To elucidate the regulatory mechanism of Dahuang Fuzi decoction (DHFZT) on hypoxia/reoxygenation (H/R)-injured intestinal epithelial cells through the solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling axis.

Twenty male Sprague-Dawley rats were randomized into DHFZT drug-containing serum (n=10) and control serum (n=10) groups. Drug-containing serum was prepared following 3-day oral gavage. Caco-2 cells were subjected to 12-hour hypoxia (1% O₂) and 2-hour reoxygenation (21% O₂) to establish the H/R model. Cell viability was quantified using CCK-8 assay. Six experimental groups were designed: Control (normal culture), H/R model, H/R+ferroptosis inhibitor Lip-1 (1μM,24h), and H/R+DHFZT serum (5%, 10%, 20%, 24h). Mitochondrial ultrastructure was analyzed by transmission electron microscopy. Oxidative stress markers (MDA, SOD, GSH), ROS, and Fe²⁺levels were measured via ELISA, DCFH-DA fluorescence, and ferrozine assay, respectively. Protein expression of HDAC3, SLC7A11, and GPX4 was assessed by Western blot.

Under transmission electron microscope, Caco-2 cells in the blank group exhibited regular morphology with abundant oval-shaped mitochondria and relatively clear mitochondrial cristae structures. In contrast, the H/R group showed disrupted cell membrane integrity, irregular surface contours, deformed cytoplasmic mitochondria, obscured mitochondrial membrane structures, and reduced or disappeared cristae. Mitochondria in the DHFZT group maintained regular morphology and orderly arrangement. Biochemical assays revealed that MDA and Fe²⁺ levels in the H/R group were significantly higher than those in the blank group (12.28±1.70 vs 9.11±0.82, q=6.47, P<0.05; 0.23±0.02 vs 0.06±0.03, q=13.21, P<0.05), while SOD and GSH levels were lower (9.09±0.41 vs 12.66±0.66, q=4.79, P<0.05; 3.33±0.22 vs 7.51±0.46, q=21.31, P<0.05). The 10% DHFZT group demonstrated reduced MDA and Fe²⁺levels compared to the H/R group (9.55±0.52 vs 12.28±1.70, q=5.57, P < 0.05; q=13.30, P<0.05) and elevated SOD and GSH levels (13.37±1.22 vs 9.09±0.41, q=5.73, P<0.05; 7.31±0.33 vs 3.33±0.22, q=20.28, P<0.05). ROS levels were significantly higher in the H/R group than in the blank group (91.67±7.37 vs 28.48±2.17, q=28.37, P<0.05), while the Lip-1 group and 10% DHFZT group showed reduced ROS levels compared to the H/R group (44.03±3.64 vs 91.67±7.37, q=21.39, P<0.05; 55.92±2.51 vs 91.67±7.37, q=16.05, P<0.05). Western blot analysis indicated that SLC7A11 and GPX4 levels in the H/R group were lower than those in the blank group (q=4.69, 5.38, P<0.05), whereas HDAC3 levels were higher (q=81.13, P<0.05). Both Lip-1 and 10% DHFZT groups exhibited upregulated SLC7A11 and GPX4 levels (q=11.05, 28.76 and q=26.79, 32.21, respectively; P<0.05) and downregulated HDAC3 levels (q=121.00 and q=59.42, P<0.05) compared to the H/R group.

Dahuang Fuzi decoction-containing serum exerts a protective effect on hypoxia-reoxygenation (H/R)-induced Caco-2 cells injury. The underlying mechanism may involve the regulation of HDAC3 expression, which subsequently activates the SLC7A11/GPX4 signaling pathway, thereby alleviating lipid peroxidation accumulation and inhibiting ferroptosis.