LPS对牦牛瘤胃上皮细胞补体C3激活和ATP生成代谢的影响

doi:10.11843/j.issn.0366-6964.2023.11.021

摘要/Abstract

摘要： 旨在通过脂多糖(LPS)诱导牦牛瘤胃上皮细胞建立炎症模型,考察瘤胃上皮细胞作为非免疫细胞是否存在细胞内补体C3激活,及其对细胞三磷酸腺苷(ATP)生成的影响,为瘤胃健康营养调控技术提供试验依据。用不同浓度的LPS处理牦牛瘤胃上皮细胞系,采用CCK-8法测定细胞活力;ELISA试剂盒测定细胞内补体C3激活产物C3a、C3b浓度以及培养基中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)浓度;化学法检测细胞内ATP浓度,线粒体红色荧光探针和流式细胞仪检测线粒体数量及膜电位;qPCR检测细胞IL-1β、IL-6、TNF-α,补体C3及其激活关键酶组织蛋白酶B(CTSB)和组织蛋白酶L(CTSL),以及ATP合成酶亚基(ATP5A和ATP5C1)等基因相对表达量。结果发现:1)不同浓度LPS处理后,牦牛瘤胃上皮细胞活力极显著下调(P<0.01),TNF-α、IL-1β和IL-6等促炎因子的基因表达量和浓度极显著上调(P<0.01),在LPS浓度为10 μg·mL^-1时,牦牛瘤胃上皮细胞炎症模型构建成功;2)炎症下,牦牛瘤胃上皮细胞内补体C3及其激活关键酶CTSB的基因表达量极显著升高(P<0.01),激活产物补体片段C3a和C3b浓度极显著升高(P<0.01);3)炎症下,牦牛瘤胃上皮细胞ATP含量极显著下降(P<0.01),线粒体膜电位显著降低(P<0.05),ATP合成酶亚基ATP5A和ATP5C1的基因表达量随着LPS处理浓度升高显著下调(P<0.01)。综上,利用LPS诱导建立了牦牛瘤胃上皮细胞炎症模型。在LPS刺激下,牦牛瘤胃上皮细胞可发生细胞内补体C3激活过程,并抑制线粒体ATP合成酶亚基ATP5A、ATP5C1和有氧呼吸关键酶ME1的基因表达,线粒体膜电位降低,抑制ATP生成。因此,LPS诱导炎症可抑制瘤胃上皮细胞ATP生成,从而导致瘤胃健康问题。

关键词: 牦牛, 瘤胃上皮细胞, 免疫炎症, 补体C3, ATP

Abstract: The aim of this study was to establish an inflammation model of yak rumen epithelial cells induced by lipopolysaccharide (LPS), and to investigate whether there is intracellular complement C3 activation in rumen epithelial cells as non-immune cells, and its influence on the production of adenosine triphosphate (ATP). To provide experimental basis for nutrition regulation techniques of rumen health. Different concentrations of LPS were used to treat yak rumen epithelial cell line. Cell viability was determined by CCK-8 method. ELISA kit was used to determine the concentrations of C3 activation products C3a and C3b in cells and the concentrations of interleukin-1β (IL-1), interleukin 6 (IL-6), tumor necrosis factor (TNF-α) in the cell culture; The intracellular ATP concentration was detected by chemical method, and the number of mitochondria and membrane potential were detected by mitochondrial red fluorescent probe and flow cytometry. The relative expression of IL-1β, IL-6, TNF-α, complement C3, cathepsin B (CTSB), cathepsin L (CTSL), and ATP synthase subunits (ATP5A and ATP5C1) were determined by q-PCR. The results showed that: 1) After different concentrations of LPS treatment, the activity of yak rumen epithelial cells was significantly decreased (P<0.01), and the gene expression and concentration of proinflammatory factors, such as TNF-α, IL-1β and IL-6 were significantly increased (P<0.01). The inflammatory model of yak rumen epithelial cells was established successfully when the concentration of LPS was 10 μg·mL^-1; 2) Under inflammation, the gene expression of complement C3 and its key activating enzyme CTSB increased significantly in yak rumen epithelial cells (P<0.01), and the concentration of activated complement fragments C3a and C3b increased significantly (P<0.01); 3) Under inflammation, the ATP content (P<0.01) and mitochondrial membrane potential (P<0.01) in yak rumen epithelial cells decreased significantly, and the gene expression of ATP synthase subunits ATP5A and ATP5C1 decreased significantly with the increasing of LPS concentration (P<0.01). In summary, the inflammation model of yak rumen epithelial cells was established by LPS. The LPS stimulation can activate the intracellular complement C3, and inhibit the gene expression of mitochondrial ATP synthase subunits ATP5A, ATP5C1 and key enzyme ME1 of aerobic respiration, thus reducing mitochondrial membrane potential and inhibiting ATP production in the yak rumen epithelial cells. LPS-induced inflammation can inhibit ATP production of rumen epithelial cells, leading to rumen health problems.

Key words: yak, rumen epithelial cells, immune inflammation, complement C3, ATP

中图分类号:

S856.4

张祺琪, 王俊梅, 岳子奇, 郭逸芯, 施丽媛, 张晓红, 邹华围, 彭全辉, 薛白, 王立志, 王之盛, 胡瑞. LPS对牦牛瘤胃上皮细胞补体C3激活和ATP生成代谢的影响[J]. 畜牧兽医学报, 2023, 54(11): 4664-4675.

ZHANG Qiqi, WANG Junmei, YUE Ziqi, GUO Yixin, SHI Liyuan, ZHANG Xiaohong, ZOU Huawei, PENG Quanhui, XUE Bai, WANG Lizhi, WANG Zhisheng, HU Rui. Effect of LPS on the Complement C3 Activation and ATP Production in the Rumen Epithelial Cells of Yak[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4664-4675.

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