The Mechanism Of Endoplasmic Reticulum Stress In Hypothalamic Neuronal Injury In Stressed Rats

Stress is a systemic non-specific response of the body owing to various external or internal threatening stimuli.With the rapid development of modern society,stress is an inevitable life experience of the organism.After a long period of evolution,organisms have developed a complete and highly conservative regulatory system;this regulatory system is composed of the immune,cardiovascular and neuroendocrine systems,which can cope with internal or external stimuli and make an adaptive respond.Moderate stress of the body is a normal physiological phenomenon,but if the stress is too long or strong,it can cause a series of damage,such as nervous system,cardiovascular,endocrine and autoimmune diseases,and even affect the body's survival.Stress can lead to a systemic endocrine response,the hypothalamus-pituitary-adrenal(HPA)axis and Locus ceruleus norepinephrine(LC/NE)axis play critical roles in the body's stress response.The hypothalamus is activated when the body is stimulated by internal or external risk factors,which activates the adrenal cortex through the HPA axis and facilitates the synthesis and release of glucocorticoids(GC).Meanwhile,the locus coeruleus is also activated,promoting the adrenal medulla to increase the secretion of adrenaline(E)and norepinephrine(NE).The secretion of GC,E,NE is conductive to the mobilization of energy and the regulation of metabolism,enhancing the body's resistance to internal and external risk factors.However,when stimuli exceed the ability of homeostasis regulation,the body will exhibit various functional,behavioral and metabolic disorders,leading to different degrees of damage.Endoplasmic reticulum stress(ERS)is essentially a self-protectionmechanism in the process of stress,but sustained ERS caused by excessive stress result in cell damage or even death.As broadly documented in the literature,ERS plays critical roles in cell death and its pathogenesis caused by various neurodegenerative diseases and traumatic brain injury.As an important organelle involved in the synthesis,folding,modification and secretion of proteins under normal conditions,the protein folding ability of the endoplasmic reticulum matches with the body's protein synthesis ability.Various types of internal and external stimuli can disturb cells through various signaling pathways,leading to an accumulation of unfolded or misfolded proteins in the endoplasmic reticulum,causing ERS.Meanwhile,the body's adaptation mechanism,the unfolded protein reaction(UPR),is activated to cope with those unfolded or misfolded protein accumulation.PKR-like endoplasmic reticulum kinase(PERK)and inositol requirement 1(IRE1)are important sensory elements and major injury pathway initiation factors of ERS.When ERS occurs,they are separated from glucose-regulated protein 78(GRP78)to activate downstream signaling pathways,then restore ER homeostasis by reducing protein translation and promoting chaperone production.Conversely,if ERS is severe or too long,activating transcription factor 4(ATF4)and apoptosis signal regulating kinase 1(ASK1)will be activated by the PERK and IRE1 signaling pathways to promote the upregulation of growth arrest and DNA-damage-inducible gene 153(CHOP)and c-Jun amino-terminal kinase(JNK).The sustained expressions of CHOP and JNK could lead to cell death.Previous studies have shown that stress may result in the neurons damage,However,it is not clear the pathological changes of neurons in hypothalamus and the detailed mechanism of the injury induced by stress.For the present study,we successfully established rat models with different durations of stress exposure by simulating the physiological and psychological state of human under stress.Detecting the changes of stress-related hormones and receptors,observing the behavioral changesof stressed rats,exploring the effect of stress on hypothalamic nerve cells in rats from the perspective of pathology,and investigating PERK-ATF4-CHOP and IRE1-ASK1-JNK pathways related protein changes,to provide morphological evidence for studying the role of the PERK-ATF4-CHOP and IRE1-ASK1-JNK pathways in pathological changes of hypothalamic neurons and the mechanism of death induced by stress,aim to provide new measures and strategies for the treatment and prevention of stress injuries.Part one Establishment of different durations of chronic morphine dependent rats and pathological observationObjective: Modes of different durations of stress rats were established.Using Enzyme-linked immunosorbent assay(Elisa),Open field experiment,HE staining,Immunohistochemical staining and special histochemical staining(Thionine and Crystal Violet Staining),the present study investigated the effect of different durations of stress on neurons in Hypothalamus.Methods:1.Different durations of stress rats were forced to swim in ice water for 5 minutes after 6 hours of daily restraint.It was confirmed that rat models of stress with different durations were successfully established by observing the changes in body weight and behavior of rats.2.Elisa was used to detect the changes of serum-related stress hormones and their receptors,HSP70 in stress rats.3.HE staining was used to observe the pathological changes of hypothalamus in stress rats.4.IBA1 immunohistochemical staining was used to observe the pathological changes of microglia of hypothalamus in stress rats.5.Thionine and Crystal Violet Staining were used to observe the changes of Nissl bodies of neurons in hypothalamus after different durations of stress.Results:1.With prolonged time,the weight of control group was obviously increased.The weight of stress rats was slightly decreased at 1d and 3d,but after that time the weight also increased.However,compared with control group,the weight of stress groups was lower and had significant difference(P < 0.05).Compared with the control group,the number of rat feces were significantly increased at 3d,7d,14 d,21d;the movement distance and the cumulative duration in the central area of spontaneous experimental were significantly reduced.2.Elisa showed that the levels of serum-related stress hormones and their receptors,HSP70 in stress rats changed markedly.3.With prolonged time,HE staining showed that tissue was edema and neuronophagia,glial cells were proliferation,and finally the nerve cells were shrinking.4.IBA1 immunohistochemical staining indicated that microglia emerged activation changes such as hyperplasia,increased volume,and reduced protrusions in hypothalamus of stress rats at 14 d and 21 d.5.Thionine and Crystal Violet Staining indicated that with prolonged of stress pressure,tissue structure was not clear,Nissl bodies were disappeared,nerve cells were pycnosis and deep dyeing.Summary: The present study has been successfully established the model of different durations of restraint combined with ice water swimming in rats.After observing by Spontaneous experiment,ELISA,Thionine staining,Crystal Violet Staining,HE staining and immunohisto-chemistry,we get the conclusion as follows: with prolonged of stress pressure,hypothalamic neurons were edema and neuronophagia,glial cells were proliferation,Nissl bodies were disappeared,and nerve cells were death.All results suggested that hypothalamic neurons were degeneration and death induced by stress.Part two Endoplasmic reticulum stress is involved in stress-induced hypothalamic neuronal injury in rats via the PERK-ATF4-CHOPand IRE1-ASK1-JNK pathwaysObjective: Through systematic observation the pathological changes of hypothalamic neurons,PERK-ATF4-CHOP and IRE1-ASK1-JNK pathways related proteins,and m RNA changes,the study was to detect whether ERS is involved in the hypothalamic neuron injury process in stressed rats and its detailed mechanism.Methods:1.Thionine Staining were used to observe the changes of Nissl bodies of hypothalamic neuron in stress rats.2.Immunohistochemistry and western blot were used to detect the effect of stress pressure on protein content of GRP78 in hypothalamic neuron.3.Immunohistochemistry and western blot were used to detect the effect of stress pressure on content of PERK-ATF4-CHOP pathway related proteins in hypothalamic neuron.4.Immunohistochemistry and western blot were used to detect the effect of stress pressure on content of IRE1?-ASK1-JNK pathway related proteins in hypothalamic neuron.5.RNAScope was used to detect the effect of stress pressure on m RNA content of CHOP and JNK in hypothalamic neuron.Results:1.Thionine Staining results in hypothalamus: compared with the control group,there were no significant changes in Nissl bodies in the GSK2606414 and KIRA6 groups;after stress stimulation,some Nissl bodies disappeared and pyknotic neurons were visible;associated damage significantly moderated in the stress+GSK2606414 and stress+KIRA6groups.2.Immunohistochemistry and Western blot results of GRP78 expression in hypothalamus: compared with the control group,GRP78 expression remained at a low level in the GSK2606414(P>0.05)and KIRA6(P>0.05)groups and was significantly upregulated in the stress(P< 0.01),stress+GSK2606414(P < 0.01)and stress+KIRA6(P < 0.01)groups.3.Immunohistochemistry and Western blot results of PERK,ATF4,CHOP expression in hypothalamus: compared with the control group,PERK,ATF4,CHOP expression remained at a low level in the GSK2606414(P>0.05)group and was significantly upregulated in the stress(P < 0.05),stress+GSK2606414(P < 0.05);compared with the stress group,PERK,ATF4,CHOP expression significantly decreased in the stress+GSK2606414(P < 0.05)group.4.Immunohistochemistry and Western blot results of IRE1,ASK1,JNK expression in hypothalamus: compared with the control group,IRE1,ASK1,JNK expression remained at a low level in the KIRA6(P>0.05)group and was significantly upregulated in the stress(P < 0.05),stress+KIRA6(P < 0.05);compared with the stress group,IRE1,ASK1,JNK expression significantly decreased in the stress+ KIRA6(P < 0.05)group.5.RNAscope results of CHOP and JNK m RNA in hypothalamus:compared with the control group,CHOP m RNA expression remained at a low level in the GSK2606414 group(P > 0.05)and significantly increased in the stress(P < 0.01)and stress+GSK2606414(P < 0.01)groups;compared with the stress group,CHOP m RNA expression significantly decreased in the stress+GSK2606414 group(P < 0.01);compared with the control group,JNK m RNA expression remained at a low level in the KIRA6 group(P > 0.05)and significantly increased in the stress(P < 0.01)and stress+KIRA6(P < 0.01)groups;compared with the stress group,JNK m RNA expression significantly decreased in the stress+KIRA6 group(P < 0.01).Summary: The application of PERK phosphorylation inhibitor and IRE1 phosphokinase inhibitor can significantly reduce the degree of hypothalamic cell damage,and the expression of PERK-ATF4-CHOP and IRE1-ASK1-JNK signaling pathways related proteins and m RNAs,suggesting that PERK-ATF4-CHOP and IRE1-ASK1-JNK pathways areinvolved in the neural injury process in the hypothalamus of stress rats.Part three ERS mechanism of glucocorticoid-induced PC12 cells injuryObjective: The study was to investigate the mechanism of GCinduced cell injury at the cellular level to further verify the overall experimental conclusions,and provide scientific evidences for the hypothalamic neural injury caused by stress and whether GC is involved.Methods:1.Immunofluorescence was used to investigate the expression of Map2 and GR in PC12 cells.2.CCK-8 was used to detect the survival rate of PC12 cells after different concentrations of GC stimulation.3.Immunofluorescence was used to investigate the expression of C-fos in PC12 cells after GC stimulation.4.Immunofluorescence was used to investigate the expression of GRP78,p-PERK,p-IRE1,ATF4,ASK1,CHOP and JNK in PC12 cells after GC stimulation.Results:1.The results of immunofluorescence staining showed that PC12 cells abundantly expressed Map2 and GR.2.CCK-8 result indicated that the survival rate of PC12 cells were decreased with the raise of GC concentration(50?M(P < 0.05),100?M(P< 0.01),200?M(P < 0.01)and 400?M(P < 0.01)),compared with the control group,the survival rate of PC12 cells was significantly decreased after the concentration of 100?M DEX effected on PC12 cells for 24,36,48h(P <0.05).3.Immunofluorescence results of C-fos expression in PC12 cells:compared with the control group,the expression of C-fos increased significantly in DEX group(P < 0.05).4.Immunofluorescence results of GRP78,p-PERK,p-IRE1,ATF4,ASK1,CHOP,JNK expression in PC12 cells: compared with the controlgroup,GRP78,p-PERK,p-IRE1,ATF4,ASK1,CHOP,JNK expression remained at a low level in the 4-PBA(P>0.05)group and was significantly upregulated in the DEX(P < 0.05)and DEX+4-PBA(P <0.05);compared with the DEX group,GRP78,p-PERK,p-IRE1,ATF4,ASK1,CHOP,JNK expression significantly decreased in the DEX+4-PBA(P < 0.05)group.Summary: PC12 cells with abundant expression of Map2 and GR are suitable for subsequent studies on the mechanism of GC-induced neuronal injury.GC can inhibit the viability of PC12 cells and even cause cell damage.Endoplasmic reticulum stress PERK-ATF4-CHOP and IRE1-ASK1-JNK pathways are involved in the above damage process.Conclusions:Based on successfully established the stress rats model of restraint combined with ice water swimming,we studied the effect of stress on behavioral changes,neuroendocrine changes and hypothalamic neuronal injury,and applied PERK phosphorylation inhibitor GSK2606414 and IRE1 phosphokinase inhibitor KIRA6 to investigate the role of ERS in the above damage process;The role of PERK-ATF4-CHOP and IRE1-ASK1-JNK pathways in GC-induced PC12 cells damage was explored by establishing a PC12 cell model.And got conclusions as follows:1.Stress could result in the damage of hypothalamic neurons,which may be related to the change in the level of GC.2.Endoplasmic reticulum stress is involved in stress-induced hypothalamic neuronal injury in rats via the PERK-ATF4-CHOP and IRE1-ASK1-JNK pathways.3.GC could result in the damage of PC12 cells,and ERS is involved in the damage process.In summary,restraint combined with ice water swimming stress could cause dynamic changes in the level of GC and its receptor,and then activate endoplasmic reticulum stress PERK-ATF4-CHOP andIRE1-ASK1-JNK pathways,which could lead to hypothalamic neurons damage or even die.