A novel mechanism of Reduning injection in sepsis treatment: Targeting inflammatory kinases TBK1 and IKKβ

Background: Sepsis-induced systemic inflammation, characterized by immune dysregulation and cytokine storms, presents significant therapeutic challenges. Reduning injection (RDN), a Traditional Chinese Medicine formulation, demonstrates clinical efficacy in sepsis management, yet its molecular mechanisms remain elusive.

Purpose: This study aimed to unravel RDN's immunomodulatory mechanisms and identify its core effective components targeting key inflammatory signaling networks in sepsis.

Methods: Initially, six complementary in vitro hyperinflammation models (macrophages, endothelial, epithelial, and intestinal barrier cells) were established, with their transcriptomes integrated with patient-derived septic data to prioritize canonical pathways. Transcriptome profiling of RDN, its 14 phytochemicals, and dexamethasone (DEX) was then performed to analyze pathway enrichment and identify key components. In vitro mechanistic validation included enzyme-linked immunosorbent assay (ELISA) for IL-6 inhibition screening; ADP-Glo™ Kinase Assay for IKKβ/TBK1 inhibition; Western blot to assess phosphorylation dynamics of tank-binding kinase 1 (TBK1), inhibitor of nuclear factor-kappa B kinase subunit beta (IKKβ), and nuclear factor-kappa B (NF-κB); quantitative real-time polymerase chain reaction (qRT-PCR) to confirm downregulation of canonical NF-κB targets; and molecular dynamics (MD) simulations to explore binding mechanisms. For in vivo studies, an LPS-rat sepsis model was used, with preliminary studies defining optimal LPS dose, induction timepoint, and RDN dose. Assessments on septic rats included histopathology (hematoxylin-eosin staining), cytokine profiling (ELISA), complete blood counts, transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) to uncover NF-κB pathway modulation, and qRT-PCR validation of transcriptomic changes.

Results: The clinically anchored approach effectively prioritized canonical pathways (NF-κB, TNF, and cytokine-cytokine receptor interactions) with strong translational relevance. Transcriptome profiling revealed RDN's broad pathway enrichment and identified cynaroside (CYN) as the principal effective component exerting multi-pathway anti-inflammatory effects. Mechanistically, CYN dual-inhibited TBK1 (IC₅₀: 8.9 μM) and IKKβ (IC₅₀: 23.3 μM), suppressing NF-κB signaling and cytokine production in macrophages. MD simulations showed stable IKKβ-CYN and TBK1-CYN complexes, with CYN occupying catalytic pockets via hydrogen bonds with key residues and minimal binding site fluctuations. In LPS-induced septic rats, RDN and CYN mitigated multi-organ injury, reduced systemic inflammation (decreased IL-6 and TNF-α), restored complete blood counts, and inhibited NF-κB activation.

Conclusion: This study advances understanding of TCM's multi-target immunomodulation in sepsis via a framework integrating high-dimensional data and experimental validation. CYN's dual-kinase inhibition highlights its potential as a precision therapeutic for sepsis and hyperinflammatory disorders, while reinforcing TCM's value in novel immunomodulatory drug discovery within network pharmacology and systems medicine paradigms.

Dual kinase inhibition; Multiple cellular models; NF-κB; Reduning injection; Sepsis; Transcriptome profiling.