Poly (ADP-ribose) polymerase-1 is a key mediator of liver inflammation and fibrosis

Abstract

Poly (ADP-ribose) polymerase 1 (PARP-1) is a constitutive enzyme, the major isoform of the PARP family, which is involved in the regulation of DNA repair, cell death, metabolism, and inflammatory responses. Pharmacological inhibitors of PARP provide significant therapeutic benefits in various preclinical disease models associated with tissue injury and inflammation. However, our understanding the role of PARP activation in the pathophysiology of liver inflammation and fibrosis is limited. In this study we investigated the role of PARP-1 in liver inflammation and fibrosis using acute and chronic models of carbon tetrachloride (CCl4 )-induced liver injury and fibrosis, a model of bile duct ligation (BDL)-induced hepatic fibrosis in vivo, and isolated liver-derived cells ex vivo. Pharmacological inhibition of PARP with structurally distinct inhibitors or genetic deletion of PARP-1 markedly attenuated CCl4 -induced hepatocyte death, inflammation, and fibrosis. Interestingly, the chronic CCl4 -induced liver injury was also characterized by mitochondrial dysfunction and dysregulation of numerous genes involved in metabolism. Most of these pathological changes were attenuated by PARP inhibitors. PARP inhibition not only prevented CCl4 -induced chronic liver inflammation and fibrosis, but was also able to reverse these pathological processes. PARP inhibitors also attenuated the development of BDL-induced hepatic fibrosis in mice. In liver biopsies of subjects with alcoholic or hepatitis B-induced cirrhosis, increased nitrative stress and PARP activation was noted.

Conclusion: The reactive oxygen/nitrogen species-PARP pathway plays a pathogenetic role in the development of liver inflammation, metabolism, and fibrosis. PARP inhibitors are currently in clinical trials for oncological indications, and the current results indicate that liver inflammation and liver fibrosis may be additional clinical indications where PARP inhibition may be of translational potential.

Fig. 1. Genetic ablation of PARP1 attenuates CCl₄-induced chronic liver injury and inflammation

Livers of mice treated exposed to CCl₄ for 4 weeks were excised and processed for histological and biochemical investigations. Part (A) shows representative H&E stained images of liver sections. The scale depicts 100 μm. (B): Serum ALT levels in the respective groups, n=6-8/group, *P<0.05 vs. WT (PARP1^+/+)/PARP1^-/- mice treated with vehicle; #P<0.05 vs. WT+CCl₄. Part (C) depicts oxidative/nitrative stress markers 4-HNE and 3-NT, n=8/group, *P<0.05 vs. WT/PARP1^-/- mice treated with vehicle; #P<0.05 vs. WT+CCl₄. Part (D) shows the mRNA expression of pro-inflammatory cytokines (TNF- α, IL1- β) and chemokines (MIP1- α, MIP2, MCP-1) in hepatic tissues following 4 weeks of CCl₄ administration. n=8/group, *P<0.05 vs. WT/PARP1^-/- mice treated with vehicle; #P<0.05 vs. WT+CCl₄.

Fig. 2. Genetic ablation of PARP1 attenuates CCl₄ induced chronic liver fibrosis

Part (A) shows representative images of paraffin embedded liver sections stained with Sirius red, a marker of fibrosis, and its quantification from n=4-7 livers/group. The scale depicts 100 μm. Part (B) shows mRNA expression of fibrosis markers (collagen-1, CTGF, TGF- β, and fibronectin) and part (C) denotes the levels of hydroxyproline in hepatic tissues from respective groups of mice exposed to CCl₄ for 4 weeks, n=8/group, *P<0.05 vs. WT(PARP1^+/+)/PARP1^-/- mice treated with vehicle; # P<0.05 vs. WT+CCl₄.

Fig. 3. Pharmacological inhibition of PARP ameliorates CCl₄-induced chronic liver injury, mitochondrial dysfunction, metabolic dysregulation and inflammation

Part (A) shows representative H&E images from the respective groups (the scale depicts 100 μm) and serum ALT levels. Part (B) shows hepatic myeloperoxidase (MPO) activities (marker of neutrophil infiltration) and mRNA expression of pro-inflammatory cytokines (TNF-α, IL1-β) and chemokines (MIP1-α, MIP2, MCP-1) in hepatic tissues following 4 weeks of chronic CCl₄ exposure, respectively. Part (C) shows determination of mitochondrial DNA content(left) and quantification of mitochondrial complex 1(I), 2(II) and 4(IV) activities(right). Part (D) shows expression of several mitochondrial respiration and uncoupling genes by Realtime PCR. Part (E) shows quantification of fatty acid oxidation (left), fatty acid synthesis (middle) and glucose metabolism (right) genes. All mRNA level is measured by Realtime PCR. n=6-12/group for experiments in panels A-E. *P<0.05 vs. vehicle; #P<0.05 CCl₄ vs. CCl₄+PJ34/AIQ.

Fig. 4. Pharmacological inhibition of PARP mitigates CCl₄-induced chronic liver fibrosis

Part (A) shows representative images of paraffin embedded liver sections stained with Sirius red, a marker of fibrosis, and its quantification from n=4-7 livers/group. The scale depicts 100 μm. Part (B) shows mRNA expression of fibrosis markers (collagen-1, CTGF, TGF-β, and fibronectin) in the hepatic tissues following 4 weeks of chronic CCl₄ exposure, n=8/group, *P<0.05 vs. vehicle/PJ34/AIQ alone; #P<0.05 CCl₄ vs. CCl₄+PJ34/AIQ. Part (C) is a representative immunoblot of α-SMA (marker of stellate cell activation) in the hepatic tissue lysates from the respective groups.

Fig.5. PARP inhibition or genetic deficiency of PARP1 protects against oxidant induced cell death in primary mouse hepatocytes and attenuates hepatic stellate cell activation

Part (A): isolated mouse primary hepatocytes from either WT (PARP1^+/+) or PARP1^-/- mice were treated with hydrogen peroxide (H₂O₂; 3 mM) in the presence or absence of PARP inhibitors (AIQ and PJ34, 10 μM) for 16hr and normal cell population and populations of cells exhibiting the marks of either necrotic or apoptotic death were simultaneously determined by flow cytometry as described in Supplemental Methods. n=6-8/group, *P<0.05 vs. vehicle/PJ34/AIQ alone or PARP1^+/+ cells treated with vehicle; #P<0.05 H₂O₂ vs. H₂O₂+PJ34/AIQ, respectively. (B): Hepatic stellate cells were isolated from WT (PARP1^+/+) or PARP1^-/- mice, and were maintained in the 10% FBS medium alone or with PARP inhibitors for 7 days and mRNA expression (Fig. 5B) or protein (Fig. 5C) of stellate cell activation and fibrosis markers (collagen-1, CTGF, TGF-β, and fibronectin) were determined, n=6-8/group, *P<0.05 vs. vehicle at Day 1 vs. Day 7 with or without PARP inhibitors; #P<0.05 vehicle at 7 days vs. AIQ/PJ34 at 7 days or PARP^+/+ vs. PARP^-/- cells at Day 7.

Fig. 6. Pharmacological inhibition of PARP attenuates the established liver injury and fibrosis

Initially mice were treated with CCl₄ or vehicle for 4 weeks. After reaching 4 weeks of CCl₄ or vehicle treatment mice continued to receive CCl₄, vehicle, PARP inhibitors alone or PARP inhibitors in combination with CCl₄ for additional 4 weeks. (A): Representative H&E images of liver sections and serum ALT levels. The scale depicts 100 μm. Part (B) shows the representative liver sections stained for Sirius red. The scale depicts 100 μm. Part (C) shows quantification of fibrosis based on Sirius red staining from 4-7livers/group. Part (D) depicts mRNA expression of fibrotic markers (collagen-1, CTGF, TGF-β, and fibronectin) and part (E) demonstrates the changes in hydroxyproline content in the hepatic tissues of mice exposed to CCl₄ for 8 weeks or CCl₄ for 8 weeks+PARP inhibitors (AIQ/PJ34) during the last 4 weeks of the CCl₄ administration, n=8/group, *P<0.05 vs. vehicle/PJ34/AIQ alone; # P< 0.05 CCl₄ vs. CCl₄ + PJ34/AIQ.

Fig. 7. Pharmacological inhibition of PARP attenuates the bile duct ligation (BDL)-induced liver fibrosis

(A): Representative liver sections stained for Sirius red 2 weeks following the induction of BDL (left) and quantification of fibrosis staining (right) from n=4-7 livers/group. The scale depicts 100 μm. Part (B) depicts mRNA expression of fibrotic markers (collagen-1, CTGF, TGF-β, and fibronectin) in the hepatic tissues of mice exposed to BDL for 2 weeks or BDL for 2 weeks+PARP inhibitors (AIQ/PJ34) during the 2 weeks period, n=8/group, *P<0.05 vs. vehicle; # P< 0.05 BDL vs. BDL+ PJ34/AIQ.

Fig. 8. PARP activation in hepatic tissues from human subjects with alcoholic and viral hepatitis B-induced cirrhosis

Part (A) shows PARP activation in hepatic tissues obtained from human subjects with HBV-induced or alcoholic cirrhosis, n=6/group, P<0.05 vs control subjects. Part (B) depicts the amounts of the oxidative/nitrative marker 3-NT levels in the respective groups, n=6, P<0.05 vs. control subjects. Part (C) shows a representative immunoblot for poly(ADP-ribose) [PAR] proteins of the hepatic tissue lysates from the respective groups. Note the enhanced PAR proteins in the samples obtained from subjects with alcoholic cirrhosis or viral hepatitis.