针刺通过活性氧/c-Jun氨基末端激酶/p53通路对创伤后应激障碍大鼠模型海马神经元凋亡的影响机制

Mechanism of acupuncture on hippocampal neuron apoptosis in rat model of post-traumatic stress disorder via the reactive oxygen species/c-Jun N-terminal kinase/p53 pathway

  • 摘要:
    目的 探讨针刺通过活性氧(ROS)/c-Jun氨基末端激酶(JNK)/p53通路对创伤后应激障碍(PTSD)大鼠模型海马神经元凋亡的影响机制。
    方法 将30只大鼠随机分为空白组(NC组)、PTSD大鼠模型组(PTSD组)和PTSD大鼠模型电针干预组(EA组),每组10只。比较各组大鼠行为学检测(旷场实验、高架十字迷宫实验)结果。采用原位缺口末端标记法(TUNEL)染色检测各组海马神经元凋亡情况。采用DHE荧光染色法检测各组脑组织中ROS水平。检测各组脑组织氧化应激指标丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)含量。采用Western blot法检测海马组织中JNK、p-JNK、p53、PUMA蛋白表达水平。
    结果 PTSD组大鼠运动总距离短于NC组,直立总次数及进入中央格次数少于NC组,差异有统计学意义(P < 0.05); EA组大鼠运动总距离长于PTSD组,直立总次数及进入中央格次数多于PTSD组,差异有统计学意义(P < 0.05)。PTSD组进入开放臂次数和时间的百分比低于NC组,焦虑指数高于NC组,差异有统计学意义(P < 0.05); EA组进入开放臂次数和时间的百分比高于PTSD组,焦虑指数低于PTSD组,差异有统计学意义(P < 0.05)。PTSD组大鼠的神经元凋亡率高于NC组,差异有统计学意义(P < 0.05); EA组神经元凋亡率低于PTSD组,差异有统计学意义(P < 0.05)。PTSD组脑组织中ROS平均荧光强度高于NC组,差异有统计学意义(P < 0.05); EA组脑组织中ROS平均荧光强度低于PTSD组,差异有统计学意义(P < 0.05)。PTSD组脑组织中MDA含量高于NC组, SOD活性和GSH含量低于NC组,差异有统计学意义(P < 0.05); EA组脑组织中MDA含量低于PTSD组, SOD活性和GSH含量高于PTSD组,差异有统计学意义(P < 0.05)。PTSD组脑组织中p-JNK/JNK和p53、PUMA蛋白表达水平高于NC组,差异有统计学意义(P < 0.05); EA组脑组织中p-JNK/JNK和p53、PUMA蛋白表达水平低于PTSD组,差异有统计学意义(P < 0.05)。
    结论 针刺可抑制PTSD大鼠海马神经元凋亡,减轻大鼠恐惧症状,其作用机制可能与ROS/JNK/p53通路的激活有关。

     

    Abstract:
    Objective To explore the mechanism of acupuncture′s effect on hippocampal neuron apoptosis in a post-traumatic stress disorder (PTSD) rat model via the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK)/p53 pathway.
    Methods Thirty rats were randomly divided into normal control group (NC group), PTSD rat model group (PTSD group) and electroacupuncture intervention group (EA group), with 10 rats in each group. The results of behavioral test (open field experiment and elevated cross maze experiment) were compared. The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining method was used to detect hippocampal neuron apoptosis in each group. DHE fluorescence staining was employed to measure ROS levels in the brain tissue of each group. The content of Ros indexmalondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH)in brain tissue of each group was detected. Western blot analysis was performed to determine the protein expression levels of JNK, p-JNK, p53 and PUMA in hippocampal tissue.
    Results The total movement distance of rats in the PTSD group was significantly shorter, and the total number of uprightand central cell entry times were significantly shorter than those in the NC group (P < 0.05). The total distance moved by rats in the EA group was significantly longer, and the total number of upright and central lattice entry times were significantly higher than those in the PTSD group (P < 0.05). The percentage of times and time of entering the open arm in the PTSD group was significantly lower, and the anxiety index was significantly higher than that in the NC group (P < 0.05). The percentage of times and time of entering the open arm in the EA group was significantly higher, and the anxiety index was significantly lower than that in the PTSD group (P < 0.05). The neuronal apoptosis rate of the PTSD group was significantly higher than that of the NC group (P < 0.05); the neuronal apoptosis rate in the EA group was significantly lower than that in the PTSD group (P < 0.05). The mean fluorescence intensity of ROS in brain tissue of the PTSD group was significantly higher than that of the NC group (P < 0.05); the mean fluorescence intensity of ROS in the EA group was significantly lower than that in the PTSD group (P < 0.05). The MDA content in brain tissue of the PTSD group was significantly higher, and SOD activity and GSH content were significantly lower than those of the NC group (P < 0.05); the MDA content in the brain tissue of the EA group was significantly lower, and the SOD activity and GSH content were significantly higher than those of the PTSD group (P < 0.05). The p-JNK/JNK and the expression levels of p53 and PUMA in brain tissue of the PTSD group were significantly higher than those of the NC group (P < 0.05); the p-JNK/JNK and the expression levels of p53 and PUMA in the brain tissue of the EA group were significantly lower than those of the PTSD group (P < 0.05).
    Conclusion Acupuncture can inhibit hippocampal neuron apoptosis and relieve fear symptoms in PTSD rats, possibly through the activation of the ROS/JNK/p53 pathway.

     

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