Inhibition of the JAK/STAT pathway by ruxolitinib ameliorates thioacetamide-induced hepatotoxicity

doi:10.1016/j.fct.2016.08.018

Food and Chemical Toxicology

Volume 96, October 2016, Pages 290-301

https://doi.org/10.1016/j.fct.2016.08.018 Get rights and content

Highlights

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Ruxolitinib effect was investigated on thioacetamide (TAA)-induced hepatotoxicity.
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Ruxolitinib combated TAA-induced hepatic injury and necroinflammation.
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Ruxolitinib reduced TAA-induced hepatic apoptosis and inflammatory cells infiltration.
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Ruxolitinib counteracted TAA-induced hepatic oxidative stress.
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Ruxolitinib dampened TAA-induced overproduction of proinflammatory cytokines.

Abstract

In an attempt to explore the role of the JAK/STAT pathway in liver inflammation, we investigated the effect of intervening this pathway by ruxolitinib in thioacetamide (TAA)-induced hepatotoxicity. Ruxolitinib treatments were administered to male mice either before or after intoxication with TAA. The hepatic histopathological and serum biochemical assessment revealed that ruxolitinib pre-treatments dose-dependently reduced TAA-induced liver injury, caspase 3 cleavage and increase in number of hepatocytes positive for the pro-apoptotic Bax, as well as inflammatory cells positive for F4/80 and myeloperoxidase activity in the liver. Ruxolitinib pre-treatments also curbed TAA-induced rise in NF-κB nuclear expression and STAT3 phosphorylation. Ruxolitinib pre-treatments also lowered TAA-induced elevation of hepatic oxidative stress parameters (total nitrate/nitrite and 4-hydroxynonenal), but did not restore the hepatic antioxidant reduced glutathione. Interestingly, ruxolitinib, especially at a dose of 200 mg/kg, dampened the overproduction of pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ, IL-23 and IL-17A), which coincided with boosting the release of the anti-inflammatory cytokine IL-10. Ruxolitinib when used as a post-treatment (1 and 3 h after TAA-insult) could still spare the liver from injury and might be clinically applicable. In conclusion, the multimechanistic-hepatoprotective activity of ruxolitinib can be linked to its ameliorative properties on cellular death, oxidative stress and inflammation machinery.

Introduction

Hepatic diseases are a major cause of worldwide morbidity and mortality. Most types of hepatic diseases are characterized by inflammatory processes with enhanced expression of various pro-inflammatory cytokines in the liver. One of the major pathways involved in the signal transductions of wide arrays of these cytokines is the Janus kinase/signal transducers and activators of transcription (JAK/STAT) cascade. The JAK/STAT pathway is a key player in many important biological processes, including broad immune and hematopoietic cell functions (Rane and Reddy, 2000). On the other hand, erratic function of this pathway is widely implicated in various types of illness, such as autoimmune diseases, hematopoietic disorders, graft rejection and inflammation, (Ghoreschi et al., 2011).

JAKs comprise a group of 4 tyrosine kinases (JAK1, JAK2, JAK3 and TYK2) that selectively associate with cytokine receptor chains and transduce signaling through phosphorylating tyrosine residues on themselves and STATs (Pesu et al., 2008). STATs subsequently become dimerized and transport to the nucleus, where they activate or suppress the gene transcription (Harrison, 2012). Thus, the pharmacological modulation of elements of this pathway may represent a novel treatment approach for inflammatory and immune-mediated diseases.

Ruxolitinib, a novel oral JAKs 1 and 2 inhibitor, was recently approved as a revolutionary therapy for patients suffering from intermediate/high risk myelofibrosis (Mascarenhas and Hoffman, 2012). Although the efficacy of ruxolitinib in myelofibrosis is now well established, data about the effect of ruxolitinib in inflammatory disorders, especially those occurring in the liver, are still limited. Most recently, we found that pre-treatment with ruxolitinib protected mice from carbon tetrachloride-induced hepatotoxicity (Hazem et al., 2014). In this study, we examined whether the capability of ruxolitinib to confer hepatoprotection is also extended to thioacetamide (TAA)-induced hepatotoxicity mouse model. Additionally, we tested administering ruxolitinib at different time-points from TAA intoxication to evaluate the post-treatment strategy and simulate the clinical application in humans.

Section snippets

Animals

Male adult BALB/c mice (33–37 g) had free access to tap water and diet and acclimatized at least 1 week prior experiments. All the mice included in this study received care according to the guidelines of NIH and Research Ethics Committee, Faculty of Pharmacy, University of Mansoura.

Experimental design and administration of drugs and chemicals

Ruxolitinib in the form of phosphate salt was supplied as a generous gift form Novartis Pharmaceuticals (Basel, Switzerland). Different concentration of ruxolitinib (0.5, 1 and 2% w/v) was freshly prepared in

Effects of ruxolitinib pre-treatments on TAA-induced hepatocellular injury and necroinflammation

The intoxication with TAA for 30 h caused a severe liver injury evidenced by significant (P < 0.001) rise in serum ALT and LDH activities (Fig. 1 A–B), compared to the control group. In addition, a pronounced pericentral necrosis and intense portal infiltration of inflammatory cells were observed in the livers of TAA-mice, as visualized by H-E staining (Fig. 2A) and quantified by necroinflammation score (Fig. 2B). At a dose of 100 mg/kg, ruxolitinib effectively reduced TAA-induced elevation of

Discussion

Therapeutics that impede the JAK/STAT cascade activation might be plausible candidates for suppressing hepatic inflammation. Here, we investigated the effect of different doses (50, 100 and 200 mg/kg) of the novel JAK inhibitor ruxolitinib on TAA-induced acute liver injury. As expected, the histopathology assessment revealed that ruxolitinib dose-dependently reduced TAA-induced pericentral necrosis and portal infiltration of inflammatory cells in the liver. However, the biochemical assessment

Conclusion

Ruxolitinib combated TAA-deleterious effects by ameliorating hepatic injury, cellular death, oxidative stress and inflammatory cytokines. Collectively, our results gave a strong evidence about the undeniable hepatoprotective effects of targeting the JAK/STAT pathway by ruxolitinib.

Conflict of interest

The authors declare no conflict of interest to disclose.

Acknowledgement

The Authors would like to thank Dr. Mohamed E. El-Mesery (Assistant Professor of Biochemistry, Faculty of Pharmacy, Mansoura University, Egypt) for providing some of the reagents used in Western blotting analysis and his technical assistance.

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Inhibition of the JAK/STAT pathway by ruxolitinib ameliorates thioacetamide-induced hepatotoxicity

Highlights

Abstract

Introduction

Section snippets

Animals

Experimental design and administration of drugs and chemicals

Effects of ruxolitinib pre-treatments on TAA-induced hepatocellular injury and necroinflammation

Discussion

Conclusion

Conflict of interest

Acknowledgement

Anal. Biochem.

Anal. Biochem.

Chest

Chem. Biol. Interact.

Exp. Toxicol. Pathol.

Nitric Oxide

Biochim. Biophys. Acta

J. Pharmacol. Methods

Eur. J. Pharmacol.