| PrefacePost-traumatic stress disorder?PTSD?is also known as post-traumatic stress disorder delayed psychogenic reaction,caused by severe and fatal psychological trauma[1].The earliest diagnosis of which presented in the United States diagnostic and statistical manual disorder published in 1980?DMS-??,what's more,PTSD was accepted in the scope of chronic and sress disorder diseases published in 2013-the fifth edition?DSM-??.The main clinical symptoms of PTSD are the repeated of traumatic experiences,increased awareness,persistent avoidance and recognitive,negative changes of emotion[2-5].The increased incidents of PTSD became a major factor impacted on the prognosis of mental diseases,presented as the long time,the poor cure rate,seriously affected the patient's daily life quality and social stability,which due to the severe natural disasters?such as earthquakes,tsunamis,floods?,serious accdidents,terrorist attacks and other stress violence[6,7].PTSD has attracted more and more attention by the worldwide experts and scholars from neuropsychology,psychiatry and neurobiology,ect.during the recent several years.However,the frequently invalid medication mainly due to the unclear pathogenesis,so it has important theoretical and practical significance to further study the pathogenesis and to formulate effective prevention and control measuers.One of the main symptoms and clinical diagnosis standard of PTSD is dysmnesia which always existed in the patients experienced wars,serious accidents,terrorist attacks.Dysmnisia include intrusive memory,fear memory,abnormal declarative memory and traumatic amnesia[8-10].The study has showed that the dysmnesia of PTSD patients had close relationship with the structure of central nervous system and change of function[11].Medial prefrontal cortex?mPFC?is one of the important edge systems and is sensitive area to corresponding stress response,which can adjust the reaction of amygdale to the fear irritant.The changes of structure and function of mPFC caused by PTSD lead to the decrease of regulate and control the amygdale and the overreaction to the stimulation,which may be the reason why PTSD patients unable to recede or degrade the fear reaction or even last the whole life[12].Meanwhile,the disturbance between mPFC,hippocampus and amygdale nuclear leaded to dysmnesia in PTSD.Imageology research have showed that compared with normal people,mPFC were smaller in size,and reduced gray matter density,this change was closely related to clinical symptoms[13].A large number of medical imaging studies have show that compared with the normal people,mPFC in PTSD patients has significantly reduced volume,gray matter density decreased and metabolic products changed,which is closely related to the clinical symptoms and severity of patients[14].Therefore,mPFC is one of the key brain regions in PTSD occurs and development,the changes of mPFC structure and functional disorder is the major reason for PTSD dysmnesia and stress.Autophagy is consider as a conservative subcellular structure degrading process of eucaryotic cells,which is characterized by the formation of autophagosome with double menbranes structure degradables substances[15].Physiological role of autophagy include:take part in cell growth and differentiation,maintain the basic energy for living cells under the nutritional deficiency or hypoxic-ischemic conditions by degradating the impaired cell organs,long-life or misfolded proteins,advoid cell from being further damaged[16].As apoptosis,autophagy also has positive and negative effects,basic level of autophagy can maintain the intracellular enviorment and banlance the metabolism by degradating long-life or misfolded proteins,cell organs to maintain homeostasis and metabolic balance,on the other side,excessive activation or depression of autophagy might lead to irreversible damage or even death,therefore,autophagy also known as type II programmed cell death[17].Autophagy is controlled by a series of autophagy-related genes?Atg?,especially the Beclin-1 and Microtubule-associated protein 1 light chain 3?MAP1-LC 3/LC 3?,which are mammal homologous of Atg6 and Atg8,play a critical role during the formation and extention of autophagosomes.Beclin-1/Atg6 composites with class III phosphatidylinositol 3-kinase?Class III PI3K?,and promotes the build of autophagia ves demilune and the glass shape double membrane[18].LC 3/Atg8 and Atg12 modifying the ubiquitinal protein system is the significant stage of autophagosomes formation,which can promote the double membrane to extending,change from glass shape,demilune shape to ring shape,completely wrap up the degrading cell organs or long-life,misfolded proteins to form autophagosome.LC 3/Atg8 has two types in cells are endochylema LC 3-I and hymenoocclusal LC 3-II,the latter one align in anteautophagosome and autophagosome,once autophagosome confluenced with cytolysosome,LC 3-II would be recruited[19,20].All in one word,Beclin-1/Atg6 and LC 3/Atg8 are all the important maker gene of autophagy.Autophagy is a fast and dynamic progress in the yeast and mammal eucaryon,which also known as autophagia flow or autophagia tide.Althogh the increase or decrease exression of autophagia genes standed for the ability of autophagia,the fracture of autophagy lysosome fused mechannism and change of cytolysosomic activity may evoke abnormal expression of Atg,while the activity of autophagia flow was still depressed.p62 protein paiticapate in the protein degradation of ubiquitin-proteasomes system?ups?and autophagia protein[21].During autophagy process,the expression of p62 protein changes with the activity of autophagy.The expression of autophagosome or autophagy related genes are altered at the same time with the expression of autophagic substrate,which can more fully explain the activation or inhibition of autophagy flow.Our precious study has discovered that the number of dead nerve cell significantly increased in PTSD rat[22],and revealed that mitochondrial pathway and endoplasmic reticulum stress pathway leaded to neuronic apoptosis in mPFC,correlative apoptosis factors such as caspase family caspase-9,caspase-3,caspase-12,and endoplasmic reticulum molecular chaperone GRP 78 and 94 expressed abnormally,that's to say,apoptosis play very important role in the nerve cells death[23].Autophagia was closely related with the nosogenesis and development of some nervous system diseases,such as cerebral trauma,Alzheimer disease,Parkinson disease,Huntington's disease[24-27].We presume that autophagy may also play a significant role in the mPFC nerve cells death in PTSD rats.The present study adopted Single-prolonged stress?SPS?to stimulate rats and establish PTSD animal models,applied Morris water maze to test learning memory and space exploratic ability of the rats,the exercise behavior,fear and anxiety state of the rats were detected by the elevated plus maze and the open field test.Transmission electron microscope?TEM?technology and immunofluorescence technology were used to observe and detect the changes of PFC nerve cells autophagy in PTSD rats.Western blotting and quantitative Real time PCR?qRT-PCR?were used to invetgated the protein and mRNA expression of Beclin-1,LC 3,p62,Bcl-2 and Bax.The present study not only supplied reliable experimental dates to further study autophagia in the field of pathogenesy of dysmnesia and fear memory induced by PTSD,but also to provide new ideas for clinical prevention and drug development of PTSD.Methods1.Internationally-recognized SPS method was used to establish the PTSD rat model in present study.Rats were randomly classified into five groups,including normal control group,1-day,4-day,7-day and 14-day group after SPS stimulation,respectively.2.General condition of rats in control group and PTSD model group was observed.Learning and spatial memory ability of rats in each groups was detected by Morris water maze test.Elevated plus maze and open field test was used to test the autonomous exercise and the behavior,fear and anxiety state of the rats in each groups.3.The ultramicrostructural changes of mPFC neuron autophagy were observed by Transmission electron microscopy?TEM?.4.Immunofluorescence and double-labeled immunofluorescent staining were employed to detect types of positive cells and expression of protein level of Beclin-1 and LC 3 in mPFC of each group rats.5.Immunohistochemistry was used to detect the expression of protein level of p62in mPFC of each group rats.6.Western blotting was used to test he change of prorein level of mPFC Beclin-1,LC 3-II,p62,Bcl-2 and Bax in mPFC of each groups rats.7.The change of Beclin-1 mRNA,LC 3-II mRNA,P62 mRNA,Bcl-2 mRNA and Bax mRNA in mPFC of each groups rats were determined by Real Time-PCR?qRT-PCR?.Results1.The changes of general states and behaviors in PTSD rats?1?The changes of general states in PTSD ratsPTSD rats displayed loss of appetite,lassitude and low physical activity after SPS stimulation.The rats also showed irritability and like to tussle after SPS stimulation.?2?Morris water maze testThe results of Morris water maze navigation experiment showed that the average escape latency time decreased in both SPS 7d group and control group,but the SPS 7d group's descending trend was lower than that in control group,and were higher than those in control group at each time points?P all<0.05?.The results of learning and spatial memory ability test showd rats in control group had shorter trajectories and could find the underwater platform in a short time,while the SPS 7d group had long and disordered trajectories,and failed to find underwater platforms within a limited time.?3?Elevated plus maze testThe results of the elevated cross maze showed that rats in control group had motion trajectories in the open arms and closed arms,while the motion trajectories in the open arms of rats in SPS 7d group were significantly reduced.Compared with the control group,the walking distance in the central area,the closed arm and the open arm was significantly shortened?P<0.05?.Compared with the control group,the number of entry in the central region,and the residence time of the central region were all decreased of rats in SPS 7d group?P<0.05?.The results of elevated cross maze test showed the fear and anxiety like behavior in SPS rats.?4?Open field testOpen field test results showed that rats in control group had motion trajectories in the central and peripheral regions,while in the SPS 7d group,there was little or no movement locus in the central area,and the trajectories were mainly distributed in the peripheral region.Compared with the control group,SPS 7d group rats were reduced in number and enter the central area of the residence time in the central region,the central region of the moving distance?P<0.05?,SPS 7d group rats were increased in the peripheral region of the moving distance,the peripheral region and the peripheral region into the number of stay between?P<0.05?.The result of the open field test showed that the rats in the SPS group were autonomously exercise,the exploration behavior decreased,and the anxiety and fear like behavior.2.Detection of neuronal autophagy in mPFC of PTSD rats?1?Autophagy in neuron of PTSD rats under TEMNeurons of mPFC in control group showed the ultrastructure of neurons was normal,endoplasmic reticulum and mitochondria was normal distributed under TEM.The cell membrane and nucleus were intact and clear.The structures such as bilateral membrane or autophagic corpuscle in neuron cells were rare.After SPS stimulation,there were some scattered vesicles and autophagosome in the mPFC neurons,it also observed that bilayer membrane structures around the autophagosome,and contained electron dense substances.?2?Autophagy in neuron of PTSD rats by immunofluorescent analysisThe nucleus of mPFC in each group was stained with DAPI,and the normal nucleus showed a uniform light blue fluorescence,and the volume was large and the shape was regular.In the control group,most nuclei of large volume,blue uniform dyeing,irregular shape or DAPI staining abnormal nuclei rare in mPFC.After SPS stimulation,the number of irregular nuclear in mPFC increased significantly,mainly for the size and irregular,and deeply stained dark blue.In SPS 7d group,nuclear with irregular shape and the largest number of nuclear staining deep bright.The positive expression of LC 3 and Beclin-1 were observed in the cytoplasm of mPFC cells.There was negative expression of LC 3 and Beclin-1 in some cells with DAPI stained dark blue and irregular shape.In SPS 7d group,the expression of Beclin-1 and LC 3 were positive and increased in the cytoplasm of mPFC cells?the fluorescence intensity,the number of dot dense fluorescence was LC 3 punctate?.The number of dot dense fluorescence was LC 3punctate which represented the increase of vesicle or autophagic structure.In control group and SPS 7d group,LC3-ir and Beclin-1 was mainly found in neurons.Colocalization of LC3/Beclin-1 and NeuN?a marker of mature neurons?increased as the number of LC3-ir-positive cells increased.By contrast,only a few GFAP-ir cells?a marker of glia cells?that exhibited LC3-ir or Beclin1-ir in the control and the SPS 7-day group were found.3.The expression of Beclin-1 and LC 3 in mPFC of PTSD rats?1?Immunofluorescence resultsFITC fluorescent labeling Beclin-1 and LC 3,Beclin-1 and LC 3 were expressed in green fluorescence,and mainly concentrated in the cytoplasm of m PFC neurons.Beclin-1 ir and LC 3 ir were expressed in the cytoplasm of normal cells and some DAPI stained slightly cells,but not present in the irregular,deep and bright blue nuclei.Compared with the control group,the positive expression of Beclin-1 and LC 3decreased in mPFC of rats in SPS 1d,and the fluorescence intensity increased gradually in SPS 4d and peaked at SPS 7d.The difference between control group and each SPS group were statistically significant?P<0.05?.The changes trend of LC 3 fluorescence spots in randomly selected field in accordance with the Beclin-1 expression in each group.?2?Western blotting resultsBeclin-1 expression and the ratio of LC 3-II/LC 3-I was significantly decreased in the mPFC of SPS 1d group in comparison to control group.Its expression was gradually increased from SPS 4d and reached the peak in the SPS 7d.It showed a little decrease in the SPS 14d,but still significantly higher than normal control group?P<0.05?.?3?Real Time resultsCompared with control group,the level of Beclin-1 mRNA and LC 3-II m RNA was significantly lower in the mPFC at SPS 1d group.It showed gradually increasing tendency from SPS 4d and reached the peak in the SPS 7d,and maintained a high level in the 14d.The changes in the levels of Beclin-1 mRNA and LC 3-II mRNA in qRT-PCR were basically the same as the trend of Western blotting results results.4.The expression of Bcl-2 and Bax in mPFC of PTSD rats?1?Western blotting resultsCompared with the control group,the protein expression of Bcl-2 was significantly increased,reached the peak at SPS 4d and declined from SPS 7d to 14d,and still slightly higher in SPS 14d.The protein expression of Bax was also significantly increased at SPS1d,and gradully increased and reached the peak in SPS 7d,slightly decreased at SPS14d.?2?Real Time PCR resultsqRT-PCR results showed that there was a certain level of Bcl-2 mRNA and Bax mRNA in mPFC of rats in the control group.After SPS stimulation,the expressions of Bcl-2 mRNA and Bax mRNA were upregulated.At SPS 4d,the expression of Bcl-2mRNA reached a peak and then declined.the expression of Bcl-2 mRNA at SPS 14d was still higher than that of control group.The expression of Bax mRNA reached a peak at SPS 7d,and then decreasd.The changes trend of Bcl-2 mRNA and Bax mRNA levels is in accordance with the Western blotting results.5.The expression of p62 in mPFC of PTSD rats?1?Immunohistochemical stainingThe positive immunoreaction of p62 was yellow or brown yellow granules,which were mainly expressed in the cytoplasm of mPFC cells in each group.Compared with the control group,the p62 positive cells in mPFC increased significantly at SPS 1d,and the expression decreased gradually from SPS 4d to 14d.?2?Western blotting resultsAfter SPS stimulation,p62 protein expression was significantly increased in the mPFC of SPS 1d group in comparison to control group.Its protein expression was gradually decreased from 4d to 14d?P<0.05?.?3?Real Time PCR resultsCompared with control group,p62 mRNA expression was significantly higher in the mPFC of SPS 1d group.It showed gradually decreasing tendency from 4d to 14d and significantly lower than control group?P<0.05?.Conclusion1.The learning and spatial memory ability of PTSD-SPS rats decreased,active exercise and inquiry behavior also decreased,and anxiety and fear state appeared at the same time.The obvious autophagy appears in the mPFC neuron cells of PTSD-SPS rats,which may be one of the reasons for the changes in the structure,function and clinical symptoms of the mPFC in PTSD.2.The abnormal expression of autophagy related gene Beclin-1 and LC 3 in mPFC of PTSD-SPS rats,which may exhibit a promoting effect in mPFC neuron apoptosis.SPS stimulation is closely related to these,and these abnormalities are molecular mechanisms leading to dysfunction of mPFC.3.There is a trend opposite relationship between p62 and autophagy related genes Beclin-1 and LC 3 in mPFC of PTSD rats,it indicate changes of autophagy flux in mPFC of PTSD rats.Abnormal expression p62 may also cause one of the important pathophysiological mechanism of PTSD. |
PDF Full Text Request