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iTRAQ-Based Quantitative Proteomic Analysis of Intestines in Murine Polymicrobial Sepsis with Hydrogen Gas Treatment
Drug Design, Development and Therapy ( IF 4.7 ) Pub Date : 2020-11-12 , DOI: 10.2147/dddt.s271191 Yi Jiang 1, 2 , Yingxue Bian 3 , Naqi Lian 1, 2 , Yaoqi Wang 1, 2 , Keliang Xie 1, 2 , Chao Qin 1, 2 , Yonghao Yu 1, 2
Drug Design, Development and Therapy ( IF 4.7 ) Pub Date : 2020-11-12 , DOI: 10.2147/dddt.s271191 Yi Jiang 1, 2 , Yingxue Bian 3 , Naqi Lian 1, 2 , Yaoqi Wang 1, 2 , Keliang Xie 1, 2 , Chao Qin 1, 2 , Yonghao Yu 1, 2
Affiliation
Objective: Sepsis-associated intestinal injury has a higher morbidity and mortality in patients with sepsis, but there is still no effective treatment. Our research team has proven that inhaling 2% hydrogen gas (H2) can effectively improve sepsis and related organ damage, but the specific molecular mechanism of its role is not clear. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics analysis was used for studying the effect of H2 on intestinal injury in sepsis.
Methods: Male C57BL/6J mice were used to prepare a sepsis model by cecal ligation and puncture (CLP). The 7-day survival rates of mice were measured. 4-kd fluorescein isothiocyanate-conjugated Dextran (FITC-dextran) blood concentration measurement, combined with hematoxylin-eosinstain (HE) staining and Western blotting, was used to study the effect of H2 on sepsis-related intestinal damage. iTRAQ-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used for studying the proteomics associated with H2 for the treatment of intestinal injury.
Results: H2 can significantly improve the 7-day survival rates of sepsis mice. The load of blood and peritoneal lavage bacteria was increased, and H2 treatment can significantly reduce it. CLP mice had significant intestinal damage, and inhalation of 2% hydrogen could significantly reduce this damage. All 4194 proteins were quantified, of which 199 differentially expressed proteins were associated with the positive effect of H2 on sepsis. Functional enrichment analysis indicated that H2 may reduce intestinal injury in septic mice through the effects of thyroid hormone synthesis and nitrogen metabolism signaling pathway. Western blot showed that H2 was reduced by down-regulating the expressions of deleted in malignant brain tumors 1 protein (DMBT1), insulin receptor substrate 2 (IRS2), N-myc downregulated gene 1 (NDRG1) and serum amyloid A-1 protein (SAA1) intestinal damage in sepsis mice.
Conclusion: A total of 199 differential proteins were related with H2 in the intestinal protection of sepsis. H2-related differential proteins were notably enriched in the following signaling pathways, including thyroid hormone synthesis signaling pathway, nitrogen metabolism signaling pathways, digestion and absorption signaling pathways (vitamins, proteins and fats). H2 reduced intestinal injury in septic mice by down-regulating the expressions of SAA1, NDRG1, DMBT1 and IRS2.
Keywords: sepsis, intestinal injury, hydrogen gas, proteomics, isobaric tags for relative and absolute quantitation, iTRAQ
中文翻译：
基于 iTRAQ 的氢气处理小鼠多微生物脓毒症肠道定量蛋白质组学分析
目的：脓毒症相关肠损伤在脓毒症患者中的发病率和死亡率较高，但目前尚无有效的治疗方法。我们的研究团队已经证明，吸入2%氢气（H 2）可以有效改善脓毒症及相关器官损伤，但其作用的具体分子机制尚不清楚。在本研究中，基于相对和绝对定量 (iTRAQ) 的定量蛋白质组学分析的等压标签用于研究 H 2对脓毒症肠道损伤的影响。
方法：雄性 C57BL/6J 小鼠通过盲肠结扎和穿刺 (CLP) 制备脓毒症模型。测量小鼠的7天存活率。采用 4-kd 异硫氰酸荧光素偶联葡聚糖 (FITC-葡聚糖) 血浓度测量，结合苏木精-伊红 (HE) 染色和蛋白质印迹法，研究 H 2对脓毒症相关肠道损伤的影响。基于 iTRAQ 的液相色谱-串联质谱 (LC-MS/MS) 分析用于研究与 H 2相关的蛋白质组学以治疗肠道损伤。
结果： H 2能显着提高脓毒症小鼠的7天存活率。血液和腹腔灌洗细菌负荷增加，H 2治疗可以显着降低。CLP 小鼠有明显的肠道损伤，吸入 2% 的氢气可以显着降低这种损伤。对所有 4194 种蛋白质进行了定量，其中 199 种差异表达的蛋白质与 H 2对脓毒症的积极作用有关。功能富集分析表明，H 2可能通过甲状腺激素合成和氮代谢信号通路的作用减轻脓毒症小鼠的肠道损伤。Western blot显示H 2通过下调恶性脑肿瘤缺失1蛋白（DMBT1）、胰岛素受体底物2（IRS2）、N-myc下调基因1（NDRG1）和血清淀粉样A-1蛋白的表达而降低。 (SAA1) 败血症小鼠的肠道损伤。
结论：共有199个差异蛋白与H 2在脓毒症肠道保护中相关。H 2相关差异蛋白在以下信号通路中显着富集，包括甲状腺激素合成信号通路、氮代谢信号通路、消化吸收信号通路（维生素、蛋白质和脂肪）。H 2通过下调SAA1、NDRG1、DMBT1和IRS2的表达来减轻脓毒症小鼠的肠道损伤。
关键词：脓毒症，肠道损伤，氢气，蛋白质组学，等压标签相对和绝对定量，iTRAQ
更新日期：2020-11-12
Methods: Male C57BL/6J mice were used to prepare a sepsis model by cecal ligation and puncture (CLP). The 7-day survival rates of mice were measured. 4-kd fluorescein isothiocyanate-conjugated Dextran (FITC-dextran) blood concentration measurement, combined with hematoxylin-eosinstain (HE) staining and Western blotting, was used to study the effect of H2 on sepsis-related intestinal damage. iTRAQ-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used for studying the proteomics associated with H2 for the treatment of intestinal injury.
Results: H2 can significantly improve the 7-day survival rates of sepsis mice. The load of blood and peritoneal lavage bacteria was increased, and H2 treatment can significantly reduce it. CLP mice had significant intestinal damage, and inhalation of 2% hydrogen could significantly reduce this damage. All 4194 proteins were quantified, of which 199 differentially expressed proteins were associated with the positive effect of H2 on sepsis. Functional enrichment analysis indicated that H2 may reduce intestinal injury in septic mice through the effects of thyroid hormone synthesis and nitrogen metabolism signaling pathway. Western blot showed that H2 was reduced by down-regulating the expressions of deleted in malignant brain tumors 1 protein (DMBT1), insulin receptor substrate 2 (IRS2), N-myc downregulated gene 1 (NDRG1) and serum amyloid A-1 protein (SAA1) intestinal damage in sepsis mice.
Conclusion: A total of 199 differential proteins were related with H2 in the intestinal protection of sepsis. H2-related differential proteins were notably enriched in the following signaling pathways, including thyroid hormone synthesis signaling pathway, nitrogen metabolism signaling pathways, digestion and absorption signaling pathways (vitamins, proteins and fats). H2 reduced intestinal injury in septic mice by down-regulating the expressions of SAA1, NDRG1, DMBT1 and IRS2.
Keywords: sepsis, intestinal injury, hydrogen gas, proteomics, isobaric tags for relative and absolute quantitation, iTRAQ
中文翻译：
基于 iTRAQ 的氢气处理小鼠多微生物脓毒症肠道定量蛋白质组学分析
目的：脓毒症相关肠损伤在脓毒症患者中的发病率和死亡率较高，但目前尚无有效的治疗方法。我们的研究团队已经证明，吸入2%氢气（H 2）可以有效改善脓毒症及相关器官损伤，但其作用的具体分子机制尚不清楚。在本研究中，基于相对和绝对定量 (iTRAQ) 的定量蛋白质组学分析的等压标签用于研究 H 2对脓毒症肠道损伤的影响。
方法：雄性 C57BL/6J 小鼠通过盲肠结扎和穿刺 (CLP) 制备脓毒症模型。测量小鼠的7天存活率。采用 4-kd 异硫氰酸荧光素偶联葡聚糖 (FITC-葡聚糖) 血浓度测量，结合苏木精-伊红 (HE) 染色和蛋白质印迹法，研究 H 2对脓毒症相关肠道损伤的影响。基于 iTRAQ 的液相色谱-串联质谱 (LC-MS/MS) 分析用于研究与 H 2相关的蛋白质组学以治疗肠道损伤。
结果： H 2能显着提高脓毒症小鼠的7天存活率。血液和腹腔灌洗细菌负荷增加，H 2治疗可以显着降低。CLP 小鼠有明显的肠道损伤，吸入 2% 的氢气可以显着降低这种损伤。对所有 4194 种蛋白质进行了定量，其中 199 种差异表达的蛋白质与 H 2对脓毒症的积极作用有关。功能富集分析表明，H 2可能通过甲状腺激素合成和氮代谢信号通路的作用减轻脓毒症小鼠的肠道损伤。Western blot显示H 2通过下调恶性脑肿瘤缺失1蛋白（DMBT1）、胰岛素受体底物2（IRS2）、N-myc下调基因1（NDRG1）和血清淀粉样A-1蛋白的表达而降低。 (SAA1) 败血症小鼠的肠道损伤。
结论：共有199个差异蛋白与H 2在脓毒症肠道保护中相关。H 2相关差异蛋白在以下信号通路中显着富集，包括甲状腺激素合成信号通路、氮代谢信号通路、消化吸收信号通路（维生素、蛋白质和脂肪）。H 2通过下调SAA1、NDRG1、DMBT1和IRS2的表达来减轻脓毒症小鼠的肠道损伤。
关键词：脓毒症，肠道损伤，氢气，蛋白质组学，等压标签相对和绝对定量，iTRAQ
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