Targeting PHKA2 by Thymol alleviates sepsis induced cardiomyocyte pyroptosis via FOXA1/KLF4-mediated macrophage...

Sepsis-induced crosstalk between macrophages and cardiomyocytes is a critical pathogenic mechanism underlying cardiac dysfunction. However, clinically effective pharmacological interventions to mitigate the associated cytokine storm remain limited.

In this study, we aimed to investigate the role of thymol, a natural small-molecule bioactive compound, in regulating macrophage function in septic cardiomyopathy (SCM).

The following methodologies were employed to investigate the cardioprotective effects of thymol in sepsis and its underlying mechanisms. For in vivo studies, a septic mouse model was established via cecal ligation and puncture (CLP), followed by thymol administration. Cardiac function and macrophage responses were assessed using survival analysis, echocardiography, hematoxylin and eosin (HE) staining, TUNEL assay, flow cytometry, and immunofluorescence staining. In vitro, a co-culture system of RAW264.7 macrophages and HL-1 cardiomyocytes stimulated with lipopolysaccharide (LPS) was used to examine macrophage‑cardiomyocyte interactions. Mechanistic exploration involved proteomic sequencing, western blotting, quantitative polymerase chain reaction (qPCR), and biochemical analyses. Key signaling pathways were further validated by chromatin immunoprecipitation (ChIP) and co‑immunoprecipitation (Co‑IP). Additionally, Krüppel-like factor 4 (Klf4) knockout mice, RNA sequencing (RNA-Seq), liquid chromatography‑tandem mass spectrometry (LC‑MS/MS)‑based proteomics, molecular docking, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and surface plasmon resonance (SPR) were utilized to identify the direct molecular target of thymol and elucidate its regulatory role in downstream signaling pathways.

Thymol treatment ameliorated cardiac dysfunction and promoted macrophage polarization toward the M2 phenotype. Furthermore, thymol reduced the secretion of tumor necrosis factor-α (TNF-α) by macrophages, thereby inhibiting cardiomyocyte pyroptosis. Mechanistically, RNA-Seq analysis revealed that thymol upregulated KLF4 expression, and macrophage-specific deletion of Klf4 abolished the beneficial effects of thymol on heart failure. We also identified phosphorylase kinase alpha 2 (PHKA2) as a direct binding target of thymol at GLU 546. This interaction enhanced PHKA2 kinase activity, leading to phosphorylation of the downstream transcription factor forkhead box A1 (FOXA1). Importantly, phosphorylated PHKA2 translocated into the nucleus and bound to the promoter region of Klf4, facilitating its transcriptional activation.

Our findings demonstrate that thymol alleviates the cytokine storm in SCM, highlighting its potential as a therapeutic candidate for this condition.