These data suggest that CCl4-induced liver fibrosis might be inhi

These data suggest that CCl4-induced liver fibrosis might be inhibited in SMP30 KO mice due to inhibition of the nuclear translocation of p-Smad2/3 and a lower level of ROS and lipid peroxidation as compared with WT mice. To GSK126 determine if activated HSCs express SMP30 in the fibrotic liver, we performed immunohistochemistry using

SMP30 antibody and α-SMA antibody on serial liver sections. As shown in Fig. 4A, nonparenchymal cells exhibited no expression of SMP30 (Fig. 4A, a, arrowheads and b, arrows), whereas hepatocytes revealed obvious nuclear and cytoplasmic expression of SMP30 (Fig. 4A, a and b, asterisk). In normal livers of WT mice, the quiescent HSCs containing lipid droplets in their cytoplasm also showed depletion of SMP30 (Fig. 4A, a, arrowhead). To confirm more clearly whether HSCs from WT mice express SMP30, we performed immunocytochemistry and RT-PCR analysis using isolated HSCs. The

isolated HSCs were cultured for 6 days in serum-containing medium and learn more the SMP30 messenger RNA (mRNA) expression was determined on day 0, day 3, and day 5. As expected, HSCs from the WT mice and SMP30 KO mice revealed obvious SMP30 deficiency (Fig. 4B,C). Immunocytochemistry also showed well-matched results with the RT-PCR analysis confirming HSCs from the WT mice and the SMP30 KO mice do not express SMP30 (Fig. 4B). These data demonstrated that SMP30 is not involved directly in the activation of HSCs, suggesting the possibility of the participation of other up-regulated or down-regulated factors affecting hepatocytes and HSCs in the liver of the SMP30 KO mice. As expected, the SMP30 KO mice liver tissue showed significantly enhanced PPAR-γ expression levels and mRNA levels compared with those of the WT mice (Fig. 5A,B). In order to compare the expression level selleck inhibitor of PPAR-γ, p-Smad2/3, α-SMA, and the activation degree of SMP30 KO HSC with WT HSC, HSCs were isolated and cultured in serum containing medium for 7 days. It was found that WT HSCs were activated faster compared with SMP30 KO HSCs until day 5 (Fig. 5C). Moreover, both the α-SMA expression and the p-Smad2/3 nuclear expression were much stronger in WT HSCs

than in SMP30 KO HSCs (Fig. 5C). Additionally, it was observed that SMP30 KO HSCs contained a greater number of cytoplasmic lipid droplets compared with WT HSCs at the same time (Fig. 5D), which was well-matched with the HSC hypertrophy morphology in vivo in our previous unpublished data. For the sake of clarity, we used an RT-PCR analysis. On day 0, day 3, and day 5 the α-SMA mRNA expression levels of SMP30 KO HSCs were significantly inhibited compared with those of WT mice HSCs (Fig. 5E). The PPAR-γ expression levels showed time-dependent decreases in both WT mice HSCs and SMP30 KO HSCs. However, SMP30 KO HSCs revealed much greater PPAR-γ expression levels compared with WT HSCs at the same time (Fig. 5E). We observed that PPAR-γ negatively down-regulated α-SMA mRNA expression levels.

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