Untargeted metabolomics and proteomics were employed to investigate the intracellular response of yak rumen epithelial cells (YRECs) to conditions mimicking subacute rumen acidosis (SARA) etiology, including exposure to short-chain fatty acids (SCFA), low pH5.5 (Acid), and lipopolysaccharide (LPS) exposure for 24 h. These treatments significantly altered the cellular morphology of YRECs. Metabolomic analysis identified significant perturbations with SCFA, Acid and LPS treatment affecting 259, 245 and 196 metabolites (VIP > 1, P < 0.05, and fold change (FC) ≥ 1.5 or FC ≤ 0.667). Proteomic analysis revealed that treatment with SCFA, Acid, and LPS resulted in differential expression of 1251, 1396, and 242 proteins, respectively (FC ≥ 1.2 or ≤ 0.83, P < 0.05, FDR < 1%). Treatment with SCFA induced elevated levels of metabolites involved in purine metabolism, glutathione metabolism, and arginine biosynthesis, and dysregulated proteins associated with actin cytoskeleton organization and ribosome pathways. Furthermore, SCFA reduced the number, morphology, and functionality of mitochondria, leading to oxidative damage and inhibition of cell survival. Gene expression analysis revealed a decrease the genes expression of the cytoskeleton and cell cycle, while the genes expression associated with inflammation and autophagy increased (P < 0.05). Acid exposure altered metabolites related to purine metabolism, and affected proteins associated with complement and coagulation cascades and RNA degradation. Acid also leads to mitochondrial dysfunction, alterations in mitochondrial integrity, and reduced ATP generation. It also causes actin filaments to change from filamentous to punctate, affecting cellular cytoskeletal function, and increases inflammation-related molecules, indicating the promotion of inflammatory responses and cellular damage (P < 0.05). LPS treatment induced differential expression of proteins involved in the TNF signaling pathway and cytokine-cytokine receptor interaction, accompanied by alterations in metabolites associated with arachidonic acid metabolism and MAPK signaling (P < 0.05). The inflammatory response and activation of signaling pathways induced by LPS treatment were also confirmed through protein interaction network analysis. The integrated analysis reveals co-enrichment of proteins and metabolites in cellular signaling and metabolic pathways. In summary, this study contributes to a comprehensive understanding of the detrimental effects of SARA-associated factors on YRECs, elucidating their molecular mechanisms and providing potential therapeutic targets for mitigating SARA.

中文翻译：

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代谢组学和蛋白质组学对亚急性瘤胃酸中毒病因及体外牦牛瘤胃上皮细胞增殖抑制的见解

采用非靶向代谢组学和蛋白质组学研究牦牛瘤胃上皮细胞 (YREC) 对模拟亚急性瘤胃酸中毒 (SARA) 病因的条件的细胞内反应，包括暴露于短链脂肪酸 (SCFA)、低 pH5.5（酸）、和脂多糖（LPS）暴露24小时。这些处理显着改变了 YREC 的细胞形态。代谢组学分析发现 SCFA、酸和 LPS 处理存在显着扰动，影响 259、245 和 196 种代谢物（VIP > 1，P < 0.05，倍数变化 (FC) ≥ 1.5 或 FC ≤ 0.667）。蛋白质组分析显示，SCFA、Acid 和 LPS 处理分别导致 1251、1396 和 242 个蛋白质的差异表达（FC ≥ 1.2 或 ≤ 0.83，P < 0.05，FDR < 1%）。 SCFA 治疗可导致与嘌呤代谢、谷胱甘肽代谢和精氨酸生物合成有关的代谢物水平升高，以及与肌动蛋白细胞骨架组织和核糖体途径相关的蛋白质失调。此外，SCFA 减少了线粒体的数量、形态和功能，导致氧化损伤和细胞存活抑制。基因表达分析显示，细胞骨架和细胞周期相关基因表达量减少，而与炎症和自噬相关的基因表达量增加（P < 0.05）。酸暴露改变了与嘌呤代谢相关的代谢物，并影响了与补体和凝血级联以及 RNA 降解相关的蛋白质。酸还会导致线粒体功能障碍、线粒体完整性改变以及 ATP 生成减少。它还导致肌动蛋白丝从丝状变为点状，影响细胞的细胞骨架功能，并增加炎症相关分子，表明促进炎症反应和细胞损伤（P < 0.05）。 LPS 处理诱导参与 TNF 信号通路和细胞因子-细胞因子受体相互作用的蛋白质差异表达，并伴有与花生四烯酸代谢和 MAPK 信号传导相关的代谢物的变化（P < 0.05）。 LPS 治疗诱导的炎症反应和信号通路激活也通过蛋白质相互作用网络分析得到证实。综合分析揭示了细胞信号传导和代谢途径中蛋白质和代谢物的共同富集。总之，本研究有助于全面了解 SARA 相关因素对 YREC 的不利影响，阐明其分子机制，并为减轻 SARA 提供潜在的治疗靶点。