Article Figures & Data
Figures
- Fig. 1.
Role of Atg5 in injury of the small intestine after IM treatment in vitro and in vivo. (A and B) Gross appearance of intestinal lesions (A) and ulcer index values (B) after IM administration in WT and conditional Atg5KO mice. Arrows indicate epithelial erosion caused by IM-induced intestinal injuries. Ulcer index values are expressed as means ± S.E. (n = 5). (C) Cytotoxicity was estimated after treatment with various concentrations of IM in IEC6 and IEC6shAtg5 cells. Open bars, IEC6 cells; closed bars, IEC6shAtg5 cells. Statistical analysis was performed using Tukey’s method. *P < 0.05.
- Fig. 2.
Induction of autophagy and subsequent apoptosis by IM treatment in IEC6 cells, not in IEC6shAtg5 cells. The cells were treated with 200 μM IM for various durations (0, 1, 2, 4, 8, and 12 hours). (A) LC3 expression levels at each time point in IEC6 cells and IEC6shAtg5 cells were determined using Western blot analyses. The arrow and arrowhead indicate LC3-I and LC3-II, respectively. (B) The cleavage of PARP1 in IM-treated IEC6 cells and IEC6shAtg5 cells was determined. The arrow and arrowhead indicate intact and cleaved PARP1, respectively.
- Fig. 3.
ROS generation before and after IM treatment in IEC6 and IEC6shAtg5 cells. (A and B) Both total ROS and superoxide were estimated before and after treatment with 200 μM IM in IEC6 and IEC6shAtg5 cells using a Total ROS/Superoxide Detection Kit (Enzo Life Sciences Inc.). The effect of N-acetyl cysteine (NAC) was evaluated to confirm that the assay was suitable. (C and D) The changing rates of relative ROS or superoxide production after various concentrations (0, 50, 100, or 200 μM) of IM treatment were estimated in both cells and compared with each other. *P < 0.05; **P < 0.01.
- Fig. 4.
Quantitative evaluation of functionally active mitochondria before and after IM treatment in IEC6 cells and IEC6shAtg5 cells. Mitochondria staining with MitoTracker Red CMXRos, a membrane potential dependent mitochondria probe. Cells were preincubated with or without 200 μM IM for 1 h; then, MitoTracker Red CMXRos was added, and further incubated for 1 h. The fluorescence was measured using GloMax (Promega). *P < 0.05; Tukey’s test.
- Fig. 5.
Changes in protein expression levels after IM treatment in IEC6 and IEC6shAtg5 cells. The cells were treated with 200 μM IM for the indicated times. (A and B) Protein expression levels of total and phosphorylated ERK (A) and Nrf2 and HO-1 (B) at each time point after IM treatment in IEC6 and IEC6shAtg5 cells were determined using Western blot analyses.
- Fig. 6.
Involvement of ROS in IM-induced autophagy, subsequent apoptosis, and the ERK/Nrf2/HO-1 pathway. After the preincubation with or without 10 μM MnTMPyP for 30 minutes, IEC6 cells were treated with 200 μM IM for another 2 hours. (A and B) Cleavage of LC3 (A) or PARP1 (B) was determined using Western blot analyses. Arrows indicate LC3-I (A) or intact PARP1 (B), and arrowheads indicates LC3-II (A) or cleaved PARP1 (B). (C) Using the same samples, the levels of ERK phosphorylation, Nrf2 stabilization, and HO-1 induction were determined using Western blot analyses. (D) After preincubation with or without 1 μM MnTMPyP for 30 minutes, IEC6 cells were treated with 200 μM IM for another 2 hours. Nrf2 promoter activity was evaluated by a luciferase assay. *P < 0.01.
- Fig. 7.
Effects of autophagy deficiency and IM administration on the ERK/Nrf2/HO-1 pathway in small intestinal mucosa. WT and Atg5 KO mice were treated with or without IM administration orally. After 24 hours, small intestines (stomach to ileum) of the euthanized animals were excised, and the mucosa was surgically scraped. The scraped tissues were collected and analyzed with Western blot analyses. *P < 0.05.
- Fig. 8.
Possible mechanism for the amelioration of IM-induced intestinal epithelial cell injury by autophagy deficiency. (A) In normal cells, oxidative stress is suppressed by autophagy; therefore, the ERK/Nrf2/HO-1 pathway is not activated under basal conditions. After IM treatment, the cells cannot fully prevent apoptosis-induced cell damage due to the increase of oxidative stress. (B) In autophagy-deficient cells, the ERK/Nrf2/HO-1 pathway is activated by oxidative stress even under basal conditions. The cells can minimize apoptosis-induced cell damage after IM treatment, because it is more resistant to oxidative stress due to the upregulated ERK/Nrf2/HO-1 pathway.
Tables
- TABLE 1
Effect of autophagy deficiency with or without IM treatment on IEC6 cells or mouse intestinal epithelial cells
Mice Epithelial Cell Damage Autophagy Apoptosis Oxidative Stress ERK/Nrf2/HO-1 WT — + + + + WT + IM +++ +++ +++ +++ ++ Atg5 KO — — + ++ ++ Atg5 KO + IM + — ++ +++ +++ — : not detectable; + : low; ++ : medium; +++ : high.
In this issue
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