Mass spectrometry-based metabolomics reveal Dendrobium huoshanense polysaccharide effects and potential mechanism of N-methyl-N'-nitro-N-nitrosoguanidine -induced damage in GES-1 cells

Abstract

Ethnopharmacological relevance: Dendrobium huoshanense C. Z. Tang et S. J. Cheng is an important edible medicinal plant that thickens the stomach and intestines, and its active ingredient, polysaccharide, can have anti-inflammatory, immunoregulatory, and antitumor effects. However, the gastroprotective effects and potential mechanisms of Dendrobium huoshanense polysaccharides (DHP) remain unclear.

Aim of the study: An N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced human gastric mucosal epithelial cells (GES-1) damage model was used in this research, aiming to investigate whether DHP has a protective effect on MNNG-induced GES-1 cell injury and its underlying mechanism based on the combination of multiple methods.

Materials and methods: DHP was extracted using water extraction and alcohol precipitation methods, and the proteins were removed using the Sevag method. The morphology was observed using scanning electron microscopy. A MNNG-induced GES-1 cell damage model was developed. Cell viability and proliferation of the experimental cells were investigated using a cell counting kit-8 (CCK-8). Cell nuclear morphology was detected using the fluorescent dye Hoechst 33342. Cell scratch wounds and migration were detected using a Transwell chamber. The expression levels of apoptosis proteins (Bcl-2, Bax, Caspase-3) in the experimental cells were detected by Western blotting. Ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was performed to investigate the potential mechanism of action of DHP.

Results: The CCK-8 kit analysis showed that DHP increased GES-1 cell viability and ameliorated GES-1 cell injury by MNNG. In addition, scratch assay and Transwell chambers results suggested that DHP improved the MNNG-induced motility and migration ability of GES-1 cells. Likewise, the results of the apoptotic protein assay indicated that DHP had a protective effect against gastric mucosal epithelial cell injury. To further investigate the potential mechanism of action of DHP, we analyzed the metabolite differences between GES-1 cells, GES-1 cells with MNNG-induced injury, and DHP + MMNG-treated cells using UHPLC-HRMS. The results indicated that DHP upregulated 1-methylnicotinamide, famotidine, N4-acetylsulfamethoxazole, acetyl-L-carnitine, choline and cer (d18:1/19:0) metabolites and significantly down-regulated 6-O-desmethyldonepezil, valet hamate, L-cystine, propoxur, and oleic acid.

Conclusions: DHP may protect against gastric mucosal cell injury through nicotinamide and energy metabolism-related pathways. This research may provide a useful reference for further in-depth studies on the treatment of gastric cancer, precancerous lesions, and other gastric diseases.

Keywords: Apoptosis; Cell metabolomics; Dendrobium huoshanense polysaccharide (DHP); Human gastric mucosal epithelial cell lines (GES-1); N-methyl-N′- nitro-N-nitrosoguanidine (MNNG).