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
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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。
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关键词:
- 针刺 /
- 创伤后应激障碍 /
- 海马神经元凋亡 /
- 活性氧/c-Jun氨基末端激酶/p53通路 /
- 氧化应激指标
Abstract:ObjectiveTo 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.
MethodsThirty 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 index[malondialdehyde (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.
ResultsThe 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).
ConclusionAcupuncture 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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图 1 各组大鼠神经元凋亡率比较
A: TUNEL检测神经元凋亡率; B: 各组大鼠神经元凋亡率比较。与NC组比较, *P < 0.05; 与PTSD组比较, #P < 0.05。
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图 1 各组大鼠神经元凋亡率比较
A: TUNEL检测神经元凋亡率; B: 各组大鼠神经元凋亡率比较。与NC组比较, *P < 0.05; 与PTSD组比较, #P < 0.05。
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图 2 各组大鼠脑组织中ROS平均荧光强度比较
A: DHE检测脑组织中ROS荧光强度; B: 各组大鼠脑组织中ROS平均荧光强度比较。与NC组比较, *P < 0.05; 与PTSD组比较, #P < 0.05。
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图 2 各组大鼠脑组织中ROS平均荧光强度比较
A: DHE检测脑组织中ROS荧光强度; B: 各组大鼠脑组织中ROS平均荧光强度比较。与NC组比较, *P < 0.05; 与PTSD组比较, #P < 0.05。
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图 3 各组大鼠脑组织中MDA、SOD、GSH水平比较
A: 脑组织中MDA含量比较; B: 脑组织中SOD活性比较; C: 脑组织中GSH含量比较。与NC组比较, *P < 0.05; 与PTSD组比较, #P < 0.05。
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图 3 各组大鼠脑组织中MDA、SOD、GSH水平比较
A: 脑组织中MDA含量比较; B: 脑组织中SOD活性比较; C: 脑组织中GSH含量比较。与NC组比较, *P < 0.05; 与PTSD组比较, #P < 0.05。
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图 4 各组大鼠脑组织中JNK、p-JNK、p53、PUMA蛋白表达比较
A: 脑组织中JNK、p-JNK、p53、PUMA蛋白条带图; B: 脑组织中p-JNK/JNK比较; C、D: 脑组织中p53、PUMA蛋白表达比较。与NC组比较, *P < 0.05; 与PTSD组比较，# P < 0.05。
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图 4 各组大鼠脑组织中JNK、p-JNK、p53、PUMA蛋白表达比较
A: 脑组织中JNK、p-JNK、p53、PUMA蛋白条带图; B: 脑组织中p-JNK/JNK比较; C、D: 脑组织中p53、PUMA蛋白表达比较。与NC组比较, *P < 0.05; 与PTSD组比较，# P < 0.05。
表 1 各组大鼠运动总距离、直立总次数及进入中央格次数比较(x±s)
组别 n 运动总距离/mm 直立总次数/次 进入中央格次数/次 NC组 10 15 678.32±815.46 23.46±2.62 7.35±0.82 PTSD组 10 3 081.47±392.68* 2.87±0.36* 1.42±0.17* EA组 10 9 532.61±620.86*# 14.92±1.75*# 5.16±0.62*# 与NC组比较, *P < 0.05; 与PTSD组比较, #P < 0.05。 
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表 1 各组大鼠运动总距离、直立总次数及进入中央格次数比较(x±s)
组别 n 运动总距离/mm 直立总次数/次 进入中央格次数/次 NC组 10 15 678.32±815.46 23.46±2.62 7.35±0.82 PTSD组 10 3 081.47±392.68* 2.87±0.36* 1.42±0.17* EA组 10 9 532.61±620.86*# 14.92±1.75*# 5.16±0.62*# 与NC组比较, *P < 0.05; 与PTSD组比较, #P < 0.05。
下载: 导出CSV
表 2 各组大鼠进入开放臂次数和时间的百分比及焦虑指数比较(x±s)
组别 n 进入开放臂次数百分比/% 进入开放臂时间百分比/% 焦虑指数 NC组 10 35.16±4.08 37.12±4.05 65.38±6.89 PTSD组 10 12.35±1.37* 6.07±0.76* 94.62±10.31* EA组 10 31.62±3.95*# 29.32±3.28*# 77.19±8.05*# 与NC组比较, *P < 0.05; 与PTSD组比较, #P < 0.05。
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表 2 各组大鼠进入开放臂次数和时间的百分比及焦虑指数比较(x±s)
组别 n 进入开放臂次数百分比/% 进入开放臂时间百分比/% 焦虑指数 NC组 10 35.16±4.08 37.12±4.05 65.38±6.89 PTSD组 10 12.35±1.37* 6.07±0.76* 94.62±10.31* EA组 10 31.62±3.95*# 29.32±3.28*# 77.19±8.05*# 与NC组比较, *P < 0.05; 与PTSD组比较, #P < 0.05。
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