampal cells apoptosis.Immunohistochemical method was used todetect the endoplasmic reticulum stress markers:protein expression of GRP78and CHOP.We observated that the effect of phentolamine treat onhippocampal apoptosis of restriant stress rats and the role of the endoplasmicreticulum stress.Methods:1Establishment of restraint stress rat modelRats was breed by independent single cage,and restraint stress device wasused by a well-ventilated cylindrical homemade plastic bottles,7.5cm indiameter,(17±1)cm in length.Rats freely turn is not advisable.during8h (8:00to16:00),daily for7days.During this time,food and water was prohibited.Atthe rest of time, food and water was free.2Establish restraint stress model in rats, throμgh brain stereotaxic andtrace injection technology,respectively in30min before retraint stress oflateral ventricle injection of NE α receptor antagonist phentolamine (Phen):1μg group,10μg group,100μg group.3Apoptotic cells of different group in vivo were determined by TUNEL,The protein levels of GRP78and CHOP were examined byimmunohistochemical method.SPSS13.0software was used for statistical analysis.All data wererepresented as mean±standard deviation.One-way ANOVA was performed todetermined whether an overall statistically significant change existed beforeusing LSD test. A value of P<0.05was considered statistically significant.Results:1Hippocampal apoptosis was detected by TUNEL Compared with normal control group,TUNEL positive expression of thesimple restraint stress7days group rats was significantly increased (P <0.05).Compared with normal saline administration before restraint stress,NE αreceptor antagonist phentolamine:(1μg group,10μg group,or100μggroup),administration before restraint stress effectively reduced the apoptoticrate detected by TUNEL (P<0.05).2The protein expression of GRP78and CHOP was detected byimmunohistochemical method:GRP78and CHOP positive signals were located in the cytoplasm.Compared with normal control group,the protein expression of GRP78andCHOP were significantly increased in the hippocampus of restraint stressgroup rats.Compared with restraint stress group or normal salineadministration before restraint stress,NE α receptor antagonist phentolamine(1μg group,10μg group or100μg group) administration before restraintstress, the positive expression GRP78and CHOP were significantly lower(P<0.05).Conclusion: Under the condition of restraint stress rats model, the rathippocampal neuron apoptosis is increasing,and GRP78and CHOP positiveexpression has increased. GRP78is the key factor of endoplasmic reticulumstress, CHOP seves as a factor of the endoplasmic reticulum stress inducedapoptosis signaling pathways, they activate the endoplasmic reticulum stresspathway and endoplasmic reticulum stress related to apoptosis. After the NE αreceptor antagonist phentolamine treating, GRP78and CHOP positiveexpression of endoplasmic reticulum stress and apoptosis become inhibition, itsuggests that phentolamine can improve rat hippocampal neuron apoptosisphenomenon, and improve the mechanism of apoptosis,and the machanismmay be related to reduce endoplasmic reticulum stress. So we think thatwhen the body is under stress state, the NE plays a role of biology throughreceptors, it can activate the LC/NE system and promote the apoptosis of hippocampal neurons. May be the machanism may be related to reduceendoplasmic reticulum stress.