Figure 6. Necrotic hepatocyte induces liver macrophage activation via NOX1.

(A) Bone marrow derived macrophages (BMDM) from WT and Nox1^−/− mice were incubated with condition medium (CM) from necrotic hepatocytes (lysed by three cycles of freezing and thawing, 5×10⁵ cells per ml) for 4 hours, and cellular RNA was extracted. Relative expression of the indicated genes was determined by quantitative RT-PCR (Q-PCR). (B) IB analysis of indicated target proteins in cell lysate collected from BMDM stimulated with CM from necrotic hepatocytes. (C) BMDMs were pretreated with MEK inhibitor U0126 (10μM) for 30min, and then incubated with CM from necrotic hepatocytes for 4 hours. Relative expression of the indicated genes was determined by Q-PCR. (D) IB analysis of indicated target proteins in cell lysate collected from BMDM pretreated with U0126 and stimulated with CM from necrotic hepatocytes. (E-F) BMDMs were pretreated with JNK inhibitor SP600125 (10μM), p38 inhibitor SB203580 (10μM), IKK inhibitor Bay11–7082 (5μM) or DPI (10μM) for 30min, and then incubated with CM from necrotic hepatocytes for 4 hours. Relative expression of the indicated genes was determined by Q-PCR. (G) BMDM from WT and Nox1^−/− mice were incubated with CM from necrotic hepatocytes for 4 hours. Cells were then stained with CM-H2DCFDA, and florescent intensity was quantified. (H) BMDMs were pretreated with isotype control or anti-HMGB1 antibody (2.5ug/ml) for 1h, and then incubated with CM from necrotic hepatocytes for 4 hours. Relative expression of the indicated genes was determined by QPCR. Results are means ± s.e.m. Student’s t-test for independent samples and unequal variances was used to assess statistical significance (*P<0.05, **P<0.01, ***P<0.001). Ordinary one-way ANOVA was used to compare between treatment groups to assess statistical significance (*P<0.05, **P<0.01, ***P<0.001).