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

doi:10.12307/2025.800

Abstract

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

CLC Number:

Zhou Ying, Tian Yong, Zhong Zhimei, Gu Yongxiang, Fang Hao. Inhibition of tumor necrosis factor receptor associated factor 6 regulates mTORC1/ULK1 signaling and promotes autophagy to improve myocardial injury in sepsis mice[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(30): 6434-6440.

Figures/Tables 5

2.1 实验动物数量分析假手术组全部存活；模型组在术后24 h内小鼠死亡2只，模型+C组死亡1只，模型+ C+3-MA组死亡2只，模型+C+M组死亡2只。为符合随机对照的原则，每组小鼠随机选取3个样本开展后续实验。 2.2 C25-140对模型小鼠心肌组织肿瘤坏死因子受体相关因子6的抑制作用与假手术组比较，模型组肿瘤坏死因子受体相关因子6蛋白及mRNA表达升高(P < 0.05)；与模型组比较，模型+C组、模型+C+3-MA组肿瘤坏死因子受体相关因子6蛋白及mRNA表达下降(P < 0.05)，见图1。 2.3 苏木精-伊红染色各组小鼠心肌组织的炎性损伤光镜下可见假手术组小鼠心肌无炎症细胞浸润；模型组心肌炎症细胞浸润，心肌纤维排列稍不规则、纤维水肿；模型+C组心肌炎性细胞浸润、纤维水肿较模型组减轻；与模型+C组比较，模型+C+3MA组见炎症细胞浸润较明显，见图2。 2.4 TUNEL染色检测小鼠心肌细胞凋亡荧光显微镜下可见假手术组小鼠心肌偶见单个细胞凋亡；模型组心肌细胞凋亡明显增多；与模型组比较，模型+C组心肌细胞凋亡明显减轻；与模型+C组比较，模型+C+3MA组心肌凋亡细胞增多，见图3。 2.5 各组小鼠心肌组织中炎症与凋亡相关蛋白的变化与假手术组比较，模型组小鼠心肌磷酸化核因子κB P65/P65、Bax表达升高，Bcl2表达下降(P < 0.05)；与模型组比较，模型+C组心肌磷酸化核因子κB P65/P65、Bax表达下降，Bcl2表达升高(P < 0.05)；与模型+C组比较，模型+C+3-MA组心肌磷酸化核因子κB P65/P65、Bax表达升高，Bcl2表达下降(P < 0.05)，见图4。 2.6 透射电镜观察各组小鼠心肌组织中自噬小体、线粒体微结构透射电镜可见假手术组小鼠心肌线粒体排列正常，无肿胀，未见自噬小体；模型组心肌线粒体明显肿胀，排列紊乱，偶见自噬小体；与模型组比较，模型+C组心肌线粒体肿胀减轻，自噬小体增多；与模型+C组比较，模型+C+3MA组心肌见自噬小体数量明显减少，线粒体肿胀有加重，见图5。 2.7 各组小鼠心肌组织中自噬相关蛋白的变化与假手术组比较，模型组小鼠心肌P62表达升高(P < 0.05)；与模型组比较，模型+C组心肌P62表达下降，Beclin-1表达升高(P < 0.05)；与模型+C组比较，模型+C+3-MA组心肌P62表达升高，Beclin-1表达下降(P < 0.05)，见图6。 2.8 抑制肿瘤坏死因子受体相关因子6后小鼠心肌组织中自噬蛋白与mTORC1/ULK1通路蛋白的变化关系为进一步探索肿瘤坏死因子受体相关因子6是否调节mTORC1/ULK1通路参与模型小鼠心肌自噬过程，采用Western blot检测了mTOR/ULK1蛋白磷酸化和自噬相关蛋白的变化。与假手术组比较，模型组小鼠心肌肿瘤坏死因子受体相关因子6、p-mTOR/mTOR、p-ULK1/ULK1表达升高(P < 0.05)；与模型组比较，模型+C组心肌肿瘤坏死因子受体相关因子6、p-mTOR/mTOR、p-ULK1/ULK1表达下降，Beclin-1、微管相关蛋白1轻链3Ⅱ/Ⅰ表达升高(P < 0.05)；与模型+C组比较，模型+C+M组心肌p-mTOR/mTOR、p-ULK1/ULK1表达升高，Beclin-1、微管相关蛋白1轻链3Ⅱ/Ⅰ表达下降(P < 0.05)，见图7。"

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