NINJ1-mediated macrophage ferroptosis impairs diabetic wound healing attenuated by Ruan Jian Qing Mai formula.

BACKGROUND

Impaired healing of diabetic foot ulcers (DFU) is a major clinical challenge. This condition is driven by unresolved inflammation and cellular dysfunction. However, the specific role of macrophage ferroptosis in this process remains poorly defined.

PURPOSE: This study aimed to investigate the contribution of macrophage ferroptosis to DFU pathogenesis. We also evaluated the therapeutic effect and underlying mechanism of the Ruan Jian Qing Mai formula (RJQM). Our investigation focused on the NINJ1-ferroptosis-inflammation axis and its role in intercellular communication.

METHODS

We combined single-cell RNA sequencing of human wound tissues with a streptozotocin-induced diabetic mouse model. The chemical composition of RJQM was characterized by HPLC-MS. For in vitro experiments, we used macrophages stimulated with high glucose and LPS to model diabetic conditions. Key markers for ferroptosis, inflammation, and signaling pathways were assessed using Western blot, qPCR, and flow cytometry. Finally, endothelial cell behavior was evaluated through migration and tube formation assays.

RESULTS

Our analysis revealed that macrophages exhibited the highest ferroptosis activity in diabetic wounds. In diabetic mice, RJQM treatment accelerated wound healing in a ferroptosis-dependent manner. We identified 87 compounds in RJQM. These compounds effectively suppressed ferroptosis by enhancing GPX4/xCT expression while reducing lipid peroxidation and iron overload. Mechanistically, RJQM inhibited a positive feedback loop involving NINJ1 and ferroptosis, and also suppressed downstream STAT3 activation. Furthermore, conditioned media from RJQM-treated macrophages promoted endothelial migration and tube formation. This pro-angiogenic effect was reversed by NINJ1 overexpression, confirming its critical role.

CONCLUSION

RJQM promotes diabetic wound repair by targeting the NINJ1-STAT3-ferroptosis pathway in macrophages. This action reduces inflammation and restores the cells' pro-angiogenic functions. Our findings support RJQM as a promising therapeutic strategy for DFU.