Liu Haiying, Liu Yuqi, Lin Xiaoxi, Fan Jianhui, Huang Zhao, Li Ao
Clinical Research Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, Guangdong, China; Department of Anesthesiology, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, Guangdong, China.
Clinical Research Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, Guangdong, China.
Int Immunopharmacol. 2024 Dec 25;143(Pt 3):113605. doi: 10.1016/j.intimp.2024.113605. Epub 2024 Nov 15.
Lipopolysaccharide (LPS) triggers the activation of nuclear factor kappa B (NF-κB) by interacting with Toll-like receptor 4 (TLR4), leading to the production of various proinflammatory enzymes and cytokines that are crucial in the development of acute lung injury (ALI). Mitoxantrone (MTX) has been demonstrated to mitigate the inflammatory response caused by LPS; however, its precise function in the context of ALI is not fully comprehended.
This study aimed to investigate the inhibitory effects and underlying mechanisms of MTX against LPS-induced ALI.
ALI was induced in C57BL/6 mice via a single intratracheal administration of LPS (5 mg/kg), followed by an intraperitoneal injection of MTX to evaluate its therapeutic potential. The effects of MTX on lung injury and the progression of inflammation in ALI mice were assessed using a comprehensive range of techniques, including hematoxylin-eosin (H&E) staining, immunohistochemistry (IHC), myeloperoxidase activity measurement, cell enumeration in bronchoalveolar lavage fluid (BALF), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Additionally, IHC, Western blotting, and co-immunoprecipitation were used to elucidate the specific signaling pathways and molecular mechanisms by which MTX exerted its anti-inflammatory effects in ALI mice. Surface plasmon resonance (SPR) and molecular docking were used to examine the target to which MTX binds directly to reduce inflammation. We also established a lung epithelial cell injury model using LPS-treated A549 cells. The polyubiquitination of IκBα and TRAF6 in LPS-induced A549 cells was detected through Western blotting following immunoprecipitation.
In mice with LPS-induced ALI, MTX exhibits anti-inflammatory effects by ameliorating histopathological abnormalities caused by LPS, reducing inflammatory cell infiltration, and decreasing the production of proinflammatory enzymes and cytokines. It has been observed that MTX directly binds to the NEDD8 activating enzyme (NAE), thereby inhibiting the transfer of NEDD8 to the substrates UBC12, Cul1, and Cul5. Consequently, the polyubiquitination of IκBα and TRAF6 is disrupted, leading to the suppression of TAK1 activation by TRAF6. This suppression of TAK1 activity hindered the phosphorylation of IKK and MAPK. By stabilizing IκBα through dephosphorylation via IKK inhibition and preventing polyubiquitination, NF-κB activation is reduced. This cascade of events ultimately leads to a reduction in the production of proinflammatory enzymes and cytokines, effectively mitigating the inflammatory response in ALI. In A549 cells, MTX reduces the LPS-induced K48-linked polyubiquitination of IκBα and K63-linked polyubiquitination of TRAF6. This process can be reversed by the overexpression of NEDD8. Additionally, treatment with MG-132, a proteasome inhibitor, can restore the polyubiquitination of IκBα that was inhibited by MTX.
These findings confirm the essential role of Cul1/5 neddylation in ALI and suggest that MTX could be a promising therapeutic agent for ALI.
脂多糖（LPS）通过与Toll样受体4（TLR4）相互作用触发核因子κB（NF-κB）的激活，导致产生各种促炎酶和细胞因子，这些在急性肺损伤（ALI）的发展中至关重要。米托蒽醌（MTX）已被证明可减轻LPS引起的炎症反应；然而，其在ALI背景下的确切功能尚未完全理解。
本研究旨在探讨MTX对LPS诱导的ALI的抑制作用及其潜在机制。
通过单次气管内给予LPS（5mg/kg）诱导C57BL/6小鼠发生ALI，随后腹腔注射MTX以评估其治疗潜力。使用多种技术评估MTX对ALI小鼠肺损伤和炎症进展的影响，包括苏木精-伊红（H&E）染色、免疫组织化学（IHC）、髓过氧化物酶活性测量、支气管肺泡灌洗液（BALF）中的细胞计数、蛋白质印迹法和酶联免疫吸附测定（ELISA）。此外，使用IHC、蛋白质印迹法和免疫共沉淀来阐明MTX在ALI小鼠中发挥抗炎作用的特定信号通路和分子机制。表面等离子体共振（SPR）和分子对接用于检查MTX直接结合以减轻炎症的靶点。我们还使用LPS处理的A549细胞建立了肺上皮细胞损伤模型。通过免疫沉淀后的蛋白质印迹法检测LPS诱导的A549细胞中IκBα和TRAF6的多聚泛素化。
在LPS诱导的ALI小鼠中，MTX通过改善LPS引起的组织病理学异常、减少炎症细胞浸润以及降低促炎酶和细胞因子的产生而表现出抗炎作用。据观察，MTX直接与NEDD8激活酶（NAE）结合，从而抑制NEDD8向底物UBC12、Cul1和Cul5的转移。因此，IκBα和TRAF6的多聚泛素化被破坏，导致TRAF6对TAK1激活的抑制。TAK1活性的这种抑制阻碍了IKK和MAPK的磷酸化。通过抑制IKK使IκBα去磷酸化并防止多聚泛素化来稳定IκBα，从而减少NF-κB的激活。这一系列事件最终导致促炎酶和细胞因子的产生减少，有效减轻了ALI中的炎症反应。在A549细胞中，MTX减少了LPS诱导的IκBα的K48连接的多聚泛素化和TRAF6的K63连接的多聚泛素化。该过程可通过NEDD8的过表达逆转。此外，用蛋白酶体抑制剂MG-132处理可恢复被MTX抑制的IκBα的多聚泛素化。
这些发现证实了Cul1/5类泛素化在ALI中的重要作用，并表明MTX可能是一种有前途的ALI治疗药物。
