Flavonoids from Apios americana Medikus Leaves Protect RAW264.7 Cells against Inflammation via Inhibition of MAPKs, Akt-mTOR Pathways, and Nfr2 Activation

Figure 1

HPLC elution profiles of compounds in Apios americana Medik leaves and effects of ALE on LPS-induced cytotoxicity and inflammation in RAW264.7 cells. (a) The liquid chromatography profile of ALE in Apios americana Medik leaves. The peak numbers were labeled according to the retention times. (b) RAW264.7 cell viability was measured by the MTT method after treatment with ALE at different concentrations for 24 h with/without LPS inducement (). (c) Cell proliferation assay via BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488. (d) Morphological changes of RAW264.7 cells after ALE treatment (). (e, f) Cell apoptosis, NO staining with Hoechst 33258, and DAF-FMDA in the presence of LPS and ALE (). (g) The quantitative data of (f). (h) NO production in cell culture medium according to the Griess reagent method (). (i) Intracellular concentration of NO according to the Griess reagent method quantified by total protein concentration (). ALE-treated cells were inoculated in different concentrations of ALE for 24 hours, and then, 250 ng/mL LPS was added for a total of 12 hours. Cells without LPS and ALE were used as a negative control group. Cells treated with LPS alone were used as a model group. Images were captured with a fluorescence microscope in the same settings. All the fluorescence images were quantified in the whole field with the background removed and represented by normalized fluorescence (-axes) via Image-Pro Plus 6.0 (). Significance analysis was carried out according to the one-way ANOVA test, and different letters in figures mean statistically significant differences among the groups (A, B, C, and D were labeled from large to small and the same letters in columns mean statistical insignificance; otherwise, they mean statistical significance ()).