Ethanol-induced gastric mucosal injury is a common gastrointestinal disorder. Polysaccharides separated from herbs have been shown to be effective for ethanol-induced gastric mucosal injury, but whether the polysaccharides from
Dendrobium officinale
Kimura & Migo leaves (LDOP-1) protected mucosa from ethanol-induced injury remains unknown. Thus, the present study carried out gastric mucosal protection and the mechanism of LDOP-1 in
vivo
and
vitro
. The chemical composition of LDOP-1 was a heteropolysaccharide comprising mannose, galacturonic acid, glucose, galactose, and arabinose at a molar ratio of 2.0:1.1:0.7:0.5:0.4. Pharmacological results showed that LDOP-1 significantly reduced gastric mucosal injury score and pathological injury, improved antioxidant capacity, reduced the level of reactive oxygen species, and reversed the apoptosis of GES-1 in
vivo
and
vitro
. Research showed that LDOP-1 pretreatment upregulated the expression level of p-AMPK, LC3β, HO-1, and Beclin-1; downregulated the expression level of p-mTOR and p62; and reversed the expression level of caspase3, Bax, and Bcl-2. This study was the first to demonstrate that LDOP-1 could protect against ethanol-induced gastric mucosal injury via the AMPK/mTOR signaling pathway in
vitro
and
vivo
.
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