Abstract
Objectives
The present study aimed to investigate the protective effect of daphnetin (dap) against high glucose and high free fatty acids (HGHF) and lipopolysaccharide(LPS)-induced injury in the aforementioned co-culture system under normal control conditions (normal glucose and no exogenous FFA supplementation), and to explore its potential molecular mechanisms.
Methods
Neutrophils were isolated from the peripheral blood of healthy individuals. Subsequently, HK-2 cells and a neutrophil suspension were co-cultured at 1x10
5
cells/mL to establish the experimental model. The non-cytotoxic concentration of dap for subsequent experiments was determined by Cell Counting Kit-8 (CCK-8) assay. Heparin-binding protein (HBP) concentration was measured using dry quantitative immunofluorescence assay. Inflammatory cytokines were quantified via cytometric bead array, and cell apoptotic rate was assessed by Annexin V-FITC-based flow cytometry.
Results
Both HGHF treatment and LPS exposure significantly elevated the levels of the inflammatory mediator HBP, cytokines (TNF-α, IFN-γ, IL-1β, IL-2, IL-6 and IL-10) and cellular apoptosis, whereas dap administration markedly attenuated these inflammatory and apoptotic alterations induced by HGHF or LPS (all
P
Conclusion
In this in vitro model, dap attenuated HGHF- and LPS-induced inflammatory and apoptotic responses. Further studies are needed to assess its relevance to diabetic kidney disease.