39% ± 0.26%) (Fig. 1C). In PAR-2 KO mice, CCl4 administration KU-57788 in vitro induced similar fibrosis to that of WT mice at 5 weeks (2.07% ± 0.26%). However, there was no progression of liver fibrosis with continued CCl4 exposure between 5 and 8 weeks in the PAR-2 KO mice (2.09% ± 0.28%). At 8 weeks, there was significantly less

hepatic fibrosis in the PAR-2 KO, compared to WT, mice (P = 0.004) (Fig. 1B,C). Histological assessment of fibrosis correlated closely with other indices of hepatic collagen content in mice given CCl4. At 8 weeks, PAR-2 KO mice showed significantly less induction of procollagen mRNA (1.8- ± 0.23-fold above untreated mice), compared with WT mice (5.9- ± 1.08-fold; P = 0.002) (Fig. 1D). After 5 weeks of CCl4 administration, similar increases in hepatic hydroxyproline were observed in WT and PAR-2 KO mice (0.45 ± 0.02 μg/mg and 0.43 ± 0.009 μg/mg, respectively) (Fig. 1E). However, after 8 weeks, whereas hepatic hydroxyproline content increased significantly in WT mice, there was no increase in PAR-2 KO mice, compared to levels at 5 weeks. PAR-2 KO mice (0.42 ± 0.026) had significantly less hepatic hydroxyproline, compared to WT mice (0.63 ±

0.03) at 8 weeks (P < 0.002). αSMA is a marker of HSC activation and myofibroblast differentiation. In WT mice, hepatic fibrosis induced by the administration of CCl4 was accompanied by a progressive increase in αSMA expression at 8 weeks, compared to untreated mice. PI3K targets In PAR-2 KO mice receiving CCl4, induction of αSMA was Racecadotril similar to WT mice treated with CCl4 at 5 weeks (Fig. 2A), but did not increase further, resulting in significantly less αSMA expression, compared to WT mice at 8 weeks (P = 0.014). CCl4-induced hepatic fibrosis was associated with up-regulation of TGFβ mRNA (3.44- ± 0.72-fold greater than control) and protein (9.2 ± 0.9 pg/mg liver, control 6.9 ± 0.19 pg/mg) in WT mice at 8 weeks. In PAR-2 KO mice, TGFβ mRNA up-regulation was significantly

reduced (1.38- ± 0.23-fold of control; P = 0.016, compared to WT) (Fig. 2B), as was TGFβ protein, which was similar to control levels (Fig. 2C). Matrix metalloproteinases (MMPs) and their specific tissue inhibitors, tissue inhibitors of metalloproteinase (TIMPs), regulate ECM composition and their expression is altered in response to liver injury. In WT mice treated with CCl4 for 8 weeks, both MMP-2 and TIMP-1 mRNA increased, consistent with active ECM remodeling during the development of hepatic fibrosis (Fig. 3A,B). Both MMP-2 and TIMP-1 mRNA expression were significantly reduced in PAR-2 KO mice, compared to WT mice, suggesting that ECM remodeling is reduced in association with the arrest in progression of fibrosis between 5 and 8 weeks in PAR-2 KO mice. The temporal pattern of PAR-1 mRNA expression was examined to investigate the potential mechanisms for the lack of early protection against hepatic fibrosis observed in PAR-2 KO mice.