Sulforaphane protects intestinal epithelial cells against lipopolysaccharide-induced injury by activating the AMPK/SIRT1/PGC-1ɑ pathway

Figures & data

Figure 1. Effects of sulforaphane (SFP) on viability of Caco-2 cells exposed to LPS. (a) Cells were treated with different concentrations of LPS (0.01–4 μg/mL) for 24 h. (b) Cells were treated with different concentrations of sulforaphane (0.1–10 μM) for 24 h. (c) Cells were treated with 1 μg/mL LPS and different concentrations of sulforaphane for 24 h. (d) Cultures treated as in panel (C) were also assayed for LDH activity. Values are mean ± SD (n = 6). Difference between two groups was performed by an independent-samples t-test, *P < 0.05 vs. control group (CN); ^#P < 0.05 vs. LPS group. The difference between different concentrations of sulforaphane was performed using one-way analysis of variance

Figure 2. Effects of sulforaphane (SFP) on the monolayer barrier function of Caco-2 cells exposed to LPS. Cells were treated for 24 h with LPS (1 μg/mL) and different concentrations of sulforaphane (0.5–5 μM). (a) TEER measurements. (b) FITC-D4 flux measurements. Values are mean ± SD (n = 6). Difference between two groups was performed by an independent-samples t-test, *P < 0.05 vs. control group (CN); ^#P < 0.05 vs. LPS group. The difference between different concentrations of sulforaphane was performed using one-way analysis of variance

Figure 3. Effects of sulforaphane (SFP) on oxidative stress in Caco-2 cells induced by LPS. Cells were exposed for 24 h to LPS (1 μg/mL) and different concentrations of sulforaphane (0.5–5 μM). (a) Mitochondrial ROS levels, based on MitoSox dye oxidation. (b) Total intracellular ROS levels, based on H2DCF oxidation. (c) MDA levels. (d) H₂O₂ levels. (e) SOD activity. (f) GPx levels. (g) CAT activity. (h) T-AOC levels. Values are mean ± SD (n = 6). Difference between two groups was performed by an independent-samples t-test, *P < 0.05 vs. control group (CN); ^#P < 0.05 vs. LPS group. The difference between different concentrations of sulforaphane was performed using one-way analysis of variance

Figure 4. Effects of sulforaphane (SFP) on inflammatory injury in Caco-2 cells induced by LPS. Cells were exposed for 24 h to LPS (1 μg/mL) and different concentrations of sulforaphane (0.5–5 μM). (a) IL-1β. (b) IL-6. (c) IL-8. and (d) TNF-ɑ. Values are mean ± SD (n = 6). Difference between two groups was performed by an independent-samples t-test, *P < 0.05 vs. control group (CN); ^#P < 0.05 vs. LPS group. The difference between different concentrations of sulforaphane was performed using one-way analysis of variance

Figure 5. Effects of sulforaphane (SFP) on LPS-induced apoptosis in Caco-2 cells. Cells were exposed for 24 h to LPS (1 μg/mL) and different concentrations of sulforaphane (0.5–5 μM). (a) Levels of caspase-3 mRNA. (b) Levels of caspase-9 mRNA. (c) Activity of caspase-3. (d) Activity of caspase-9. Values are mean ± SD (n = 6). Difference between two groups was performed by an independent-samples t-test, *P < 0.05 vs. control group (CN); ^#P < 0.05 vs. LPS group. The difference between different concentrations of sulforaphane was performed using one-way analysis of variance

Figure 6. Effects of sulforaphane (SFP) on levels of p-AMPK, SIRT1, and PGC-1ɑ in LPS-treated Caco-2 cells. Cells were exposed for 24 h to LPS (1 μg/mL) and different concentrations of sulforaphane (0.5–5 μM). (a) Representative Western blot. (b-d) Quantitation of Western blots against p-AMPK, SIRT1 and PGC-1ɑ. Values are mean ± SD (n = 3). Difference between two groups was performed by an independent-samples t-test, *P < 0.05 vs. control group (CN); ^#P < 0.05 vs. LPS group. The difference between different concentrations of sulforaphane was performed using one-way analysis of variance

Figure 7. Sulforaphane (SFP) protects Caco-2 cells against LPS-induced injury by activating AMPK. Cells were exposed for 24 h to LPS (1 μg/mL) and SFP (1 μM). (a) Representative Western blot. (b-c) Relative levels of SIRT1 and PGC-1ɑ. (d) Cell viability. (e) Mitochondrial ROS levels, based on MitoSox dye oxidation. (f) IL-1β levels. Values are mean ± SD (n = 3). *P < 0.05 vs. control group (CN); ^#P < 0.05 vs. LPS group; ^$P < 0.05 vs. LPS+SFP group

Figure 8. Sulforaphane (SFP) protects Caco-2 cells against LPS-induced injury by activating the AMPK/SIRT1/PGC-1ɑ pathway. Cells were exposed for 24 h to LPS (1 μg/mL) and SFP (1 μM) in the presence or absence of a SIRT1 inhibitor (EX527) or PGC-1ɑ inhibitor (SR-18,292). (a) Cell viability. (b) Mitochondrial ROS levels, based on MitoSox dye oxidation. (c) IL-1β levels. Values are mean ± SD (n = 3). *P < 0.05 vs. control group (CN); ^#P < 0.05 vs. LPS group; ^$P < 0.05 vs. LPS+SFP group