Cystic fibrosis transmembrane conductance regulator prevents ischemia/reperfusion induced intestinal apoptosis via inhibiting PI3K/AKT/NF-κB pathway

Figure 1 Apoptosis was induced in Caco2 cells after hypoxia/reoxygenation treatment. A: Cell viability was tested using the CCK8 kit on different times after the hypoxia/reoxygenation (H/R) treatment, ^aP < 0.01 and ^bP < 0.001 vs control (n = 3); B: The quantification data of apoptosis tested by the TUNEL at different times after the H/R treatment, ^bP < 0.001 vs control (n = 3); C: Representative Western blotting and quantification data for cell apoptotic proteins Bcl-2 and Bax at different times after the H/R treatment. Data are presented as mean ± SD, ^aP < 0.01 and ^bP < 0.001 vs control (n = 3). NS: No significant difference; H/R: Hypoxia/reoxygenation.

Figure 2 Cystic fibrosis transmembrane conductance regulator can protect the Caco2 cells from apoptosis induced by the hypoxia/reoxygenation. A: Representative Western blotting and quantification data for cystic fibrosis transmembrane conductance regulator (CFTR) after the hypoxia/reoxygenation (H/R) treatment, ^bP < 0.001 vs control (n = 3); B: Representative Western blotting and quantification data for CFTR after the LV-CFTR transfection, ^bP < 0.001 vs control (n = 3); C: Cell viability was tested using the CCK8 kit after the LV-CFTR transfection, ^bP < 0.001 vs control (n = 3); D: Representative Western blotting and quantification data for cell apoptotic proteins Bcl-2 and Bax after the LV-CFTR transfection. Data are presented as mean ± SD. ^bP < 0.001 vs control (n = 3). CFTR: Cystic fibrosis transmembrane conductance regulator; H/R: Hypoxia/reoxygenation.

Figure 3 PI3K/AKT/NF-κB pathway was involved in hypoxia/reoxygenation-induced apoptosis. A: Representative western blotting and quantification data for PI3K, p-AKT^ser473, p-AKT^Thr308, NF-κB P65, and β-actin at different time points after hypoxia/reoxygenation (H/R) treatment. ns, no significant difference, ^aP < 0.01 and ^bP < 0.001 vs control (n = 3); B: Representative images of immunofluorescence staining for NF-κB P65 in Caco2 cells before (Con) and after the H/R treatment at different time, Bars = 100-μm (n = 3); C: Representative Western blotting and quantification data for cell apoptotic proteins Bcl-2, and Bax after NF-κB inhibitor BAY11, AKT activator IGF-1 and PI3K activator 740 Y-P treatment, ^aP < 0.01 and ^bP < 0.001 vs control (n = 3); D: Representative Western blotting and quantification data for NF-κB P65 after AKT activator IGF-1 and PI3K activator 740 Y-P treatment, ^aP < 0.01 and ^bP < 0.001 vs control (n = 3); E: Representative Western blotting and quantification data for PI3K, p-AKT^ser473, and p-AKT^Thr308, after NF-κB inhibitor BAY11 treatment. Data are presented as mean ± SD. ^bP < 0.001 vs control (n = 3). NS: No significant difference.

Figure 4 Cystic fibrosis transmembrane conductance regulator protected the Caco2 cells from hypoxia/reoxygenation-induced apoptosis by PI3K/AKT/NF-κB pathway. A: Representative images of immunofluorescence staining for NF-κB P65 in Caco2 cells after the LV-cystic fibrosis transmembrane conductance regulator (CFTR) transfection (n = 3); B: Representative western blotting and quantification data for PI3K, p-AKT^ser473, p-AKT^Thr308, and NF-κB P65 after LV-CFTR transfection, ^aP < 0.01 and ^bP < 0.001 vs control (n = 3); C: Cell viability was tested using the CCK8 kit after LV-CFTR transfection and treatment with NF-κB activator Betulinic acid (BA), AKT inhibitor GSK690693 and PI3K inhibitor LY294002, ^bP < 0.001 vs control (n = 3); D: Representative Western blotting and quantification data for cell apoptotic proteins Bcl-2 and Bax after LV-CFTR transfection and treatment with NF-κB activator BA, AKT inhibitor GSK690693 and PI3K inhibitor LY294002. Data are presented as mean ± SD. ^bP < 0.001 vs control (n = 3).

Figure 5 Cystic fibrosis transmembrane conductance regulator protects the intestinal cell from apoptosis induced by ischemia/ reperfusion by inhibiting PI3K/AKT/NF-κB pathway in vivo. A: Representative Western blotting and quantification data for cell apoptotic proteins Bcl-2 and Bax at different time after the ischemia/reperfusion (I/R) treatment, ^bP < 0.001 vs control (n = 3); B: Representative Western blotting and quantification data for PI3K, p-AKT^ser473, p-AKT^Thr308, and NF-κB P65 at different time after I/R treatment, ^aP < 0.01 and ^bP < 0.001 vs control (n = 3); C: Representative Western blotting and quantification data for cell apoptotic proteins Bcl-2 and Bax after the FSK treatment in I/R treatment animal model, ^aP < 0.01 and ^bP < 0.001 vs control (n = 3); D: Representative Western blotting and quantification data for PI3K, p-AKT^ser473, p-AKT^Thr308, and NF-κB P65 after FSK treatment in the I/R treatment animal model. Data are presented as mean ± SD. ^aP < 0.01 and ^bP < 0.001 vs control (n = 3).