Volume 120, Issue 2, Pages 460-469 (February 2001) Endogenous PPARγ mediates anti-inflammatory activity in murine ischemia-reperfusion injury  Atsushi Nakajima, Koichiro Wada, Hiroshi Miki, Naoto Kubota, Noriko Nakajima, Yasuo Terauchi, Shin Ohnishi, Lawrence J. Saubermann, Takashi Kadowaki, Richard S. Blumberg, Ryozo Nagai, Nobuyuki Matsuhashi  Gastroenterology  Volume 120, Issue 2, Pages 460-469 (February 2001) DOI: 10.1053/gast.2001.21191 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Effects of PPARγ deficiency on intestinal I/R injury. The superior mesenteric artery in mice was clamped for 30 minutes and reperfused for 60 minutes, and samples were collected. (A) Messenger RNA levels of PPARγ in intestine from wild-type (2) and heterozygous PPARγ-deficient (+/−) mice (■). Normalized mRNA levels of PPARγ in intestine from heterozygous-deficient mice were significantly lower than those from wild-type mice. Each point represents mean ± SEM from 9 experiments. **P < 0.01 vs. wild-type. (B) Macroscopic or (C) microscopic images of intestine after I/R from heterozygous (+/−) or wild-type (+/+) mice. Histologically, profound intestinal injury including extensive loss of epithelial cells was observed in PPARγ-deficient mice vs. wild-type mice. (D) Macroscopic injury score of intestinal injury in PPARγ-deficient or wild-type mice after I/R. Each column represents mean ± SEM from 5 experiments. (E) Loss of LDH of intestinal tissues from PPARγ-deficient and wild-type mice after intestinal I/R. LDH loss in I/R intestinal tissues of PPARγ-deficient mice was significantly greater than that of wild-type mice. Each column represents mean ± SEM from 5 experiments. *P < 0.05 vs. wild-type. (F) MPO activity in intestinal tissues of PPARγ-deficient mice (measured as described in Materials and Methods) was significantly greater than that of wild-type mice. Each column represents mean ± SEM from 5 experiments. **P < 0.01 vs. wild-type. Gastroenterology 2001 120, 460-469DOI: (10.1053/gast.2001.21191) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Effects of BRL-49653 on intestine and lung pathology in I/R injury. (A) Macroscopic, (B) stereomicroscopic and (C) microscopic, observations of intestine from sham–, I/R control– (I/R + Vehicle), and I/R plus BRL–(I/R + BRL-49653; 30 mg/kg) treated groups. The magnifications of microscopic pictures are 100×. The I/R procedure caused destruction of epithelial cells, loss of villi, hemorrhage, and inflammatory cell infiltration, which were attenuated by treatment with BRL-49653. Microscopic pictures of lung sections (Figure 2D) after intestinal I/R were prepared and stained by H&E. Intestinal I/R caused marked increases in infiltration of inflammatory cells in I/R control lung; these were inhibited by treatment with BRL-49653. Gastroenterology 2001 120, 460-469DOI: (10.1053/gast.2001.21191) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Effects of BRL-49653 on intestine and lung in I/R injury. (A) Injury score; (B) loss of LDH from intestinal tissue; infiltration of leukocytes into (C) intestine and (D) lung. (A) Macroscopic injury scores of intestinal tissue were performed blindly by a pathologist. Score index for intestine from sham (○), I/R plus vehicle (●), or I/R plus BRL-49653 (●) is shown. **P < 0.01 vs. control (I/R plus vehicle). (B) Loss of intestinal tissue LDH activity after I/R. LDH activity of tissue homogenate was measured and expressed as units per milligram wet weight of tissue. Each point represents mean ± SEM from 5–9 different experiments. *P < 0.05, **P < 0.01 vs. control (I/R plus vehicle). The tissue LDH for the sham-operated group is indicated as an open circle. Results with I/R plus vehicle or I/R plus BRL-49653 are shown as closed circle. (C) Effect of BRL-49653 on leukocyte infiltration into intestinal tissue after I/R. MPO activity of intestinal tissues from sham (○), I/R plus vehicle (●), or I/R plus BRL-49653 (●) was measured and expressed as milliunits per milligram wet weight of tissue. Each point represents mean ± SEM from 5–9 experiments. **P < 0.01 vs. I/R vehicle control. (D) Effect of BRL-49653 on leukocyte infiltration into lung tissue after intestinal I/R. MPO activity of lung tissues from sham (○), I/R plus vehicle (●), or I/R plus BRL-49653 (●) was measured and expressed as milliunits per milligram of wet tissue. Each point represents mean ± SEM from 5–9 experiments. *P < 0.05, vs. I/R vehicle control. Gastroenterology 2001 120, 460-469DOI: (10.1053/gast.2001.21191) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Pretreatment with BRL-49653 prevents adhesion molecule expression and proinflammatory cytokine mRNA levels in vivo in association with inhibition of NF-κB pathway. (A) Immunohistochemical staining of ICAM-1 molecules in intestinal tissues from either I/R or I/R plus BRL-49653. Positive staining of ICAM-1 expression was observed in endothelial cells in the submucosa of I/R intestine. This staining was abrogated by treatment with BRL-49653. (B) Expression of TNF-α mRNA in I/R intestine. TNF-α mRNA levels were measured as described in Materials and Methods. The increase in TNF-α mRNA levels observed in I/R intestine was significantly suppressed by pretreatment with BRL-49653. *P < 0.05 vs. I/R control. (C) Electrophoretic mobility shift assays (EMSA) of intestinal tissues were performed as described in Materials and Methods. I/R caused a marked increase in the nuclear translocation of NF-κB in intestine (lane 2) compared with the sham-operated group (lane 1). This translocation was inhibited by BRL-49653 (lane 3). Lane 4 shows the HeLa cell nuclear extract as a positive control. (D) Decrease in phosphorylated IκB by treatment with BRL-49653 in intestinal tissue after I/R exposure. A phosphorylation state–specific antibody for IκB was applied by immunohistochemistry to intestinal tissue sections from I/R control– and I/R plus BRL-49653–(30 mg/kg) treated mice. Positive staining was observed in endothelial cells in submucosa of I/R intestine. This staining was abrogated by treatment with BRL-49653. Gastroenterology 2001 120, 460-469DOI: (10.1053/gast.2001.21191) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 PPARγ ligand mediates rapid transcriptional regulation of IL-1β–induced IL-8 mRNA expression. (A) HT-29 human colon cancer cell line was treated with 30 μmol/L BRL-49653 for 60 minutes before stimulation with IL-1β. Lanes 1, 2, 3, and 4 represent 0, 1, 2, and 12 hours of incubation with BRL-49653, respectively. GAPDH was used as an internal control. An RNA protection assay demonstrates that BRL-49653 attenuates the IL-8 mRNA levels as early as 2 hours after IL-1β stimulation. (B) Time course of IL-8 mRNA levels in arbitrary units in HT-29 cells in vehicle-treated control (○) and BRL-49653–treated cell line (●). Gastroenterology 2001 120, 460-469DOI: (10.1053/gast.2001.21191) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Western blot analysis of PPARγ protein expression in different organs. Lane 1, liver; lane 2, small intestine; lane 3, stomach; lane 4, colon. Samples (50 μg of each) were applied to SDS-PAGE, and visualization was performed by anti-PPARγ polyclonal antibody and enhanced chemiluminescence. Gastroenterology 2001 120, 460-469DOI: (10.1053/gast.2001.21191) Copyright © 2001 American Gastroenterological Association Terms and Conditions