大肠杆菌感染奶牛乳腺上皮细胞和小鼠乳腺组织致其线粒体损伤的机制研究

doi:10.11843/j.issn.0366-6964.2024.02.038

摘要/Abstract

摘要： 旨在探究大肠杆菌(Escherichia coli,E. coli)感染奶牛乳腺上皮细胞和小鼠乳腺组织致其线粒体损伤的机制。本试验以奶牛乳腺上皮细胞和小鼠乳腺为研究对象,按试验要求分别随机分成3组:空白对照(Control)组、大肠杆菌(E.coli)组和脂多糖(lipopolysaccharide,LPS)组。Control组奶牛乳腺上皮细胞和小鼠乳腺未被E.coli感染;E.coli组奶牛乳腺上皮细胞和小鼠乳腺被感染复数为5或10⁶ CFU的E.coli感染6或24 h,LPS组奶牛乳腺上皮细胞和小鼠乳腺经1 μg·mL^-1或20 mg·kg^-1体重LPS处理6或24 h,奶牛乳腺上皮细胞每组3个重复,小鼠乳腺每组6只小鼠。结果表明:1)奶牛乳腺上皮细胞胞浆中角18蛋白被染成绿色且均匀分布在细胞浆内,细胞核被DAPI染成蓝色。2)正常培养的奶牛乳腺上皮细胞内线粒体结构完整,细胞连接紧密;经E.coli感染的奶牛乳腺上皮细胞细胞间隙增大,线粒体肿胀,线粒体嵴缺失且部分模糊消失。3)E.coli感染或LPS处理使小鼠乳腺腺泡内出现中性粒细胞,且使奶牛乳腺上皮细胞和小鼠乳腺线粒体能量代谢(D(520 nm)吸光值、ATP的含量、线粒体电子传递链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ活性降低)、线粒体的融合与分裂(Drp1、Fis1、Mfn1、Mfn2和OPA1 mRNA表达减少)和线粒体的生物发生(PGC-1α、NRF1、TFAM和D-Loop的基因表达下降)极显著降低(P<0.05,P<0.01)。上述研究表明,E.coli感染主要通过LPS造成奶牛乳腺上皮细胞和小鼠乳腺线粒体能量代谢紊乱、抑制线粒体分裂与融合以及减少线粒体的生物发生,进而造成线粒体损伤,最终导致乳腺炎的发生。因此,确认E.coli感染是通过诱导线粒体损伤造成奶牛乳腺炎,且线粒体损伤可能是造成乳腺损伤的重要原因之一。

关键词: 奶牛乳腺炎, 大肠杆菌, 线粒体, 奶牛乳腺上皮细胞, 小鼠乳腺

Abstract: This experiment was conducted to study the mechanism of Escherichia coli (E. coli) infection on mitochondrial damage of mammary epithelial cells and mouse mammary gland. In this study, bovine mammary epithelial cells and mouse mammary gland were used as the research object. Bovine mammary epithelial cells and mouse mammary gland were randomly divided into three groups, including Control group, E.coli group and lipopolysaccharide (LPS) group, respectively. No treatment was conduceted in the Control group, boving mammary epithelial cells and mouse mammary gland tissue in the E.coli group were infected with E.coli (MOI=5) for 6 h or 10⁶ CFU E. coli for 24 h, respectively. In the LPS group they were treated with 1 μg·mL^-1 lipopolysaccharide (LPS) for 6 h or 20 mg·kg^-1 body weight LPS for 24 h, respectively. with three replicates in each group, 6 mice in each group. The results showed that: 1) Cytokeratin 18 in bovine mammary epithelial cells was dyed green and evenly distributed in the cytoplasm, and the nucleus was dyed blue by DAPI. 2) The mitochondria in untreated-bovine mammary epithelial cells were intact and the cell connections were tight. In E.coli group, the space between bovine mammary epithelial cells was increased, and mitochondria were swollen with disappeared mitochondrial cristae and blurred partial mitochondria cristae. 3) E. coli infection or LPS treatment caused the appearance of neutrophils in the mammary glands of mice. E.coli infection or LPS significantly decreased (P<0.05 or P<0.01) mitochondrial energy metabolism (decreased D(520 nm) absorption value, ATP concentration, and the activities of mitochondrial electron transport chain complex Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ), mitochondrial fusion and division (reduced Drp1, Fis1, Mfn1, Mfn2, and OPA1 mRNA expressions) and mitochondrial biogenesis (inhibited PGC-1α, NRF1, TFAM, and D-Loop gene expression). These results indicated that E.coli infection caused energy metabolism disorder of mitochondria, inhibited mitochondrial division and fusion, and reduced mitochondrial biogenesis mainly through LPS in bovine mammary epithelial cells and mouse mammary gland, thus causing mitochondrial damage and eventually leading to mastitis. Therefore, it can be inferred that E.coli infection could cause bovine mastitis through inducing mitochondrial damage, which play an important role in mammary gland inflammation.

Key words: bovine mastitis, Escherichia coli, mitochondrion, bovine mammary epithelial cell, mouse mammary gland

中图分类号:

S852.612

庄翠翠, 韩博. 大肠杆菌感染奶牛乳腺上皮细胞和小鼠乳腺组织致其线粒体损伤的机制研究[J]. 畜牧兽医学报, 2024, 55(2): 822-833.

ZHUANG Cuicui, HAN Bo. Mechanism of Mitochondrial Damage in Bovine Mammary Epithelial Cells and Mouse Mammary Gland Infected with Escherichia coli Isolated from Bovine Mastitis[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 822-833.

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