Puerarin modulates the P62-Keap1-NRF2 pathway and enhances CA7 function to inhibit ferroptosis in ethanol-induced gastric mucosal injury

Abstract

Gastric mucosal injury caused by ethanol (EtOH) exposure is a complex pathological process involving oxidative stress, inflammation, and ferroptosis. However, the effective therapeutic strategies remain scarce. The protective mechanisms of puerarin (Pu) were investigated by a combination of in vivo, in vitro and computational approaches. Pu reduced reactive oxygen species and malondialdehyde levels, restoring oxidative balance by enhancing antioxidant enzymes, including superoxide dismutase and glutathione. These antioxidative effects were coupled with an anti-inflammatory response, as Pu decreased IL-1β and TNF-α levels while increasing IL-10. In addition to regulating oxidative stress and inflammation, Pu suppressed ferroptosis. It upregulated GPX4, reduced COX2 expression, and restored mitochondrial membrane potential. Activation of the P62-Keap1-NRF2 pathway further bolstered antioxidant defenses, mitigating the damage caused by EtOH. Notably, computational and experimental analyses identified Carbonic Anhydrase 7 (CA7) as a novel target of Pu. Pu enhanced CA7's CO₂-binding capacity, reversing EtOH-induced intracellular acidification. This acid-base regulation played a critical role in suppressing ferroptosis. Molecular dynamics simulations revealed that Pu stabilized CA7 through a unique “Puerarinization” mechanism, improving its catalytic efficiency and acid-buffering capacity. This study establishes Pu's ability to address gastric injury by targeting interconnected pathways, including oxidative stress, inflammation, ferroptosis, and acid-base regulation. The findings not only provide mechanistic insights into Pu's therapeutic effects but also highlight its potential for treating gastric mucosal injury and other diseases linked to ferroptosis and acid-base imbalance.