To further elucidate the molecular mechanisms of the HMGB1- and ASC-mediated inflammatory response in liver IRI, we used an LPS-stimulated BMM cell culture system. As shown in Fig. 4A, western blot–assisted expression of HMGB1 was reduced in ASC-deficient BMMs (0.9-1.2 AU) versus WT controls (2.0-2.2 AU). In contrast to LPS-stimulated WT BMMs (2.1-2.3 AU), the expression of TLR4 (0.2-0.4 AU)

and NF-κB (0.3-0.5 AU) was decreased selleck screening library in ASC-deficient BMMs. Moreover, LPS-stimulated ASC-deficient BMMs showed reduced expression of phospho-p38 MAPK (0.1-0.2 AU) versus WT BMMs (2.7-2.9 AU). Next, we analyzed HMGB1 gene expression with qRT-PCR (Fig. 4B). The mRNA-level coding for HMGB1 was decreased in LPS-stimulated BMMs of ASC KO mice versus LPS-stimulated WT cells (P < 0.05). These findings were confirmed by decreased caspase-1 activity in ASC-deficient BMMs but not WT BMMs (0.51 ± 0.357 versus 3.55 ± 0.19 U, P < 0.005; Fig. 4C). To investigate the role of ASC/caspase-1/IL-1–mediated inflammatory responses, we analyzed the production of IL-1β in BMMs by ELISA. As shown in Fig. 4D, LPS-stimulated ASC-deficient BMMs revealed decreased IL-1β levels in comparison with WT BMMs (186.5 ± 108.7 versus 1722.7 ± 125.9 pg/mL, P < 0.005). Furthermore, Palbociclib our qRT-PCR results showed that IL-1β

and IL-18 decreased in LPS-stimulated, ASC-deficient BMMs versus WT BMMs (P < 0.005; Fig. 4E). Having demonstrated that ASC/caspase-1/IL-1β contributes to the IR inflammation response, we next investigated the role of IL-1β by using a neutralizing anti–IL-1β mAb in our model. The disruption of IL-1β signaling alleviated IR liver damage, as evidenced by diminished sALT levels (11,300 ± 4595.5 versus 33,626 ± 5156.6 and 32,617 ± 3859.4 IU/L, P < 0.0001; Fig. 5A) and well-preserved liver histology (Suzuki's score = 1.1 ± 0.5; Supporting Fig. 1 and Fig. 5B). In contrast, livers in phosphate-buffered saline and IgG groups revealed moderate to severe

edema (Suzuki’s score = 3.6 ± 0.5) and extensive hepatocellular necrosis (Suzuki’s score = 3.7 ± 0.48, P < 0.0001; Fig. 5B). In agreement with these data, MPO activity was suppressed in the anti–IL-1β mAb–treated group versus the phosphate-buffered saline and IgG controls [0.34 ± 0.1 versus 3.13 ± 0.72 (P < 0.05) and 3.08 ± 0.11 U/g (P < 0.005); Fig. 5C]. 上海皓元 To investigate the mechanism by which an anti–IL-1β mAb treatment may exert anti-inflammatory effects, we analyzed the expression of NF-κB, COX2, and inflammatory mediators in IR livers. As shown in Fig. 6A, the anti–IL-1β Ab depressed western blot–assisted expression of NF-κB (0.4-0.6 AU) and COX2 proteins (0.1-0.2 AU) in comparison with WT (2.6-2.8 and 1.4-1.6 AU, respectively) and IgG controls (2.0-2.2 and 1.6-1.8 AU, respectively).