畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (4): 1919-1933.doi: 10.11843/j.issn.0366-6964.2025.04.039
收稿日期:
2024-05-20
出版日期:
2025-04-23
发布日期:
2025-04-28
通讯作者:
徐童
E-mail:wanchenhouneau@163.com;tongxu@neau.edu.cn
作者简介:
侯宛辰(2002-), 女, 黑龙江哈尔滨人, 本科生, 主要从事药理毒理学研究, E-mail: wanchenhouneau@163.com
基金资助:
Received:
2024-05-20
Online:
2025-04-23
Published:
2025-04-28
Contact:
XU Tong
E-mail:wanchenhouneau@163.com;tongxu@neau.edu.cn
摘要:
本研究旨在探究大麻二酚(cannabidiol, CBD)通过BRD4/AMPK/mTOR信号通路缓解双酚A(bisphenol A, BPA)导致的猪肠上皮细胞(pig intestinal epithelial cells, IPEC-J2)的凋亡和自噬的作用机制。首先通过CCK-8法测定细胞活力,筛选出BPA对IPEC-J2细胞的半数抑制浓度和CBD对BPA的最适拮抗剂量。用分组比较方法,将试验分为空白对照组、BPA组(150 μmol·L-1 BPA)、BPA+CBD组(150 μmol·L-1 BPA+10 μmol·L-1 CBD)、BPA+CBD+CQ组(150 μmol·L-1 BPA+10 μmol·L-1 CBD+20 μmol·L-1 CQ)和CBD(10 μmol·L-1 CBD)组。通过AO/EB染色法以及流式细胞术检测IPEC-J2细胞的凋亡率;通过DCFH-DA法检测细胞中ROS水平;通过氧化应激试剂盒检测氧化应激水平;通过免疫荧光技术检测LC3-Ⅱ蛋白表达水平;采用实时荧光定量PCR(qRT-PCR)和蛋白质印迹法(Western blot)来检测细胞凋亡、自噬、肠紧密连接蛋白和BRD4/AMPK/mTOR信号通路相关基因的表达水平。结果表明,经150 μmol·L-1 BPA处理,IPEC-J2细胞凋亡和自噬水平均升高,经10 μmol·L-1 CBD处理后,细胞凋亡和自噬水平均降低;CBD可以显著下调由BPA引起的ROS和MDA水平升高(P < 0.05),上调BPA引起的GSH-Px活性降低(P < 0.05);CBD可以显著下调由BPA引起的细胞凋亡相关基因(Bax和caspase-3)、细胞自噬相关基因(P62、LC3-Ⅱ/LC3-Ⅰ和ATG5)和BRD4/AMPK/mTOR通路(AMPK)相关基因的表达水平升高(P < 0.05),上调BPA引起的LAMP1、Bcl-2、BRD4、mTOR、ZO-1和Claudin-1的表达水平降低(P < 0.05);免疫荧光结果表明,CBD可以显著降低由BPA所诱导的IPEC-J2细胞LC3-Ⅱ的表达与分布(P < 0.05)。综上所述,CBD可以缓解BPA导致的IPEC-J2细胞氧化应激,进而通过BRD4/AMPK/mTOR信号通路拮抗BPA诱导的IPEC-J2细胞凋亡和自噬。
中图分类号:
侯宛辰, 徐童. 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[J]. 畜牧兽医学报, 2025, 56(4): 1919-1933.
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.
表 1
引物序列"
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 |
表 2
Western blot中使用的抗体信息"
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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