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Abstract：
BACKGROUND: 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.
RESULTS: 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.
CONCLUSIONS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
摘要：
背景：非靶向代谢组学和蛋白质组学用于研究牦牛瘤胃上皮细胞（YRECs）对亚急性瘤胃酸中毒（SARA）病因学的细胞内反应，包括接触短链脂肪酸（SCFA），低pH5.5（酸性），和脂多糖（LPS）暴露24小时。
结果：这些处理显著改变了YRECs的细胞形态。代谢组学分析确定了SCFA的显著扰动，酸和LPS处理影响259、245和196代谢物（VIP>1，P<0.05，倍数变化（FC）≥1.5或FC≤0.667）。蛋白质组学分析显示，用SCFA治疗，酸,和LPS导致1251、1396和242蛋白的差异表达，分别为(FC≥1.2或≤0.83，P<0.05，FDR<1%)。用SCFA治疗诱导嘌呤代谢相关的代谢物水平升高，谷胱甘肽代谢，和精氨酸生物合成，以及与肌动蛋白细胞骨架组织和核糖体途径相关的失调蛋白。此外，SCFA减少了数量，形态学,线粒体的功能，导致氧化损伤和抑制细胞存活。基因表达分析显示，细胞骨架和细胞周期的基因表达降低，而与炎症和自噬相关的基因表达增加(P<0.05)。酸暴露改变了与嘌呤代谢有关的代谢物，以及与补体和凝血级联反应以及RNA降解相关的受影响蛋白质。酸也会导致线粒体功能障碍,线粒体完整性的改变，减少ATP的产生。它还导致肌动蛋白丝从丝状变为点状，影响细胞骨架功能，增加炎症相关分子，提示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的差异表达,伴随着与花生四烯酸代谢和MAPK信号相关的代谢物的改变(P<0.05)。通过蛋白质相互作用网络分析也证实了LPS处理诱导的炎症反应和信号通路的激活。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富集。
结论：总之，这项研究有助于全面了解SARA相关因素对YRECs的有害影响，阐明其分子机制，并为缓解SARA提供潜在的治疗靶点。
结果：这些处理显著改变了YRECs的细胞形态。代谢组学分析确定了SCFA的显著扰动，酸和LPS处理影响259、245和196代谢物（VIP>1，P<0.05，倍数变化（FC）≥1.5或FC≤0.667）。蛋白质组学分析显示，用SCFA治疗，酸,和LPS导致1251、1396和242蛋白的差异表达，分别为(FC≥1.2或≤0.83，P<0.05，FDR<1%)。用SCFA治疗诱导嘌呤代谢相关的代谢物水平升高，谷胱甘肽代谢，和精氨酸生物合成，以及与肌动蛋白细胞骨架组织和核糖体途径相关的失调蛋白。此外，SCFA减少了数量，形态学,线粒体的功能，导致氧化损伤和抑制细胞存活。基因表达分析显示，细胞骨架和细胞周期的基因表达降低，而与炎症和自噬相关的基因表达增加(P<0.05)。酸暴露改变了与嘌呤代谢有关的代谢物，以及与补体和凝血级联反应以及RNA降解相关的受影响蛋白质。酸也会导致线粒体功能障碍,线粒体完整性的改变，减少ATP的产生。它还导致肌动蛋白丝从丝状变为点状，影响细胞骨架功能，增加炎症相关分子，提示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的差异表达,伴随着与花生四烯酸代谢和MAPK信号相关的代谢物的改变(P<0.05)。通过蛋白质相互作用网络分析也证实了LPS处理诱导的炎症反应和信号通路的激活。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富集。
结论：总之，这项研究有助于全面了解SARA相关因素对YRECs的有害影响，阐明其分子机制，并为缓解SARA提供潜在的治疗靶点。
