Heme oxygenase-1 protects rat liver against warm ischemia/reperfusion injury via TLR2/TLR4-triggered signaling pathways

Figure 1 Serum concentrations of liver enzymes and myeloperoxidase activity. The hepatocellular function, as evidenced by serum aspartate aminotransferase (ALT) (A), aspartate aminotransferase (AST) (B) and lactate dehydrogenase (LDH) (C) levels were decreased in cobalt protoporphyrin (CoPP) treatment group, compared with zinc protoporphyrin (ZnPP) treatment. The myeloperoxidase (MPO) activity (D) was decreased in livers subjected to CoPP, compared with ZnPP. Data are shown as mean ± SE, (n = 4 per group).

Figure 2 Representative histological findings in rat liver after 75 min of warm ischemia followed by 6 h of reperfusion. Results are representative of four to six rats per group. A: Sham; B: Ischemia/reperfusion (I/R); C: Cobalt protoporphyrin (CoPP); D: Zinc protoporphyrin (ZnPP); original magnification, × 100; E: Histological injury score based on Suzuki’s criteria at 6 h after reperfusion was quantified (n = 4 per group).

Figure 3 Accumulation of neutrophils, T cells and macrophages in ischemic liver at 6 h of reperfusion after 75 min of ischemia. A: Lymphocyte antigen 6G (Ly-6G); B: CD3; C: CD4; D: CD11b. Left panel: Representative liver sections stained by immunohistology (magnification × 200); Right panel: Cell quantification/HPF (n = 4 per group). CoPP: Cobalt protoporphyrin; ZnPP: Zinc protoporphyrin.

Figure 4 Heme oxygenase-1 changes activation of Toll-interleukin-1R- containing adaptor inducing interferon-β and myeloid differentiation factor 88 dependent pathways. A: Whole cell lysates were prepared, immunoprecipitated with anti-Toll-interleukin (IL)-1R-containing adaptor inducing interferon-β (TRIF), and immune complexes were analyzed by immunoblotting with the indicated antibodies. Shown are data of representative (n = 4) experiments; B: Expression levels of heme oxygenase (HO)-1, Toll-like receptor (TLR)-2, TLR3 and TLR4 were analyzed in whole cell lysates by immunoblotting with the respective Abs. n = 4-5 per group; C: The cell lysates were subjected to immunoprecipitation with anti-myeloid differentiation factor 88 (MyD88), and then the immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies; n = 4 per group. TBK: TANK binding kinase; IRAK: IL-1R-associated kinase; TRAF: Tumor necrosis factor receptor-associated factor; I/R: Ischemia/ reperfusion; CoPP: Cobalt protoporphyrin; ZnPP: Zinc protoporphyrin.

Figure 5 Phosphorylation of multiple kinases at 6 h after reperfusion. A: Phosphorylation of interferon regulatory factor (IRF)-3, p38, inhibitor of nuclear factor-κB (NF-κB) (IκB)-α and NF-κB were evaluated in Western blot using hepatic cytoplasmic or nuclear protein at 6 h after reperfusion. B: Phosphorylated (p)-to-total (t) protein kinase ratios were quantified by densitometry. Data are mean ± SE of 4 independent experiments (n = 5 per group). I/R: Ischemia/reperfusion; CoPP: Cobalt protoporphyrin; ZnPP: Zinc protoporphyrin.

Figure 6 Quantitative reverse transcription polymerase chain reaction-assisted detection of proinflammatory and chemokines at 6 h of reperfusion after 75 min of ischemia. Data were normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT) gene expression. Mean ± SE are shown (n = 5 per group). TNF: Tumor necrosis factor; IL: Interleukin; IFN: Interferon; CXCL: Chemokine CXC ligand; I/R: Ischemia/reperfusion; CoPP: Cobalt protoporphyrin; ZnPP: Zinc protoporphyrin.

Figure 7 Expression of negative regulators of TLR signaling. A: Expression of Toll-interacting protein (Tollip), interleukin-1R-associated kinase (IRAK-M), suppressor of cytokine signaling (SOCS)-1 and Src homology 2 domain-containing inositol-5-phosphatase (SHIP-1) in liver at 6 h after reperfusion were evaluated by Western blotting assay using whole cell lysates; B: Results was quantified and expressed by the ratio to β-actin. Mean ± SE are shown (n = 4/group). I/R: Ischemia/reperfusion; CoPP: Cobalt protoporphyrin; ZnPP: Zinc protoporphyrin.