In response to TAT-ARC pretreatment, survival was significantly improved in both PLX4032 chemical structure models compared to PBS or TAT-βgal-pretreated hepatocytes. TAT-ARC-mediated protection of hepatocytes was in both models comparable to that of JNK-inhibitor SP600125 pretreated hepatocytes (Fig. 5A). TNF/AcD or TNF/GalN stimulation of hepatocytes resulted in JNK activation that was inhibited by TAT-ARC or JNK-inhibitor pretreatment (Fig. 5B). In both models, ConA- and GalN/LPS-induced hepatitis, TNF-α levels have been shown

to be critical for hepatocyte killing and high mortality of the animals. Thus, we examined the effect of TAT-ARC on serum TNF-α levels in murine models of hepatitis caused by ConA and GalN/LPS. Importantly, in both models TAT-ARC significantly reduced serum TNF-α levels (Fig. 5C). Together, these data suggest that TAT-ARC prevents TNF-mediated hepatitis by inhibiting TNF-α expression, e.g., in nonparenchymal cells, but also directly protects hepatocytes from apoptosis. The crucial role of JNK signaling in TNF-dependent ALF

was demonstrated by Maeda et al.,21 who showed that mice lacking either JNK1 or JNK2 are highly resistant to ConA-induced ALF. Thus, we investigated JNK activation by immunoblot analysis using liver lysates from both ConA- and GalN/LPS-treated mice. As shown in Fig. 6A, both p46- and p54-JNK phosphorylation, which are essential steps for JNK activation, were significantly Z-VAD-FMK manufacturer induced after ConA or GalN/LPS stimulation. Interestingly, both p46- and p54-JNK phosphorylation were strongly reduced in TAT-ARC-treated mice following ConA stimulation and were completely abrogated after GalN/LPS administration (Fig. 6A). Because activated JNK translocates from cytosol to mitochondria to trigger cell death JNK translocation was assessed by subcellular fractionation and immunoblotting of liver lysates.22 TAT-ARC application completely blocked JNK activation

and subsequent mitochondrial translocation selleck chemicals llc following ConA or GalN/LPS, respectively (Fig. 6B). Because the death-promoting function of JNK-signaling in the liver is antagonized by p38 signaling, p38α phosphorylation was analyzed23 (Fig. 6A). Although no relevant activation of p38-signaling was detected 4 hours after GalN/LPS stimulation when JNK was already activated, TAT-ARC-mediated hepatoprotection following ConA stimulation was associated with a substantial concomitant activation of p38α signaling. It remains unclear whether p38α activation following ConA and TAT-ARC treatment plays a causal role for the observed protective effect seen or is rather a secondary phenomenon. JNK specifically regulates the proapoptotic activity of BH3-only proteins Bax and Bim, which cooperate with Bid in hepatocyte killing.