Targeted inhibition of METTL3 reprograms alveolar echinococcosis progression: attenuating ferroptosis by downregulating ACSL4 and reversing macrophage M2 polarization

Background: Alveolar echinococcosis (AE) is a severe parasitic disease caused by larval-stage Infection of Echinococcus multilocularis (Em), characterized by high mortality and recurrence rates. The interplay between Ferroptosis and macrophage polarization is critically implicated in sustaining parasitic Infection and disease progression. This study aims to investigate the role of acyl-CoA synthetase long-chain family member 4 (ACSL4) in AE pathogenesis, with particular emphasis on its mechanistic involvement in Ferroptosis and macrophage polarization.

Methods: Differential gene expression in AE patients was analyzed using the GEO database, with ferroptosis-related genes screened and identified through GO functional enrichment analysis. Expression levels of the key target gene ACSL4 and Other ferroptosis-related genes were validated in liver tissues from AE patients and Em-infected mouse models using immunohistochemistry (IHC), quantitative Real-Time PCR (qRT-PCR), and Western blotting (WB). The METTL3-specific inhibitor STM2457 was administered to established Em-infected mouse models to delineate the impact of METTL3 inhibition on Ferroptosis and macrophage polarization and its regulatory mechanisms. In vitro models, including human normal hepatocytes (LO2) and a macrophage (RAW264.7)/hepatic stellate cell (JS1) co-culture system simulating the hepatic microenvironment, were established. Following treatment with Echinococcus antigen protein (EmP) and STM2457, intracellular Reactive Oxygen Species (ROS) levels were measured using ROS detection kits, macrophage invasive capacity was assessed, and mitochondrial alterations in macrophages were examined via transmission electron microscopy (TEM) to elucidate cellular-level mechanisms.

Results: ACSL4 and ferroptosis-related genes were significantly upregulated in liver lesions from both AE patients and Em-infected mice. Targeted intervention with the METTL3 Inhibitor STM2457 markedly downregulated ferroptosis-related gene expression, substantially alleviated hepatic fibrosis, and redirected macrophage polarization from pro-fibrotic M2 toward anti-pathogen M1 phenotypes. Further cellular-level analyses revealed that STM2457 treatment significantly reduced ROS fluorescence intensity, ameliorated characteristic Ferroptosis mitochondrial pathologies in macrophages (including mitochondrial shrinkage, reduced volume, and cristae degeneration), and enhanced macrophage invasive and migratory capacities.

Conclusion: The METTL3 Inhibitor STM2457 reverses hepatic fibrosis in AE by suppressing the ACSL4-mediated Ferroptosis pathway, redirects macrophages from M2 to M1 anti-pathogen phenotypes, and concurrently mitigates mitochondrial damage while enhancing cellular invasive functions.

Alveolar echinococcosis; Ferroptosis; Macrophage polarization.