Notch1通过TAK1对APAP诱导的肝损伤作用机制研究
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南京医科大学第一附属医院

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江苏省自然科学基金青年基金(BK20161059);中国肝炎防治基金会一天晴肝病研究基金(CFHPC20132071)


To study the mechanism of Notch1 on APAP-induced liver injury through TAK1
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Department of Infectious Diseases,The First Affiliated Hospital with Nanjing Medical University

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Jiangsu Provincial Natural Science Foundation for Youth ( BK20161059 ); China Hepatitis Prevention and Control Foundation-Tianqing Liver Disease Research Fund ( CFHPC20132071);

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    摘要:

    目的:探讨Notch1信号通过TAK1调控对乙酰氨基酚(acetyl-para-aminophenol,APAP)诱导的肝损伤(APAP induced liver injury,AILI)的作用机制。方法:将24只Notch1FL/FL,Notch1M-KO小鼠随机分成Notch1FL/FL + PBS组(n=6)、Notch1FL/FL + APAP组(n=6)、Notch1M-KO + PBS组(n=6)和Notch1M-KO+ APAP组(n=6),通过APAP腹腔注射构建AILI模型。留取小鼠血清标本,用全自动生化分析仪及酶联免疫反应分析法检测肝功能和细胞因子。留取小鼠肝组织标本,HE染色后使用Suzuki评分评估肝组织损伤程度,用免疫印迹法检测Notch1、转化生长因子β激活激酶1(转化生长因子β激活激酶1,TAK1)、p-TAK1、p65、p-p65、半胱氨酸酶-8(Caspase-8,Casp-8)、受体相互作用蛋白激酶1 (Receptor-interacting protein kinase 1, RIPK1)、p-混合线状激酶域类样 (mixed lineage kinase domain-like, MLKL)的表达,用免疫荧光法观察CD11b、p-TAK1、ROS的表达。结果:与Notch1FL/FL对照组相比,经APAP腹腔注射的Notch1M-KO小鼠血清ALT、AST明显升高,血清炎性因子水平上升,HE染色显示肝细胞水肿明显,窦道淤血,炎性细胞浸润增加,DCF探针显示细胞内ROS表达上升。肝组织TAK1表达升高,激活p-P65,RIPK1、p-MLKL表达上升,抑制Casp-8的表达,从而促进肝细胞坏死性凋亡的发生,加重肝损伤。结论:在AILI中,巨噬细胞Notch1特异性敲除可激活TAK1表达,从而抑制Casp-8水平,激活RIPK1-MLKL坏死性凋亡通路,加重肝损伤的发生。

    Abstract:

    Objective: To investigate the mechanism by which Notch1 signaling regulates APAP-induced liver injury (AILI) via TAK1. Methods: Twenty-four C57BL/6, floxed Notch1(Notch1FL/FL), and Myeloid-specific Notch1 knockout(Notch1M-KO)mice were randomly divided into four groups: control (Notch1FL/FL) + PBS (n=6), control + APAP (n=6), Notch1M-KO + PBS (n=6), and Notch1M-KO + APAP (n=6), and APAP was injected intraperitoneally to establish the AILI model. Mouse serum and tissue samples were retained for serological examination, HE staining, immunofluorescence, Western blot(WB), and enzyme-linked immunoassay analysis, respectively. Results: Compared with the Notch1FL/FL group, the Notch1M-KO group displayed significantly higher serum levels of ALT, AST, and cytokines; HE staining reveals significant hepatocyte edema, sinusoidal stasis, and increased macrophage accumulation; the DCF probe demonstrated increased intracellular ROS expression. Elevated TAK1 activated p-P65 and increased RIPK1, and p-MLKL expression, thus promoting the development of necroptosis in hepatocytes and aggravating the liver injury. At the same time, caspase-8 expression was suppressed. Conclusion: In AILI, myeloid-specific Notch1 knockout activates TAK1 expression, which also decreases Caspase-8 levels and promotes the RIPK1-MLKL necroptosis pathway, aggravating the liver injury.

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  • 收稿日期:2022-10-09
  • 最后修改日期:2023-08-14
  • 录用日期:2023-10-18
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