姜黄素影响铁死亡途径缓解LPS诱导牛乳腺上皮细胞炎性反应的分析

doi:10.11843/j.issn.0366-6964.2025.09.049

摘要/Abstract

摘要： 本研究旨在从铁死亡途径探究姜黄素(curcumin, Cur)对脂多糖(LPS)诱导的牛乳腺上皮细胞(MAC-T)炎性反应的影响。将处于对数生长期的MAC-T细胞按照试验要求随机分组并进行相应的处理，每组3个重复。采用MTT法测定不同浓度的LPS/Cur对MAC-T细胞存活率的影响；RT-PCR法检测炎症相关基因(TNF-α、IL-6、IL-1β、iNOS、CXCL2、CCL2)与铁死亡相关基因(PTGS2、SLC7A11、GPX4、FTH1、ACSL4)的转录水平；BODIPY 581/591 C11染色检测细胞脂质过氧化水平；试剂盒检测细胞中MDA含量、LPO浓度、GSH含量、GSH/GSSG比值；Ferro-Orange染色和Fe²⁺检测试剂盒测定细胞中Fe²⁺含量；ELISA试剂盒检测细胞中TNF-α、IL-6、IL-1β浓度；Western blot检测GPX4、ACSL4、TNF-α、IL-6、IL-1β蛋白的相对表达水平。结果显示，与CON组相比，10 μg·mL^-1 LPS或20 μmol·L^-1 Cur处理对MAC-T细胞存活率无显著影响。LPS处理后细胞中促炎相关基因(TNF-α、IL-6、IL-1β、iNOS、CXCL2、CCL2)、促铁死亡相关基因(PTGS2、ACSL4)以及相关蛋白(ACSL4)的表达水平极显著升高(P<0.01)；抗铁死亡相关基因(SLC7A11、GPX4、FTH1)和蛋白(GPX4)的表达水平极显著下降(P<0.01)；MDA含量和LPO浓度均极显著升高(P<0.01)；GSH含量和GSH/GSSG比值均极显著下降(P<0.01)；Fe²⁺含量极显著升高(P<0.01)；TNF-α、IL-6、IL-1β的浓度和蛋白的表达水平均极显著升高(P<0.01)。与LPS单独处理组相比，LPS与Cur-H共处理组促炎相关基因、促铁死亡相关基因以及相关蛋白的表达水平极显著下降(P<0.01)；抗铁死亡相关基因和蛋白的表达水平极显著升高(P<0.01)；脂质过氧化水平、MDA含量和LPO浓度均极显著下降(P<0.01)；GSH含量和GSH/GSSG比值均极显著升高(P<0.01)；Fe²⁺含量极显著下降(P<0.01)；TNF-α、IL-6、IL-1β的浓度和蛋白的表达水平均极显著下降(P<0.01)。此外，铁死亡激活剂Erastin显著抑制了Cur对LPS诱导炎症的缓解作用。综上所述，Cur可通过抑制铁死亡缓解LPS诱导的MAC-T细胞炎性反应。

关键词: 铁死亡, 姜黄素, 脂多糖, 牛乳腺上皮细胞(MAC-T)

Abstract: This study aimed to explore the effects of curcumin (Curcumin, Cur) on the inflammatory response of lipopolysaccharide (LPS)-induced bovine mammary epithelial cells (MAC-T) from the ferroptosis pathway. MAC-T cells in the logarithmic growth phase were randomly grouped according to the experimental requirements and underwent corresponding treatments, with three replicates in each group. The MTT method was used to determine the effects of different concentrations of LPS/Cur on the survival rate of MAC-T cells; RT-PCR was employed to detect the transcription levels of inflammation-related genes (TNF-α, IL-6, IL-1β, iNOS, CXCL2, CCL2) and ferroptosis-related genes (PTGS2, SLC7A11, GPX4, FTH1, ACSL4); BODIPY 581/591 C11 staining was utilized to detect the lipid peroxidation level of cells; kits were used to measure the contents of MDA, LPO, GSH, and the GSH/GSSG ratio in cells; Ferro-Orange staining and the Fe²⁺ detection kit were used to determine the Fe²⁺ content in cells; ELISA kits were used to detect the concentrations of TNF-α, IL-6, and IL-1β in cells; Western blot was used to detect the relative expression levels of TNF-α, IL-6, and IL-1β proteins. The results revealed that compared with the CON group, treatments with 10 μg·mL^-1 LPS or 20 μmol·L^-1 Cur had no significant effect on the survival rate of MAC-T cells. After LPS treatment, the expression levels of pro-inflammatory related genes (TNF-α, IL-6, IL-1β, iNOS, CXCL2, CCL2), ferroptosis-related genes (PTGS2, ACSL4), and the related protein (ACSL4) in cells were significantly elevated (P<0.01); the expression levels of anti-ferroptosis genes (SLC7A11, GPX4, FTH1) and protein (GPX4) were significantly decreased (P<0.01); the MDA content and LPO concentration were all significantly increased (P<0.01); the GSH content and GSH/GSSG ratio were both extremely decreased (P<0.01); the Fe²⁺ content was extremely significantly increased (P<0.01); the concentrations and protein expression levels of TNF-α, IL-6, and IL-1β were all extremely increased (P<0.01). Compared with the LPS alone treatment group, the expression levels of pro-inflammatory related genes, ferroptosis-related genes and related proteins in the LPS and Cur-H co-treatment group were extremelydecreased (P<0.01); the expression levels of anti-ferroptosis genes and proteins were extremely increased (P<0.01); the lipid peroxidation level, MDA content, and LPO concentration were all extremely decreased (P<0.01); the GSH content and GSH/GSSG ratio were both significantly increased (P<0.01); the Fe²⁺ content was significantly decreased (P<0.01); the concentrations and protein expression levels of TNF-α, IL-6, and IL-1β were all extremely decreased (P<0.01). Additionally, the ferroptosis activator Erastin significantly inhibited the alleviating effect of Cur on LPS-induced inflammation. In conclusion, Cur can alleviate LPS-induced inflammatory responses in MAC-T cells by inhibiting ferroptosis.

Key words: ferroptosis, curcumin, lipopolysaccharide, bovine mammary epithelial cell (MAC-T)

中图分类号:

S857.26

杨文哲, 王锦浩, 赵子琛, 赵彤, 潘飞龙, 陈芳芳, 邵雯琪, 刘克祥, 赵树臣, 赵立佳. 姜黄素影响铁死亡途径缓解LPS诱导牛乳腺上皮细胞炎性反应的分析[J]. 畜牧兽医学报, 2025, 56(9): 4730-4740.

YANG Wenzhe, WANG Jinhao, ZHAO Zichen, ZHAO Tong, PAN Feilong, CHEN Fangfang, SHAO Wenqi, LIU Kexiang, ZHAO Shuchen, ZHAO Lijia. Analysis of the Impact of Curcumin on the Ferroptosis Pathway in Alleviating the Inflammatory Response Induced by LPS in Bovine Mammary Epithelial Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4730-4740.

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