乌灵菌粉对创伤后应激障碍大鼠行为学及海马神经元自噬与凋亡的调节作用

Effect of Xylaria Nigripes on Behaviors,Hippocampal Neuronal Apoptosis and Autophagy in PDST Rat

目的探讨乌灵菌粉(xylaria nigripes,XN)对创伤后应激障碍(post-traumatic stress disorder,PTSD)大鼠行为学以及海马神经元自噬与凋亡的调节作用。方法90只SD大鼠随机分为对照组、PTSD组和XN组(0.8g/kg)。采用单一连续应激联合足底电刺激方法构建PTSD模型并通过旷场实验、高架十字迷宫和僵立实验观察大鼠行为学变化;Tunel法检测海马凋亡神经元;免疫荧光法观察海马Beclin1、LC3Ⅱ、caspase3表达;Western blot法和RT-PCR法分别检测海马Beclin1、LC3Ⅱ、caspase3、mTOR、p-mTOR蛋白和mRNA水平。结果与PTSD组比较,XN组大鼠在旷场箱内运动总路程、直立总次数和进入中央格次数均显著增加(P<0.01),进入开臂时间和次数的百分比显著增多(P<0.01),木僵率显著降低(P<0.01),海马Tunel、Beclin1、LC3Ⅱ、caspase3阳性神经元减少,Beclin1、LC3Ⅱ、caspase3、mTOR蛋白和mRNA水平降低,p-mTOR/mTOR蛋白比值升高。结论大鼠PTSD的发生可能与海马神经元自噬和凋亡增强有关;XN改善大鼠PTSD样行为的可能机制是促进海马mTOR蛋白磷酸化,抑制海马神经元过度自噬和凋亡。

Objective To investigate the effect of Xylaria nigripes(XN)on post-traumatic stress disorder(PTSD)rats behaviors and the regulation mechanism on autophagy and apoptosis in hippocampal neurons.Methods A total of 90 SD rats were randomly divided into the XN group(0.8g/kg),the control group and the PTSD group,with 30 rats in each group.The PTSD model was established by a combination of single continuous stress and plantar electric shock.Behavioral changes evaluated through open field,elevated plus maze and stiff behavior experiment.A Tunel assay was performed to detect neuronal apoptosis.Beclin1,LC3Ⅱand caspase3 expression was detected by Immunofluorescence.The protein and mRNA expression of Beclin1,LC3Ⅱ,caspase3,mTOR and p-mTOR were detected by Western blot analysis and RT-PCR,respectively.Results Compared with the PTSD group,the XN group showed a significant increase in the total distance,standing times and crossing number of central grid in the open field test(P<0.01).The percentages of entries and time into the open arm were significantly increased in the elevated plus maze experiment(P<0.01)and the stiff behavior was reduced(P<0.01).Significant reductions were observed in apoptosis of hippocampal neurons,hippocampal Beclin1,LC3Ⅱand caspase3 positive neurons numbers.The protein and mRNA expression levels of Beclin1,LC3Ⅱ,caspase3 and mTOR were decreased by treatment with XN,while p-mTOR/mTOR were increased.Conclusion The occurrence of PTSD in rats may be related to the enhancement of autophagy and apoptosis of hippocampal neurons.XN could regulate abnormal autophagy and apoptosis levels of hippocampal neurons by increasing the phosphorylation level of mTOR in hippocampus,and ameliorate PTSD-like behavior in rats.