Huangqi Guizhi Wuwu Decoction inhibits ferroptosis to improve cyclophosphamide induced immunosuppression through regulation of arachidonic acid metabolism

Abstract

Huangqi Guizhi Wuwu Decoction (HGWD) has shown laboratory efficacy in autoimmune diseases, however its effectiveness and mechanism in addressing the decline of immune function remain unclear. We first established a cyclophosphamide-induced mouse model of immunosuppression and evaluated various immune indicators to determine the efficacy of HGWD in improving the immune function of CTX-immunosuppressed mice. Next, we conducted serum non-targeted metabolomics analysis to investigate HGWD’s effects on serum differential metabolites and used KEGG pathway enrichment analysis to identify the key pathways through which HGWD improves immune function. Finally, we validated HGWD’s impact on arachidonic acid (AA) metabolism and ferroptosis. HGWD treatment significantly improves the number of immune cells, ameliorates thymus and spleen tissue pathology, and restores the immune function. Non-targeted metabolomics analysis indicated that AA metabolism was a common pathway among the control group, CTX group, and H-HGWD group. HGWD intervention resulted in downregulation of serum levels of 15(S)-HpETE, 16(R)-HETE, Prostaglandin H2, and Prostaglandin G2 in CTX-immunosuppressed mice, while upregulating the level of 12(S)-HETE. RT-qPCR and Western blot analyses revealed that HGWD intervention significantly downregulated the expressions of ALOX15, CYP2C, ALOX12, and COX1, while upregulating GPX4 expression. Furthermore, HGWD intervention reduced TUNEL-positive expression in spleen tissue, improved levels of ferroptosis-related factors (total iron, MDA, 4-HNE, GSH/GSSG), and modulated expressions of ferroptosis-related proteins (FTL, FTH, TRF, and ACSL4). Our research has confirmed the significant potential of HGWD in improving the immune function of CTX-immunosuppressed mice. Specifically, HGWD may improve immune function by regulating AA metabolism to inhibit ferroptosis.