BI 113823 reduced CCl4-induced liver fibrosis and PH, and improves survival in mice
Liver fibrosis in mice was evaluated by HE and sirius red staining, and hydroxyproline assay at 6 weeks after the initial CCl4 challenge. HE staining of liver sections from CCl4-treated mice showed prominent hepatic steatosis, necrosis, fibrotic septa formation, disruption of tissue architecture, and inflammatory cell infiltration (Fig. 1A). Liver sections from BI 113823-treated mice showed relatively normal architecture (Fig. 1A). Treatment with BI 113823 significantly reduced CCl4-induced liver fibrosis, as demonstrated by reduction in sirius red staining for collagen and reduction of hydroxyproline content in liver (Fig. 1A and B). HSC activation and differentiation, as assessed by immunofluorescence staining for α-SMA, showed marked increase in CCl4-treated mice, and this was correlated with marked induction of B1R expression in CCl4-induced fibrotic liver (Fig. 1A). The expression of α-SMA and B1Rs in liver tissue was significantly reduced in mice treated with BI 113823 (Fig. 1A, G and H). B2R expression was not changed (Fig. 1H). Immunofluorescence double staining results showed that cells expressing the B1Rs co-localized with those expressing α-SMA (Fig. 1A). These findings indicate B1R signalling mediates CCl4-induced liver fibrosis via activation of HSCs.
Six weeks after the initial CCl4 challenge, bodyweight growth was slightly lower compared to the sham control (not significantly different, Fig. 1D). However, liver/body weight ratio and portal vein pressure were significantly increased in CCl4-challenged mice, compared to the sham control (Fig. 1C). CCl4-induced liver fibrosis and PH were associated with high mortality rate in those mice (12/22 death, Fig. 1F). In contrast, mice that received BI 113823 showed significantly lower liver/body weight ratio, lower portal vein pressure and, most importantly, improved survival (Fig. 1C, E and F).
BI 113823 reduced BDL-induced liver fibrosis and PH, and improved survival in mice
In our next model, liver fibrosis in mice was evaluated 3 weeks after BDL. Liver sections from BDL mice showed marked hepatic steatosis, necrosis, inflammatory cell infiltration and fibrotic septa formation, as well as extensive collagen deposition as accessed by HE and sirius red staining, and hydroxyproline assay (Fig. 2A and B). Similarly, liver fibrosis in BDL mice was significantly reduced in mice treated with BI 113823, compared to vehicle controls (Fig. 2A). Liver fibrosis in BDL mice was also associated with marked increase of α-SMA expression, as well as co-localized induction of B1Rs. BI 113823 reduced the expression of α-SMA and B1Rs in BDL mice (Fig. 2A, G and H). These findings further support the hypothesis that B1R signalling mediates chronic liver fibrosis via activation of HSCs.
Three weeks after BDL, liver/body weight ratio was not significantly different among groups (Fig. 2C) and body weight was significantly decreased in vehicle-treated BDL mice, compared to sham control (Fig. 2D). However, the decrease of body weight after BDL was less in BI 113823-treated mice (Fig. 2D). BDL resulted in severe PH, and BI 113823 significantly decreased portal vein pressure (Fig. 2E) and improved survival (Fig. 2F).
Altogether, these data demonstrate that, in experimental models of CLD, B1R inhibition with BI 113823 attenuates hepatic fibrosis formation, PH and, most importantly improves survival.
BI 113823 reduced the expression of profibrogenic mediators in liver after chronic CCl4 challenge and BDL
We next determined the expression of profibrotic mediators in CCl4- and BDL-induced liver fibrosis. Western blot analysis showed an extensive increase in the expression of profibrotic mediators α-SMA, collagen I, VEGF and proliferating cell nuclear antigen (PCNA) in CCl4- and BDL-induced liver fibrosis (Fig. 3A and B, Additional file 1: Fig. S2A and B). Compared to vehicle-treated mice, expression of α-SMA, collagen I, VEGF and PCNA were all significantly reduced in mice treated with BI 113823 (Fig. 3A and B). Furthermore, hepatic expression of phosphorylated Akt protein was significantly increased in CCl4- and BDL-induced liver fibrosis (Fig. 3A and B), and BI 113823 reduced this expression (Fig. 3A and B, Additional file 1: Fig. S2A and S2B).
RT-PCR showed that hepatic mRNA expressions of growth factors PDGF, TGF and CTGF, as well as ECM molecules Col-1, Col-3 and Col-4 increased significantly in CCl4- and BDL-induced liver fibrosis, and all were reduced in mice treated with BI 113823 (Fig. 3C and D). Together, these findings further demonstrate that BI 113823 attenuates hepatic fibrosis via inhibition of HSC activation and proliferation, downregulates the expression of fibrogenic mediators via inhibition of the Akt signalling pathway.
BI 113823 reduced inflammatory responses in CCl4- and BDL-induced liver fibrosis
Chronic CCl4 challenge and BDL resulted in marked hepatic inflammatory cell infiltration in mice (Fig. 4A and B). Immunohistochemical staining of liver sections showed a marked increase of CD68 positive macrophages and neutrophils (positive for neutrophil elastase) in both CCl4- and BDL-induced liver fibrosis (Fig. 4A and B). BI 113823 reduced hepatic inflammatory cell infiltration of macrophages and neutrophils, and hepatocyte apoptosis (Fig. 4A and B).
To provide further evidence that B1Rs mediate inflammatory cell infiltration and inflammatory mediator production in CCl4- and BDL-induced liver fibrosis, we assessed protein expression of inflammatory cell markers CD68 and neutrophil elastase, and chemoattractant markers MCP-1 and COX-2 in liver tissues. Western blot of liver lysates showed that inflammatory molecules, COX-2, MCP-1, CD68, and neutrophil elastase were strongly increased in CCl4- and BDL-induced liver fibrosis, compared to sham control mice (Fig. 5A, B, Additional file 1: Fig. S3A and B). BI 113823 significantly reduced hepatic expression of these inflammatory mediators (Fig. 5A, B, Additional file 1: Fig. S3A and B).
RT-PCR analyses supported these observations by showing that mRNAs of inflammatory mediators, IL-1β, IL-6, MCP-1, MCP-3 and TIMP-1 increased in CCl4- and BDL-induced liver fibrosis, and decreased in BI 113823-treated mice (Fig. 5C and D). These findings indicate that B1R-mediated inflammatory responses are highly implicated in the pathogenesis of liver fibrosis.
BI 113823 reduced human inflammatory cell migration and activation
We next determined the effect of B1R-mediated inflammatory responses and activation in human monocytes and neutrophils. BI 113823 inhibited TNF-α-induced monocyte and neutrophil transmigration (Fig. 6A and D), reduced LPS-induced TNF-α production in monocytes and reduced LPS-stimulated MPO activity in neutrophils (Fig. 6B and E). LPS treatment resulted in a significant increase in activation of human monocytes and neutrophils, as evidenced by an increase in cell surface molecule CD11 and CD18 expression (Fig. 6C and F, Additional file 1: Fig. S4A and S4B). LPS-induced monocyte and neutrophil activation were inhibited by BI 113823 treatment (Fig. 6C and F, Additional file 1: Fig. S4A and B).
BI 113823 reduced hHSC activation, contraction, migration and fibrosis protein expression
TGF-β is a major profibrogenic cytokine responsible for initiation and perpetuation of HSC activation. In this study, TGF-β stimulated strong intracellular expression of α-SMA in LX2 hHSCs, indicating HSC activation switching from a quiescent state to a myofibroblast phenotype, which was inhibited by BI 113823 (Fig. 7A). In collagen gel contraction assays, BI 113823 reduced TGF-β-stimulated hHSC contractility measured at 48 h by final gel area (Fig. 7C and D). Next, we determined the downstream effect of myofibroblast transdifferentiation, specifically fibrosis molecules production. Western blot analysis showed that TGF-β stimulated a significant increase in the expression of profibrogenic proteins α-SMA, Col-1, VEGF and MCP-1, as well as the expression of B1Rs and B2Rs in LX-2 hHSCs. Treatment of hHSCs with BI 113823 reduced the expression of profibrotic proteins and B1Rs (Fig. 7B). Furthermore, BI 113823 treatment inhibited TGF-β stimulated phosphorylation of Akt in LX-2 hHSCs (Fig. 7B).
HSCs can migrate to the sites of tissue injury during fibrogenesis and differentiate into contractile myofibroblasts that promote liver stiffness and PH. We further examined the effects of BI 113823 using migration (Fig. 7E) and wound-healing assays (Fig. 7F and G), where the selective B1R agonist [21] DBK stimulated hHSCs migration in a dose-dependent manner (Fig. 7F). BI 113823 treatment significantly inhibited FBS- and DBK-stimulated migration in LX-2 hHSCs (Fig. 7E–G).
BI 113823 inhibits DBK stimulated HSC proliferation, arrests cell cycle from G1 to S phase transition, and blocks of PI3K/AKT signalling pathway
In this study, we found that the selective B1R agonist DBK enhances the proliferation of hHSCs (Fig. 8A). BI 113823 dose-dependently inhibited DBK-stimulated hHSC proliferation (Fig. 8B) but did not affect non-DBK-stimulated hHSC growth or apoptosis (Fig. 8C and D). Flow cytometry analysis showed that DBK (2 µM) stimulated the entry of hHSCs into S phase of the cell cycle. At 1 µM, BI 113823 reduced DBK-stimulated G1 to S phase cell cycle transition by 64% (Fig. 8E, Additional file 1: Fig. S5A). Taken together, the present study provides direct evidence that DBK induces hHSC proliferation and migration via activation of B1Rs.
Western blot analysis showed that B1Rs were weakly expressed in serum-free quiescent or growing hHSCs, but were strongly upregulated by DBK (Fig. 8F, Additional file 1: Fig. S5B). DBK also stimulated phosphorylation of P70S6 kinase, PI3K, GSK3, ERK and increased the expression of PCNA in HSCs (Fig. 8F, Additional file 1: Fig. S5B). Interestingly, phosphorylation of P70S6 kinase was not detected in serum-free quiescent HSCs with or without DBK, but DBK stimulated strong phosphorylation of P70S6 kinase in HSCs with 1% FBS. BI 113823 treatment inhibited phosphorylation of P70S6 kinase, PI3K, GSK3 and ERK, blocked B1R induction, and reduced expression of PCNA (Fig. 8F, Additional file 1: Fig. S5B). Together, these findings demonstrate that DBK-stimulated B1R signalling mediates HSC proliferation and migration via activation of the PI3K/Akt pathway.