Nuezhenoside G13 from Osmanthus fragrans fruit ameliorates Concanavalin A-induced autoimmune hepatitis by regulating the NF-κB/MAPK pathway

Abstract

Ethnopharmacological relevance: Osmanthus fragrans fruit (OFF) exhibits hepatoprotective function, and it is consumed as food and used in traditional medicine in China. Nuezhenoside G13 (G13) is present in the highest levels in OFF. Autoimmune hepatitis (AIH) is a manifestation of liver disease and seriously endangers health. However, it remains unclear whether G13 affects AIH.

Aim of the study: To clarify the effect of G13 on AIH and its exact underlying mechanism from a new perspective.

Materials and methods: We used a Concanavalin A-induced AIH mouse model and lipopolysaccharide-treated Raw264.7 cells to quantify serum biochemical indicators and confirm whether G13 exhibited protective effects in the AIH mice. Furthermore, we evaluated the effect of G13 via hematoxylin and eosin and immunohistochemical staining. We used enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction to quantify the inflammatory factors. We confirmed that G13 inhibited apoptosis via terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Molecular docking, immunofluorescence, and western blotting experiments of G13 and key proteins of the NF-κB/MAPK pathway revealed that G13 alleviated inflammation. In addition, Cell Counting Kit-8, ELISA, NO detection, and western blotting assays were performed. Finally, we used an inhibitor of the p38 MAPK to verify that G13 reduced inflammation through the NF-κB/MAPK pathway in Raw264.7 cells.

Results: The in vivo experiments revealed that G13 improved oxidative stress and apoptosis. In addition, G13 decreased the expression levels of CD4⁺, CD8⁺, F4/80⁺, and Ly6G and the secretion of inflammatory factors. Interestingly, G13 reduced the phosphorylation levels of IκBα, NF-κB, JNK, ERK1/2, and p38. Additionally, the in vitro experiments revealed that G13 alleviated inflammation through the NF-κB/MAPK pathway in lipopolysaccharide-treated Raw264.7 cells. Furthermore, molecular docking demonstrated that the binding fraction of G13 with these proteins was high.

Conclusion: G13 suppressed oxidative stress, apoptosis, and inflammation in a Concanavalin A-induced AIH mouse model. Furthermore, G13 exerted its effect through the NF-κB/MAPK pathway.

Keywords: Apoptosis; Inflammation; Liver injury; NF-κB/MAPK pathway; Oxidative stress.