7B). Additionally, we determined that this difference was not the result of decreased hepatocyte death and passive HMGB1 release by determining supernatant levels of lactate dehydrogenase (LDH) and β-actin (Fig. 7B). The effect of this website the JNK inhibitor on HMGB1 release in vivo after I/R was also investigated. Efficacy of the JNK inhibitor was first confirmed by decreased phosphorylation of c-Jun, compared to vehicle control, on western blotting analysis (Fig. 7C). With administration of the inhibitor given before I/R, there was a significant decrease in serum levels of HMGB1 after I/R (Fig. 7D). We, again, confirmed that this decrease in HMGB1 was not solely the result of decreased hepatocellular

injury with JNK inhibition by determining that sALT screening assay levels were unchanged at 3 hours of reperfusion (Supporting Fig. 3), in addition to histologic analysis (data not shown). The p38 inhibitor, SB203580, was also studied both in vitro and in vivo similar to the JNK inhibitor. With administration of the p38 inhibitor before hypoxia exposure in vitro and before I/R in vivo, there was no inhibitory effect noted on HMGB1 release (data not shown), suggesting that p38 does not play a major role in TLR4-mediated HMGB1 release. Therefore, it seems that activation of JNK, but not p38, is required

for the extracellular release of HGMB1, both after hypoxic stress in vitro and I/R in vivo. Hepatic I/R is dependent on the pattern recognition receptors

(PRRs) to sense and initiate the sterile inflammatory response. Although the central role of the PRR, TLR4, in this process had been previously demonstrated,5, 6 the role of TLR4 on individual cell types, specifically, parenchymal versus NPC, during the sterile inflammatory response was conflicted. Therefore, in this study, we describe the novel use of Cre-loxP technology to knock out TLR4 in HCs, myeloid cells, and DCs and elucidate their individual role in I/R injury. The key and novel findings include the following: (1) Both HC and myeloid cell TLR4 is required for maximal I/R-associated injury; (2) DC TLR4−/− worsens injury after I/R and is associated with decreased IL-10 expression; (3) HCs are a major source of circulating selleckchem HGMB1 after I/R; (4) HCs respond to hypoxia with increased phosphorylation of MAP kinases (JNK and p38) in a TLR4-dependent fashion; and (5) hypoxia-induced HMGB1 release from HCs is dependent on the function of JNK. Previous work to define the function of TLR4 on individual cellular populations was limited to the use of chimeras. Although we have shown that there was not a significant difference in hepatic I/R-induced injury with lack of TLR4 on non-BM-derived cells, there was a trend toward an effect and others have subsequently shown that both BM and non-BM-derived populations have a role in mediating I/R injury.