Co-fermentation of honeysuckle-Cassia seeds by Lactobacillus acidophilus and Bacillus subtilis: A new approach to attenuate alcohol-induced acute gastric mucosal damage and modulate immune response.

Alcohol-induced acute gastric mucosal damage (AGMD) remains a significant health concern, driven by oxidative stress and inflammatory responses. Current therapeutic approaches (e.g., acid-suppressive agents, mucosal protectants) are limited by side effects and suboptimal bioavailability, while conventional extraction of herbal medicines (e.g., Honeysuckle-Cassia seeds) yields low bioactive compound solubility. This study explored the potential of Lactobacillus acidophilus and Bacillus subtilis co-fermented Honeysuckle-Cassia seed extracts in mitigating AGMD. In vitro antioxidant assays revealed that mixed bacterial fermentation extract (MBF) exhibited superior scavenging activity against superoxide anion (86.14 %), ABTS (88.11 %), and DPPH (42.96 %) radicals compared to unfermented aqueous extract (AE) and single-strain fermented extract (LAF). In vivo experiments in ethanol-induced AGMD mice showed that MBF significantly restored gastric mucosal redox balance, reducing MDA levels by 60.92 % and enhancing SOD activity and GSH content. Mechanistically, MBF suppressed neutrophil infiltration (MPO ↓61.36 %) and pro-inflammatory cytokines (IL-1β, TNF-α, IL-6), while upregulating tight junction proteins (ZO-1, Claudin-1, Occludin) to protect mucosal integrity. These protective effects were mainly mediated by modulation of the MAPK signaling axis-specifically, inhibiting JNK/p38 and activating ERK-thereby enhancing antioxidant defenses and maintaining tight junction integrity. These findings indicate that probiotic fermentation significantly enhances the therapeutic potential of herbal formulations. Importantly, given its safety and efficacy, MBF may be further developed as a functional food, nutraceutical, or adjunctive dietary therapy to prevent alcohol-related gastric injury, offering a sustainable and food-based preventive strategy.