Figures index : Effect of n-Hexane Extract of Camellia Oleifera Fruit Hull on Inflammatory Responses in LPS-Induced RAW264.7 Cells : Science and Education Publishing

Figures index

Journal of Food and Nutrition Research. 2023, 11(4), 301-309 doi:10.12691/jfnr-11-4-4

Figure 1. Effects of n-hexane extract of C. oleifera fruit hull on NO production and cell viability in LPS-induced RAW264.7 cells. Cells were pretreated with the indicated concentrations of n-hexane extract (0, 12.5, 25, 50, and 100 µg/mL) for 2 h following treatment of LPS (1 µg/mL) for 24 h. Cell supernatants were collected for the detection of NO production by Griess assay (a). Cell viability was evaluated by MTT reagent (b). Cell morphology and density were pictured and identified through microscope (c). The data are presented as means ± SD of three separate experiments. ****p<0.0001 compared with control group, ####p<0.0001 compared with LPS group

Figure 2. Effects of n-hexane extract of C. oleifera fruit hull on the release of proinflammatory factors in LPS-induced RAW264.7 cells. Cells were pretreated with the indicated concentrations of n-hexane extract (0, 25, 50, 100, and 200 µg/mL) for 2 h following treatment of LPS (1 µg/mL) for 24 h. Cell supernatants were collected and the releases of PGE2 (a), TNFα (b), IL-6 (c), and IL-1β (d) were determined using ELISA. The data are presented as means ± SD of three independent experiments. *p<0.05, ****p<0.0001 compared with control group, #p<0.05 compared with LPS group

Figure 3. Effects of n-hexane extract of C. oleifera fruit hull on the expression of inflammatory proteins in LPS-induced RAW264.7 cells. Cells were pretreated with the indicated concentrations of n-hexane extract (0, 25, 50, 100, and 200 µg/mL) for 2 h following treatment of LPS (1 µg/mL) for 24 h. Cell lysates were obtained and the protein expressions of iNOS, COX2, and GAPDH were analyzed by western blotting. (a). The representative results of western blotting bands. (b-c). The relative protein expressions of iNOS (b) and COX2 (c) were quantified using image J and normalized to GAPDH. The data are presented as means ± SD of three independent experiments. ****p<0.0001 compared with control group, ##p<0.01, ###p<0.001, ####p<0.0001 compared with LPS group

Figure 4. Effects of n-hexane extract of C. oleifera fruit hull on the mRNA level of proinflammatory genes in LPS-induced RAW264.7 cells. Cells were pretreated with the indicated concentrations of n-hexane extract (0, 25, 50, 100, and 200 µg/mL) for 2 h following treatment of LPS (1 µg/mL) for 24 h. Cell lysates were obtained for RNA extraction and the mRNA levels of iNOS, COX2, TNFα, IL-1β, IL-6, and GAPDH were analyzed using fast PCR. (a). The representative results of agarose gel eletrophoresis bands. (b-f). The relative mRNA levels of iNOS (b), COX2 (c), TNFα (d), IL-1β (e), and IL-6 (f) were quantified using image J and normalized to GAPDH. The data are presented as means ± SD of three independent experiments. *p<0.05, **p<0.01, ****p<0.0001 compared with control group, ##p<0.01, ###p<0.001, ####p<0.0001 compared with LPS group

Figure 5. Effects of n-hexane extract of C. oleifera fruit hull on the phosphorylation level of MAPKs in LPS-induced RAW264.7 cells. Cells were pretreated with n-hexane extract (200 µg/mL) for 2 h following treatment of LPS (1 µg/mL) for the indicated times (1, 2, 6 h). Cell lysates were collected and protein expression and phosphorylation of ERK, P38, and JNK were analyzed using western blotting. (a). The representative results of western blotting bands. (b-d). The relative phosphorylation levels of ERK (b), P38 (c), and JNK (d) were quantified using image J and normalized to that of the unphosphorylated form. The data are presented as means ± SD of three independent experiments. *p<0.05, ****p<0.0001

Figure 6. Effects of n-hexane extract of C. oleifera fruit hull on the activation and nuclear translocation of NF-κB in LPS-induced RAW264.7 cells. Cells were pretreated with n-hexane extract (200 µg/mL) for 2 h following treatment with LPS (1 µg/mL) for 6 h. Cell supernatants were discarded and cells were used for the detection of NF-κB nuclear translocation via immunofluorescence. The representative graphs of immunofluorescence were presented from three separate experiments