Activation of Slit2-Robo1 signaling promotes liver fibrosis.

The secretory protein Slit2 and its receptor Robo1 are believed to regulate cell growth and migration. Here, we aimed to determine whether Slit2-Robo1 signaling mediates the pathogenesis of liver fibrosis. Serum levels of Slit2 in patients with liver fibrosis were determined by ELISA. Liver fibrosis was induced in wild-type (WT), Slit2 transgenic (Slit2-Tg) and Robo1(+/-)Robo2(+/-) double heterozygotes (Robo1/2(+/-)) mice by carbon tetrachloride (CCl4). The functional contributions of Slit2-Robo1 signaling in liver fibrosis and activation of hepatic stellate cells (HSCs) were investigated using primary mouse HSCs and human HSC cell line LX-2. Significantly increased serum Slit2 levels and hepatic expression of Slit2 and Robo1 were observed in patients with liver fibrosis. Compared to WT mice, Slit2-Tg mice were much more vulnerable to CCl4-induced liver injury and more readily develop liver fibrosis. Development of hepatic fibrosis in Slit2-Tg mice was associated with a stronger hepatic expression of collagen I and α-smooth muscle actin (α-SMA). However, liver injury and hepatic expression of collagen I and α-SMA were attenuated in CCl4-treated Robo1/2(+/-) mice in response to CCl4 exposure. In vitro, Robo1 neutralizing antibody R5 and Robo1 siRNA downregulated phosphorylation of Smad2, Smad3, PI3K, and AKT in HSCs independent of TGF-β1. R5 and Robo1 siRNA also inhibited the expression of α-SMA by HSCs. Finally, the protective effect of R5 on the CCl4-induced liver injury and fibrosis was further verified in mice. Slit2-Robo1 signaling promotes liver injury and fibrosis through activation of HSCs.

Chang J ，Lan T ，Li C ，Ji X ，Zheng L ，Gou H ，Ou Y ，Wu T ，Qi C ，Zhang Q ，Li J ，Gu Q ，Wen D ，Cao L ，Qiao L ，Ding Y ，Wang L ... -  《-》

Slit2-Robo2 signaling modulates the fibrogenic activity and migration of hepatic stellate cells.

Slit/Robo signaling was originally identified as a repulsive guidance cue in regulating axon branching and neuronal migration. Hepatic stellate cells (HSCs) are the key fibrogenic cells in the liver, which are migratory when activated, and express neural crest markers. The aim of the present study was to investigate the functional significance of Slit/Robo signaling in liver fibrogenesis and in HSCs. By transcriptomic analysis it was found that axon guidance signaling pathways were significantly upregulated in both diethylnitrosamine (DEN) and thioacetamide (TAA)-induced experimental liver fibrosis. The up-regulation of the ligand Slit2 and membrane receptor Robo2 genes within this pathway was further validated in TAA-induced fibrotic livers. By immunofluorescence staining, Robo2 was localized in fibrotic septa of fibrotic liver and on the surface of HSCs. By Western blot analysis, recombinant Slit2 (rSlit2) was found to promote fibrogenic protein expression in JS1 cells, an immortalized mouse HSC line, while activating PI3K/Akt signaling pathway. This effect was abrogated by LY294002, a PI3K/Akt pathway inhibitor. In addition, rSlit2 stimulation markedly inhibited JS1 cells migration in transwell migration assays, which was abrogated by small interfering RNA (siRNA) knockdown of Robo2 in the cells. The present study provides evidence that Slit2/Robo2 signaling mediates the pathogenesis of hepatic fibrogenesis and regulates HSCs biology, thus providing potential markers for HSCs, and therapeutic and diagnostic target toward liver fibrosis.

Zeng Z ，Wu Y ，Cao Y ，Yuan Z ，Zhang Y ，Zhang DY ，Hasegawa D ，Friedman SL ，Guo J ... -  《-》

Slit2 signaling contributes to cholestatic fibrosis in mice by activation of hepatic stellate cells.

Liver Cholestasis is a widespread disease of broad etiologies and ultimately results in fibrosis, which is still lacking effective therapeutic strategies. Activation of hepatic stellate cells (HSCs) is the key event of liver fibrosis. Here, we aimed to investigate the effect and mechanism of the Slit2 signaling in cholestasis-induced liver fibrosis. Our findings revealed that the serum levels and hepatic expression of Slit2 were significantly increased in patients with primary biliary cirrhosis (PBC). Additionally, Slit2-Tg mice were much more vulnerable to BDL-induced liver injury and fibrosis compared to WT control. Slit2 up-regulation by Slit2 recombinant protein induced proliferation, and inhibited apoptosis of human HSCs cell line LX-2 via p38 and ERK signaling pathway, resulting in the activation of HSCs. In contrast, Slit2 down-regulation by siRNA silencing inhibit the activation of HSCs. In conclusion, Slit2 is involved in the activation of HSCs and liver fibrogenesis, highlighting Slit2 as a potential therapeutic target for liver fibrosis.

Li C ，Yang G ，Lin L ，Xuan Y ，Yan S ，Ji X ，Song F ，Lu M ，Lan T ... -  《-》

Involvement of the nuclear high mobility group B1 peptides released from injured hepatocytes in murine hepatic fibrogenesis.

This study investigated the pro-fibrogenic role of high mobility group box 1 (HMGB1) peptides in liver fibrogenesis. An animal model of carbon tetrachloride (CCl4)-induced liver fibrosis was used to examine the serum HMGB1 levels and its intrahepatic distribution. The increased serum HMGB1 levels were positively correlated with elevation of transforming growth factor-β1 (TGF-β1) and collagen deposition during fibrogenesis. The cytoplasmic distribution of HMGB1 was noted in the parenchymal hepatocytes of fibrotic livers. In vitro studies confirmed that exposure to hydrogen peroxide and CCl4 induced an intracellular mobilization and extracellular release of nuclear HMGB1 peptides in clone-9 and primary hepatocytes, respectively. An uptake of exogenous HMGB1 by hepatic stellate cells (HSCs) T6 cells indicated a possible paracrine action of hepatocytes on HSCs. Moreover, HMGB1 dose-dependently stimulated HSC proliferation, up-regulated de novo synthesis of collagen type I and α-smooth muscle actin (α-SMA), and triggered Smad2 phosphorylation and its nuclear translocation through a TGF-β1-independent mechanism. Blockade with neutralizing antibodies and gene silencing demonstrated the involvement of the receptor for advanced glycation end-products (RAGE), but not toll-like receptor 4, in cellular uptake of HMGB1 and the HMGB1-mediated Smad2 and ERK1/2 phosphorylation as well as α-SMA up-regulation in HSC-T6 cells. Furthermore, anti-RAGE treatment significantly ameliorated CCl4-induced liver fibrosis. In conclusion, the nuclear HMGB1 peptides released from parenchymal hepatocytes during liver injuries may directly activate HSCs through stimulating HSC proliferation and transformation, eventually leading to the fibrotic changes of livers. Blockade of HMGB1/RAGE signaling cascade may constitute a therapeutic strategy for treatment of liver fibrosis.

Kao YH ，Lin YC ，Tsai MS ，Sun CK ，Yuan SS ，Chang CY ，Jawan B ，Lee PH ... -  《-》

Physalin D attenuates hepatic stellate cell activation and liver fibrosis by blocking TGF-β/Smad and YAP signaling.

Hepatic fibrosis is considered integral to the progression of chronic liver diseases, as it leads to the development of cirrhosis and hepatocellular carcinoma. The activation of hepatic stellate cells (HSCs) is the dominant event in hepatic fibrogenesis. The transforming growth factor-β1 (TGF-β1) and Yes-associated protein (YAP) pathways play a pivotal role in HSC activation, hepatic fibrosis and cirrhosis progression. Therefore, targeting the TGF-β/Smad and YAP signaling pathways is a promising strategy for antifibrotic therapy. The present study investigated the protective effects of Physalin D (PD), a withanolide isolated from Physalis species (Solanaceae), against liver fibrosis and further elucidated the mechanisms involved in vitro and in vivo. We conducted a series of experiments using carbon tetrachloride (CCl4)- and bile duct ligation (BDL)-induced fibrotic mice and cultured LX-2 cells. Serum markers of liver injury, and the morphology, histology and fibrosis of liver tissue were investigated. Western blot assays and quantitative real-time PCR were used to investigate the mechanisms underlying the antifibrotic effects of PD. PD decreased TGF-β1-induced COL1A1 promoter activity. PD inhibited TGF-β1-induced expression of Collagen I and α-smooth muscle actin (α-SMA) in human hepatic stellate LX-2 cells. PD significantly ameliorated hepatic injury, including transaminase activities, histology, collagen deposition and α-SMA, in CCl4- or BDL-induced mice. Moreover, PD markedly decreased the expression of phosphorylated Smad2/3 in vitro and in vivo. Furthermore, PD significantly decreased YAP protein levels, and YAP knockdown did not further enhance the effects of PD, namely α-SMA inhibition, Collagen I expression and YAP target gene expression in LX-2 cells. These results clearly show that PD ameliorated experimental liver fibrosis by inhibiting the TGF-β/Smad and YAP signaling pathways, indicating that PD has the potential to effectively treat liver fibrosis.

Xiang D ，Zou J ，Zhu X ，Chen X ，Luo J ，Kong L ，Zhang H ... -  《-》