Zhilining formula suppresses ferroptosis in colonic epithelial cells by inhibiting ALOX15/15(S)-HPETE to repress colorectal tumorigenesis and progression.

[BACKGROUND] Ferroptosis has been established as a significant contributor to intestinal epithelial cell death, which potentially contributes to colorectal tumorigenesis and progression. Zhilining Formula (ZLN), composed of Andrographis herba (AH), Sophorae flavescentis radix (SFA), and Aucklandia radix (AR), possesses anti-inflammatory and intestinal barrier-protective effects. However, its effects and molecular mechanisms in colorectal cancer (CRC) remain unclear.

[OBJECTIVE] To investigate the effect of ZLN in CRC and elucidate its underlying molecular mechanism.

[METHODS] The therapeutic effect of ZLN on CRC was evaluated using an AOM/DSS-induced colitis to CRC progression mouse model. Time-series metabolomics mapped the metabolic network during disease progression, and characterize the regulatory effects of ZLN. Ferroptosis PCR array analysis was integrated to ascertain the mechanism by which ZLN mitigates colonic epithelial cells (CEC) ferroptosis. Further in vitro loss/gain-of-function cell and molecular biology experiments were conducted to elucidated the molecular mechanism of ZLN.

[RESULTS] ZLN significantly suppressed colorectal tumorigenesis and progression, accompanied by reduced inflammation and oxidative stress, and protected intestinal epithelial barrier in AOM/DSS-treated mice. An integrated analysis of the time-series metabolomics and ferroptosis PCR array revealed that the inhibition of lipid peroxidation induced by ALOX15/15(S)-HPETE represents a key mechanism through which ZLN reduces colonic epithelial cells (CEC) ferroptosis, thereby repressing colorectal tumorigenesis and progression. Besides, an in vitro Alox15 silencing/overexpression experiments confirmed that ZLN's inhibition of Alox15 effectively reduced CEC ferroptosis, and 15(S)-HPETE was identified as a trigger of CEC ferroptosis, with ZLN effectively counteracting its pro-ferroptosis effects.

[CONCLUSION] ZLN suppresses CEC ferroptosis by inhibiting ALOX15/15(S)-HPETE to repress colorectal tumorigenesis and progression, providing a rationale for employing ZLN as a potential therapeutic approach.