Protease-Activated Receptor-1 Antagonist Protects Against Lung Ischemia/Reperfusion Injury

Figures

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  FIGURE 1. SCH530348 alleviated IR-induced acute lung edema. (A) LW gain, (B) Kf, (C) LW/BW ratio, (D) W/D weight ratio, and (E) protein concentration in BALF significantly increased in the IR group. The increases in these parameters were significantly ameliorated by SCH530348. Compared with other doses, 5 mg/kg SCH530348 was more effective in decreasing lung edema. The data are expressed as the mean ± SD (n = 6 per group). *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group; + p < 0.05, ++ p < 0.01, +++ p < 0.001, compared with the IR group.

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  FIGURE 2. SCH530348 decreased IR-induced PAR-1 expression. (A) PAR-1 protein levels were determined by western blotting with specific antibodies. (B) IHC analysis of lung slices revealed that PAR-1 expression (indicated by arrowheads) was increased by IR injury compared with that of the control group. SCH530348 treatment decreased PAR-1 expression in the context of IR injury. The data are expressed as the mean ± SD (n = 4 per group). ***p < 0.001, compared with the control group; +++ p < 0.001, compared with the IR group.

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  FIGURE 3. SCH530348 ameliorated IR-induced proinflammatory cytokine production. (A) CINC-1, (B) IL-6, and (C) TNF-α levels in BALF were measured using ELISA kits. CINC-1, IL-6 and TNF-α levels were all significantly elevated in the IR group compared with the control group. SCH530348 treatment significantly attenuated the production of these cytokines in the context of IR injury. The data are expressed as the mean ± SD (n = 6 per group). **p < 0.01, ***p < 0.001, compared with the control group; ++ p < 0.01, +++ p < 0.001, compared with the IR group.

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  FIGURE 4. SCH530348 mitigated IR-induced acute lung injury. (A) HE staining of lung tissue showed thickening of alveolar septa and neutrophil infiltration in the interstitial and alveolar spaces after IR injury compared with that of the sham control. Alveolar damage and neutrophil recruitment after IR injury were attenuated by SCH530348 treatment. (B) The number of infiltrated neutrophils per high-power field (400x magnification) was elevated in the IR group, while additional SCH530348 significantly reduced the number of neutrophils in the context of IR injury. (C) SCH530348 significantly decreased IR-induced elevations in lung injury scores. Cell counting was performed at 400 x magnification; scale bar = 100 μm. The data are expressed as the mean ± SD (n = 6 per group). **p < 0.01, ***p < 0.001, compared with the control group; +++ p < 0.001, compared with the IR group.

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  FIGURE 5. SCH530348 decreased IR-induced MPO activity. Serial paraffin-embedded sections from IR rats were stained for the neutrophil marker MPO. MPO-positive cells are indicated by arrowheads (200× magnification). The number of MPO-positive cells in lung tissue was increased following IR injury, and SCH530348 treatment markedly attenuated this increase.

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  FIGURE 6. SCH530348 downregulated IR-induced thrombin activity. (A) TAT levels in BALF were measured by ELISA. (B) Representative images showing IHC staining of thrombin in lung sections from the vehicle or IR group (200× magnification). Scale bars, 50 μm. TAT levels in BALF and thrombin-positive cells in lung tissue (indicated by arrowheads) were increased in the IR group compared with the control group. SCH530348 treatment mitigated these increases. The data are expressed as the mean ± SD (n = 6 per group). *p < 0.05, compared with the control group; ++ p < 0.01, compared with the IR group.

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  FIGURE 7. SCH530348 reduced IR-induced lung apoptosis. Western blot analysis of (A) Bcl-2 and (B) cleaved caspase-3 protein expression in lung tissue. β-actin served as a loading control. Representative blots are shown. IR significantly decreased Bcl-2 and induced cleaved caspase-3 protein expression in lung tissue. SCH530348 treatment significantly reversed these IR-induced apoptotic effects. The data are expressed as the mean ± SD (n = 4 per group). *p < 0.05, ***p < 0.001, compared with the control group; +++ p < 0.001, compared with the IR group.

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  FIGURE 8. SCH530348 suppressed IR-induced activation of PIK3 and NF-κB signaling. The protein levels of (A) IκB, (B) NF-κB, (C) Akt and (D) PI3K were detected by western blotting. Lamin B1 and β-actin served as loading controls for nuclear and cytoplasmic proteins, respectively. The protein levels of NF-κB, Akt and PI3K in lung tissue significantly increased and the protein level of IκB significantly decreased following exposure to IR. In addition, SCH530348 significantly attenuated the nuclear translocation of NF-κB, the degradation of IκB, and Akt and PI3K protein expression in the context of IR injury. The data are expressed as the mean ± SD (n = 4 per group). **p < 0.01, ***p < 0.001, compared with the control group; + p < 0.05, +++ p < 0.001, compared with the IR group.

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  FIGURE 9. SCH530348 attenuated IR-induced MAPK signaling activation. Western blot analysis showing that the levels of (A) phosphorylated ERK, (B) JNK and (C) p38 were upregulated in lung tissue after IR injury compared with those of the control group. The levels of phosphorylated ERK, JNK and p38 were downregulated after SCH530348 treatment following IR injury. The data are expressed as the mean ± SD (n = 4 per group). *p < 0.05, ***p < 0.001, compared with the control group; ++ p < 0.01, +++ p < 0.001, compared with the IR group.

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  FIGURE 10. SCH530348 attenuated HR-induced chemokine production and NF-κB signaling activation in MLE-12 cells. Mouse lung epithelial cells were subjected to HR for 3 h and were treated with vehicle or 10 µM SCH530348. (A) HR-induced MLE-12 cells produced higher CXCL1/KC levels than control group cells, and SCH530348 significantly decreased chemokine production following HR for 3 h (B,C) SCH530348 significantly reduced HR-induced NF-κB p65 phosphorylation and IκB-α degradation. β-actin was used as an internal control. The data are expressed as the mean ± SD (n = 3 per group). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group; ##p < 0.01, ###p < 0.001, compared with the HR group.