Role Of Beclin1 Signaling Pathway-mediated Autophagy In The Injury Of Amygdala Nerve Cells Of Stressed Rats

Objective: In the practice of forensic medicine,there are often cases in which sustained and serious stress events lead to mental symptoms or even death of the parties concerned.Due to the lack of diagnostic criteria or diagnostic system for stress injury,it brings great difficulties to forensic medicine identification.As the emotional center,amygdala plays an important role in emotional regulation.Earlier studies found that endoplasmic reticulum stress is widely involved in the process of damage to the nerve cells outside the base of the amygdala,but unfolded or misfolded proteins of endoplasmic reticulum stress activate autophagy.Autophagy is a common life phenomenon in eukaryotic cells.The activation of autophagy plays a double-edged sword role.Moderate autophagy is helpful for cell growth and development,while excessive autophagy will lead to cell damage.The Class III PI3K-Beclin-1-Vpsl5 complex pathway is a key pathway for the formation of autophagy precursors in the early stages of autophagy,which can initiate autophagy and positively regulate autophagy.Autophagy inhibitor 3-methyladenine(3-MA)is a specific inhibitor of Class? PI3 K.It is not clear how autophagy plays a role in stress-induced nerve cell injury.Therefore,this study intends to establish a rat model of restraint stress,and observe the expression of Class ? PI3K-Beclin1 signal pathway in neurons of basolateral nucleus of amygdala from the overall animal level.Through the use of Class? PI3K-Beclin1 signaling pathway specific inhibitor 3-MA,comparative observation was made to clarify the mechanism of Class? PI3K-Beclin1 signaling pathway in stress-induced damage to amygdala basolateral nucleus nerve cells.So as to provide scientific theoretical support for forensic medicine identification of mental injury or death caused by stress.Methods: The restraint stress model of rats was established.Theexperimental groups were Control group(21 days),1-day group,3-day group,7-day group,14-day group and 21-day group.Then,the expression of autophagy-related proteins in amygdala of rats was detected,and the time point of strongest autophagy(7 days)was selected as the time point of autophagy inhibitor(3-MA).Control group(7 days),autophagy inhibitor group(3-MA group),restraint Stress 7-day model group(stress group),restraint stress 7-day +autophagy inhibitor group(Stress+3-MA group).The changes of anxiety behavior in rats and the success of the evaluation model were observed through weight changes,elevated cross maze experiment and open field experiment.The pathological changes of nerve cells in BLA region of rat amygdala were observed by HE staining and toluidine blue staining.The expressions of autophagy-related proteins Class ? PI3 K,Beclin1,LC3(LC3? / ?),and P62 in nerve cells of BLA region of amygdala were detected by immunohistochemistry,immunofluorescence technology and Western blot.Results processing and statistical analysis: SPSS23.0 was used for statistical analysis,and all data were expressed as means±SD.When the variances are equal,the independent t test is used to compare the two groups,the one-way ANOVA is used to compare the multiple groups,and the Tukey post hoc test is used to compare each other;when the variance is not at the same time,the non-parametric test Kruskal–Wallis test was used,and Dunn's multiple comparison test was used for pairwise comparison.All statistical results were statistically different with P<0.05.Results:1.Compared with the control group,the weight of restraint stress rats increased slowly.2.The elevated cross maze and open field experiments observed:(1)Compared with the control group,the stressed rats showed obvious anxiety-like behaviors,and the symptoms gradually worsened with the extension of stress time.(2)Compared with the stress 7-day group,the stress7-day + 3-MA group significantly reduced the degree of anxiety-like behavior;but compared with the control 7-day group and 3-MA group,there was still anxiety-like behavior change.3.The results of HE staining showed that:(1)with the extension of stress time,BLA neurons in the amygdala gradually appeared neurocytoedema,loose tissue structure,shrinkage of neurons,proliferation of microglia,satellite phenomenon and neurophilic phenomenon.(2)The number of nerve cells in the BLA region of the amygdala in the Stress7 days + 3-MA group was significantly reduced compared with the Stress7 group.4.Toluidine blue staining results showed that:(1)With the extension of stress time,the amygdala BLA nerve cells gradually showed unclear structure,irregular and disappeared Nissl body arrangement,cell shrinkage and deep staining,and satellite phenomenon.(2)The degeneration of nerve cells in the BLA region of the amygdala in the Stress 7 days + 3-MA group was reduced compared with the Stress 7 days group.It showed that 3-MA,an autophagy inhibitor,can improve nerve cell damage in the BLA region of the amygdala.5.The results of immunohistochemical staining,immunofluorescence staining and Western blot showed that:(1)Compared with the control group,PI3 K,Beclin-1,LC-3(LC3-? / ?)The protein expression showed a trend of increasing first and then decreasing with the extension of stress time.The stress group 1d,3d and 7d gradually increased and reached a peak on the 7th day of stress.The positive expression in the 14 d and 21 d groups gradually decreased,but it was still higher than that in the control group.The expression of P62 protein decreased first and then increased with the extension of stress time.The expression of P62 protein decreased gradually in stress group 1d,3d and 7d,and reached the lowest value in stress 7 days.The positive expression in groups 14 d and 21 d increased gradually,but was still lower than that in the control group of 7 days.(2)Compared with the Stress 7-day group,the positive expression of PI3 K,Beclin-1,LC-3(LC3-II/I)protein in Stress 7-day+3-MA group was significantly decreased,but still higher than that in the control 7-day group and 3-MA group.The expression of P62 protein was significantly increased,but it was still lower than the control group of 7 daysand 3-MA group.It is suggested that restraint stress induces activation of autophagy Class ? PI3K-Beclin1 pathway,and leads to increase of LC3(?)and decrease of P62 in downstream.However,after class ? PI3 K Beclin 1pathway inhibitor 3-m A was given,the expression level of class ? PI3 K Beclin1 pathway related proteins decreased,which inhibited the activation of class ? PI3 K Beclin 1 pathway.Conclusions:1.Restraint stress can induce the damage of rat BLA nerve cells.2.Class ? PI3K-Beclin1 signaling pathway mediated autophagy is invo lved in the damage of neurons in basolateral amygdala caused by chronic restraint stress.