电针通过Bcl-2/Bax/caspase-3信号通路修复海马线粒体损伤改善创伤后应激障碍大鼠的焦虑症状

doi:10.12122/j.issn.1673-4254.2025.11.10

摘要/Abstract

摘要：

目的观察电针治疗对创伤后应激障碍（PTSD）大鼠海马线粒体超微结构及Bcl-2、Bax、caspase-3基因和蛋白表达的影响，探讨其治疗作用及机制。方法 40只SPF级SD雄性大鼠随机分为空白对照组、模型组、假针刺组、帕罗西汀组、电针组，8只/组。采用连续单一应激联合足底电刺激建立PTSD模型，空白组和模型组不予以治疗，电针组针刺“百会”“神庭”及双侧“肝俞”“肾俞”，假针刺组大鼠浅刺腧穴旁开5 mm 处，不连接电针仪，15 min/次，5次/周，持续3周。帕罗西汀组给予帕罗西汀混悬液（10 mg/kg）灌胃，5次/周，持续3周。治疗后通过旷场实验和高架十字迷宫实验评估行为学变化，HE染色和尼式染色观察海马病理及神经细胞变化，电镜观察线粒体超微结构，实时荧光定量PCR和免疫荧光检测Bcl-2、Bax、caspase-3的mRNA及蛋白表达。结果与空白组相比，模型组大鼠旷场活动总距离、高架十字迷宫开臂区域活动路程及时间明显减少（P<0.05）；海马神经细胞数量减少，细胞皱缩、空泡增多、血管周围水肿，线粒体肿胀、膜破损、嵴减少；海马神经细胞数量明显减少（P<0.0001）；Bcl-2表达下降，Bax、caspase-3表达上升（P<0.0001）；与模型组相比，帕罗西汀组和电针组旷场活动总距离、高架十字迷宫开臂区域路程明显增加（P<0.01）；尼氏染色神经细胞数量增加（P<0.05）；海马神经元皱缩减少，细胞形态更规则、排列更整齐，且神经元结构染色较均匀；线粒体结构较为完整；Bcl-2表达上升，Bax、caspase-3表达下降（P<0.001）；帕罗西汀组高架十字迷宫开臂区域活动时间增加，电针组仅呈上升趋势（P>0.05）；假针刺组无明显改善。结论电针可能通过上调Bcl-2、下调Bax和caspase-3，改善海马线粒体损伤，发挥对PTSD大鼠的治疗作用。

关键词: 创伤后应激障碍, 电针, 线粒体, 神经细胞, caspase-3, 焦虑障碍

Abstract:

Objective To investigate the mechanism underlying the therapeutic effect of electroacupuncture (EA) on post-traumatic stress disorder (PTSD) in rats. Methods Forty male SD rats were randomized equally into blank control group, PTSD model group, sham-acupuncture group, paroxetine group, and EA group. In the latter 3 groups, the rat models of PTSD, induced by continuous single-prolonged stress and plantar electrical stimulation, were treated with EA at GV20, GV24, BL18 and BL23 acupoints for 15 min (5 times a week for 3 weeks), sham-acupuncture without electrical stimulation, or gavage with paroxetine suspension on the same schedule. Behavioral changes of the rats were evaluated using open field test (OFT) and elevated plus maze (EPM) test. Hippocampal pathologies and neuronal changes were examined with HE and Nissl staining, and mitochondrial ultrastructure was examined using electron microscopy. The mRNA and protein expression levels of Bcl-2, Bax, and caspase-3 were detected by RT-qPCR and immunofluorescence staining. Results The rat models of PTSD showed significantly reduced total distance traveled in OFT and distance and time spent in the open arms of the EPM, with decreased hippocampal neurons, obvious neuronal and mitochondrial pathologies, decreased hippocampal expression of Bcl-2, and increased Bax and caspase-3 expressions. Treatments with paroxetine and EA both significantly improved behavioral changes of the rat models, increased the number of Nissl-stained neurons, obviously alleviated pathologies in the hippocampal neurons and mitochondrial ultrastructure, increased hippocampal Bcl-2 expression, and lowered caspase-3 expressions. Paroxetine showed significantly better effect than EA for improving performance of the rats in EPM test, whereas sham-acupuncture did not produce any significant improvement. Conclusion EA alleviates PTSD in rats possibly by upregulating Bcl-2 and downregulating Bax and caspase-3, thereby ameliorating hippocampal mitochondrial damage.

Key words: post-traumatic stress disorder, electroacupuncture, mitochondria, neurons, caspase-3, anxiety disorder

马丹丹, 程洁, 张虹, 刘广, 宋凯. 电针通过Bcl-2/Bax/caspase-3信号通路修复海马线粒体损伤改善创伤后应激障碍大鼠的焦虑症状[J]. 南方医科大学学报, 2025, 45(11): 2375-2384.

Dandan MA, Jie CHENG, Hong ZHANG, Guang LIU, Kai² SONG. Electroacupuncture improves post-traumatic stress disorder in rats by alleviating hippocampal mitochondrial injury via regulating Bcl-2/Bax/caspase-3 signaling[J]. Journal of Southern Medical University, 2025, 45(11): 2375-2384.

图/表 8

表1 引物序列、产物长度

Tab.1 Primer sequences for RT-qPCR and product lengths

Target gene	Primer sequences	Product Length (bp)
Bcl-2	CTGGTGGACAACATCGCTCT(F) GCATGCTGGGGCCATATAGT(R)	115
Bax	CAACATGGAGCTGCAGAGGA(F) GGAAAGGAGGCCATCCCAG (R)	274
Caspase-3	GAGCTTGGAACGCGAAGAAA(F) TTGCGAGCTGACATTCCAGT (R)	221
GAPDH	CCTCGTCCCGTAGACAAAATG (F) TGAGGTCAATGAAGGGGTCGT (R)	133

图1 各组大鼠焦虑行为学比较

Fig.1 Anxiety-like behaviors of the rats in the 5 groups (Mean±SD, n=8). A: Motion trajectory in open field test. B: Statistics of total distance traveled. ****P<0.0001 vs Control group; ####P<0.0001 vs Model group.

图2 各组大鼠焦虑行为学比较

Fig.2 Anxiety-like behaviors of the rats in the 5 in elevated plus maze test (Mean±SD, n=8). A: Representative movement trajectories in the elevated plus maze test. B: Statistics of open arm total distance. C: Open arm total time. *P<0.05, ****P<0.0001 vs Control group; ##P<0.01, ####P<0.0001 vs Model group.

图3 各组大鼠海马神经元病理形态特征比较

Fig.3 Pathological and morphological characteristics of hippocampal neurons of the rats in the 5 rats (HE staining, scale bar=20 μm). Yellow arrows: Normal neurons; Black arrows: Shrunken neurons; Green arrows: Vacuoles; Red arrows: Perivascular edema.

图4 各组大鼠海马区尼式染色结果

Fig.4 Comparison of neuronal structure in the hippocampal region of the rats among the 5 groups (Nissl staining, scale bar=50 μm). A: Representative photomicrographs.B: Neuronal cell count (n=3).****P<0.0001 vs Control group; #P<0.05 vs Model group.

图5 各组大鼠海马神经元线粒体形态比较

Fig.5 Comparison of mitochondrial morphology in the hippocampal neurons among the 5 groups (scale bar=500 nm). Red arrows indicate mitochondria.

图6 各组大鼠海马Bcl-2、Bax、Caspase-3 mRNA表达情况

Fig.6 Expression levels of Bcl-2 (A), Bax (B), and caspase-3 (C) mRNA in the hippocampus of the rats in the 5 groups (Mean±SD, n=3). ****P<0.0001 vs Control group; ###P<0.001, ####P<0.0001 vs Model group.

图7 各组大鼠海马区Bcl-2、Bax、caspase-3平均荧光强度表达情况

Fig.7 Mean fluorescence intensity of Bcl-2, Bax, and caspase-3 in the hippocampal region of the rats in the 5 group (Original magnification:×400). A: Hippocampal Bcl-2 fluorescence intensity. B: Hippocampal Bax fluorescence intensity. C: Hippocampal caspase-3 fluorescence intensity. D: Quantitative analysis of hippocampal Bcl-2 fluorescence intensity. E: Quantitative analysis of hippocampal Bax fluorescence intensity. F: Quantitative analysis of hippocampal caspase-3 fluorescence intensity. ***P<0.001 vs Control group; #P<0.05, ##P<0.01 vs Model group.

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