Huanglian muxiang decoction alleviates FTH-mediated ferroptosis in colitis by inhibiting the PI3K-AKT pathway

Abstract

Background: Dysregulated iron metabolism and subsequent ferroptosis play a critical role in the pathogenesis of ulcerative colitis (UC), highlighting iron homeostasis as a therapeutic target. Huanglian muxiang decoction (HMD), a traditional Chinese herbal formula, is widely utilized for gastrointestinal disorders such as colitis. However, its underlying molecular mechanisms remain unclear.

Purpose: To identify the active compounds in HMD and elucidate its molecular mechanisms of action in alleviating UC, specifically concerning iron metabolism and ferroptosis.

Study design: Mice were given drinking water containing 3 % dextran sulfate sodium (DSS) for a period of 7 days. After a 3-day interval, mice were randomly assigned to treatment groups and orally administered HMD (4.29, 8.58, or 17.16 g/kg) or sulfasalazine (SASP; 250 mg/kg) once daily for 7 days. Additionally, intestinal-specific FTH knockout mice (FTH^flox/flox; Villin-Cre) were treated with low-dose HMD (4.29 g/kg) using the same protocol. Network pharmacology was employed to predict potential therapeutic targets of HMD against UC, followed by validation in both in vivo and in vitro models.

Methods: The active compounds in HMD were identified using UHPLC-HRMS. A mouse model of colitis was employed to evaluate the effects of HMD on disease symptoms (body weight, fecal occult blood, survival rate, disease activity index), colon length, inflammation, histopathology, and tight junction protein expression (Occludin, ZO-1). Network pharmacology and functional analyses were used to predict involved signaling pathways. Mechanistic investigations included assessments of mitochondrial structure, iron overload, and the levels of ferroptosis-related proteins (GPX4, FTH). The critical role of FTH was further validated using intestinal-specific FTH knockout mice. Additionally, co-immunoprecipitation (co-IP), cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR) analyses were performed to explore the PI3K-AKT-FTH pathway, while immunoprecipitation-mass spectrometry (IP-MS) was used to identify potential intermediary effectors.

Results: Multiple active compounds, including alkaloids, flavonoids, and phenylpropanoids, were identified in HMD. HMD treatment significantly alleviated colitis symptoms, improved intestinal integrity, and increased tight junction protein expression, with a low dose showing effects comparable to SASP. Mechanistically, HMD was found to regulate iron metabolism and inhibit ferroptosis via the PI3K-AKT pathway, as evidenced by restored mitochondrial structure, reduced iron overload, and increased expression of GPX4 and FTH. Intestinal-specific knockout of FTH aggravated colitis and attenuated the protective effects of HMD, confirming FTH as a key mediator. Further assays suggested that p-AKT indirectly regulates FTH, potentially through transcription factors or kinases, supporting a PI3K-AKT-FTH regulatory axis.

Conclusion: Our findings demonstrate that HMD attenuates colitis by modulating ferroptosis via the PI3K-AKT-FTH pathway.This study provides novel insights into the therapeutic mechanisms of HMD and highlights its potential as a natural remedy for UC treatment.

Keywords: FTH; Ferroptosis; Huanglian muxiang decoction; Iron accumulation; PI3K–AKT signaling pathway; Ulcerative Colitis.