FIGURES AND TABLES

Figure 1.

(a) The susceptibility of hepatocytes isolated from WT and IL-1ra KO mice to APAP or NAPQI. Hepatocytes were isolated from WT and IL-1ra KO mice, and cultured in the presence of APAP (5 mM) or NAPQI (400 M) for 4 h, as described in 'Materials and Methods'. Each value represents means.e.m. (n=6 animals). ^** P<0.01, WT vs IL-1ra KO mice. (b) Western blotting analysis of APAP adducts in hepatocyte homogenates from WT and IL-1ra KO mice after APAP treatment. Representative results from six independent experiments are shown here. (c) Western blotting analysis of APAP adducts in the livers of WT and IL-1ra KO mice administered with APAP (100–300 mg/kg). Representative results from six independent experiments are shown here. (d) Blood acetaminophen concentrations in WT and IL-1ra KO mice after APAP challenge (200 mg/kg). Each value represents means.e.m. (n=6 animals). ^** P<0.01, WT vs IL-1ra KO mice. (e) Intrahepatic GSH levels in WT and IL-1ra KO mice after APAP challenge (200 mg/kg). Each value represents means.e.m. (n=6 animals).

Figure 2.

RT–PCR analysis for gene expression of CYP1A2, CYP2E1, and CYP3A11 in the livers of WT and IL-1ra KO mice before or after APAP treatment (200 mg/kg). Representative results from six independent experiments are shown in (a). The ratios of CYP1A2 (b), CYP2E1 (c), and CYP3A11 (d) to -actin in the livers of WT and IL-1ra KO mice are shown here. Each value represents means.e.m. (n=6 animals). ^* P<0.05, WT vs IL-1ra KO mice.

Figure 3.

Western blotting analysis of CYP1A2, CYP2E1, and CYP3A in the livers of WT and IL-1ra KO mice before or after APAP treatment (200 mg/kg). Representative results from six independent experiments are shown in (a). The ratios of CYP1A2 (b), CYP2E1 (c), and CYP3A (d) to -actin in the livers of WT and IL-1ra KO mice are shown in (d). Each value represents means.e.m. (n=6 animals). ^* P<0.05, ^** P<0.01, WT vs IL-1ra KO mice.

Figure 4.

(a) The effects of exogenous IL-1 and IL-1ra on DNA-binding activity of the nuclear NF-B p65 in WT-derived hepatocytes. All values represent the means.e.m. (n=6 independent experiments). ^* P<0.05 vs vehicle treatment. (b) Western blotting analysis of CYP1A2, CYP2E1, and CYP3A in WT-derived hepatocytes after IL-1 and IL-1ra treatment. Representative results from six independent experiments are shown here.

Figure 5.

(a, b) Western blotting analysis of NF-B p65 in cytosol and nuclear extracts of untreated WT and IL-1ra KO mice. Representative results from six independent experiments shown are in (a). The ratios of NF-B p65 to -actin were calculated and are shown in (b). All values represent the means.e.m. (n=6 animals). ^* P<0.05, WT vs IL-1ra KO mice. (c) DNA-binding activity of the nuclear NF-B p65 in the livers of WT and IL-1ra KO mice before and after APAP treatment (200 mg/kg). All values represent the means.e.m. (n=6 animals). ^* P<0.05, WT vs IL-1ra KO mice.

Figure 6.

(a) Analysis of serum ALT levels in WT (n=10) and IL-1ra KO (n=10) mice at 2, 6, 10, and 24 h after APAP challenge. All values represent the means.e.m. ^** P<0.01, WT vs IL-1ra KO mice. (b, c) Histopathological analysis on livers of WT and IL-1ra KO mice after APAP challenge (H&E staining, original magnification, 200). Representative results from six independent experiments are shown in (b). Histological changes were scored as described in 'Materials and Methods' and shown in (c; n=6). All values represent the means.e.m. ^** P<0.01, WT vs IL-1ra KO mice.

Figure 7.

(a, b) Immunohistochemical detection of apoptotic hepatocytes using anti-ssDNA Abs in the livers from WT and IL-1ra KO mice at 2 h after APAP challenge. Representative results from six animals are shown in (a) (original magnification; 200). The numbers of apoptotic hepatocytes per high-power microscopic field ( 400) were enumerated and are shown in (b). All values represent meanss.e.m. (n=6 animals). ^* P<0.05, WT vs IL-1ra KO mice.

Figure 8.

Leukocyte recruitment into the liver of WT and IL-1ra KO mice after APAP challenge. Immunohistochemical detection of neutrophils and macrophages in the livers was performed as described in 'Materials and Methods'. Representative results on neutrophils (a, b) and macrophages (c, d) from six independent experiments are shown here (original magnification; 200). The numbers of neutrophils (e) and macrophages (f) in the livers were determined at the indicated time intervals after APAP challenge (n=6). All values represent the means.e.m. ^* P<0.05, ^** P<0.01, WT vs IL-1ra KO mice.

Figure 9.

RT–PCR analysis for gene expression of IL-1, IL-1, CXCL1/KC, CXCL2/MIP-2, CCL2/MCP-1, and CCL3/MIP-1 in the livers of WT and IL-1ra KO mice at the indicated time intervals after APAP challenge. Representative results from six independent experiments are shown in (a). The ratios of IL-1 (b), IL-1 (c), CXCL1/KC (d), CXCL2/MIP-2 (e), CCL2/MCP-1 (f) and CCL3/MIP-1 (g) to -actin in the livers of WT and IL-1ra KO mice are shown here. Each value represents means.e.m. (n=6 animals). ^* P<0.05; ^** P<0.01, WT vs IL-1ra KO mice.

Figure 10.

The effects of IL-1 pretreatment on APAP-induced liver injury and intrahepatic expression of CYPs, and APAP adduct. WT mice were intraperitoneally administered recombinant IL-1 at 12 h before APAP challenge. (a) Analysis of serum ALT levels in IL-1-treated WT (n=10) and PBS-treated WT (n=10) mice at 2, 6, 10, and 24 h after APAP challenge. All values represent the means.e.m. ^* P<0.05; ^** P<0.01, IL-1 treatment vs PBS treatment. (b) Western blotting analysis of CYP1A2, CYP2E1, and CYP3A in the livers before and after APAP treatment. Representative results from six independent experiments are shown here.

Table 1 - Sequences of the primers used for RT–PCR.