Mitoxantrone attenuates lipopolysaccharide-induced acute lung injury via inhibition of NEDD8 activating enzyme.

BACKGROUND

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.

PURPOSE

This study aimed to investigate the inhibitory effects and underlying mechanisms of MTX against LPS-induced ALI.

METHODS

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.

RESULTS

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.

CONCLUSIONS

These findings confirm the essential role of Cul1/5 neddylation in ALI and suggest that MTX could be a promising therapeutic agent for ALI.