IL-10R activation of the STAT3 pathway increases expression of STAT3 responsive genes, such as SOCS3 and HO-1.2 Culture of Kupffer cells with gAcrp increased the expression of SOCS3 and HO-1 mRNA (Fig. 5A/B). Consistent with the increased gAcrp-stimulated IL-10 expression and phosphorylation of STAT3 after chronic ethanol feeding, gAcrp treatment increased HO-1 and SOCS3 mRNA expression to a greater extent in Kupffer cells from ethanol-fed compared with pair-fed rats (Fig. 5A/B). gAcrp increased HO-1 protein expression

in Kupffer cells from ethanol-fed rats (Fig. 5C) but not in Kupffer cells from pair-fed rats. Despite the increase in SOCS3 mRNA, SOCS3 protein was not significantly https://www.selleckchem.com/products/ABT-737.html increased in response to gAcrp in Kupffer cells from either ethanol-fed or pair-fed rats (Fig. 5C). Because HO-1 is a critical mediator of the anti-inflammatory effects of IL-10,15 we Proteases inhibitor further investigated the mechanisms by which gAcrp

increased HO-1 expression in Kupffer cells. To test whether gAcrp induces HO-1 expression through an IL-10–dependent pathway, Kupffer cells were transfected with siRNA against IL-10 to knockdown IL-10 expression. When IL-10 expression was inhibited, gAcrp-stimulated HO-1 mRNA expression was suppressed in Kupffer cells from both pair-fed and ethanol-fed rats (Fig. 6A). Scrambled siRNA administration had no effect on gAcrp-stimulated HO-1 mRNA expression (Fig. 6A). The signaling pathways downstream of gAcrp-stimulated IL-10 expression were investigated with the use of selective inhibitors. The gAcrp-stimulated HO-1 mRNA expression

was attenuated when Kupffer cells were pretreated with a selective inhibitor of STAT3 (JSI-124) (Fig. 6B). Finally, IL-10–stimulated HO-1 mRNA expression was suppressed in Kupffer cells transfected with siRNA against STAT3; scrambled siRNA had no effect on IL-10–dependent HO-1 expression (Fig. 6C). The siRNA knockdown of STAT3 decreased STAT3 protein expression (Supporting Fig. 1C). Taken together, these data demonstrate that gAcrp induces HO-1 expression via an IL-10/STAT3–dependent pathway. Phospholipase D1 Because HO-1 has potent anti-oxidant and anti-inflammatory activity, we investigated the role of HO-1 in mediating the effect of gAcrp using both biochemical and siRNA knockdown strategies. First, when Kupffer cells were treated with zinc protoporphyrin, a competitive inhibitor of HO-1 activity, before culture with gAcrp, the inhibitory effect of gAcrp on LPS-stimulated TNF-α expression was ameliorated (Fig. 7A). Similar results were obtained using an siRNA strategy. When Kupffer cells were transfected with siRNA against HO-1, expression of HO-1 protein was decreased compared with Kupffer cells transfected with scrambled siRNA (Supporting Fig. 1B). Knockdown of HO-1 with siRNA prevented the inhibitory effect of gAcrp on LPS-stimulated TNF-α mRNA, whereas scrambled siRNA had no effect (Fig. 7B).