| Posttraumatic stress disorder(PTSD)is a psychological trauma and subsequently delayed persistent mental disorders caused by a catastrophic event or a serious threat.PTSD is characterized by high incidence,poor curative effect,and long disease course and becomes one of the typical stress disorders as well as current research hotspot.The core symptoms of PTSD include re-experiencing invasive trauma,heightened alertness,and responsive numbness.Previous studies showed that the hippocampus and amygdala of PTSD patients were reduced in volume,suggesting abnormal loss and death of the hippocampus neurons in PTSD patients.The results of animal experiments showed that the neuronal apoptosis phenomenon occurred in these regions.Beclin1(expression product of autophagy gene Beclin1)participates in the early formation process of autophagy and can positively regulate the occurrence of autophagy.Beclin1 existed in the hippocampus of adult rats,suggesting that the cell autophagy phenomenon might occur in the region.The abnormal loss and death of hippocampal neurons in the pathogenesis of PTSD was related to neuronal apoptosis and cell autophagy.The phosphatase and tension homolog(PTEN),which is absent in the10 th chromosome,has the phosphatase activity to dephosphorylate PIP3 to generate PIP2 and can negatively regulate thePI3K/Akt signaling pathway,weaken the signal intensity from the upstream of PI3K/Akt/mTOR signal transduction pathways,inhibit cell proliferation,and promote apoptosis.In previous studies,the expression level of PTEN in PTSD rats was increased,indicating that PTEN was closely related to the autophagy and apoptosis of hippocampal neurons of PTSD rats.However,the relationship was seldom reported.The role of PTEN in the autophagy and apoptosis of hippocampal neurons of PTSD rats and related signaling pathway should be further studied.In this study,the rat model of PTSD was established by using the modified single-prolonged stress & shock(SPS&S stress)to explore the effects of PTEN on autophagy and apoptosis of hippocampal neurons in PTSD rats as well as related signaling mechanisms.Part One Behavior test and morphological changes of hippocampal neurons in PTSD ratsObjective: In this part aims to study the behavioral changes and morphological changes of hippocampal neurons in PTSD rats and explore whether the abnormal function existed in hippocampal neurons of PTSD rats from behavioral and morphological aspects.The rat model of PTSD was established by using the modified single-prolonged stress & shock(SPS&S stress)to explore the pathogenesis mechanism of PTSD and morphological changes of hippocampal neurons in PTSD rats.Moreover,the behavior changes of PTSD rats were tested through the shuttle box,open field experiments,and Morris water maze experiment.Methods:1 In the experiment,60 SPF-grade adult male Sprague-Dawley rats(180-200 g)after 7-day breeding were randomly divided into control group and model group.The model group rats were used to establish PTSD model.The PTSD rat model was prepared according to a modified SPS&S procedure,which included 2 hours of imprison and 15-min forced swimming,ether anesthesia to coma,and 10-s inevitable foot shock.After rats rested for 7 days,the model preparation procedure was completed.The breeding conditions were controlled as follows: 22 to 25 °C,circadian rhythm,and free access to both water and food.All animal experiments in this study were performed according to the animal ethics committee guidelines.In 1 day after the model preparation,we started the behavior test including shuttle box experiments,open field test and Morris water maze tests.2 In the shuttle box test,the two groups of rats were subjected to an acousto-optic stimulus for 10 seconds.After 5 seconds,a continuous foot shock of 8mA was performed for 10 seconds.Testing parameters,such asactive avoidance time of rats and trajectories,were recorded.This test was performed for 5 consecutive days and each test included 20 cycles.After4-day training,the measurement data obtained on the fifth day were recorded.3 In the field test,we recorded the active time and total distance of every rat and the times of standing up.The testing time was 5 min.4 Morris water maze experiment includes the hidden platform experiment and space exploration test.In the hidden platform test,we put the rats into water from any one of the four quadrants and in every test the rat swam for 90 sec to search for the hidden platform.We recorded the searching time as the escape latency(EL).In the space exploration test,we removed the platform,put the rats into water in the same entry point,and recorded the swimming path in 60 sec,the staying time in original platform and the times for passing through original platform.5 we observed morphological changes in hippocampal neurons of two groups of rats.All experimental data were expressed in the form of x?±s.We performed statistical analysis in SPSS16.0 statistical software and compared the results with independent T tests.If P< 0.05,the difference is statistically significant.Results:1 The behavior results showed that the PTSD rat model was prepared successfully.2 Behavior changes:The active avoidance response of PTSD rats was delayed and the learning memory ability became worse than before.The alert level and anxiety state of PTSD rats become higher and the ability of environmental adaptation was worse than before.The ability of learning memory in space of the PTSD rats was damaged.3 Morphological changes: Model group rats showed the decreased hippocampal neurons with disordered arrangement,nuclear pyknosis and hyperchromatic nucleus. summary: The learning memory ability of PTSD rats became worse than before.Hippocampal neurons with disordered arrangement,nuclear pyknosisand hyperchromatic nucleus.Part Two PTEN expression in PTSD rats hippocampal neurons and its relationship with autophagy and apoptosisObjective: To test the expression of PTEN in PTSD rats hippocampal neurons,and explore the function of PTEN gene in PTSD rats hippocampal neurons of autophagy and apoptosis.Methods:1 The expression of PTEN gene in hippocampal neurons of PTSD rats.1)Firstly,90 SPF-grade of adult male SD rats were selected and then randomly divided into control group and model group.Model group SPS&S was used to establish PTSD model.After the model rats were established,the two groups were randomly tested at 5 time points(Day 1,Day 3,Day 7,Day14,and Day 28).Each test was performed with 9 rats in every group.2)PTEN protein expression of two groups of rats was analyzed with Western blotting.With the Bio-rad gel imaging analysis system,the semi-quantitative analysis of the ratio of the average grey degree of target protein was performed to the average grey degree of internal control protein;3)The PTEN mRNA expression was tested with the real-time PCR technique and the PCR results were analyzed with the relative determination of CT value;4)PTEN protein expression was detected with the immunofluorescence chemical technology.In Olympus F1000 laser confocal microscopy,a 488-nm laser was used to stimulate green light.Data acquisition and imaging were completed by computers.2 The function of PTEN gene on autophagy and apoptosis of hippocampal neurons in PTSD rats was explored.1)Firstly,the PTEN gene recombination adenovirus was established.2)The 80 SPF-grade adult male SD rats were randomly divided into control group,model group,transfection group,and PBV group.Control group was not subjected to any treatment.Before preparing model rats,we respectively injected the empty virus(Ad-GFP)into hippocampus in Modelgroup rats and transfected PTEN recombinant adenovirus(Ad-PTEN-GFP)into hippocampus in Transfection group rats or PTEN protein inhibitor(BPV)into hippocampus in PBV group rats.3)Morris water maze test was performed to detect the behavior changes of the rats of every group.4)The expression levels of autophagy-related proteins LC3-? and Beclin1 in rat hippocampal neurons was analyzed with Western blotting technique and the change in the ratio of LC3-?/LC3-I was calculated.We analyzed the contents of the hippocampal neuron apoptosis-related proteins such as Bax and Bcl-2 in the rats of every group with the western blotting technique and calculated the changes in the Bax/Bcl-2 ratio.5)The mRNA levels of LC3 and Beclin1 in rats' hippocampal neurons were analyzed with Real-Time PRC technique.6)Apoptosis cells of hippocampal neurons in the rats of each group were detected by TUNEL method and the positive cells,the total cell number and the apoptosis index were calculated.7)The autophagy body,autophagy-lysosome,and nerve cell apoptosis as well as ultrastructure changes in hippocampal neurons of the rats of all the groups were observed by transmission electron microscopy.All experimental data were expressed in the form of x?±s.We performed the statistical analysis in SPSS16.0 statistical software and compared the experimental data through independent T tests.If P<0.05,the difference is statistically significant.Results:1 The results of the PTEN gene expression in hippocampal neurons of PTSD rats.1)The model group showed the significantly increased expression level of hippocampal PTEN protein.The expression level significantly increased on Day 3,peaked on Day 7,and then gradually declined,but it was still higher than that in the control group on Day 28.2)The model group showed the obviously increased hippocampal PTENmRNA level.The hippocampal PTEN mRNA level in the model group was1.12 times of that in control group on Day 1,1.48 times of that in control group on Day 3,and 3.38 times of that in control group on Day 7.The hippocampal PTEN mRNA level in the model group peaked on Day 7,then decreased to 1.21 times of that in control group and 1.05 times of that in control group on Day 28.3)The immunofluorescence staining results of PTEN.The expression of PTEN in hippocampal neurons in the model group was obviously higher than that in the control group.The expression of PTEN in the model group rose on Day 3,peaked on Day 7,and then gradually declined,but it was still higher than that in control group on Day 28.2 The results of behavior tests of the influences of PTEN gene on autophagy and apoptosis of hippocampal neurons in PTSD rats.1)According to the MWM experimental results,the EL of various groups were decreased in the following sequence: transfection group>model group>bpv group>control group;the residence time of original platform quadrant and the times of passing through original platform of various groups were decreased in the following sequence: control group>bpv group>model group>transfection group.2)According to analysis results of western blotting,LC3-?/LC3-?ratios of various groups were decreased in the following sequence: transfection group>model>BPV>controls;Beclin1 protein expression levels of various groups were decreased in the following sequence: transfection group>model>BPV>control group.According to the results of the expression levels of apoptosis-related proteins,such as Bcl-2 and Bax,the expression levels of Bcl-2 protein of various groups were decreased in the following sequence: the control group>BPV>model group>transfection group.The expression levels of Bax protein of various groups were decreased in the following sequence: transfection group> model group>BPV>control groups.3)According to the mRNA levels measured by real-time PRC technique,the mRNA ratios of LC3-?/LC3-?in hippocampus of various groups weredecreased in the following sequence: transfection group>model>BPV>control groups.Beclin1 mRNA expression levels of various groups were decreased in the following sequence: transfection group>model>BPV>control groups.4)According to the results of TUNEL method,in the apoptosis neurons,the cells were stained into tan and the chromatin was condensed.In the control group,there are a few scattered TUNEL-positive cells and in the CA1 area,AI was 5.90%;in the CA1 area in model group,the AI was 26.08%;in CA1 area of PBV group,AI was 19.98%;in the CA1 area in the Transfection group,AI was 38.08%,which was the highest among the four groups.5)TEM observation results.In the model group,the microscopic structures of hippocampal neurons were changed and condensed chromatin cell shrinkage were observed.In BPV group,the damaged microscopic structures of hippocampal neurons were improved.In the transfection group,cracked nucleus and apoptotic bodies were observed and the microscopic structures of hippocampal neurons were seriously damaged.In control group,bubbles and autophagosome were occasionally observed.In model group,BPV group,and transfection group,autophagosome and autophagy-lysosome were visible.There are more cytolysosome and autophagy-lysosome in transfection group.All the above results are statistically significant(P<0.05).summary:1 PTSD rats showed the significantly increased expression level of hippocampal PTEN protein.2 Autophagy and apoptosis of hippocampal neurons occur in PTSD rats.3 PTEN regulates the PTSD autophagy and apoptosis of hippocampal neurons.Part Three PTEN regulation of autophagy and apoptosis in hippocampal neurons in PTSD ratsObjective: To explore the pathways of signal transduction of PTEN gene regulation of apoptosis and autophagy of hippocampal neurons in PTSD rats.Methods:1 The changes in the PI3K/ PTEN/Akt/mTOR signal pathways ofhippocampus of PTSD rats were determined.1)Firstly,50 SPF adult male SD rats were selected and then randomly divided into control group and model group.Control group was not subjected to any treatment.PTSD model was established.After the construction of the PTSD model,the two groups were tested randomly at 5 time points(Day 1,Day 3,Day 7,Day 14,and Day 28).Each test was performed with 5 rats in every group.2)The key proteins of PTEN signal pathways of PI3K/PTEN/Akt/mTOR were determined with Western blotting technique,including p-Akt,p-mTOR,p-4E-BP1,p-p70S6 K,p-eIF4 B,and p-eIF4 E.2 To determine whether the PI3K/Akt/mTOR signal pathways were involved in PTEN regulation of apoptosis and autophagy of hippocampal nerve cells in PTSD rats.1)Firstly,60 SPF adult male SD rats were selected and then randomly divided into control group,model group,LY294002 group,and rapamycin group.Before giving SPS&S,PI3K/Akt inhibitor LY294002 was injected into the hippocampus of LY294002 group.Before giving SPS&S,mTOR rapamycin inhibitor was injected into hippocampus in the rats of Rapamycin group.2)The expression level of hippocampal neuron autophagy-related protein LC3 in the rats of every group was determined by Western blotting technique and the change in the ratio of LC3-?/LC3-?was calculated.3)The apoptosis rate of hippocampal neuron in the rats of every group was determined by the flow cytometry technique.3 The correlation between abnormal apoptosis and autophagy of hippocampal neurons in PTSD rats.We performed the co-positioning of Beclin1 and Caspase-3 proteins of hippocampal neurons in the rats of every group and analyzed the correlation between apoptosis and autophagy by the doubleimmunofluorescence labeling technology.Results:1 The expression levels of key proteins in PI3K/PTEN/Akt/ mTOR signalpathways in hippocampal neurons in PTSD rats:The expression levels of PTEN,p-AKT,p-mTOR,p-4E-BP1,p-p70S6 K,and p-eIF4 B in hippocampal neurons in control group were low and showed no significant difference among various time points.However,the above proteins in hippocampal neurons in model group were significantly increased compared with those in control group(P<0.05).Among the 5 time points,the above proteins in hippocampal neurons in model group increased firstly and then decreased.The expression level of PTEN peaked on Day 7,whereas the expression level of p-AKT peaked on Day 1.The expression levels of p-eIF4 B in hippocampal neurons in both control group and model group were low and the difference control group and model group was not significant(P>0.05).Moreover,the expression levels of p-eIF4 B showed no significant difference among various time points.2 Results of the PTEN regulation apoptosis and autophagy of hippocampal neurons in PTSD rats via the PI3K/Akt/mTOR signal pathway.According to the results obtained by Western blotting technique,the LC3-?/LC3-? ratio in the hippocampus the model group was higher.LC3-?/LC3-? ratio in the hippocampus LY294002 and rapamycin groups was higher than that in the model group.According to the activity determination result of apoptosis proteins,the expression level of Caspase3 in the hippocampus the model group was higher.The expression level of Caspase3 in the hippocampus LY294002 and rapamycin groups was higher than that in the model group.The determination results of rats' hippocampal neurons apoptosis by the flow cytometry technique.The apoptosis rates of hippocampal neuron were respectively 5.83±0.10,9.24±0.46,27.24±1.27,and 21.22±1.82 in control group,model group,LY294002 group,and rapamycin group.All the results are statistically significant(P<0.05).3 Correlation between abnormal apoptosis and autophagy of hippocampal neurons in PTSD rats.According to the determination results by the double-immunoflu-orescence labeling technique,the small amounts of Beclin1 and Caspase3 existed in the control group.In the model group,the expressions of Beclin1 protein and Caspase3 protein were enhanced and the colocalization phenomenon of Beclin1 and Caspase3 were observed.Compared with the model group,LY294002 and rapamycin groups showed the colocalization phenomenon of partial Beclin1 and Caspase3 and the significantly increased expressions of Beclin1 and Caspase3.summary:1 The regulation function of PTEN on autophagy and apoptosis of PTSD after hippocampal neurons is realized by PI3K/Akt/mTOR.2 Autophagy and apoptosis of neurons show a co-localization relationship in the pathogenesis of PTSD.Conclusions:1 Autophagy and apoptosis of hippocampal neurons occur in PTSD rats and learning and memory ability of the PTSD rats declined,as indicated by behavior tests.The alert level and anxiety state are increased.2 PTEN regulates the PTSD autophagy and apoptosis of hippocampal neurons.The expression level of PTEN in PTSD rats' hippocampal neurons is elevated,thus stimulating the occurrence of autophagy and apoptosis of hippocampal neurons.Inhibition of the PTEN activity can decrease the autophagy and apoptosis of hippocampal neurons after PTSD.3 The regulation function of PTEN on autophagy and apoptosis of PTSD after hippocampal neurons is realized by PI3K/Akt/mTOR.PTEN protein can regulate the autophagy and apoptosis of hippocampal neurons.The regulation function can be also weakened by the mTOR inhibitors(rapamycin)or PI3K/Akt inhibitor(LY294002).4 Autophagy and apoptosis of neurons show a colocalization relationship in the pathogenesis of PTSD. |
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