大麻二酚通过BRD4/AMPK/mTOR信号通路拮抗双酚A诱导的猪肠上皮细胞凋亡和自噬

doi:10.11843/j.issn.0366-6964.2025.04.039

Abstract

Abstract:

This study aimed to explore the mechanism of cannabidiol (CBD) in alleviating the apoptosis and autophagy of pig intestinal epithelial cells (IPEC-J2) induced by bisphenol A (BPA) through BRD4/AMPK/mTOR signaling pathway. In this study, the cell viability was measured by CCK-8 method, and the half inhibitory concentration of BPA on IPEC-J2 cells and the optimal dose of CBD antagonist for BPA were screened. Using the group comparison method, The experiment was divided into blank control group, BPA group (150 μmol·L^-1 BPA), BPA+CBD group (150 μmol·L^-1 BPA+10 μmol·L^-1 CBD), BPA+CBD+CQ group (150 μmol·L^-1 BPA+10 μmol·L^-1 CBD+20 μmol·L^-1 CQ) and CBD group (10 μmol·L^-1 CBD). The apoptosis rate of IPEC-J2 cells was detected by AO/EB staining and flow cytometry. The ROS levels in cells were detected by DCFH-DA method. Oxidative stress was detected by oxidative stress kit. The expression of LC3-Ⅱ protein was detected by immunofluorescence technique. Quantitative Real-time PCR (qRT-PCR) and Western blot were used to detect the expression of genes related to apoptosis, autophagy, intestinal tight-junction protein and BRD4/AMPK/mTOR signaling pathway. The results showed that apoptosis and autophagy levels of IPEC-J2 cells were increased after 150 μmol·L^-1 BPA treatment, and decreased after 10 μmol·L^-1 CBD treatment. CBD could significantly down-regulate the increase of ROS and MDA levels caused by BPA (P < 0.05), and up-regulate the decrease of GSH-Px activity (P < 0.05); CBD significantly down-regulated the increase of expression levels of apoptosis related genes (Bax and caspase-3), autophagy related genes (P62, LC3-Ⅱ/LC3-Ⅰ and ATG5) and BRD4/AMPK/mTOR path-related genes (AMPK) induced by BPA (P < 0.05). The expression levels of LAMP1, Bcl-2, BRD4, mTOR, ZO-1 and Claudin-1 were increased (P < 0.05); Immunofluorescence results showed that CBD could significantly reduce the expression and distribution of LC3-Ⅱ in IPEC-J2 cells induced by BPA (P < 0.05). In conclusion, CBD could antagonize BPA-induced apoptosis and autophagy of IPEC-J2 cells through BRD4/AMPK/mTOR signaling pathway.

Key words: bisphenol A, cannabidiol, porcine intestinal epithelial cells, apoptosis, autophagy, BRD4/AMPK/mTOR

CLC Number:

S859.7

HOU Wanchen, XU Tong. Cannabidiol Antagonizes BPA-induced Apoptosis and Autophagy in Porcine Intestinal Epithelial Cells through the BRD4/AMPK/mTOR Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1919-1933.

Figures/Tables 8

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引物名称 Primer name	上游引物(5′→3′) Forward primer	下游引物(5′→3′) Reverse primer
Bcl-2	TCTTTGAGTTCGGTGGGGTC	CTCCACAAAGGCATCCCAGC
Bax	GGCCCTTTTGCTTCAGGGTTTC	GTCCACGGCTGCGATCAT
caspase-3	GCTGTAGAACTCTAACTGGCAAACC	CCCACTGTCCGTCTCAATCCC
Nrf2	TAAGGGTGCTCCTTTGCGAG	CGGGCTAAGTTCAGTCGCTT
Keap-1	CCATGAAGCATCGGCGAAGC	CGCTCCAGGTGTCCGTGTC
LC3	TGTCAACATGAGCGAGTTGGT	CTGCTCATAGATGTCCGCGAT
P62	AAGAACGTAGGGGAGAGTGTG	TTGCTAGGGTCGGAAGAGCA
LAMP1	CTCCACAAAGGCATCCCAGC	TCTTGCTGGCTGAAGCTGTT
BRD4	ACAGCAACACAAGCAAGAAGGA	GCTGCCGCTTTTCATCATAACTCA
AMPK	CGACAGCCGAGAAGCAGAAAC	TTTGCCAACCTTCACTTTGCC
mTOR	ACTGTGCGGATCACTTCCTG	GGTTATCCCAACCACGAGCA
β-actin	GACGATATTGCTGCGCTCGTG	TCAGGATGCCTCTCTTGCTCT

抗体 Antibodies	稀释比例 Dilution ratio	公司 Company
Bcl-2	1∶1 000	Wanleibio
Bax	1∶1 000	Wanleibio
caspase-3	1∶1 000	Wanleibio
Nrf2	1∶800	Wanleibio
Keap1	1∶1 000	Wanleibio
LC3	1∶1 000	ABclonal
P62	1∶1 000	ABclonal
ATG5	1∶1 000	Wanleibio
LAMP1	1∶1 000	Wanleibio
BRD4	1∶400	ABclonal
AMPK	1∶1 000	ABclonal
mTOR	1∶1 000	ABclonal
β-actin	1∶1 000	ABclonal

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Cannabidiol Antagonizes BPA-induced Apoptosis and Autophagy in Porcine Intestinal Epithelial Cells through the BRD4/AMPK/mTOR Signaling Pathway

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