电针血清通过激活PI3K/Akt信号通路改善OGD/R模型HT22神经元突触可塑性

doi:10.12122/j.issn.1673-4254.2026.06.20

摘要/Abstract

摘要：

目的探讨电针血清对 OGD/R 损伤HT22神经元的保护作用，并初步探索 PI3K/Akt 信号通路在其中的可能参与机制。方法通过公共数据库进行生物信息学分析，筛选卒中相关差异通路；在此基础上采用线栓法建立大脑中动脉缺血再灌注（MCAO/R）大鼠模型，制备电针“神庭”“百会”穴血清及模型血清。体外培养HT22神经元并建立OGD/R模型后，分为对照组、模型组、电针血清组、PI3K抑制剂组（LY294002，20 μmol/L）及电针血清+PI3K抑制剂组。采用CCK-8检测细胞活力，TUNEL染色评估细胞凋亡，尼氏染色观察神经元结构变化；Western blotting检测PI3K、Akt总蛋白及其磷酸化水平（p-PI3K、p-Akt），并检测其下游相关蛋白p-mTOR/mTOR及抗凋亡蛋白Bcl-2的表达变化，同时检测突触可塑性相关蛋白NMDAR1、PSD-95的表达；RT-qPCR法检测PI3K、Akt、NMDAR1、PSD-95的mRNA表达水平。结果与对照组相比，模型组HT22神经元活力下降、凋亡增加、尼氏体数量减少且染色变浅（P<0.05）。电针血清干预可提高细胞存活率、降低凋亡并改善神经元形态（P<0.05）；PI3K抑制剂LY294002则进一步加重神经元损伤。与电针血清组相比，电针血清+PI3K抑制剂组的保护作用有所减弱；但与单独抑制剂组相比，其细胞状态仍得到改善（P<0.05）。在分子水平上，与对照组相比，模型组HT22神经元中p-PI3K/PI3K、p-Akt/Akt及p-mTOR/mTOR比值均降低（P<0.05），Bcl-2蛋白表达亦明显下调（P<0.05）；同时，突触可塑性相关蛋白NMDAR1和PSD-95的表达亦降低（P<0.05）。电针血清能上调这些蛋白的表达，而LY294002则呈现抑制作用。电针血清+PI3K抑制剂组的蛋白表达水平介于电针血清组与抑制剂组之间。RT-qPCR检测显示，PI3K、Akt、NMDAR1及PSD-95的mRNA表达变化趋势与其相应蛋白水平一致。结论电针“神庭”“百会”穴血清对OGD/R所致HT22神经元损伤具有一定的保护作用，其效应可能与 PI3K/Akt 信号通路的激活及其下游相关分子的参与，并伴随突触可塑性相关分子的上调有关。

关键词: 卒中后认知障碍, 电针血清, PI3K/Akt, 突触可塑性, HT22神经元

Abstract:

Objective To investigate the protective effects of sera from rats receiving electroacupuncture (EA) against oxygen and glucose deprivation/reoxygenation (OGD/R) injury in HT22 neurons and explore the involvement of the PI3K/Akt signaling pathway in this protective mechanism. Methods Bioinformatics analysis was performed using public databases to identify stroke-related pathways. A rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) were treated with EA at "Shenting" and "Baihui" to prepare EA rat serum. HT22 neurons were subjected to OGD/R and treated with MCAO rat serum, EA rat serum, LY294002, or EA rat serum combined with LY294002. The changes in cell viability, apoptosis, and neuronal morphology were assessed using CCK-8 assay, TUNEL staining, and Nissl staining. Western blotting was used to detect PI3K/Akt phosphorylation and the expressions of the downstream proteins (p-mTOR/mTOR and Bcl-2) and synaptic proteins (NMDAR1 and PSD-95); the mRNA expressions of PI3K, Akt, NMDAR1, and PSD-95 were detected using RT-qPCR. Results HT22 neurons with OGD/R injury treated with MCAO rat serum showed significantly decreased cell viability, increased apoptosis, and impaired neuronal morphology, which were obviously improved following treatment with EA rat serum but aggravated after LY294002 treatment. The protective effect of EA rat serum was significantly attenuated by application of LY294002. HT22 neurons treated with MCAO rat serum showed decreased p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR ratios with lowered expressions of Bcl-2, NMDAR1 and PSD-95, which were obviously upregulated by EA rat serum treatment but reduced after LY294002 treatment. The cells with the combined treatment showed intermediate expression levels of these molecules at both the mRNA and protein levels. Conclusion Serum from rats receiving EA at "Shenting" and "Baihui" shows a protective effect against OGD/R injury in HT22 neurons, mediated possibly by activation of the PI3K/Akt signaling pathway and its downstream effectors and upregulation of synaptic plasticity-related molecules.

Key words: post-stroke cognitive impairment, electroacupuncture serum, PI3K/Akt, synaptic plasticity, HT22 neurons

史雪珂, 陈婷玉, 高静, 吴明莉, 李瑞青, 苏凯奇, 吕转, 宋晓磊, 冯晓东. 电针血清通过激活PI3K/Akt信号通路改善OGD/R模型HT22神经元突触可塑性[J]. 南方医科大学学报, 2026, 46(6): 1407-1415.

Xueke SHI, Tingyu CHEN, Jing GAO, Mingli WU, Ruiqing LI, Kaiqi SU, Zhuan LÜ, Xiaolei SONG, Xiaodong FENG. Serum from rats with electroacupuncture activates the PI3K/Akt signaling pathway to improve synaptic plasticity in HT22 neurons with oxygen and glucose deprivation[J]. Journal of Southern Medical University, 2026, 46(6): 1407-1415.

图/表 11

表1 引物序列

Tab.1 Primer sequence for RT-qPCR

Gene name		Sequence (5' to 3')
PI3K	Forward	CTCCCCAGCTGCTGTAACTC
PI3K	Reverse	AGGTTCCACAAGTCCAGGTG
Akt	Forward	ACTCATTCCAGACCCACGAC
Akt	Reverse	CCGGTACACCACGTTCTTCT
NMDAR1	Forward	CTGCGACCCCAAGATTGTCA
NMDAR1	Reverse	TATTGGCCTGGTTTACTGCCT
PSD-95	Forward	TTGCGGCTAGAGGCATCCA
PSD-95	Reverse	GTCCCCCTGGTTTAGGAATCC

图1 KEGG富集分析

Fig.1 KEGG enrichment analysis.

图2 不同浓度LY294002对HT22神经元的抑制作用

Fig.2 Inhibitory effect of different concentrations of LY294002 on HT22 neurons (n=4).

图3 各组HT22神经元存活率

Fig.3 Cell viability of HT22 neurons in each group (n=4). *P<0.05 vs Control group; ^P<0.05 vs Model group; △P<0.05 vs EA-serum group; #P<0.05 vs LY294002 group.

图4 HT22神经元TUNEL染色结果

Fig.4 TUNEL staining results of HT22 neurons (n=3). A: TUNEL staining results of HT22 neurons in each group (Original magnification: ×100); B: Quantitative analysis of the relative TUNEL fluorescence ratio of HT22 neurons in each group. *P<0.05 vs Control group; ^P<0.05 vs Model group; △P<0.05 vs EA-serum group; #P<0.05 vs LY294002 group.

图5 各组HT22神经元尼氏染色结果

Fig.5 Nissl staining results of HT22 neurons in each group (Scale bar=50 μm).

图6 各组HT22神经元p-PI3K、p-Akt蛋白相对表达量的比较

Fig.6 Comparison of the relative expression levels of p-PI3K and p-Akt proteins in HT22 neurons in each group (n=3). A: Expressions of PI3K and Akt proteins in HT22 neurons in each group detected by Western blotting. B: Quantitative analysis of the p-PI3K/PI3K ratio. C: Quantitative analysis of the p-Akt/Akt ratio. *P<0.05 vs Control group; ^P<0.05 vs Model group; △P<0.05 vs EA-serum group; #P<0.05 vs LY294002 group.

图7 各组HT22神经元PI3K、Akt mRNA相对表达量的比较

Fig.7 Comparison of the relative mRNA expression levels of PI3K and Akt in HT22 neurons in each group (n=3). A: Quantitative analysis of PI3K mRNA relative expression by RT-qPCR. B: Quantitative analysis of Akt mRNA relative expression by RT-qPCR. *P<0.05 vs Control group; ^P<0.05 vs Model group; △P<0.05 vs EA-serum group; #P<0.05 vs LY294002 group.

图8 各组HT22神经元p-mTOR、Bcl-2蛋白相对表达量的比较

Fig.8 Comparison of relative expression levels of p-mTOR and Bcl-2 proteins in HT22 neurons in each group (n=3). A: Expression of p-mTOR and Bcl-2 proteins in HT22 neurons in each group detected by Western blotting. B: Quantitative analysis of the p-mTOR/mTOR ratio. C: Quantitative analysis of the Bcl-2/GAPDH ratio. *P<0.05 vs Control group; ^P<0.05 vs Model group; △P<0.05 vs EA-serum group; #P<0.05 vs LY294002 group.

图9 各组HT22神经元NMDAR1、PSD-95蛋白相对表达量的比较

Fig.9 Comparison of the relative expression levels of NMDAR1 and PSD-95 proteins in HT22 neurons in each group (n=3). A: The expression of NMDAR1 and PSD-95 proteins in HT22 neurons in each group detected by Western blotting. B: Quantitative analysis of the NMDAR1/GAPDH ratio. C: Quantitative analysis of the PSD-95/GAPDH ratio. *P<0.05 vs Control group; ^P<0.05 vs Model group; △P<0.05 vs EA-serum group; #P<0.05 vsLY294002 group.

图10 各组HT22神经元NMDAR1、PSD-95 mRNA相对表达量的比较

Fig.10 Comparison of relative mRNA expression levels of NMDAR1 and PSD-95 in HT22 neurons in each group (n=3). A: Quantitative analysis of NMDAR1 mRNA relative expression by RT-qPCR. B: Quantitative analysis of PSD-95 mRNA relative expression by RT-qPCR. *P<0.05 vs Control group; ^P<0.05 vs Model group; △P<0.05 vs EA-serum group; #P<0.05 vs LY294002 group.

参考文献 30

[1]	Lim JS, Lee JJ, Woo CW. Post-stroke cognitive impairment: pathophysiological insights into brain disconnectome from advanced neuroimaging analysis techniques[J]. J Stroke, 2021, 23(3): 297-311. doi：10.5853/JOS.2021.02376
[2]	El Husseini N, Katzan IL, Rost NS, et al. Cognitive impairment after ischemic and hemorrhagic stroke: a scientific statement from the American heart association/American stroke association[J]. Stroke, 2023, 54(6): 272-91. doi：10.1161/str.0000000000000430
[3]	He AN, Wang Z, Wu X, et al. Incidence of post-stroke cognitive impairment in patients with first-ever ischemic stroke: a multicenter cross-sectional study in China[J]. Lancet Reg Health West Pac, 2023, 33: 100687. doi：10.1016/j.lanwpc.2023.100687
[4]	Li ZX, Wang HY, Xiao GX, et al. Recovery of post-stroke cognitive and motor deficiencies by Shuxuening injection via regulating hippocampal BDNF-mediated Neurotrophin/Trk Signaling[J]. Biomed Pharmacother, 2021, 141: 111828. doi：10.1016/j.biopha.2021.111828
[5]	Magee JC, Grienberger C. Synaptic plasticity forms and functions[J]. Annu Rev Neurosci, 2020, 43: 95-117. doi：10.1146/annurev-neuro-090919-022842
[6]	Dai XM, Lin AL, Zhuang LP, et al. Targeting SIK3 to modulate hippocampal synaptic plasticity and cognitive function by regulating the transcription of HDAC4 in a mouse model of Alzheimer's disease[J]. Neuropsychopharmacology, 2024, 49(6): 942-52. doi：10.1038/s41386-023-01775-1
[7]	Weaver NA, Kuijf HJ, Aben HP, et al. Strategic infarct locations for post-stroke cognitive impairment: a pooled analysis of individual patient data from 12 acute ischaemic stroke cohorts[J]. Lancet Neurol, 2021, 20(6): 448-59. doi：10.1016/S1474-4422(21)00060-0
[8]	Sánchez-Castillo C, Cuartero MI, Fernández-Rodrigo A, et al. Functional specialization of different PI3K isoforms for the control of neuronal architecture, synaptic plasticity, and cognition[J]. Sci Adv, 2022, 8(47): eabq8109. doi：10.1126/sciadv.abq8109
[9]	Qin ZS, Zheng Y, Zhou XD, et al. Shexiang Baoxin pill, a proprietary multi-constituent Chinese medicine, prevents locomotor and cognitive impairment caused by brain ischemia and reperfusion injury in rats: a potential therapy for neuropsychiatric sequelae of stroke[J]. Front Pharmacol, 2021, 12: 665456. doi：10.3389/fphar.2021.665456
[10]	Cao H, Zuo CC, Gu ZY, et al. High frequency repetitive transcranial magnetic stimulation alleviates cognitive deficits in 3xTg-AD mice by modulating the PI3K/Akt/GLT-1 axis[J]. Redox Biol, 2022, 54: 102354. doi：10.1016/j.redox.2022.102354
[11]	Choi J, Kim D, Jeong H, et al. A novel miR-4536-3p inhibition ameliorates Alzheimer's disease by reducing Aβ accumulation and tau phosphorylation[J]. Alzheimers Res Ther, 2025, 17(1): 189. doi：10.1186/s13195-025-01834-3
[12]	Hu GT, Zhou CH, Wang J, et al. Electroacupuncture treatment ameliorates depressive-like behavior and cognitive dysfunction via CB1R dependent mitochondria biogenesis after experimental global cerebral ischemic stroke[J]. Front Cell Neurosci, 2023, 17: 1135227. doi：10.3389/fncel.2023.1135227
[13]	孙可心, 白艳杰, 王岩, 等. 基于缺氧诱导因子-1α/丙酮酸脱氢酶激酶1通路探讨通督醒神针刺法调控糖酵解改善脑卒中后认知障碍的作用机制[J/OL]. 针刺研究, 2025, 50(12): 1387-95.
[14]	张慧, 杨帆, 龙学平, 等. 针刺血清对无镁培养的新生大鼠海马神经元微管相关蛋白2和生长相关蛋白43表达的影响[J/OL]. 针刺研究, 2021, 46(12): 1029-35, 1066.
[15]	苏凯奇, 吕转, 吴明莉, 等. 电针对缺血再灌注后学习记忆障碍大鼠 BDNF/TrkB/PI3K/akt通路的影响及对海马神经元保护作用研究[J]. 中国全科医学, 2023, 26(33): 4187-93.
[16]	谢亚威, 赵永辰, 李轶璠, 等. 针灸干预卒中后认知障碍的作用机制研究进展[J/OL]. 中华中医药杂志, 2024, 39(7): 3571-4.
[17]	苏凯奇, 吕转, 吴明莉, 等. 电针后留针联合认知训练治疗脑卒中后认知功能障碍：多中心随机对照试验[J]. 中国针灸, 2023, 43(11): 1221-5.
[18]	林如辉, 夏金言, 马小涵, 等. 电针通过促进突触再生改善脑缺血再灌注损伤大鼠的学习记忆功能[J]. 南方医科大学学报, 2024, 44(12): 2317-26.
[19]	吕转, 陈玉龙, 王亚敏, 等. 通督醒神针刺调节肠道微生物改善缺血性脑卒中后认知障碍的临床机制研究[J]. 世界科学技术-中医药现代化, 2025, 27(2): 545-55.
[20]	Lin BB, Wang MX, Chen XC, et al. Involvement of P2X7R-mediated microglia polarization and neuroinflammation in the response to electroacupuncture on post-stroke memory impairment[J]. Brain Res Bull, 2024, 212: 110967. doi：10.1016/j.brainresbull.2024.110967
[21]	张浛芮, 吕鹤群, 曾春利, 等. 基于lncRNA H19的m6A甲基化调控S1PR2/TLR4/NLRP3 通路探讨电针血清对缺血性脑卒中后血脑屏障的作用机制[J/OL]. 世界科学技术-中医药现代化, 2024, 26(10): 2716-25.
[22]	陈露露, 阮晓迪, 高静, 等. 电针血清对氧糖剥夺/复氧糖后HT22神经元铁死亡的影响[J]. 海南医学院学报, 2025, 31(24): 1851-61.
[23]	Torres-López C, Cuartero MI, García-Culebras A, et al. Ipsilesional hippocampal GABA is elevated and correlates with cognitive impairment and maladaptive neurogenesis after cortical stroke in mice[J]. Stroke, 2023, 54(10): 2652-65. doi：10.1161/strokeaha.123.043516
[24]	Zhang LJ, Cui HZ, Hu WD, et al. Targeting MAD2B as a strategy for ischemic stroke therapy[J]. J Adv Res, 2025, 72: 369-85. doi：10.1016/j.jare.2024.07.003
[25]	袁宏伟, 刘云霞, 张含, 等. “通督醒神”法针灸联合认知训练治疗卒中后轻度认知障碍：随机对照试验[J]. 中国针灸, 2022, 42(8): 839-43. doi：10.13703/j.0255-2930.20210811-0005
[26]	Lang JW, Luo JC, Lang JC, et al. Electroacupuncture alleviated post-stroke cognitive impairment via the mTOR/NLRP3-mediated autophagy-inflammatory pathway[J]. Eur J Med Res, 2024, 29(1): 532. doi：10.1186/s40001-024-02131-9
[27]	Yu J, Li JL, Matei N, et al. Intranasal administration of recombinant prosaposin attenuates neuronal apoptosis through GPR37/PI3K/Akt/ASK1 pathway in MCAO rats[J]. Exp Neurol, 2024, 373: 114656. doi：10.1016/j.expneurol.2023.114656
[28]	Ugalde-Triviño L, Díaz-Guerra M. PSD-95: an effective target for stroke therapy using neuroprotective peptides[J]. Int J Mol Sci, 2021, 22(22): 12585. doi：10.3390/ijms222212585
[29]	于云红, 谢炜, 李慧. 柴胡疏肝汤通过上调ASIC1蛋白调节NMDAR通道改善大鼠癫痫后的认知障碍[J]. 南方医科大学学报, 2025, 45(7): 1506-12.
[30]	Wright WJ, Hedrick NG, Komiyama T. Distinct synaptic plasticity rules operate across dendritic compartments in vivo during learning[J]. Science, 2025, 388(6744): 322-8. doi：10.1126/science.ads4706