To support these data and further define the mechanism by which R

To support these data and further define the mechanism by which Ron may regulate cytokine production, the human macrophage cell line THP-1 was utilized. Differentiated THP-1 cells express abundant levels of Ron (data not shown). As shown in Fig. 3B, LPS stimulation induces the phosphorylation of NF-κB at Ser536, a verified marker of NF-κB transcriptional activity,22

in THP-1 cells. Treatment with HGFL decreases this phosphorylation. In addition, the stimulation of IκB kinase (IKK) phosphorylation, an upstream kinase that regulates NF-κB activation, is reduced by HGFL treatment, similar to recently reported findings.21 Similar results are also observed with Kupffer cells find more that exhibit less NF-κB and IKK phosphorylation in response to HGFL treatment than that observed with LPS alone (Fig. AZD1208 mouse 3C and data not shown). Given the significant decrease in hepatocyte apoptosis observed in TK−/− mice in response to LPS/GalN treatment in vivo16 and the alteration of cytokine signaling observed in the TK−/− Kupffer cells ex vivo, we initially hypothesized that the altered cytokine milieu produced by the TK−/− Kupffer cells was capable of affording protection to hepatocytes during the induction of acute liver failure. To test this hypothesis, we isolated Kupffer cells from TK+/+ and TK−/− mice

and stimulated them ex vivo with LPS or vehicle. Equal amounts of conditioned media were collected after 2 hours and were applied to the AML12 hepatocyte cell line in the presence of the transcriptional inhibitor actinomycin D (ActD) to emulate conditions in vitro of hepatocyte death observed

in previous in vivo experiments. As shown in Fig. 4, whereas the viability of hepatocytes exposed to control media or conditioned media from either untreated TK+/+ or TK−/− Kupffer cells did not significantly differ from one another, the percent hepatocyte death was significantly increased in the presence of conditioned media from LPS-treated TK−/− Kupffer cells compared to similarly treated TK+/+ Kupffer cells. These results demonstrate that the LPS-induced cytokine milieu from the TK−/− 上海皓元医药股份有限公司 Kupffer cells is capable of inducing increased hepatocyte death compared to control cells under these conditions. In Fig. 4C, neutralization of TNF-α in the TK−/− Kupffer cell conditioned media restored hepatocyte viability to near 100%, suggesting that TNF-α has a prominent role in inducing hepatocyte death in ActD-treated hepatocytes. Given that our prior studies in TK−/− mice demonstrated lessened hepatocyte apoptosis in vivo, despite elevated serum levels of TNF-α, following the induction of acute liver failure by LPS/GalN treatment, we next sought to determine if Ron signaling regulates hepatocyte survival.

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