BDL is an established animal model of cholestasis with complete biliary obstruction and accumulation of multiple primary bile acids in liver and serum [19
]. The changes that are observed in the BDL model of cholestasis parallel those that occur in cholestasis in humans [20
]. A previous study has indicated that TGFβ1 plays a prominent role in stimulating liver fibrogenesis through myofibroblasts that are derived from HSCs [10
] during the development of chronic liver injury, including inflammation, fibrosis, and regeneration. In this study, IHQD significantly ameliorated the cholestatic liver fibrosis in BDL rats, and IHQD inhibited the protein expression of TGFβ1.
TGFβ1, a prototype of multifunctional cytokines, has been proposed to be a "master switch" in the induction of fibrosis [21
]. Upregulation of TGFβ1 expression is a consistent feature of most fibrotic disease [22
]. Cells, such as HSCs, Kupffer cells, myofibroblasts, endothelial cells, and invading mononuclear cells, could synthesize and release TGFβ1 [12
]. Many studies have created a strong rationale for an antifibrotic strategy in which the principal objective of treatment is blocking TGFβ1.
Our work suggested that hepatocytes, cholangiocytes, and myofibroblasts could synthesize and release TGFβ1. In return, TGFβ1 promoted reduction of hepatocytes, proliferation of cholangiocytes, and activation of myofibroblasts (Figures , , ). IHQD suppressed TGFβ1 protein expression and thus suppressed the effects that TGFβ1 protein expression had on these cell types. These results suggested that TGFβ1 was a plausible candidate that may play a central role in mediating cholestatic fibrosis, and that suppression of TGFβ1 expression may be an effective target of IHQD antifibrotic injury.
TGFβ1 signaling occurs through heteromeric complexes of type I and type II receptors (TβRI, TβRII) and cytoplasmic protein mediators belonging to the Smad family [25
]. The activation of the receptor complexes occurs when TβRII transphosphorylates the GS domain of TβRI. The activated TβRI associates transiently with, and also phosphorylates, the receptor-regulated Smads (R-Smads), Smad2 and Smad3. Once phosphorylated, R-Smads dissociate from the receptor, bind to Smad4, and enter the nucleus. The activated Smad complex binds to target promoters in association with DNA-binding cofactors, and recruits coactivators to activate transcription. Alternatively, activated Smad complex can also recruit corepressors, which in turn bind histone deacetylases. As a result, Smads can either positively or negatively regulate the transcription of specific genes in response to TGFβ1 signaling [25
]. Antagonistic Smad7 competes with R-Smads for binding sites to activate TβRI and thus prevents the phosphorylation of R-Smads, resulting in receptor degradation [28
]. Therefore, Smad7 terminates or reduces the strength of the TGFβ1-R-Smads signal in a negative feedback loop.
In this study, focusing on the roles of Smad3 and antagonistic Smad7, we investigated the differential regulatory mechanisms of the TGFβ1 signal in rats during chronic cholestatic liver injury. Our data suggested that in the cholestatic liver injury, hepatocytes, cholangiocytes, and myofibroblasts released TGFβ1 and activated TGFβ1 receptors. TGFβ1 bound its receptor, phosphorylated Smad3, and accelerated liver fibrosis. A remarkable finding noted in other data was that cholestasis induced TGFβ signaling via Smad3 in vivo [29
]. In liver wound healing, Smad3 is required for hepatic stellate cell matrix production and matrix interactions [9
], as well as maximal type I collagen induction [31
]. In addition, high-level expression of Smad7 protein was also observed. In the process of cholestatic liver fibrosis, Smad7 antagonized Smad3-mediated TGFβ1 signal in a negative feedback loop without interfering with fibrogenesis. This finding is consistent with previous work [29
]. IHQD markedly inhibited the protein expression of Smad3 without changing Smad7 expression, suggesting that IHQD ameliorated cholestatic liver fibrosis by preventing Smad3-mediated TGFβ1 signal in a positive feedback loop.
Recent findings have suggested a more complex paradigm of TGFβ1 signaling wherein Smads interact with other signaling cascades, including the MAPK pathway [32
]. ERK is an important member of the MAPK family. A study demonstrated that TGFβ signaling that was activated after BDL was mediated through ERK activation. The decrease of TGFβ1-induced transcriptional activity by the ERK blockade results from direct suppression of R-Smad dependent transcriptional activation. In addition, differential inhibition of phosphorylation at different Smad serines suggests mechanisms of crosstalk between the Smad and MAPK pathways, which would account for partial inhibition of TGFβ/Smad signaling by MAPK pathway inhibitors [33
]. These results strongly suggest a synergizing role for ERK signal in Smad-signaling that is initiated by TGFβ1.
In this study, we found that ERK1/2 was activated after BDL, and crosstalk between ERK1/2 and the Smad-signaling pathway enhanced TGFβ1-dependent responses in cholestatic fibrosis caused by BDL. IHQD inhibited ERK1/2 activation after BDL. IHQD may exert its suppressive effects on cholestatic liver fibrosis, at least in part, through suppression of ERK1/2 activation and crosstalk between ERK1/2 and the Smad-signaling pathway.
The multifunctional characteristics of TGFβ1 indicate a need for tight control of its signaling. Indeed, both positive and negative regulatory mechanisms have been observed at nearly every step in the TGFβ1 signaling cascade, from release of biologically active ligands to the Smad-mediated transcriptional effects [34
]. Our results demonstrated that TGFβ1-mediated induction of Smad3, Smad7, and ERK1/2 was involved in this tight regulation of its fibrosis signals in cholestatic liver fibrosis. Smad7 expression increased but did not interfere with fibrogenesis. The TGFβ1 signal through phosphorylation of Smad3 and activation of ERK1/2 was constantly propagated throughout hepatic biliary injury.
Understanding the differential regulatory mechanisms of the TGFβ1 signal between physiologic and pathologic situations will be essential in the design of new therapeutic approaches for various diseases caused by a deregulation of the TGFβ1 signal. Thus, antagonists of the TGFβ1 signal could be applied in cholestatic liver fibrosis. Our findings suggested that IHQD suppressed the cholestatic liver fibrosis by inhibiting TGFβ1 signal-mediated activation of Smad3 and ERK1/2. This provides scientific evidence for the clinical application of Huangqi decoction in treatment of cholestatic liver fibrosis.