Elsevier

Journal of Autoimmunity

Volume 46, October 2013, Pages 25-34
Journal of Autoimmunity

STAT3-mediated attenuation of CCl4-induced mouse liver fibrosis by the protein kinase inhibitor sorafenib

https://doi.org/10.1016/j.jaut.2013.07.008Get rights and content

Highlights:

  • Sorafenib attenuates liver fibrosis and injury.

  • Sorafenib increases STAT3 phosphorylation in hepatocytes.

  • Sorafenib upregulates IL-6 expression in Kupffer cells.

  • Anti-fibrotic effect suggests sorafenib as a therapeutic agent for liver fibrosis.

Abstract

There have been major advances in defining the immunological events associated with fibrosis in various chronic liver diseases. We have taken advantage of this data to focus on the mechanisms of action of a unique multi-kinase inhibitor, coined sorafenib, on CCl4-induced murine liver fibrosis, including the effects of this agent in models of both acute and chronic CCl4-mediated pathology. Importantly, sorafenib significantly attenuated chronic liver injury and fibrosis, including reduction in liver inflammation and histopathology as well as decreased expression of liver fibrosis-related genes, including α-smooth muscle actin, collagen, matrix metalloproteinases and the tissue inhibitor of metalloproteinase-1. Furthermore, sorafenib treatment resulted in translocation of cytoplasmic STAT3 to the nucleus in its active form. Based on this observation, we used hepatocyte-specific STAT3 knockout (STAT3Hep−/−) mice to demonstrate that hepatic STAT3 was critical for sorafenib-mediated protection against liver fibrosis, and that the upregulation of STAT3 phosphorylation was dependent on Kupffer cell-derived IL-6. In conclusion, these data reflect the clinical potential of the multi-kinase inhibitor sorafenib for the prevention of fibrosis as well as the treatment of established liver fibrosis and illustrate the immunological mechanisms that underlie the protective effects of sorafenib.

Introduction

Liver fibrosis is a significant health problem resulting from response of the liver to injury [1], [2]. Following injury, hepatocytes, biliary cells, Kupffer cells and T cells respond to create an inflammatory milieu [3], which stimulates hepatic stellate cells (HSCs) to transform from a quiescent vitamin A-storing cell lineage to an activated myofibroblast (α-SMA-expressing) and thereby acquires fibrogenic properties producing extracellular matrix proteins (ECM) [2], [4], [5], [6], [7], [8]. Other cell types also contribute to this pathological process and complex interplay amongst hepatic cell lineages occurs during fibrogenesis [3], [9]. Hepatocytes are the major targets in liver fibrosis [10]. Damaged hepatocytes release ROS as well as fibrogenic mediators and induce the recruitment of white blood cells by inflammatory cells. Apoptosis of damaged hepatocytes also stimulate the fibrogenic actions of liver myofibroblasts [11]. Thus, drugs which protect hepatocytes from injury are potentially treatment options for human liver fibrosis.

There are several pathways that can potentially be modulated to alter fibrosis. Amongst potential inhibitors is an oral multi-kinase inhibitor well known for its influence on tumor signaling and vasculature, coined Sorafenib (BAY43-9006, Nexavar) [12], [13], [14]. Signaling pathways affected by sorafenib include Raf, platelet-derived growth factor (PDGF), c-kit and vascular endothelial growth factor (VEGF). Sorafenib blocks receptor tyrosine kinase signaling (VEGFR, PDGFR, c-Kit and RET) and inhibits downstream Raf serine/threonine kinase activity to prevent tumor growth by antiangiogenic, antiproliferative and/or pro-apoptotic effects [15].

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates cell growth and survival by modulating the expression of many target genes [16]. Sorafenib has the potential to suppress STAT3 phosphorylation in human neoplasia [17], [18], [19]. However, due to the hepatoprotective and proliferative functions of STAT3, STAT3 in hepatocytes plays a protective role in preventing liver fibrosis [20], [21], [22], [23].

Recent data has shown a beneficial effect of sorafenib in patients with hepatocellular carcinoma (HCC) [12], [13], [14]. Other studies suggest that sorafenib attenuates the hypertensive syndrome in partial portal vein ligated rats [24], [25]. There are also data that note that sorafenib has antifibrotic activity, including in vitro effects of sorafenib on inhibition of activation, growth and collagen accumulation of HSC [26], [27]. Moreover, sorafenib influences paracrine signaling between HSCs and LECs and thereby regulates matrix and vascular changes associated with bile duct ligation-induced liver injury in rats [28]. In hepatocytes, in vitro experiments demonstrated that sorafenib suppresses TGF-β1-induced EMT [29]. In this study, we focused on the effect of sorafenib on cross talk between hepatocytes and Kupffer cells during liver fibrosis. We used CCl4 induced murine liver fibrosis model and three independent acute liver injury models to illustrate that sorafenib attenuates liver fibrosis and injury through up-regulation STAT3 phosphorylation in hepatocytes which dependent on Kupffer cell-derived IL-6.

Section snippets

Mice

C57BL/6 (B6) mice were purchased from the Experimental Animal Center, Chinese Academy of Science (Shanghai, China). Hepatocyte-specific signal transducer and activator of transcription 3 (STAT3) knockout (STAT3Hep−/−) mice (AlbCre+/− STAT3flox/flox) were provided by Dr. Xin-Yuan Fu. B6.129S6-Il6tm1Kopf (IL-6−/−) mice were purchased from JAX (Bar Harbor, Maine, USA). All mice were housed in a specific pathogen-free and controlled environment. All animal experiments conformed to the guidelines

Sorafenib attenuates CCl4-induced chronic liver injury and liver fibrosis

We first studied CCl4 induced chronic liver injury by injecting mice with CCl4 for 4 weeks and treating daily with sorafenib or control vehicle (Fig. 1A). As shown in Fig. 1B, ALT levels were significantly lower in sorafenib-treated mice compared to controls at 48 and 72 h (P < 0.001). Liver sections revealed less vacuolated cells (Fig. 1C) and significantly improved portal inflammation and hepatocellular degeneration in the sorafenib-treated mice compared to controls (P < 0.001) (Fig. 1D). The

Discussion

Liver fibrosis results from chronic damage to the liver in conjunction with the accumulation of ECM proteins, which is a characteristic of chronic liver diseases [1], [3]. Although experimental data has suggested targets that prevent fibrosis in rodents [8], the efficacy of most treatments has not been recapitulated in humans [3]. Sorafenib, the first oral multikinase inhibitor that blocks multiple signaling pathways, was initially approved as an oral agent for treating advanced renal cell

Conclusions

We found that sorafenib significantly attenuates CCl4-induced liver fibrosis and injury, with activated STAT3 playing a critical role in this therapeutic effect. And further study has proved that Kupffer cell-derived cytokine IL-6 is required for the sorafenib-mediated phosphorylation of STAT3. These results suggest that sorafenib is a potential therapeutic agent for liver fibrosis.

Conflict of interest

The authors have declared that no conflict of interest exists.

Acknowledgments

Financial support provided by the National Basic Research Program of China (973 Program-2010CB945300, 2013CB944900), the National Natural Science Foundation of China (30972738, 31021061, 81130058), and the Hundred Talents Program of the Chinese Academy of Sciences.

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    Both authors contributed equally to the manuscript.

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