1. Volume 118, Issue 2, Pages 404-421 (February 2000)
NO-aspirin protects from T cell–mediated liver injury by inhibiting caspase-dependent processing of Th1-like cytokines 
Stefano Fiorucci, Luca Santucci, Elisabetta Antonelli, Eleonora Distrutti, Giuseppe del Sero, Olivia Morelli, Luigina Romani, Barbara Federici, Piero del Soldato, Antonio Morelli 
Gastroenterology 
Volume 118, Issue 2, Pages (February 2000)
DOI: /S (00)70223-X
Copyright © 2000 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Molecular structure of the NO-aspirin derivative NCX-4016 [2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester]. The molecular weight of this compound is ; the molecular weight of aspirin is 180.
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3. Fig. 2
(A) NCX-4016 protects from con A–induced hepatitis. BALB/c mice were injected with 0.3 mg con A per mouse alone or in combination with 100 mg/kg NO-aspirin and killed at indicated time points. Data are mean ± SE of 5 mice for each data point. Squares denote AST, circles ALT. (B) Protection against con A–induced liver damage by NO-aspirin is dose-dependent. Aminotransferase (2, AST; ■, ALT) plasma levels were measured 24 hours after con A injection. Data are mean ± SE of 5 mice/group. *P < vs. control. **P < 0.01 vs. con A alone.
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4. Fig. 2
(A) NCX-4016 protects from con A–induced hepatitis. BALB/c mice were injected with 0.3 mg con A per mouse alone or in combination with 100 mg/kg NO-aspirin and killed at indicated time points. Data are mean ± SE of 5 mice for each data point. Squares denote AST, circles ALT. (B) Protection against con A–induced liver damage by NO-aspirin is dose-dependent. Aminotransferase (2, AST; ■, ALT) plasma levels were measured 24 hours after con A injection. Data are mean ± SE of 5 mice/group. *P < vs. control. **P < 0.01 vs. con A alone.
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5. Fig. 3
Light microscopy of liver sections from control mice and mice treated with con A alone or in combination with increasing doses of NO-aspirin. BALB/c mice were given a single IP inoculation of NO-aspirin 5 minutes after con A (0.3 mg/mouse) injection. (A) Liver section from a control mouse (H&E; original magnification 125×). (B) Immunohistochemical detection of CD4+ T cells in control animals showing only few CD4+ T cells infiltrating the liver. CD4+ T cells were stained with a rabbit anti-mouse CD4 antibody followed by fluorescein-labeled rat anti-rabbit IgG (125×). (C) Histopathology of the liver 24 hours after con A injection showing widespread areas of necrosis and inflammation within the liver lobules and around the central veins and portal tract (H&E; 125×). (D) At higher magnification, extensive necrotic and apoptic changes were seen in the liver of mice 24 hours after con A injection (H&E; 400×). (E and F) Immunohistochemical detection of CD4+ T cells in liver collected 24 hours after con A administration (125× [E] and 1000× [F]). (G) Histopathology of the liver from a mouse treated with 25 mg/kg NO-aspirin 24 hours after con A administration (H&E; 125×). (H) Histopathology of the liver from a mouse treated with 50 mg/kg NO-aspirin 24 hours after con A administration. Liver damage was markedly reduced, and liver histology shows minor necrotic phenomena (H&E; 125×). (I) Histopathology of the liver from a mouse treated with 100 mg/kg NO-aspirin 24 hours after con A administration. Liver damage was completely prevented, and liver histology shows no necrosis or inflammation. In these sections, apoptosis of liver cells could not be seen and liver histology was almost normal (H&E; 125×). (J) Immunohistochemical detection of CD4+ T cells in animals treated with 100 mg/kg NO-aspirin 24 hours after con A injection. Only few CD4+ T cells were found (100×). (K) Immunohistochemical detection of FasL expression on the liver of control mice. FasL was undetectable on the liver of untreated BALB/c mice (eosin counterstaining; 100×). (L) Con A administration induced FasL expression in the liver of BALB/c mice. FasL-positive cells were localized in the area of con A–induced apoptosis/necrosis (eosin counterstaining; 125×). (M) FasL expression (eosin counterstaining; 400×). (N) NO-aspirin (100 mg/kg) prevented FasL up-regulation induced by con A (eosin counterstaining; 100×).
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6. Fig. 4
Effect of Fas gene ablation on con A–induced liver toxicity. Fas-deficient mice (lpr/lpr) on an MRL genetic background were protected against con A–induced damage. (A and B) Aminotransferase plasma levels after con A injection in BALB/c (•), control MRL mice (■), and Fas-deficient MRL, lpr/lpr, mice (○). Data represent the mean ± SE of aminotransferase activity in plasma from 5 mice. *P < 0.05 vs. basal. (C) NO-aspirin prevents liver toxicity induced by con A injection in wild-type MRL mice. ■, Con A alone; 2, Con A + NCX Data represent the mean ± SE of aminotransferase activity in plasma from 5 mice. *P < 0.05 vs. basal.
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7. Fig. 4
Effect of Fas gene ablation on con A–induced liver toxicity. Fas-deficient mice (lpr/lpr) on an MRL genetic background were protected against con A–induced damage. (A and B) Aminotransferase plasma levels after con A injection in BALB/c (•), control MRL mice (■), and Fas-deficient MRL, lpr/lpr, mice (○). Data represent the mean ± SE of aminotransferase activity in plasma from 5 mice. *P < 0.05 vs. basal. (C) NO-aspirin prevents liver toxicity induced by con A injection in wild-type MRL mice. ■, Con A alone; 2, Con A + NCX Data represent the mean ± SE of aminotransferase activity in plasma from 5 mice. *P < 0.05 vs. basal.
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8. Fig. 4
Effect of Fas gene ablation on con A–induced liver toxicity. Fas-deficient mice (lpr/lpr) on an MRL genetic background were protected against con A–induced damage. (A and B) Aminotransferase plasma levels after con A injection in BALB/c (•), control MRL mice (■), and Fas-deficient MRL, lpr/lpr, mice (○). Data represent the mean ± SE of aminotransferase activity in plasma from 5 mice. *P < 0.05 vs. basal. (C) NO-aspirin prevents liver toxicity induced by con A injection in wild-type MRL mice. ■, Con A alone; 2, Con A + NCX Data represent the mean ± SE of aminotransferase activity in plasma from 5 mice. *P < 0.05 vs. basal.
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9. Fig. 5
NO-aspirin protects from DNA fragmentation induced by con A. DNA fragmentation of liver cells was monitored (A) by assessing histone ELISA as described in Materials and Methods or (B) by DNA laddering on agarose gel. (A) The rate of DNA fragmentation increases over time after con A administration (■). Cotreatment of mice with 100 mg/kg NO-aspirin (2) resulted in an almost complete prevention of DNA fragmentation induced by con A. *P < 0.01 vs. control. (B) Measurement of DNA fragmentation by DNA laddering on agarose gel. Soluble DNA was extracted from liver lysates and separated on an agarose gel and visualized by ethidium bromide staining. Lane 1, molecular mass markers; lane 2, control liver; lane 3, liver 8 hours after con A administration; lane 4, liver 24 hours after con A administration; lane 5, liver 24 hours after administration of con A in combination with 100 mg/kg NO-aspirin. (C) NO-aspirin prevents liver caspase activation. Assessment of caspase 1–like (2) and caspase 3–like (■) activity on liver collected 24 hours after injection of con A alone or in combination with 100 mg/kg NO-aspirin. Caspase 1– and 3–like activities were measured using YVAD.AMC and DEVD.AFC peptides as substrates. Specific activities in control livers were 1.9 ± 0.7 and 2.9 ± 1.1 pmol/mg protein per 20 minutes. Data are mean ± SE of 5 livers. Each assay was carried out in triplicate. *P < 0.01 vs. control; **P < 0.01 vs. con A alone.
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10. Fig. 5
NO-aspirin protects from DNA fragmentation induced by con A. DNA fragmentation of liver cells was monitored (A) by assessing histone ELISA as described in Materials and Methods or (B) by DNA laddering on agarose gel. (A) The rate of DNA fragmentation increases over time after con A administration (■). Cotreatment of mice with 100 mg/kg NO-aspirin (2) resulted in an almost complete prevention of DNA fragmentation induced by con A. *P < 0.01 vs. control. (B) Measurement of DNA fragmentation by DNA laddering on agarose gel. Soluble DNA was extracted from liver lysates and separated on an agarose gel and visualized by ethidium bromide staining. Lane 1, molecular mass markers; lane 2, control liver; lane 3, liver 8 hours after con A administration; lane 4, liver 24 hours after con A administration; lane 5, liver 24 hours after administration of con A in combination with 100 mg/kg NO-aspirin. (C) NO-aspirin prevents liver caspase activation. Assessment of caspase 1–like (2) and caspase 3–like (■) activity on liver collected 24 hours after injection of con A alone or in combination with 100 mg/kg NO-aspirin. Caspase 1– and 3–like activities were measured using YVAD.AMC and DEVD.AFC peptides as substrates. Specific activities in control livers were 1.9 ± 0.7 and 2.9 ± 1.1 pmol/mg protein per 20 minutes. Data are mean ± SE of 5 livers. Each assay was carried out in triplicate. *P < 0.01 vs. control; **P < 0.01 vs. con A alone.
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11. Fig. 5
NO-aspirin protects from DNA fragmentation induced by con A. DNA fragmentation of liver cells was monitored (A) by assessing histone ELISA as described in Materials and Methods or (B) by DNA laddering on agarose gel. (A) The rate of DNA fragmentation increases over time after con A administration (■). Cotreatment of mice with 100 mg/kg NO-aspirin (2) resulted in an almost complete prevention of DNA fragmentation induced by con A. *P < 0.01 vs. control. (B) Measurement of DNA fragmentation by DNA laddering on agarose gel. Soluble DNA was extracted from liver lysates and separated on an agarose gel and visualized by ethidium bromide staining. Lane 1, molecular mass markers; lane 2, control liver; lane 3, liver 8 hours after con A administration; lane 4, liver 24 hours after con A administration; lane 5, liver 24 hours after administration of con A in combination with 100 mg/kg NO-aspirin. (C) NO-aspirin prevents liver caspase activation. Assessment of caspase 1–like (2) and caspase 3–like (■) activity on liver collected 24 hours after injection of con A alone or in combination with 100 mg/kg NO-aspirin. Caspase 1– and 3–like activities were measured using YVAD.AMC and DEVD.AFC peptides as substrates. Specific activities in control livers were 1.9 ± 0.7 and 2.9 ± 1.1 pmol/mg protein per 20 minutes. Data are mean ± SE of 5 livers. Each assay was carried out in triplicate. *P < 0.01 vs. control; **P < 0.01 vs. con A alone.
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12. Fig. 6
(A–C) NO-aspirin (100 mg/kg) prevents cytokine release induced by con A. Animals were killed at the indicated time points after con A injection (0.3 mg/mouse). Data points represent the mean ± SE for 5 mice/group. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone.
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13. Fig. 6
(A–C) NO-aspirin (100 mg/kg) prevents cytokine release induced by con A. Animals were killed at the indicated time points after con A injection (0.3 mg/mouse). Data points represent the mean ± SE for 5 mice/group. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone.
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14. Fig. 6
(A–C) NO-aspirin (100 mg/kg) prevents cytokine release induced by con A. Animals were killed at the indicated time points after con A injection (0.3 mg/mouse). Data points represent the mean ± SE for 5 mice/group. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone.
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15. Fig. 7
RT-PCR analysis of IL-1β, IFN-γ, and IL-18 mRNA transcripts on spleen lysates examined at different time points after con A administration. In each panel, lane 1 is con A alone; lane 2 is con A mg/kg NO-aspirin. Con A administration increased cytokine mRNA accumulation in a time-dependent manner. NO-aspirin failed to prevent cytokine mRNA accumulation. Expression of HPRT mRNA was used as a control.
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16. Fig. 8
(A–C) NO-aspirin (100 mg/kg) prevents Fas, FasL, and IL-2R expression up-regulation induced by con A administration on spleen lymphocytes. , Control; •, con A; ○, con A + NO-aspirin. Data points represent the mean ± SE for 5 mice/group. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone.
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17. Fig. 8
(A–C) NO-aspirin (100 mg/kg) prevents Fas, FasL, and IL-2R expression up-regulation induced by con A administration on spleen lymphocytes. , Control; •, con A; ○, con A + NO-aspirin. Data points represent the mean ± SE for 5 mice/group. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone.
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18. Fig. 8
(A–C) NO-aspirin (100 mg/kg) prevents Fas, FasL, and IL-2R expression up-regulation induced by con A administration on spleen lymphocytes. , Control; •, con A; ○, con A + NO-aspirin. Data points represent the mean ± SE for 5 mice/group. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone.
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19. Fig. 9
NO-aspirin (100 mg/kg) prevents (A) IL-12 and (B) IL-18 release induced by con A. Data points represent the mean ± SE for 5 mice/group. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone.
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20. Fig. 9
NO-aspirin (100 mg/kg) prevents (A) IL-12 and (B) IL-18 release induced by con A. Data points represent the mean ± SE for 5 mice/group. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone.
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21. Fig. 10
IL-18 immunoneutralization protects from liver damage induced by con A. Each mouse (5 mice/group) was treated with 200 μg rabbit anti–IL-18 antibody or 200 μg normal rabbit IgG polyclonal antibody (control) 2 hours before con A injection and killed 8 hours after plant lectin administration. (A) Liver histology from con A–treated mice (H&E; original magnification 125×). (B) Liver section from mouse treated with anti–IL-18. Liver histology shows no necrosis or inflammation (H&E; 125×). IL-18 immunoneutralization prevented (C) aminotransferase release and caused a significant reduction of (E) TNF-α and (F) IFN-γ release induced by con A. In contrast, IL-18 immunoneutralization had no effect on (D) IL-1β and (G) IL-12 release induced by con A. Each bar is the mean ± SE of 5 animals. *P > 0.01 vs. control; **P < 0.01 vs. con A alone.
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22. Fig. 11
(A) Time course of caspase 1–like activity on spleen lysates from a mouse treated with con A and NO-aspirin. Caspase 1 activity in basal samples was 2.2 ± 1.1 pmol/mg protein per 20 minutes. Data are mean ± SE of 5 mice. Each assay was carried out in triplicate. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone. (B) Time course of caspase 3–like activity on spleen lysates from a mouse treated with con A and NO-aspirin. Caspase 3 activity in basal samples was 4.2 ± 0.8 pmol/mg protein per 20 minutes. Data are mean ± SE of 5 mice, with each assay carried out in triplicate. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone. (C) Exposure of spleen lysates to increasing concentrations of DTT reverted inhibition of caspase 1– and 3–like activity induced by in vivo administration of 100 mg/kg NO-aspirin. Data are mean ± SE of 5 mice, with each assay carried out in triplicate. *P < 0.01 vs. basal. (D) Exposure of spleen lysates obtained from con A–treated mice to increasing concentrations of NO-aspirin caused a concentration-dependent inhibition of caspase activity. The effect was prevented by 20 mmol/L DTT. Data are mean ± SE of 5 spleens, with each assay carried out in triplicate. Caspase 1– and 3–like activities in spleen samples obtained from con A–treated mice were 7.2 ± 1.9 and 19.5 ± 3.7 pmol/mg protein per 20 minutes, respectively. *P < 0.01 vs. basal. (E) HgCl2 (5 mmol/L) inhibits caspase 1– and 3–like activity on spleen lymphocytes. HgCl2-induced inhibition was prevented by DTT (20 mmol/L). Data are mean ± SE of 5 experiments, with each assay carried out in triplicate. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone. ○P < 0.01 vs. HgCl2 alone. (F) HgCl2 (5 mmol/L) releases NO from spleen homogenates obtained from mice treated with con A + NO-aspirin. Data are mean ± SE of 5 experiments, with each assay carried out in triplicate. *P < 0.01 vs. control; **P < 0.01 vs. con A alone.
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23. Fig. 11
(A) Time course of caspase 1–like activity on spleen lysates from a mouse treated with con A and NO-aspirin. Caspase 1 activity in basal samples was 2.2 ± 1.1 pmol/mg protein per 20 minutes. Data are mean ± SE of 5 mice. Each assay was carried out in triplicate. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone. (B) Time course of caspase 3–like activity on spleen lysates from a mouse treated with con A and NO-aspirin. Caspase 3 activity in basal samples was 4.2 ± 0.8 pmol/mg protein per 20 minutes. Data are mean ± SE of 5 mice, with each assay carried out in triplicate. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone. (C) Exposure of spleen lysates to increasing concentrations of DTT reverted inhibition of caspase 1– and 3–like activity induced by in vivo administration of 100 mg/kg NO-aspirin. Data are mean ± SE of 5 mice, with each assay carried out in triplicate. *P < 0.01 vs. basal. (D) Exposure of spleen lysates obtained from con A–treated mice to increasing concentrations of NO-aspirin caused a concentration-dependent inhibition of caspase activity. The effect was prevented by 20 mmol/L DTT. Data are mean ± SE of 5 spleens, with each assay carried out in triplicate. Caspase 1– and 3–like activities in spleen samples obtained from con A–treated mice were 7.2 ± 1.9 and 19.5 ± 3.7 pmol/mg protein per 20 minutes, respectively. *P < 0.01 vs. basal. (E) HgCl2 (5 mmol/L) inhibits caspase 1– and 3–like activity on spleen lymphocytes. HgCl2-induced inhibition was prevented by DTT (20 mmol/L). Data are mean ± SE of 5 experiments, with each assay carried out in triplicate. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone. ○P < 0.01 vs. HgCl2 alone. (F) HgCl2 (5 mmol/L) releases NO from spleen homogenates obtained from mice treated with con A + NO-aspirin. Data are mean ± SE of 5 experiments, with each assay carried out in triplicate. *P < 0.01 vs. control; **P < 0.01 vs. con A alone.
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24. Fig. 11
(A) Time course of caspase 1–like activity on spleen lysates from a mouse treated with con A and NO-aspirin. Caspase 1 activity in basal samples was 2.2 ± 1.1 pmol/mg protein per 20 minutes. Data are mean ± SE of 5 mice. Each assay was carried out in triplicate. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone. (B) Time course of caspase 3–like activity on spleen lysates from a mouse treated with con A and NO-aspirin. Caspase 3 activity in basal samples was 4.2 ± 0.8 pmol/mg protein per 20 minutes. Data are mean ± SE of 5 mice, with each assay carried out in triplicate. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone. (C) Exposure of spleen lysates to increasing concentrations of DTT reverted inhibition of caspase 1– and 3–like activity induced by in vivo administration of 100 mg/kg NO-aspirin. Data are mean ± SE of 5 mice, with each assay carried out in triplicate. *P < 0.01 vs. basal. (D) Exposure of spleen lysates obtained from con A–treated mice to increasing concentrations of NO-aspirin caused a concentration-dependent inhibition of caspase activity. The effect was prevented by 20 mmol/L DTT. Data are mean ± SE of 5 spleens, with each assay carried out in triplicate. Caspase 1– and 3–like activities in spleen samples obtained from con A–treated mice were 7.2 ± 1.9 and 19.5 ± 3.7 pmol/mg protein per 20 minutes, respectively. *P < 0.01 vs. basal. (E) HgCl2 (5 mmol/L) inhibits caspase 1– and 3–like activity on spleen lymphocytes. HgCl2-induced inhibition was prevented by DTT (20 mmol/L). Data are mean ± SE of 5 experiments, with each assay carried out in triplicate. *P < 0.01 vs. basal; **P < 0.01 vs. con A alone. ○P < 0.01 vs. HgCl2 alone. (F) HgCl2 (5 mmol/L) releases NO from spleen homogenates obtained from mice treated with con A + NO-aspirin. Data are mean ± SE of 5 experiments, with each assay carried out in triplicate. *P < 0.01 vs. control; **P < 0.01 vs. con A alone.
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25. Fig. 12
Caspase 1 is not strictly required to produce damage and IFN-γ release in mice treated with con A. C57BL/6 ICE−/− mice and wild-type, ICE+/+, C57BL/6, and BALB/c mice were injected with 0.3 mg/kg con A and killed 8 hours later. (A) Histopathology of the liver obtained from ICE−/− C57BL/6 mice 8 hours after con A injection showing widespread areas of necrosis and inflammation within the liver lobules and around the central veins and portal tract (H&E; original magnification 125×). (B and C) Histopathology of the liver obtained from (B) ICE+/+ C57BL/6 and (C) BALB/c mice 8 hours after con A injection showing widespread areas of necrosis and inflammation within the liver lobules and around the central veins and portal tract (H&E; 125×). (D) Aminotransferase plasma levels 8 hours after con A injection in ICE−/− mice and ICE+/+ (C57BL/6 and BALB/c) mice. Data are mean ± SE of 5 mice/group. *P < vs. control. (E and F) IL-18 and IFN-γ plasma levels 8 hours after con A injection in ICE−/− C57BL/6 mice and ICE+/+ (C57BL/6 and BALB/c mice). Data are mean ± SE of 5 mice/group. *P < vs. control.
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26. Fig. 13
(A–E) Effect of selective and nonselective caspase inhibitors on liver damage and cytokine release induced by con A. Animals were treated with con A alone or in combination with 5 mg/kg of each inhibitor (Ac.YVAD.CHO, Ac.DEVD.CHO, and zVAD.FMK). Data are mean ± SE of 5 mice/group. *P < vs. control; **P < 0.01 vs. con A alone. Control animals were treated with PBS alone. (F) Histopathology of the liver obtained from mice 8 hours after con A injection showing widespread areas of necrosis and inflammation within the liver lobules and around the central veins and portal tract. (G) Extensive liver injury in animals treated with Ac.YVAD.CHO. (H) Ac.DEVD.CHO reduced the extent of liver damage induced by con A. (I) zVAD.FMK fully protected the liver from damage induced by con A. (F–I: H&E; original magnification 125×).
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27. Fig. 14
(A–C) Effect of ICE gene ablation, caspase inhibitors, and NO donors on IFN-γ release from spleen lymphocytes incubated with 1 μg/mL con A (2) or 10 μg/mL LPS (■). *P < 0.01 vs. control; **P < 0.01 vs. con A or LPS. Data are mean ± SE of 4 experiments. Control cells were incubated with medium alone.
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28. Fig. 14
(A–C) Effect of ICE gene ablation, caspase inhibitors, and NO donors on IFN-γ release from spleen lymphocytes incubated with 1 μg/mL con A (2) or 10 μg/mL LPS (■). *P < 0.01 vs. control; **P < 0.01 vs. con A or LPS. Data are mean ± SE of 4 experiments. Control cells were incubated with medium alone.
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29. Fig. 14
(A–C) Effect of ICE gene ablation, caspase inhibitors, and NO donors on IFN-γ release from spleen lymphocytes incubated with 1 μg/mL con A (2) or 10 μg/mL LPS (■). *P < 0.01 vs. control; **P < 0.01 vs. con A or LPS. Data are mean ± SE of 4 experiments. Control cells were incubated with medium alone.
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