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

doi:10.12122/j.issn.1673-4254.2025.06.13

Abstract

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

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.

Figures/Tables 10

Tab.1 Primer sequence of qRT-PCR

Gene

Forward Primer

Reverse Primer

PERK

eIF2α

CHOP

TACAGTGGACGGCGATGATG

AAAGCTACTGCTGTGCTGGT

CCTCGCTCTCCAGATTCCAG

CTGGGGTCCTCCTTACTGGA

GTCGCAATGTAGTGCAGTGT

AGCTGTGCCACTTTCCTCTC

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).

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.

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.

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.

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.

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.

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.

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

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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