| Objective: The core feature of post-traumatic stress disorder(PTSD)is abnormal fear memory.Abnormal fear memory is the impaired inhibition of fear in a safe environment,fear learning that is more easily generalized,or inappropriate fear responses that cannot be suppressed.However,at present,the molecular mechanism underlying fear memory abnormalities is still unclear.Studies have shown that PTSD is closely related to brain structures of the fear memory loop brain regions such as the hippocampus,medial prefrontal cortex(m PFC)and amygdala.Stathmin protein is a ubiquitous cytosolic phosphoprotein,a member of the microtubule instability regulatory protein family and has the ability to promote microtubule depolymerization.Nerve cells all have a stathmin-like domain(SLD).The stathmin protein family can form a tight trimeric T2 S complex between the alpha-helix and α/β heterodimers of microtubules,thereby inhibiting microtubules.Polymerization plays a role in regulating tubulin.Between the N-terminus and the C-terminus is the ligation domain.When stathmin is phosphorylated,the T2 S complex disintegrates,releasing microtubule dimers and inhibiting the binding of stathmin to α/β tubulin,and the stathmin protein depolymerizes microtubules,thereby promoting microtubule formation and elongation.The phosphorylated stathmin protein promotes microtubule polymerization,whereas dephosphorylated stathmin promotes microtubule depolymerization.Therefore,the phosphorylation site of the stathmin protein family has an important relationship with its function.The downstream target of stathmin protein is tubulin.The protein that stabilizes microtubules is microtubule associated protein(MAP).Microtubule dimers can bind to MAP to stabilize microtubules.Stathmin plays an important regulatory role in fear memory.Therefore,we hypothesize that stathmin and its phosphorylation anomalies are associated with fear memory loop abnormalities caused by PTSD.In this study,we established a rat model of PTSD by exposing rats to single prolonged stress(SPS).Then,we detected the expression of stathmin,p-stathmin, β-tubulin and MAP-1B in the hippocampus,m PFC and amygdala of PTSD rats the fear memory loop brain regions at different times by immunohistochemistry,Western blotting and q RT-PCR.We discussed the temporal variation of stathmin,p-stathmin,β-tubulin and MAP-1B in the hippocampus,m PFC and amygdala after SPS.We elucidated the molecular mechanism of the involvement of stathmin in PTSD-induced fear memory abnormalities and provided experimental evidence for identifying effective drug targets for PTSD treatment.Methods: 1.Experimental animal Groups: A total of 175 Wistar rats were randomly divided into 5 groups:(1)normal control group;(2)SPS 1 day group;(3)SPS 4 days group;(4)SPS 7 days group;(5)SPS 14 days group.There were 35 rats in each group.2.Establishment of a Rat Model of PTSD: A rat model of PTSD was established by the SPS method described at the International PTSD Scientific Conference held by the Ministry of Education of Japan in 2005.At the set time point,experimental rats were selected and tested accordingly.3.Immunohistochemical staining: The SABC method was used to immunohistochemically stain the brain slices of each group of rats to detect the morphological expression of stathmin,β-tubulin and MAP-1B in the hippocampus,m PFC and amygdala of each group of rats.The Meta Morph/DPIO/BX41 morphological image analysis system was used to measure the optical density values of the stained cells in each field of view and for a semiquantitative analysis.4.Western blotting technology: Western blotting was used to detect stathmin,p-stathmin(Ser16),p-stathmin(Ser25),p-stathmin(Ser38),p-stathmin(Ser63),β-tubulin and MAP-1B proteins.The expression changes of hippocampus,m PFC and amygdala of each group of rats were analyzed by gel image processing system for net optical density values of target protein(stathmin,etc.)bands and internal reference(GAPDH)bands.The optical density ratio of stathmin etc./GAPDH of each group of samples was used to observe the relative expression changes of proteins such as stathmin and to perform semiquantitative analysis.5.q RT-PCR technology: The q RT-PCR method was used to detect stathmin,β-tubulin and MAP-1B m RNA expression changes.q RT-PCR uses GAPDH as an internal reference,normalizes the target genes,and uses the 2-ΔΔCt method to compare the differences in the relative expression levels of stathmin,β-tubulin and MAP-1B m RNA in each group of samples.6.All data obtained from the experiments were expressed as the mean ± standard deviation.Differences among the five groups were analyzed by one-way ANOVA using SPSS 19.0 statistical software,and t test was used to compare the differences between the two groups data.P < 0.05 was considered statistically significant.Results: 1.Stathmin expression in hippocampus,m PFC and amygdala of PTSD rats: Immunohistochemical staining showed that stathmin expression was indicated by brown-yellow particles,which were mainly distributed in the neuronal cell bodies of rat hippocampus,m PFC and amygdala.In the control group,the expression of stathmin was strong in rat hippocampus,m PFC and amygdala neurons.One and 4 days after SPS,the expression of stathmin gradually decreased,and the expression was minimal on 7th day,and then gradually increased.Western blotting was used to detect the expression of stathmin protein in the hippocampus,m PFC and amygdala of rats of the normal control group and SPS groups.Compared with that in the normal control group,the expression of stathmin protein in the SPS 1 day group and the SPS 4 days group decreased gradually,and the SPS 7 days group exhibited the lowest expression.On the 14 th day,the level was restored to that of the normal control group.The results of q RT-PCR analysis showed that the level of stathmin m RNA in the SPS 1 day and SPS 4 days groups was lower than that in the normal control group,the SPS 7 days group reached the lowest level,and then recovered.2.p-stathmin(Ser16),p-stathmin(Ser25),p-stathmin(Ser38)and p-stathmin(Ser63)expression results in hippocampus,m PFC and amygdala of PTSD rats: Western blotting was used to detect p-stathmin(Ser16),p-stathmin(Ser25),p-stathmin(Ser38)and p-stathmin(Ser63)protein expression in the hippocampus of the normal control group and SPS groups.The results showed that the p-stathmin(Ser25)and p-stathmin(Ser38)proteins were expressed in the hippocampus of the normal rats.The expression levels of the p-stathmin(Ser25)and p-stathmin(Ser38)proteins increased one day after SPS,peaked on the 7th day,and then decreased.However,p-stathmin(Ser16)and p-stathmin(Ser63)were not expressed in the normal group or in the SPS groups.Western blotting was used to detect the expression of p-stathmin(Ser16),p-stathmin(Ser25),p-stathmin(Ser38),p-stathmin(Ser63)protein in the m PFC of the control group and SPS groups.The results showed that the protein expression of p-stathmin(Ser63)increased gradually compared with that in the control group,peaked on the 7th day after SPS,and then decreased.However,p-stathmin(Ser16),p-stathmin(Ser25),and p-stathmin(Ser38)were not expressed in any of the groups.3.β-tubulin and MAP-1B expression results in hippocampus,m PFC and amygdala of PTSD rats: β-Tubulin immunoreactivity was indicated by yellow particles expressed in neuron cell dendrites,axons and bodies.In the normal control group,β-tubulin was slightly expressed in rat hippocampal neurons.The expression of β-tubulin was increased 1 day after SPS,and the expression was highest on the 7th day after SPS,and then decreased.Western blotting was used to detect the expression of β-tubulin and MAP-1B in the hippocampus of the normal control group and SPS groups.The expression of the β-tubulin protein was increased 1 day after SPS,peaked on the 7th day,and then decreased.The MAP-1B protein expression peaked in the SPS 1 day group,and reached the lowest level in the SPS 4 days group and then gradually increased.q RT-PCR was used to detect the m RNA expression of β-tubulin and MAP-1B in the hippocampus of the control group and SPS groups.The results showed that the β-tubulin m RNA levels in the SPS 4 days group and the SPS 7 days group were significantly higher than those in the normal control group.The MAP-1B m RNA level was increased 1 day after SPS,peaked at 7 days,and then decreased.Immunohistochemical staining showed that β-tubulin and MAP-1B were coexpressed in m PFC neuron cell bodies,dendrites and axons,and positive staining was indicated by yellow particles.In the normal control group,rat m PFC neurons were positive for β-tubulin and MAP-1B.On the 7th day after SPS,β-tubulin and MAP-1B positive expression reached a peak,and then the expression decreased.Western blotting was used to detect the expression of β-tubulin and MAP-1B in the m PFC of the normal control group and SPS groups.The results showed that the protein expression of β-tubulin and MAP-1B increased gradually compared with that in the normal control group,peaked on the 7th day after SPS,and then decreased.q RT-PCR was used to detect the m RNA expression of β-tubulin and MAP-1B in the m PFC of the normal control group and SPS groups.The results showed that the expression level of β-tubulin m RNA in the SPS 7 days group was significantly higher than that in the normal control group.The expression level of MAP-1B m RNA was increased 1 day after SPS,peaked on the 7th day,and then decreased.Western blotting was used to detect the expression of β-tubulin and MAP-1B in the amygdala of the normal control group and the SPS groups.The results showed that the protein expression of β-tubulin and MAP-1B increased gradually compared with that in the normal control group,peaked at SPS 7 days,and then decreased.The m RNA expression of β-tubulin and MAP-1B in the amygdala of rat in the normal control group and SPS groups by q RT-PCR showed that β-tubulin m RNA level in SPS 7 days group was significantly higher than that in the normal control group.The expression level of MAP-1B m RNA was increased 1 day after SPS,peaked in the SPS 7 days group,and then decreased.Conclusion: 1.The expression changes of hippocampus,m PFC and amygdala brain regions stathmin in PTSD rats caused by SPS affected the stability of microtubules and may regulate the abnormal memory of fear in PTSD rats.2.In the hippocampus of PTSD rats,the phosphorylation of p-stathmin(Ser25)and p-stathmin(Ser38)may be involved in the regulation of microtubule stability.3.Stathmin-β-tubulin induced biphasic changes in the fear memory loop brain regions-hippocampus,m PFC and amygdala of PTSD rats caused by SPS,which provided the basis for the changes of microtubule stability.4.The results of this experiment confirmed that MAP-1B was involved in regulating microtubule stability during PTSD caused by SPS. |
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