Active components and potential mechanisms of Wuzhuyu decoction (吴茱萸汤) in the treatment of ethanol-induced acute gastric mucosal injury: a network pharmacology and experimental verification

doi:10.19852/j.cnki.jtcm.2026.03.007

Abstract

Abstract:

OBJECTIVE: To investigate the underlying mechanisms and active components of Wuzhuyu decoction (吴茱萸汤, WD) in alleviating ethanol-induced acute gastric mucosal injury (GMI) using an integrated approach of network pharmacology and experimental verification.

METHODS: Sprague-Dawley rats were randomly divided into six groups: control (Con), model (Mod), bismuth potassium citrate (BPC), WD at low (WD-L), medium (WD-M), and high (WD-H) doses. Following seven days of continuous intragastric administration of the respective treatments, an ethanol-induced gastric mucosal injury model was established in all groups except the control group by oral gavage of anhydrous ethanol. The gastric mucosal injury index was evaluated, and pathological changes were assessed viahematoxylin and eosin (HE) staining. Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were measured by enzyme-linked immunosorbent assay (ELISA). The chemical composition was identified by ultra-performance liquid chromatography-tandem mass spectrometry. Active compounds were screened using the Swiss-absorption, distribution, metabolism, and excretion database, and their potential targets were predicted using the Swiss Target Prediction database and bioinformatics annotation database for molecular mechanism. Simultaneously, disease targets related to GMI were retrieved from the online mendelian inheritance in man and GeneCards databases. A protein-protein interaction (PPI) network was constructed, and functional enrichment analyses of gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed using the Metascape database. Key predictions from the network pharmacology analysis were subsequently verified through animal experiments. Protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Cleaved Caspase-3, and Cleaved Caspase-9 were analyzed by Western blot. Finally, molecular docking was performed using AutoDock Vina to investigate the interactions between the active components and core targets.

RESULTS: WD treatment significantly reduced the gastric mucosal injury index and the levels of TNF-α, IL-1β, MDA, while it increased the activities of SOD and GSH-Px. Histopathological examination revealed marked improvement in gastric tissue morphology. A total of 145 compounds were identified in WD. Network pharmacology analysis identified 440 overlapping targets between WD and GMI. GO and KEGG enrichment analyses highlighted the apoptosis signaling pathway as a key mechanism for WD's protective effect against ethanol-induced GMI. Experimental validation demonstrated that WD treatment reduced the apoptosis of gastric mucosal epithelial cells, promoted the expression of Bcl-2, and inhibited the expression of Bax, Cleaved Caspase-3 and Cleaved Caspase-9. Molecular docking results indicated that dehydroevodiamine, rutaecarpine, evodiamine, hexahydrocurcumin, and isorhamnetin are potential active components in WD that contribute to the inhibition of apoptosis.

CONCLUSIONS: WD alleviates ethanol-induced acute GMI, at least in part, by inhibiting the apoptosis. The primary active components responsible for this effect are dehydroevodiamine, rutaecarpine, evodiamine, hexahydrocurcumin, and isorhamnetin.

Key words: ethanol-induced acute gastric mucosal injury, active components, mechanism, apoptosis, network pharmacology, Wuzhuyu decoction

HUANG Huahua, TU Mengzhen, XU Jie, WU Xuebing, CHEN Qiaofeng. Active components and potential mechanisms of Wuzhuyu decoction (吴茱萸汤) in the treatment of ethanol-induced acute gastric mucosal injury: a network pharmacology and experimental verification[J]. Journal of Traditional Chinese Medicine, 2026, 46(3): 617-628.

Figures/Tables 6

Figure 1 Protective effects of WD on ethanol-induced GMI A: macroscopic observation of gastric mucosal tissue; B: comparison of the gastric mucosal damage index; C: histopathological changes in gastric mucosa observed via HE staining (× 100): local defects (green arrow), atrophy of glands in the lamina propria (red arrow), and capillary dilation and congestion (yellow arrow); D: comparison of TNF-α and IL-1β; E: comparison of MDA; F: comparison of SOD, and GSH-Px; A1, C1: Con ; A2, C2: Mod; A3, C3: BPC; A4, C4: WD-L; A5, C5: WD-M; A6, C6: WD-H. Con and Mod groups treated with distilled water; BPC group treated with bismuth potassium citrate (0.36 g/kg); WD-L, WD-M, and WD-H groups treated with WD decoctions 4.5, 9 and 18 g/kg, respectively. Administration was performed once daily for 7 consecutive days. Con: control; Mod: model; BPC: bismuth potassium citrate; WD-L: WD at low doses; WD-M: WD at medium doses; WD-H: WD at high doses; HE: hematoxylin and eosin; TNF-α: tumor necrosis factor-alpha; IL-1β: interleukin-1 beta; MDA: malondialdehyde; SOD: superoxide dismutase; GSH-Px: glutathione peroxidase. Data are presented as mean ± standard deviation (n = 8). Group comparisons were performed using one-way analysis of variance. aP < 0.01, vs the Con group; bP < 0.01, cP < 0.05, vs the Mod group.

Figure 2 Results of the UPLC-MS/MS A: TIC in positive ion mode (n = 3); B: TIC in negative ion mode (n = 3); C: distribution chart of component classifications; D: top 15 component content in positive ion mode; E: top 15 component content in negative ion mode. UPLC-MS/MS: ultra-performance liquid chromatography-tandem mass spectrometry; TIC: total ion chromatogram.

Figure 3 Results of network analysis A: Venn diagram; B: Top 15 targets; C: GO enrichment analysis; D: KEGG enrichment analysis. AKT1: AKT serine/threonine kinase 1; IL6: interleukin 6; TNF: tumor necrosis factor; ACTB: actin beta; TP53: tumor protein p53; ALB: albumin; IL1B: interleukin 1 beta; STAT3: signal transducer and activator of transcription 3; EGFR: epidermal growth factor receptor; CTNNB1: catenin beta 1; BCL2: B-cell lymphoma 2; CASP3: Caspase-3; HIF1A: hypoxia inducible factor 1 subunit alpha; SRC: SRC proto-oncogene, non-receptor tyrosine kinase; JUN: Jun proto-oncogene, AP-1 transcription factor subunit; GO: gene ontology; KEGG: kyoto encyclopedia of genes and genomes.

Figure 4 Apoptosis of gastric mucosal cells and expression of relevant proteins A: TUNEL staining of gastric mucosal cells (×100); A1-A6: nuclear staining images; A7-A12: apoptotic cell labeling images; A13-A18: merged images; A1, A7, A13: Con; A2, A8, A14: Mod; A3, A9, A15: BPC; A4, A10, A16: WD-L; A5, A11, A17: WD-M; A6, A12, A18: WD-H; B: effects of WD on gastric mucosal cell apoptosis; C: Western blot bands of relevant proteins; D: expression of Bcl-2; E: expression of Bax; F: expression of Cleaved Caspase-3; G: expression of Cleaved Caspase-9. Con and Mod groups treated with distilled water; BPC group treated with bismuth potassium citrate (0.36 g/kg); WD-L, WD-M, and WD-H groups treated with WD decoctions 4.5, 9 and 18 g/kg, respectively. Administration was performed once daily for 7 consecutive days. Con: control; Mod: model; BPC: bismuth potassium citrate; WD-L: WD at low doses; WD-M: WD at medium doses; WD-H: WD at high doses; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; Bcl-2: B-cell lymphoma-2; Bax: Bcl-2-associated X protein. Data are presented as mean ± standard deviation (n = 8). Group comparisons were performed using one-way analysis of variance. aP < 0.01, cP < 0.05, vs the Con group; bP < 0.01, vs the Mod group.

Table 1 Pocket coordinates and grid box sizes

Target gene	PDB ID	Protein pocket coordinates	Grid box size
BCL2	8HTS	center_x = 7.647, center_y = 11.371, center_z = 14.514	size_x = 41.0, size_y = 31.0, size_z = 38.0
CASP3	2DKO	center_x = 30.952, center_y = 19.826, center_z = 49.131	size_x = 51.0, size_y = 52.0, size_z = 59.0

Table 2 Results of molecular docking

PubChem CID	Compound	Target protein	PDB ID	Affinity (kcal/mol)
445858	(E)-ferulic acid	BCL2	8HTS	－6.2
445858	(E)-ferulic acid	CASP3	2DKO	－5.7
168114	[8]-gingerol	BCL2	8HTS	－6.1
168114	[8]-gingerol	CASP3	2DKO	－6.2
5317587	[6]-gingerdiol 3,5-diacetate	BCL2	8HTS	－5.9
5317587	[6]-gingerdiol 3,5-diacetate	CASP3	2DKO	－5.6
73337	Magnoflorine	BCL2	8HTS	－6.9
73337	Magnoflorine	CASP3	2DKO	－6.8
5281654	Isorhamnetin	BCL2	8HTS	－7.5
5281654	Isorhamnetin	CASP3	2DKO	－6.9
9796015	[6]-dehydrogingerdione	BCL2	8HTS	－5.9
9796015	[6]-dehydrogingerdione	CASP3	2DKO	－5.8
5318039	Hexahydrocurcumin	BCL2	8HTS	－6.5
5318039	Hexahydrocurcumin	CASP3	2DKO	－7.5
11068834	Octahydrocurcumin	BCL2	8HTS	－6.4
11068834	Octahydrocurcumin	CASP3	2DKO	－7.3
932	Naringenin	BCL2	8HTS	－7.0
932	Naringenin	CASP3	2DKO	－6.7
442793	[6]-gingerol	BCL2	8HTS	－6.0
442793	[6]-gingerol	CASP3	2DKO	－5.8
1548943	Capsaicin	BCL2	8HTS	－7.2
1548943	Capsaicin	CASP3	2DKO	－6.6
107982	Dihydrocapsaicin	BCL2	8HTS	－6.9
107982	Dihydrocapsaicin	CASP3	2DKO	－6.2
65752	Rutaecarpine	BCL2	8HTS	－8.4
65752	Rutaecarpine	CASP3	2DKO	－7.7
442088	Evodiamine	BCL2	8HTS	－8.1
442088	Evodiamine	CASP3	2DKO	－7.8
9817839	Dehydroevodiamine	BCL2	8HTS	－8.5
9817839	Dehydroevodiamine	CASP3	2DKO	－7.7

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