大麻二酚经PERK-eIF2α-ATF4-CHOP通路减轻多重脑震荡大鼠的神经元内质网应激和凋亡

doi:10.12122/j.issn.1673-4254.2025.06.13

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (6): 1240-1250.doi: 10.12122/j.issn.1673-4254.2025.06.13

大麻二酚经PERK-eIF2α-ATF4-CHOP通路减轻多重脑震荡大鼠的神经元内质网应激和凋亡

杨毓甲¹(), 杨丽芳¹^,²(), 吴雅玲¹, 段兆达¹, 于春泽¹, 吴春云¹, 于建云³(), 杨力¹()

^1.昆明医科大学，基础医学院人体解剖与组织胚胎学系，云南昆明 650500
^2.昆明医科大学，法医学院，云南昆明 650500
^2.朔州市妇幼保健院超声科，山西朔州 036000

收稿日期:2025-01-03 出版日期:2025-06-20 发布日期:2025-06-27
通讯作者: 于建云,杨力 E-mail:yujiayang1172@163.com;sophiay0717@163.com;jianyunyu@sina.com;yanglikm@163.com
作者简介:杨毓甲，在读硕士研究生，E-mail: yujiayang1172@163.com
杨丽芳，硕士，住院医师，E-mail: sophiay0717@163.com
第一联系人：杨毓甲、杨丽芳共同为第一作者
基金资助:
国家自然科学基金(81960223);云南省昆明医科大学联合专项(202301AY070001-163);云南省教育厅科学研究基金研究生项目(2024Y206)

Cannabidiol inhibits neuronal endoplasmic reticulum stress and apoptosis in rats with multiple concussions by regulating the PERK-eIF2α-ATF4-CHOP pathway

Yujia YANG¹(), Lifang YANG¹^,²(), Yaling WU¹, Zhaoda DUAN¹, Chunze YU¹, Chunyun WU¹, Jianyun YU³(), Li YANG¹()

^1.Department of Human Anatomy and Histology & Embryology, Faculty of Basic Medical Sciences, Shuozhou Maternal and Child Health Hospital, Shuozhou 036000, China
^2.Faculty of Forensic Medicine, Kunming Medical University, Kunming 650500, China, Shuozhou Maternal and Child Health Hospital, Shuozhou 036000, China
^3.Department of Ultrasound, Shuozhou Maternal and Child Health Hospital, Shuozhou 036000, China

Received:2025-01-03 Online:2025-06-20 Published:2025-06-27
Contact: Jianyun YU, Li YANG E-mail:yujiayang1172@163.com;sophiay0717@163.com;jianyunyu@sina.com;yanglikm@163.com
Supported by:
National Natural Science Foundation of China(81960223)

摘要/Abstract

摘要：

目的探究大麻二酚（CBD）对多重脑震荡后神经元内质网应激及其介导的神经元凋亡的作用。方法将SD大鼠分为假手术组（sham组）、多重脑震荡组（MCC组）、多重脑震荡溶剂组（MCC+TW组，1% tween20）、低剂量CBD-10组（10 mg/kg）和高剂量CBD-40组（40 mg/kg），用金属单摆打击装置制成大鼠MCC模型，给药各组于造模成功后连续腹腔给药2周。采用qRT-PCR、Western blotting和免疫荧光染色检测大鼠脑组织中PERK、eIF2α、ATF4、CHOP、TRIB3、p-AKT、Pro-caspase-3的表达变化。通过网络药理学筛选CBD治疗创伤性脑损伤的核心靶点，经Autodock可视化分析CBD与内质网应激和凋亡相关因子的分子对接情况。结果 MCC后大脑皮质内质网应激因子PERK、eIF2α、CHOP mRNA表达水平升高（P<0.05）。MCC组大脑皮质中PERK、eIF2α、ATF4、CHOP、TRIB3、p-AKT、Pro-caspase-3蛋白表达水平较sham组升高（P<0.05）；CBD治疗后，p-AKT表达水平进一步升高（P<0.05），而其余因子表达水平均降低（P<0.05），且CBD-40组降低更显著。网络药理学分析显示，CBD治疗TBI的核心靶点与内质网应激和脑损伤因子存在蛋白互作关系；分子对接显示CBD，与多个内质网应激和凋亡因子具有较高的结合能。结论大鼠多重脑震荡诱发神经元内质网应激和细胞凋亡，CBD可通过抑制内质网应激和抗凋亡发挥神经保护作用，且高剂量CBD的保护作用更明显。

关键词: 多重脑震荡, 大麻二酚, 内质网应激, 神经元凋亡, PERK通路

Abstract:

Objective To explore the effects of cannabidiol on endoplasmic reticulum stress and neuronal apoptosis in rats with multiple concussions (MCC). Methods SD rats were randomized into sham group, MCC group, 1% tween20 (TW) treatment group, and low-dose (10 mg/kg) and high-dose (40 mg/kg) cannabidiol treatment groups. In all but the sham group, MCC models were established using a metal pendulum percussion device, after which the rats received daily intraperitoneal injections of the corresponding agents for 2 weeks. The expressions of PERK, eIF2α, ATF4, CHOP, TRIB3, p-Akt and pro-caspase-3 in the brain tissue of the rats were detected with qRT-PCR, Western blotting and immunofluorescence staining. The core targets of cannabidiol in treatment of traumatic brain injury (TBI) were identified by network pharmacology analysis, and molecular docking was carried out to simulate the interaction of cannabidiol with the factors related to endoplasmic reticulum stress and apoptosis. Results Compared with the sham-operated rats, the rat models of MCC showed significantly increased mRNA expressions of PERK, eIF2α and CHOP and protein expressions of PERK, eIF2α, ATF4, CHOP, TRIB3, p-AKT and pro-caspase-3 in the cerebral cortex. CBD treatment, especially at the high dose, obviously increased the expression of p-Akt and lowered the expression levels of the other factors tested in the rat models. Network pharmacology analysis indicated interactions of the core targets of CBD with the factors related to endoplasmic reticulum stress and TBI, and molecular docking study showed a high binding energy of CBD with multiple factors pertaining to endoplasmic reticulum stress and apoptosis. Conclusion MCC induce endoplasmic reticulum stress and apoptosis in rat brain tissues, for which CBD, especially at a high dose, provides neuroprotective effects by inhibiting endoplasmic reticulum stress and cell apoptosis.

Key words: multiple cerebral concussions, cannabidiol, endoplasmic reticulum stress, neuronal apoptosis, PERK signaling pathway

杨毓甲, 杨丽芳, 吴雅玲, 段兆达, 于春泽, 吴春云, 于建云, 杨力. 大麻二酚经PERK-eIF2α-ATF4-CHOP通路减轻多重脑震荡大鼠的神经元内质网应激和凋亡[J]. 南方医科大学学报, 2025, 45(6): 1240-1250.

Yujia YANG, Lifang YANG, Yaling WU, Zhaoda DUAN, Chunze YU, Chunyun WU, Jianyun YU, Li YANG. Cannabidiol inhibits neuronal endoplasmic reticulum stress and apoptosis in rats with multiple concussions by regulating the PERK-eIF2α-ATF4-CHOP pathway[J]. Journal of Southern Medical University, 2025, 45(6): 1240-1250.

图/表 10

表1 qRT-PCR引物序列

Tab.1 Primer sequence of qRT-PCR

Gene

Forward Primer

Reverse Primer

PERK

eIF2α

CHOP

TACAGTGGACGGCGATGATG

AAAGCTACTGCTGTGCTGGT

CCTCGCTCTCCAGATTCCAG

CTGGGGTCCTCCTTACTGGA

GTCGCAATGTAGTGCAGTGT

AGCTGTGCCACTTTCCTCTC

图1 MCC后大鼠大脑皮质PERK、eIF2α和CHOP mRNA的变化

Fig.1 Changes of PERK (A), eIF2α (B) and CHOP (C) mRNA expressions levels in rat cerebral cortex after multiple concussion (MCC). *P<0.05, **P<0.01 (n=3).

图2 MCC后大鼠脑皮质中PERK、eIF2α、CHOP、TRIB3和ATF4的蛋白表达

Fig.2 Protein expressions of PERK, eIF2α, CHOP, TRIB3 and ATF4 in the cerebral cortex of rats after MCC. A, F: Western blotting for detecting the taget proteins. B, C, D, E, G: Quantitative analysis of the expression levels of PERK, eIF2α, CHOP, TRIB3 and ATF4. *P<0.05, **P<0.01.

图3 MCC后大鼠脑皮质中PERK、eIF2α、ATF4、CHOP和TRIB3免疫荧光染色

Fig.3 Immunofluorescence staining of PERK, eIF2α, ATF4, CHOP and TRIB3 in the cerebral cortex of rats after MCC. A, C, E, G, I: Immunofluorescence staining images of PERK, eIF2α, ATF4, CHOP and TRIB3, respectively (Original magnification: ×400). B, D, F, H, J: Quantitative analysis of PERK, eIF2α, ATF4, CHOP and TRIB3 expressions, respectively; *P<0.05, **P<0.01.

图4 MCC后大鼠脑皮质中Pro-caspase-3表达变化

Fig.4 Expression of pro-caspase-3 in rat brain after MCC. A, B: Western blotting for analyzing pro-caspase-3 expression levels. C, D: Immunofluorescence double-label staining for detecting Pro-caspase-3 expression levels (scale bar=20 µm). *P<0.05, **P<0.01.

图5 MCC后大鼠脑皮质中p-AKT的表达变化

Fig.5 Expression of p-AKT in rat brain after MCC. A, B: Expression of p-AKT detected by Western blotting. C, D: Expression of p-AKT detected by immunofluorescence double-label staining (scale bar=20 µm). *P<0.05, **P<0.01.

图6 CBD治疗TBI网络药理学分析韦恩图和蛋白互作图

Fig.6 Venn map of network pharmacology analysis of CBD for treatment of traumatic brain injury (TBI) and the protein-protein interaction network. A: Venn diagram of CBD targets and TBI disease targets. B: Visualization of interactions between CBD and TBI intersection target proteins. C: Protein-protein interactions of the core targets of cannabidiol in treatment of TBI diseases with PERK, eIF2α, ATF4, CHOP, TRIB3, AKT, and caspase-3.

图7 CBD调节TBI潜在靶点GO和KEGG富集分析

Fig.7 GO and KEGG pathway enrichment analyses of the potential targets of CBD in modulating TBI. A: GO enrichment analysis bubble chart. B: KEGG enrichment analysis bar chart.

表2 CBD 与内质网应激和凋亡相关蛋白分子对接结合能

Tab.2 Moleculare docking binding energy of CBD with endoplasmic reticulum stress- and apoptosis-related proteins

AKT

PERK

CHOP

eIF2α

caspase-3

TRIB3

ATF4

3oiw

4g31

2zfy

4v0x

3gjt

3gj0

4ut3

CBD

-7.1

-6.91

-6.68

-6.2

-5.38

-4.68

-4.46

图8 CBD 与ERS和凋亡相关蛋白分子对接

Fig.8 Molecular docking of CBD and endoplasmic reticulum stress- and apoptosis-related proteins AKT (A), PERK (B), CHOP (C), eIF2α (D), caspase-3 (E), TRIB3 (F) and ATF4 (G).

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大麻二酚经PERK-eIF2α-ATF4-CHOP通路减轻多重脑震荡大鼠的神经元内质网应激和凋亡

Cannabidiol inhibits neuronal endoplasmic reticulum stress and apoptosis in rats with multiple concussions by regulating the PERK-eIF2α-ATF4-CHOP pathway

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