Elsevier

Journal of Hepatology

Volume 66, Issue 3, March 2017, Pages 589-600
Journal of Hepatology

Research Article
PARP inhibition protects against alcoholic and non-alcoholic steatohepatitis

https://doi.org/10.1016/j.jhep.2016.10.023Get rights and content

Background & Aims

Mitochondrial dysfunction, oxidative stress, inflammation, and metabolic reprograming are crucial contributors to hepatic injury and subsequent liver fibrosis. Poly(ADP-ribose) polymerases (PARP) and their interactions with sirtuins play an important role in regulating intermediary metabolism in this process. However, there is little research into whether PARP inhibition affects alcoholic and non-alcoholic steatohepatitis (ASH/NASH).

Methods

We investigated the effects of genetic deletion of PARP1 and pharmacological inhibition of PARP in models of early alcoholic steatohepatitis, as well as on Kupffer cell activation in vitro using biochemical assays, real-time PCR, and histological analyses. The effects of PARP inhibition were also evaluated in high fat or methionine and choline deficient diet-induced steatohepatitis models in mice.

Results

PARP activity was increased in livers due to excessive alcohol intake, which was associated with decreased NAD+ content and SIRT1 activity. Pharmacological inhibition of PARP restored the hepatic NAD+ content, attenuated the decrease in SIRT1 activation and beneficially affected the metabolic-, inflammatory-, and oxidative stress-related alterations due to alcohol feeding in the liver. PARP1−/− animals were protected against alcoholic steatohepatitis and pharmacological inhibition of PARP or genetic deletion of PARP1 also attenuated Kupffer cell activation in vitro. Furthermore, PARP inhibition decreased hepatic triglyceride accumulation, metabolic dysregulation, or inflammation and/or fibrosis in models of NASH.

Conclusion

Our results suggests that PARP inhibition is a promising therapeutic strategy in steatohepatitis with high translational potential, considering the availability of PARP inhibitors for clinical treatment of cancer.

Lay summary

Poly(ADP-ribose) polymerases (PARP) are the most abundant nuclear enzymes. The PARP inhibitor olaparib (Lynparza) is a recently FDA-approved therapy for cancer. This study shows that PARP is overactivated in livers of subjects with alcoholic liver disease and that pharmacological inhibition of this enzyme with 3 different PARP inhibitors, including olaparib, attenuates high fat or alcohol induced liver injury, abnormal metabolic alteration, fat accumulation, inflammation and/or fibrosis in preclinical models of liver disease. These results suggest that PARP inhibition is a promising therapeutic strategy in the treatment of alcoholic and non-alcoholic liver diseases.

Introduction

Chronic alcoholism is a leading cause of liver disease worldwide. The development of alcoholic steatohepatitis involves alcohol and acetaldehyde-induced direct hepatocyte injury and death by increasing reactive oxygen and nitrogen species (ROS/RNS) production, lipid peroxidation and oxidative DNA injury, coupled with overactivation of hepatic inflammatory processes (both innate and adaptive immunity) and reprograming of lipid metabolism by promoting hepatic lipid accumulation [1], [2], [3], [4]. Non-alcoholic fatty liver disease (NAFLD) may also progress to steatohepatitis (non-alcoholic steatohepatitis; NASH). Oxidative stress, inflammation and hepatocyte injury are also often characteristic features of NASH. Chronic and sustained liver injury, inflammation, and fat accumulation promote scarring, and in susceptible subjects eventually lead to the development of cirrhosis.

Poly(ADP-ribose) polymerases (PARPs) are key enzymes involved in DNA repair processes. PARP1 is the most abundant nuclear enzyme and the predominant isoform of the PARP enzyme family. Upon DNA damage (for example induced by ROS/RNS or ionizing radiation), PARP1 activity increases and the ADP-ribose part of NAD+ is transferred to proteins, giving rise to formation of poly(ADP-ribose) polymers [5]. This post-translational modification alters the function of many enzymes and structural proteins and may initiate caspase-independent cell death [5]. PARP1 is also a co-activator of key pro-inflammatory transcription factors [6], including NF-κB [7]. In the recent years, several additional PARP functions have been discovered, involving the regulation of oxidative stress, mitochondrial function and intermediary metabolism [6], [8]. In experimental models of tissue injury and inflammation, PARP inhibition exerts marked protective effects in cells/tissues [5], [9]. In contrast, in cancers with selective defects in homologous recombination repair (cancer cells frequently harbor defects in DNA repair pathways leading to genomic instability), inactivation of PARPs directly causes cell demise [10]. This principle resulted in the development and Food and Drug Administration (FDA) approval of the PARP inhibitor, olaparib (AZD-2281; Lynparza), for the treatment of BRCA-1 and 2 (tumor suppressor genes) negative ovarian cancer.

Recently, we described PARP activation in livers of subjects with advanced cirrhosis induced by hepatitis B or excessive alcohol consumption, and demonstrated anti-inflammatory and anti-fibrotic effects of PARP inhibition using experimental models of liver fibrosis, as well as utilizing isolated hepatic stellate cells [11].

In this study, we investigated the effects of pharmacological inhibition of PARP with structurally different inhibitors or genetic deletion of PARP1 on hepatocellular injury, mitochondrial function, inflammation, metabolic reprograming and lipid accumulation in the liver, utilizing in vivo models of early alcoholic steatohepatitis, as well as isolated Kupffer cells stimulated with bacterial lipopolysaccharide in vitro. To enhance the translational potential of our study, the effects of PARP inhibition on hepatic lipid accumulation, metabolic reprograming and/or inflammation were also evaluated on mouse models of high fat (HF) or methionine and choline deficient (MCD) diet-induced NAFLD/NASH.

Section snippets

Materials and methods

Further detailed description of methods can be found in the Supplementary materials.

Increased hepatic PARP and decreased SIRT1 activity and NAD+ content in steatohepatitis. Pharmacological inhibition of PARP restores attenuated NAD+ content and SIRT1 activity

Using a mouse model of chronic and binge ethanol feeding (model of early steatohepatitis) [12], [14], alcohol fed mice showed significantly increased hepatic PARP activation (Fig. 1A) that was associated with depleted NAD+ levels and decreased SIRT1 activity (Fig. 1A, C) and mRNA expression (Fig. 1B). Treatment of ethanol-fed mice with PARP inhibitors AIQ and PJ34 significantly decreased alcohol feeding-induced PARP activation, and prevented both the alcohol-induced drop in hepatic NAD+

Discussion

The key findings of our study demonstrate that: a) hepatic PARP activity is increased, coupled with decreased NAD+ content and SIRT1 activity/expression, in a mouse model of early steatohepatitis induced by chronic and binge ethanol feeding, and likewise in human liver specimens with alcoholic steatohepatitis; b) pharmacological inhibition of PARP restores the hepatic NAD+ content, attenuates the decrease in SIRT1 activation/expression in mice exposed to ethanol feeding; c) pharmacological

Financial support

This study was supported by Intramural Research Program of NIH/NIAAA (to P.P.). ZVV is supported by the Rosztoczy Foundation. P.B. is supported by the NKFI (Hungary) 108308 and GINOP-2.3.2-15-2016-00006 grants. J.A. is the Nestlé Chair in Energy Metabolism. The research in the J.A. laboratory is supported by École Polytechnique Fédérale de Lausanne, National Institute of Health(RO1AG043930), Krebsforschung Schweiz/SwissCancerLeague(KFS-3082-02-2013), Systems X(51RTP0-151019),

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Authors’ contributions

P.M., B.H., M.R., Z.V.V., K.G., D.R., Z.C., E. H., O.P., Z.Z., M.J.X., W.W., G.G., J.P., B.T.N., Y.P., L.L., G. H., P. B., J.A., B.G. and P.P. designed or conducted experiments, analyzed or interpreted data or edited the manuscript. A.H.B. provided knockout mice. P.M., Z.V.V., P.B., P.P. wrote the draft; P.P. supervised experiments and obtained funding.

Acknowledgments

P.M. dedicates this work to his beloved father, Sri Mrityunjoy Mukherjee, who passed away during the study. The authors are indebted to Dr. George Kunos, the Scientific Director of NIAAA, for continuous support.

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