| Objective The purpose of this study was to investigate the mechanism of microtubule depolymerization protein Stathmin on apoptosis of amygdala neurons in PTSD rats.Methods Male SD rats aged 8 weeks(200-240 g)were randomly divided into 5 groups: normal control group(Control),model group(SPS),Stathmin gene overexpression group(Stathmin-OE),virus negative control group(Stathmin-NC).PI3K/AKT pathway inhibitor group(Stathmin-OE+LY294002).PTSD rat model was established by single continuous stress(SPS)method.PTSD rats were injected with 5 μL of Stathmin gene over-expressed adenovirus solution.After modeling,the anxiety and fear states of rats in each group were evaluated by the mine field test,elevated plus maze and conditioned fear test.Immunofluorescence was used to detect the co-localization and expression of Stathmin and Neu N.The apoptosis of amygdala neurons was detected by TUNEL staining.Western blot and RT-q PCR were used to detect the expression of Stathmin,PI3 K,p-AKT and Cleaved caspase 3 in the amygdala.Results After PTSD rats were established,the movement time of SPS rats was significantly longer than that of control rats(P<0.01),and the stationary time was significantly reduced(P<0.01).The number of closed arm entry and the total number of arm entry increased in rats(P<0.01),but significantly decreased in the open-arm distance,the percentage of open-arm distance to total distance,the open-arm dwell time,and the percentage of open-arm dwell time to total dwell time in each arm(P<0.01).There were significant increases in the distance traveled on the closed arm,the percentage of distance traveled on the closed arm,the time spent on the closed arm,and the percentage of time spent on the closed arm to the total time spent on the closed arm(P<0.01).There was no significant difference in the total distance between the open arm and the closed arm(P>0.05).During the adaptation period before the conditioned stimulus in the test phase,the stiffening behavior of rats in each group was less,and there was no significant difference between the two groups(P>0.05).During conditioned stimulation,stiffening behavior increased in SPS group(P<0.01).However,overexpression of Stathmin significantly reversed the above phenomena.In addition,compared with the Stathmin overexpression group,the PI3K/AKT pathway inhibitor group significantly increased the spontaneous activity and anxiety of the rats.The results of immunofluorescence and TUNEL staining showed that compared with the control group,the expression of Stathmin in SPS group decreased,the number of neurons decreased(P<0.01),and the TUNEL positive cells in amygdala increased significantly(P<0.01).Compared with SPS group,the expression of Stathmin in Stathmin-OE group increased,the number of neurons increased(P<0.01),and the TUNEL positive cells in amygdala decreased significantly(P<0.01).Compared with the Stathmin-OE group,the expression of Stathmin and the number of neurons in the Stathmin-OE+LY294002 group decreased(P<0.01),and the TUNEL positive cells in the amygdala increased significantly(P<0.01).WB and RT-q PCR results showed that compared with the control group,the protein expression levels and amounts of Stathmin,PI3 K and p-AKT in the SPS group were decreased,and the protein expression levels and amounts of Cleaved caspase 3 were increased(P<0.01).Compared with SPS,the protein expression levels and amounts of Stathmin,PI3 K and p-AKT in overexpression group were increased,and the protein expression levels and amounts of Cleaved caspase 3 were decreased(P<0.01).Compared with the overexpression group,the expression levels and amounts of PI3 K and p-AKT proteins in the inhibitor group were decreased,and the expression levels and amounts of Stathmin and Cleaved caspase 3 proteins were increased(P<0.01).Conclusions Overexpression of Stathmin inhibited the apoptosis of amygdala neurons in PTSD rats,which may be related to the activation of PI3K/Akt signal pathway. |
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