表儿茶素没食子酸酯对MAC-T和小鼠乳腺炎症与细胞焦亡及NF-κB通路和NLRP3炎性小体的抑制效应

doi:10.11843/j.issn.0366-6964.2025.01.039

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (1): 430-441.doi: 10.11843/j.issn.0366-6964.2025.01.039

表儿茶素没食子酸酯对MAC-T和小鼠乳腺炎症与细胞焦亡及NF-κB通路和NLRP3炎性小体的抑制效应

王浩磊¹(), 刘梦燕¹(), 龙泉¹, 李曼曼¹, 吕祥¹, 林涛¹^,²^,*(), 蒋曹德¹^,*()

1. 西南大学动物科学技术学院重庆市高校草食动物工程研究中心, 重庆 400715
2. 乐山师范学院, 竹类病虫防控与资源开发四川省重点实验室, 乐山 641000

收稿日期:2024-01-24 出版日期:2025-01-23 发布日期:2025-01-18
通讯作者: 林涛,蒋曹德 E-mail:yipianche@qq.com;798163059@qq.com;727826660@qq.com;jcdpjx@swu.edu.cn
作者简介:王浩磊(1999-)，男，上海人，硕士生，主要从事动物营养与动物炎症治疗研究，E-mail: yipianche@qq.com
刘梦燕(1998-)，女，山西汾阳人，硕士生，主要从事动物分子遗传育种研究，E-mail: 798163059@qq.com
第一联系人：
王浩磊和刘梦燕为同等贡献作者
基金资助:
国家自然科学基金(32171673)

Inhibition of Epicatechin Gallate on Inflammation and Pyroptosis as Well as NF-κB Pathway and NLRP3 Inflammasome in MAC-T Cells and Mouse Mammary Glands

WANG Haolei¹(), LIU Mengyan¹(), LONG Quan¹, LI Manman¹, LÜ Xiang¹, LIN Tao¹^,²^,*(), JIANG Caode¹^,*()

1. Chongqing University Herbivore Engineering Research Center, College of Animal Science and Technology, Southwest University, Chongqing 400715, China
2. Sichuan Key Laboratory of Bamboo Disease Control and Resource Development, Leshan Normal University, Leshan 614000, China

Received:2024-01-24 Online:2025-01-23 Published:2025-01-18
Contact: LIN Tao, JIANG Caode E-mail:yipianche@qq.com;798163059@qq.com;727826660@qq.com;jcdpjx@swu.edu.cn

摘要/Abstract

摘要：

本研究旨在探究表儿茶素没食子酸酯(epicatechin gallate, ECG)对牛乳腺上皮细胞NF-κB(nuclear factor kappa B)炎性通路和NOD样受体3(NOD-like receptor protein 3, NLRP3)炎症小体的抑制作用。利用脂多糖(lipopolysaccharide, LPS)处理牛乳腺上皮细胞系(MAC-T)和小鼠乳腺；采用CCK-8法筛选ECG处理MAC-T细胞的最佳浓度，ELISA法检测促炎因子、氧化应激指示因子以及焦亡指示因子的表达水平；苏木精-伊红染色和Annexin V-FITC/PI法检测小鼠乳腺组织炎性变化和细胞膜破裂情况；Western blot检测NF-κB、NLRP3、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein, ASC)、caspase-1和Gasdermin NH₃ 端(GSDMD-N)的表达水平；免疫荧光检测p65核转移情况。结果发现：1)ECG在10、20、40 mg·kg^-1浓度下均可减轻LPS诱导的乳腺炎症和炎性细胞浸润；2)ECG在体内和体外均可显著且剂量依赖性地降低LPS诱导的促炎因子(IL-1β、IL-6、TNF-α)和氧化应激指示因子(COX-2、iNOS、MPO)表达水平(P＜0.05)；3)ECG剂量依赖性地降低了LPS诱导的细胞死亡率以及ROS、IL-18和LDH焦亡指示因子表达水平(P＜0.05)；4)ECG极显著地抑制LPS诱导的NLRP3、ASC、GSDMD-N、caspase-1、磷酸化p65的表达与核转移(P＜0.05)。综上表明，ECG抑制MAC-T细胞和小鼠乳腺炎症与细胞焦亡以及NF-κB通路和NLRP3炎性小体。本研究为利用ECG替代抗生素防治牛乳腺炎提供了新的数据。

关键词: 表儿茶素没食子酸酯, NF-κB, 乳腺炎, NLRP3炎性小体, 焦亡

Abstract:

The aim of this study was to investigate the inhibitory effects of epicatecin gallate (ECG) on nuclear factor κB (NF-κB) signaling pathway and NOD-like receptor 3 (NLRP3) inflammasome in bovine mammary epithelial cells. Lipopolysaccharide (LPS) was used to treat bovine mammary alveolar cells (MAC-T) and mouse mammary glands. The treatment concentration of ECG in MAC-T cell was screened by the CCK-8 method. The expression levels of proinflammatory factors, oxidative stress indicators and pyroptosis indicators were detected by ELISA. Hematoxylin-eosin staining and Annexin V-FITC/PI were used to detect the inflammatory changes in mouse mammary tissues and cell membrane rupture. The expression levels of NF-κB, NLRP3, apoptosis-associated speck-like protein (ASC), caspase-1 and Gasdermin NH₃ end (GSDMD-N) were detected by Western blot. Nuclear transfer of p65 was detected by immunofluorescence. The results showed that: 1) ECG at 10, 20 and 40 mg·kg^-1 could reduce LPS-induced inflammatory changes and inflammatory cell infiltration; 2) ECG significantly and dose-dependent decreased LPS-induced levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α) and oxidative indicators (COX-2, iNOS) in both in vivo and in vitro systems (P < 0.05); 3) ECG dose-dependently reduced of LPS-induced cell death and the levels of ROS, IL-18 and LDH (P < 0.05); 4) ECG significantly inhibited the levels of NLRP3, ASC, GSDMD-N, caspase-1 and the phosphorylation and nuclear translocation levels of p65 (P < 0.05). The results indicate that ECG inhibits MAC-T cell and mouse mammary gland inflammation and pyroptosis as well as the NF-κB pathway and NLRP3 inflammasome. This study provides a new idea for the prevention and treatment of bovine mastitis.

Key words: epicatechin gallate, NF-κB, bovine mastitis, NLRP3 inflammasome, pyroptosis

中图分类号:

S857.26

王浩磊, 刘梦燕, 龙泉, 李曼曼, 吕祥, 林涛, 蒋曹德. 表儿茶素没食子酸酯对MAC-T和小鼠乳腺炎症与细胞焦亡及NF-κB通路和NLRP3炎性小体的抑制效应[J]. 畜牧兽医学报, 2025, 56(1): 430-441.

WANG Haolei, LIU Mengyan, LONG Quan, LI Manman, LÜ Xiang, LIN Tao, JIANG Caode. Inhibition of Epicatechin Gallate on Inflammation and Pyroptosis as Well as NF-κB Pathway and NLRP3 Inflammasome in MAC-T Cells and Mouse Mammary Glands[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 430-441.

图/表 9

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表儿茶素没食子酸酯对MAC-T和小鼠乳腺炎症与细胞焦亡及NF-κB通路和NLRP3炎性小体的抑制效应

Inhibition of Epicatechin Gallate on Inflammation and Pyroptosis as Well as NF-κB Pathway and NLRP3 Inflammasome in MAC-T Cells and Mouse Mammary Glands

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