PTEN-induced kinase 1-induced dynamin-related protein 1 Ser637 phosphorylation reduces mitochondrial fission and protects against intestinal ischemia reperfusion injury

Figure 1 Expression of proteins in the intestine after various reperfusion times. A-C: Dynamin-related protein 1, dynamin-related protein 1 Ser637 and PTEN-induced kinase 1 expression at different reperfusion time points (n = 6). ^bP < 0.01, ^cP < 0.001 compared with the sham group. DRP1: Dynamin-related protein 1; PINK1: PTEN induced kinase 1.

Figure 2 Mdivi-1 treatment ameliorates tissue injury and apoptosis in intestinal ischemia/reperfusion. A: Hematoxylin and eosin staining of the small intestine (scale bar = 100 μm); B: Chui’s pathological score for the small intestine (n = 6); C: TUNEL staining of the small intestine (scale bar = 100 μm. ^bP < 0.01 compared with the sham group; ^cP < 0.05 compared with the ischemia/reperfusion group. I/R: Ischemia/reperfusion.

Figure 3 Mdivi-1 treatment protects cell viability and decreases apoptosis in Caco-2 hypoxia/reoxygenation cells. A: Cell viability was detected by the CCK8 method (n = 6); B: Representative Western blotting of cleaved caspase-3 protein expression in Caco-2 cells (n = 3); C: Effect of mdivi-1 on hypoxia/reoxygenation-induced cell apoptosis detected by TUNEL assay (scale bar = 100 μm); D: TUNEL positive cell ratio in hypoxia/reoxygenation-induced cell apoptosis (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 compared with the control group; ^dP < 0.05, ^eP < 0.01 compared with the hypoxia/reoxygenation group. c-caspase 3: Cleaved caspase 3; H/R: Hypoxia/reoxygenation; TUNEL: Terminal-deoxynucleotidyl Transferase Mediated Nick End Labeling; DAPI: 4',6-diamidino-2-phenylindole.

Figure 4 Mdivi-1 administration inhibits mitochondrial fission and reactive oxygen species generation in Caco-2 hypoxia/reoxygenation cells. A: Deep Red FM in Caco-2 cells (scale bar = 10 μm); B, C: Dynamin-related protein 1 and dynamin-related protein 1 Ser637 expression upon mdivi-1 administration (n = 3); D: MitoSOX assay in Caco-2 cells (scale bar = 10 μm). ^cP < 0.001 compared with the control group; ^eP < 0.01 compared with the hypoxia/reoxygenation group. DRP1: Dynamin-related protein 1; PINK1: PTEN induced kinase 1; H/R: Hypoxia/reoxygenation.

Figure 5 Inhibition of PTEN-induced kinase 1 aggravates mitochondrial fission, reactive oxygen species accumulation and apoptosis in Caco-2 hypoxia/reoxygenation cells. A: The protein expression of PTEN- induced kinase 1 (PINK1) by Western blotting after PINK1 Small interfering RNA (siRNA) transfection (n = 3); B: Cell viability was detected by CCK-8 assay (n = 6); C: Deep Red FM in Caco-2 cells (scale bar = 10 μm); D: MitoSOX assay in Caco-2 cells (scale bar = 10 μm); E: PINK1 siRNA transfection increases intestinal cell apoptosis according to a TUNEL assay (length of the bar scale: 100 μm); F: PINK1 siRNA transfection increased intestinal cell apoptosis, as shown by the TUNEL positive cell ratio (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 compared with the control group; ^eP < 0.01 compared with the H/R group. DRP1: Dynamin-related protein 1; PINK1: PTEN induced kinase 1; ROS: Reactive oxygen species; CCK-8: Cell Counting Kit-8; H/R: Hypoxia/reoxygenation; siRNA: Small interfering RNA; TUNEL: Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling; DAPI: 4',6-diamidino-2-phenylindole.

Figure 6 Promotion of PTEN-induced kinase 1 expression decreases mitochondrial fission, reactive oxygen species accumulation and apoptosis in Caco-2 hypoxia/reoxygenation cells. A: Representative Western blotting of PTEN-induced kinase 1 (PINK1) protein expression in Caco-2 cells (n = 3); B: PINK1 overexpression increased Caco-2 cell viability according to CCK-8 assay (n = 6); C: PINK1 overexpression decreases mitochondrial fission during Hypoxia/reoxygenation (H/R) in Caco-2 cells according to MitoTracker staining (scale bars = 10 μm); D: PINK1 overexpression decreases reactive oxygen species levels detected by MitoSOX during H/R in Caco-2 cells (scale bars = 10 μm) (n = 3); E: TUNEL assay showing that PINK1 overexpression decreases apoptosis (scale bar = 100 μm); F: PINK1 overexpression decreases apoptosis, asindicated by the TUNEL-positive cell ratio (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 compared with the control group; ^eP < 0.01 compared with the H/R group. PINK1: PTEN induced kinase 1; ROS: Reactive oxygen species; CCK-8: Cell Counting Kit-8; H/R: Hypoxia/reoxygenation; TUNEL: Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling; DAPI: 4',6-diamidino-2-phenylindole.

Figure 7 PTEN-induced kinase 1 inhibits dynamin-related protein 1 recruitment on mitochondria by binding to Ser637. A, C: Mitochondrial localization of Dynamin-related protein 1 (DRP1) assessed under confocal a laser scanning microscope (green). Mitochondrial morphology was stained with MitoTracker Deep Red FM (red), and nuclei were stained with DAPI (blue) (scale bar = 10 μm); B, D: Representative Western blotting of DRP1 Ser637 protein expression in Caco-2 cells (n = 3); E: Coimmunoprecipitation assay of PTEN-induced kinase 1 with DRP1 Ser637. Representative immunoblot revealing the interaction of DRP1 Ser637 with PTEN-induced kinase 1. The input represents the total protein extracts used in immunoprecipitation. IB, immunoblotting; IP, immunoprecipitation, IgG, negative control. ^bP < 0.01 compared with the control group; ^dP < 0.01 compared with the H/R group. DRP1: Dynamin-related protein 1; PINK1: PTEN-induced kinase 1; H/R: hypoxia/reoxygenation; DAPI: 4',6-diamidino-2-phenylindole; IB: Immunoblot; IP: Immunoprecipitation.