Huotan Jiedu Tongluo Decoction targets Nrf2-mediated macrophage autophagy to inhibit ferroptosis and reduce atherosclerotic lesions

Abstract

Background

Atherosclerosis (AS) serves as a fundamental pathological basis for cardiovascular diseases (CVDs), with its progression intimately linked to ferroptosis-mediated disruptions in intracellular iron homeostasis and lipid peroxidation. Huotan Jiedu Tongluo Decoction (HTJDTLD), an effective compound formulafor treating AS based on traditional Chinese medicine (TCM) theory, has exhibited remarkable efficacy in stabilizing plaques and delaying disease progression. However, its underlying mechanism remains unclear.

Purpose

This study sought to clarify the therapeutic function and molecular pathways of HTJDTLD in addressing AS, with particular emphasis on its ability to inhibit ferroptosis by influencing macrophage autophagy.

Materials

We employed UPLC-HRMS to analyze HTJDTLD constituents, identified key ingredients. An atherosclerotic model was established using ApoE^-/- mice, alongside an in vitro foam cell model derived from RAW264.7 macrophages. A combination of histopathological staining, biochemical assays, dihydroethidium (DHE) staining, transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence (IF), western blot (WB) analysis, and assessments of lipid peroxidation and mitochondrial membrane potential using BODIPY 581/591 C11 and JC-1 fluorescent probes was employed to systematically investigate the molecular mechanisms of HTJDTLD against AS by activating autophagy and inhibiting ferroptosis. The Nrf2 inhibitor ML385 was employed to validate the core mechanism.

Results

UPLC-HRMS analysis revealed that HTJDTLD contains 537 chemical components, among which 24 prototype components were detected in the blood, indicating its potential to exert pharmacological effects via bloodstream absorption. In vivo studies demonstrated that HTJDTLD activated the Nrf2 signaling pathway, thereby upregulating autophagy- and ferroptosis-related proteins, significantly improving lipid metabolism, suppressing inflammatory cytokine release, reducing iron ion accumulation, and decreasing reactive oxygen species (ROS) production in ApoE^-/- mice, ultimately enhancing plaque stability. In vitro findings corroborated that HTJDTLD exerted anti-ferroptotic effects by mitigating lipid peroxidation, restoring mitochondrial function, and engaging multiple antioxidant pathways. Mechanism studies further confirmed that inhibition of Nrf2 eliminated the protective effect of HTJDTLD on autophagy and the inhibition of ferroptosis.

Conclusion

This study represents the initial exploration of a new mechanism through which HTJDTLD reduces macrophage ferroptosis and improves AS by stimulating the Nrf2/autophagy axis. These findings establish an essential theoretical basis for utilizing TCM in the prevention and management of AS.