中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (30): 6434-6440.doi: 10.12307/2025.800

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

抑制TRAF6调节mTORC1/ULK1信号通路促进自噬改善脓毒症小鼠的心肌损伤

周  颖1，2，田  勇2，钟芝梅1，古雍翔2，方  昊3   

  1. 1贵州省黔西南州人民医院呼吸科，贵州省兴义市  562400；2贵州医科大学临床医学院，贵州省贵阳市  550004；3贵州医科大学附属医院内科ICU，贵州省贵阳市  550004
  • 收稿日期:2024-09-28 接受日期:2024-11-11 出版日期:2025-10-28 发布日期:2025-03-27
  • 通讯作者: 方昊，副主任医师，贵州医科大学附属医院内科ICU，贵州省贵阳市 550004
  • 作者简介:周颖，男，1987年生，贵州省兴仁市人，汉族，2024年贵州医科大学毕业，硕士，副主任医师，主要从事老年呼吸危重症、脓毒症的研究。
  • 基金资助:
    贵州省卫生健康委科学技术基金项目(gzwkj2023-112)，项目负责人：方昊；贵州省卫生健康委科学技术基金项目(gzwkj2024-276)，项目负责人：周颖；贵州省黔西南州科技计划项目(2022-2-08；2023-3-12)，项目负责人：周颖

Inhibition of tumor necrosis factor receptor associated factor 6 regulates mTORC1/ULK1 signaling and promotes autophagy to improve myocardial injury in sepsis mice

Zhou Ying1, 2, Tian Yong2, Zhong Zhimei1, Gu Yongxiang2, Fang Hao3   

  1. 1Department of Respiratory Medicine, People’s Hospital of Qianxinan Prefecture, Xingyi 562400, Guizhou Province, China; 2School of Clinical Medicine, Guizhou Medical University, Guiyang 550004, Guizhou Province, China; 3Department of MICU, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China

  • Received:2024-09-28 Accepted:2024-11-11 Online:2025-10-28 Published:2025-03-27
  • Contact: Fang Hao, Associate chief physician, Department of MICU, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • About author:Zhou Ying, MS, Associate chief physician, Department of Respiratory Medicine, People’s Hospital of Qianxinan Prefecture, Xingyi 562400, Guizhou Province, China; School of Clinical Medicine, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • Supported by:
    Science and Technology Fund Project of Health Commission of Guizhou Province, No. gzwkj2023-112 (to FH); Science and Technology Fund Project of Health Commission of Guizhou Province, No. gzwkj2024-276 (to ZY); Science and Technology Plan Project of Qianxinan Prefecture of Guizhou Province, No. 2022-2-08; 2023-3-12 (to ZY)

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摘要：


文题释义：
肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor associated factor 6，TRAF6)：是一种存在于细胞质的衔接蛋白，目前认为是肿瘤坏死因子受体家族和Toll 样受体/白细胞介素1受体家族中唯一的信号转导连接分子。TRAF6具有独特的特异性结合受体参与调节先天性和适应性免疫、自噬等病理生理过程。
哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)：是一种丝氨酸/苏氨酸激酶，分为mTOR复合体1和复合体2(mTORC1，mTORC2)，具有调节细胞生长和代谢等作用。mTORC1作为自噬的主要调节因子发挥负调节作用。

背景：研究发现抑制肿瘤坏死因子受体相关因子6能改善脓毒症心肌功能并促进心肌自噬水平，但具体机制尚不明确。
目的：探索抑制肿瘤坏死因子受体相关因子6调节mTORC1/ULK1自噬信号通路对脓毒症小鼠心肌损伤的作用。
方法：雄性昆明小鼠30只，随机分为5组，分别为假手术组、盲肠结扎穿刺术组(模型组) 、模型+肿瘤坏死因子受体相关因子6特异性抑制剂C25-140(模型+C)组、模型+C25-140+自噬抑制剂3-甲基腺嘌呤(模型+C+3-MA)组、模型+C25-140+mTORC1特异性激动剂MHY1485(模型+C+M)组。假手术组小鼠盲肠不结扎穿刺；其余各组小鼠行盲肠结扎穿刺术建立小鼠脓毒症模型，按分组术后0.5 h分别腹腔注射C25-140、3-甲基腺嘌呤、MHY1485。术后24 h取心肌组织，苏木精-伊红染色评估心肌炎症病变；透射电镜观察心肌细胞自噬小体、线粒体微结构变化；TUNEL检测心肌细胞凋亡，PCR检测肿瘤坏死因子受体相关因子6 mRNA的相对表达量，Western blot检测相关蛋白的表达。
结果与结论：①与假手术组比较，模型组小鼠心肌炎症细胞浸润、纤维水肿；细胞线粒体明显肿胀，偶见自噬小体；心肌细胞凋亡显著增加；肿瘤坏死因子受体相关因子6、磷酸化核因子κB P65/P65、p-mTOR/mTOR、p-ULK1/ULK1、P62、Bax蛋白表达升高，Bcl2蛋白表达下降(P < 0.05)。②与模型组比较，模型+C组小鼠心肌炎症缓解、纤维水肿减轻；心肌线粒体肿胀减轻，自噬小体增多；心肌细胞凋亡减少；磷酸化核因子κB P65 /核因子κB P65、p-mTOR/mTOR、p-ULK1/ULK1、P62、Bax蛋白表达下降，Beclin-1、Bcl2蛋白表达升高(P < 0.05)。③与模型+C比，模型+C+3-MA组心肌自噬小体减少、心肌线粒体肿胀较明显；心肌炎症有加重；心肌细胞凋亡增多；磷酸化核因子κB65/核因子κB65、P62、Bax蛋白表达升高，Beclin-1、Bcl2蛋白表达下降(P < 0.05)。④与模型+C比，模型+C+M组p-mTOR/mTOR、p-ULK1/ULK1表达升高，Beclin-1、微管相关蛋白1轻链3Ⅱ/Ⅰ蛋白表达下降(P < 0.05)。结论：抑制肿瘤坏死因子受体相关因子6可以调节mTORC1/ULK1自噬信号促进心肌自噬参与改善脓毒症心肌损伤。 
https://orcid.org/0009-0007-2333-4985(周颖)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 肿瘤坏死因子受体相关因子6, 自噬, 脓毒症, 心肌损伤, 哺乳动物雷帕霉素靶蛋白, 工程化组织构建

Abstract: BACKGROUND: Studies have found that inhibition of tumor necrosis factor receptor-associated factor 6 improves myocardial function and promotes myocardial autophagy in sepsis, but the specific mechanism is not clear. 
OBJECTIVE: To explore the effect of inhibiting tumor necrosis factor receptor-associated factor 6-regulated mTORC1/ULK1 autophagy signaling pathway on myocardial injury in sepsis mice.
METHODS: Thirty male Kunming mice were randomly divided into sham operation group, cecal ligation and puncture group (model group), model+tumor necrosis factor receptor-associated factor 6 specific inhibitor C25-140 (model+C) group, model+C25-140+autophagy inhibitor 3-methyladenine (model+C+3-MA) group, and model+C25-140+mTORC1-specific agonist MHY1485 (model+C+M) group. The cecum of mice in the sham operation group was not ligated or punctured. The mice in the other groups underwent cecum ligation and puncture to establish the mouse sepsis model. C25-140, 3-methyladenine, and MHY1485 were intraperitoneally injected 0.5 hours after surgery according to the grouping. Myocardial tissue was obtained 24 hours after surgery. Hematoxylin-eosin staining was used to evaluate myocardial inflammatory lesions. Transmission electron microscopy was used to observe the changes in the autophagic bodies and mitochondrial microstructures of myocardial cells. TUNEL assay was used to detect myocardial cell apoptosis. PCR was used to detect the relative expression of tumor necrosis factor receptor-associated factor 6 mRNA. Western blot assay was used to detect the expression of related proteins.
RESULTS AND CONCLUSION: (1) Compared with sham operation group, myocardial inflammatory cell infiltration and fibrous edema were observed in the model group. The mitochondria of the cells were obviously swollen, and autophagosomes were occasionally seen; cardiomyocyte apoptosis increased significantly; the expression of tumor necrosis factor receptor-associated factor 6, phosphorylated nuclear factor κB P65/P65, p-mTOR/mTOR, p-ULK1/ULK1, P62 and Bax protein increased, and the expression of Bcl2 protein decreased (P < 0.05). (2) Compared with the model group, myocardial inflammation and fibrous edema were alleviated in the model+C group. Myocardial mitochondrial swelling was reduced and autophagosomes increased; cardiomyocyte apoptosis decreased; the expression of phosphorylated nuclear factor κB P65/nuclear factor-κB P65, p-mTOR/mTOR, p-ULK1/ULK1, P62, and Bax protein decreased, while the Beclin-1 and Bcl2 protein increased (P < 0.05). (3) Compared with the model+C group, myocardial autophagosomes decreased and myocardial mitochondrial swelling was more obvious in the model+C+3-MA group. Myocardial inflammation was aggravated; myocardial cell apoptosis increased; the expression of phosphorylated nuclear factor κB P65/nuclear factor κB P65, P62, and Bax protein increased, and the Beclin-1 and Bcl2 protein decreased (P < 0.05). (4) Compared with the model+C group, the expression of p-mTOR/mTOR and p-ULK1/ULK1 in the model+C+M group increased, and the Beclin-1 and microtubule-associated protein 1 light chain 3 II/I protein expression decreased (P < 0.05). It is concluded that inhibition of tumor necrosis factor receptor-associated factor 6 regulates mTORC1/ULK1 autophagy signal to promote myocardial autophagy and participate in the protection of myocardial injury in sepsis.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: tumor necrosis factor receptor-associated factor 6, autophagy, sepsis, myocardial injury, mammalian target of rapamycin, engineered tissue construction

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