Immunomodulatory Effect of Physalis pubescens L. Fruit Extract in RAW264.7 Cells
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Abstract:
To investigate the immunomodulatory effect of Physalis pubescens L. fruit extract (PPFE) in RAW264.7 cells, the cytotoxicity and proliferative activity were determined by the thiazole blue colorimetric method (MTT). Griess assay, enzyme-linked immunosorbent assay (ELISA), fluorescent probe technique and neutral red phagocytosis assay were used to measure the contents of nitric oxide (NO), tumor factor-α (TNF-α), IFN-γ, IL-6 and reactive oxygen species (ROS) as well as the phagocytosis rate. The changes in cell cycle and apoptosis were measured by flow cytometry, and the mRNA expression levels of cytokines such as TNF-α, IFN-γ and IL-6 were determined by fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results showed that UHPLC-Q-TOF-MS was used to determine the chemical composition of PPFE, and a total of 14 flavonoids were identified. The safe concentration of PPFE for RAW264.7 cells was in the range of 25~250 μg/mL. Compared with the blank group, the immunoenhancing effect was the strongest at 25 μg/mL, with the corresponding cell proliferation rate, content of released NO, phagocytosis rate, relative ROS level, and contents of secreted TNF-α, IFN-γ and IL-6 were 130.53%, 27.79 μmol/L, 189.88%, 137.75%, 150.54 pg/mL, 119.36 pg/mL, 15.41 pg/mL, respectively. The percentages of G0/G1 phase, S phase and G2/M phase of the cell cycle were 53.08%, 17.40%, and 29.52%, respectively. Compared with the blank group, the apoptosis rate was reduced by 52.80%, while the expressions of TNF-α, IFN-γ and IL-6 were significantly up-regulated (p<0.01) for the PPFE treated group. In summary, PPFE can enhance the immunomodulatory effect of RAW264 cells by regulating the phagocytosis, secretion of immune factors, cell cycle distribution and apoptosis, as well as up-regulating the mRNA expression levels of cytokines.
