reperfusion injury in mice

Journal of Hepatology 47 (2007) 784–792 www.elsevier.com/locate/jhep

Importance of peroxisome proliferator-activated receptor-c in hepatic ischemia/reperfusion injury in miceq Takahiro Akahori1, Masayuki Sho1,*, Kaoru Hamada2, Yasue Suzaki2, Yukiyasu Kuzumoto1, Takeo Nomi1, Shinji Nakamura1, Koji Enomoto1, Hiromichi Kanehiro1, Yoshiyuki Nakajima1 2

1 Department of Surgery, Nara Medical University, Nara, Japan Second Department of Internal Medicine, Nara Medical University, Nara, Japan

Background/Aims: Peroxisome proliferator-activated receptor-c (PPARc) is a transcriptional factor belonging to the nuclear receptor superfamily. Recent studies have suggested that PPARc regulates inflammatory responses and PPARc specific agonists have beneficial effects on several disease conditions in the various organs. However, the precise role of PPARc in acute liver injury remains unknown. Methods: We investigated the pathophysiological role of PPARc and the effect of the selective PPARc agonist, pioglitazone, on the hepatic ischemia/reperfusion (I/R) injury. Results: PPARc expression in the liver was upregulated after reperfusion following ischemia. Pioglitazone treatment significantly inhibited hepatic I/ R injury as determined by serological and histological analyses. The protective effect was associated with downregulation of the local expression of several potent proinflammatory cytokines, chemokines and adhesion molecules after reperfusion. The neutrophil accumulation was also inhibited by the treatment. Furthermore, the treatment inhibited the induction of apoptosis on hepatocytes. Finally, pioglitazone significantly improved the mouse survival in a lethal model of hepatic I/R injury. Conclusions: PPARc plays an inhibitory role in hepatic I/ R injury and the stimulation by selective agonist has a significant beneficial effect. Thus, PPARc may be a new therapeutic target for the protection of the liver against acute injury.  2007 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Ischemia/reperfusion injury; Neutrophil; Cytokine; Chemokine; Adhesion molecule; Apoptosis 1. Introduction Hepatic ischemia/reperfusion (I/R) injury is the main cause of hepatic damage and is inevitable after hepatic surgery, liver transplantation, shock, trauma, and so on [1,2]. Hepatic I/R leads to an acute inflammatory Received 12 March 2007; received in revised form 2 July 2007; accepted 20 July 2007, available online 4 October 2007 Associate Editor: P.-A. Clavien q The authors declare that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. * Corresponding author. Tel.: +81 744 29 8863; fax: +81 744 24 6866. E-mail address: [email protected] (M. Sho).

response, causing significant hepatocellular damage and organ dysfunction. A number of studies have shown that the mechanisms of hepatic I/R injury involve complex and multiple pathways, including the direct ischemic cellular damage as well as the cell injury due to the activation of inflammatory response after reperfusion [3–6]. The initial phase is associated with the generation of toxic free oxygen species, the activation of Kupffer cells, and an initial response of neutrophil activation. Activated Kupffer cells release various metabolites that cause cellular damage including superoxide radicals, nitric oxide, eicosanoids, proteases, and proinflammatory cytokines. Furthermore, the activation and secretion of such cytokines induces the increased expression of adhesion molecules on sinusoidal endothelial

0168-8278/$32.00  2007 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2007.07.030

T. Akahori et al. / Journal of Hepatology 47 (2007) 784–792

cells, thereby promoting the infiltration of inflammatory cells into the liver. The accumulation of inflammatory cells contributes to the progression of parenchymal injury. Despite the recent improvements in liver preservation and surgical techniques, hepatic I/R injury remains an important clinical complication. To control this complicated physiological as well as pathological process, further studies are required to find out the key pathway and a novel therapeutic approach. Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-dependent transcription factors [7,8]. The PPAR subfamily comprises three members, PPARa, PPARb, and PPARc [9]. PPARc was reported to be highly expressed in adipocytes and to play a critical role in their differentiation [10–13]. The ligands of the PPARc, such as pioglitazone, troglitazone, and rosiglitazone, act to enhance insulin sensitivity and reduce serum glucose in diabetic patients, without significant alternations in serum glucose of non-diabetic animals or humans [14–17]. Therefore, they are now widely used as antidiabetic agents. Besides the antidiabetic activity, it has been recently recognized to have other various physiological roles including inflammation [18,19]. In particular, PPARc may be a protective regulator against ischemic damage [18–20]. To date, it has been reported that PPARc agonists have beneficial effects on the kidney [21], the small intestine [22], the lung [23], the brain [24], and the heart [25,26] of ischemic injury. In the liver, it has been suggested that PPARc agonists are protective against liver injury in chronic disease conditions such as cirrhosis and fibrosis [27–29]. However, it remains unknown whether PPARc plays some roles in acute phase of hepatic damage and its specific agonists have beneficial effects on hepatic I/R injury. In this study, we investigated the pathophysiological role of PPARc and also explored the therapeutic efficacy of the PPARc agonist in an established murine hepatic I/R model. To this end, we employed a potent PPARc agonist, pioglitazone.

2. Materials and methods 2.1. Mice Male C57BL/6 mice were obtained from CLEA JAPAN, Inc. (Tokyo, Japan) ranging in age from 8 to 12 weeks and used for all experiments. All mice were maintained under specific pathogen-free conditions in the animal facility at Nara Medical University. All experiments were conducted under a protocol approved by our Institutional Review Board.

2.2. Hepatic ischemia/reperfusion procedure We used a murine model of 70% partial hepatic ischemia for 60 min as previously reported [30]. Briefly, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg; Dainoppon

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Pharmaceutical Co., Osaka, Japan), and a midline laparotomy was performed. Then, the left lateral and median lobes of the liver were clamped at its base using an atraumatic clip. After 60 min of ischemia, the clip was removed, initiating hepatic reperfusion. Mice were sacrificed at 2 or 6 h after reperfusion, and then blood and liver samples were collected for analysis. In some mice, to assess animal survival, the non-ischemic shunt liver lobes were surgically removed at the end of 105 min of ischemia of the left and median liver lobes. The selective PPARc agonist, pioglitazone (20 mg/kg weight; Takeda Pharmaceutical Co., Osaka, Japan) was administered orally 1.5 h before the initiation of ischemia. Control mice received normal saline in the same amount. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were used as established markers of hepatocyte injury. At 6 h after reperfusion following 60 min of ischemia, blood samples were obtained by cardiac puncture, immediately centrifuged at 3000g for 10 min, and stored at 20 C until analysis. Serum ALT, AST, and LDH activities were measured using a standard clinical automatic analyzer.

2.3. Histopathological examination and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay of liver sections For histological analysis, tissue samples were fixed in 4% formaldehyde-phosphate-buffered saline overnight at 4 C. The samples were dehydrated and embedded in paraffin. Six-micrometer sections were stained with hematoxylin-eosin (H&E). Alterations of neutrophil accumulation in the ischemic liver were detected by standard immunohistochemical techniques using the antimyeloperoxidase (MPO) polyclonal antibody (NeoMarkers, Fremont, California, USA) on paraffin sections. The vectastain ABC kit (Vector Laboratories, Burlingame, California, USA) was used with 3,3 0 -diaminobenzidine substrate kit (Vector Laboratories) according to manufacturer’s instructions for detection. In the immunostained sections, the number of neutrophils in the liver was counted in 10 randomly chosen visual fields (magnification, 400·) of the sections, and the average of 10 selected microscopic fields was calculated in a blind manner. For TUNEL assays we used the In Situ Apoptosis Detection Kit (CHMICON, Billerica, Massachusetts, USA). After incubation with proteinase K, sections were stained as described in the protocol provided by the manufacturer. The number of TUNEL positive cells was assessed for each liver by counting TUNEL positive cells in three sections in a blind manner. For each section 10 fields (magnification, 400·) were examined.

2.4. Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis Expression of several cytokines (TNF-a, IL-1b, IFN-c, IL-10), chemokines (MCP-1, MIP-2, IP-10), adhesion molecules (ICAM-1, E-selectin), NO synthase (iNOS, eNOS), caspase 3 and PPARc mRNA in the naive and ischemic livers was analyzed by quantitative real-time RT-PCR. In brief, amplification and detection were done with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, California, USA) with the following profile: 10 min at 95 C, and 40 cycles at 95 C for 15 s and 60 C for 1 min. All primers and probes were purchased from Applied Biosystems (Foster city, California, USA). Each gene expression of cytokines, chemokines, and adhesion molecules was normalized to b2-microgloblin before the fold change was calculated. The fold increase in each gene expression in the ischemic liver was calculated.

2.5. Statistical analysis The means and standard deviation of the mean (SD) were calculated for all parameters determined in this study. Statistical significance between two groups of parametric data was evaluated by using an unpaired Student’s t test. The survival curve by the Kaplan–Meier method was analyzed by a log-rank test. P values less than 0.05 were considered as significant.

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as comparatively preserved lobular architecture were seen in the liver treated with pioglitazone (Fig. 3c and d).

3. Results 3.1. PPARc mRNA expression in the process of hepatic ischemia/reperfusion injury First, we examined the local expression of PPARc during hepatic I/R injury by real-time RT-PCR analysis. PPARc was increased at 2 and 6 h after reperfusion compared to the naı¨ve liver and non-ischemic shunt liver (Fig. 1). There was no significant difference in PPARc expression between the naı¨ve and non-ischemic shunt liver. 3.2. Pathophysiological function of PPARc agonist in hepatic ischemia/reperfusion injury To explore the function of PPARc in hepatic injury induced by I/R, we utilized a pharmacological approach using highly specific agonist, pioglitazone. Serum levels of ALT, AST and LDH were measured after 6 h of reperfusion following 60 min of ischemia. The treatment of pioglitazone significantly reduced all serum levels compared to control (Fig. 2). 3.3. Protective effect of PPARc agonist on the ischemic liver tissue To further confirm the protective effect of PPARc agonist on hepatic I/R injury, sections of the liver obtained from the ischemic lobe at 6 h after reperfusion were evaluated for histopathological analysis. In the control liver, massive cellular infiltration and extensive hepatic cellular necrosis were observed (Fig. 3a and b). In contrast, mild cellular infiltration, few necrosis as well

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To clarify the underlying mechanisms for the protective effect of PPARc agonist on hepatic I/R injury, we first evaluated the local expressions of several cytokines, chemokines and adhesion molecules in the liver at 2 and 6 h after reperfusion following 60 min of ischemia using quantitative real-time RT-PCR analysis. PPARc agonist treatment significantly downregulated the local expression of several potent proinflammatory cytokines including TNF-a and IL-1b compared to control (Fig. 4a). Pioglitazone also inhibited the local expression of several chemokines. In particular, MCP-1, MIP-2 and IP-10 were significantly downregulated by the treatment at 2 and 6 h after reperfusion (Fig. 4b). In addition, a potent adhesion molecule, E-selectin, was significantly downregulated at 6 h after reperfusion by the treatment (Fig. 4c). Taken together, the protective effect of PPARc agonist on hepatic I/R injury might be associated with the inhibition of local immune activation. Furthermore, we assessed the expression of eNOS and iNOS in liver tissues. The expression of eNOS at 6 h after reperfusion was significantly lower in the pioglitazone-treated liver than in the control ischemic liver (Fig. 5). iNOS expression in the liver was also significantly downregulated by the treatment. 3.5. PPARc agonist inhibits neutrophil accumulation in the liver Since it is widely recognized that neutrophils play a central role in hepatic I/R injury, we directly examined the neutrophil accumulation in the ischemic liver using MPO staining analysis. In the control liver at 6 h after reperfusion following 60 min of ischemia, a considerable number of neutrophils were identified (Fig. 6a). In contrast, relatively few neutrophils were observed in the liver treated with PPARc agonist (Fig. 6b). By counting the stained cells, we found the significant reduction of the accumulated neutrophils in the PPARc agonist-treated liver compared to the control liver (Fig. 6c).

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Reperfusion time (hr) Fig. 1. Peroxisome proliferator-activated receptor-c (PPARc) mRNA expression in the liver during ischemia/reperfusion (I/R) injury. PPARc expression in the ischemic liver increased over time at 2 and 6 h after 60 min of ischemia compared to the naı¨ve liver determined by quantitative real-time polymerase chain reaction (PCR) analysis (*P < 0.05). Black bar, ischemic liver; white bar, non-ischemic shunt liver.

To further define the underlying mechanisms, we evaluated apoptosis in the liver at 6 h after reperfusion using TUNEL assay. In the control liver at 6 h after reperfusion following 60 min of ischemia, a considerable number of TUNEL-positive cells were identified mainly in hepatocytes (Fig. 7a). In contrast, comparatively few TUNEL-positive hepatocytes were identified

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Fig. 2. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in mice treated with peroxisome proliferator-activated receptor-c agonist, pioglitazone, at 6 h after reperfusion following 60 min of ischemia. Pioglitazone treatment significantly decreased all serum levels compared to control (*P < 0.05).

in the liver treated with PPARc agonist (Fig. 7b). The difference was found to be significant by counting the stained cells (Fig. 7c). Next, we analyzed the local expressions of caspase 3 in the liver at 2 and 6 h after reperfusion following 60 min of ischemia using quantitative real-time RT-PCR analysis. PPARc agonist treatment significantly downregulated the local expression of caspase 3 at 2 h after reperfusion compared to control (Fig. 7d). Furthermore, the TUNEL-positive inflammatory cells infiltrating into the injured liver were observed around the vessels (Fig. 7b). Taken together, while the treatment inhibited the induction

of apoptosis in hepatocytes in the process of hepatic I/R injury, it might induce the apoptosis of pathological inflammatory cells. 3.7. PPARc agonist improves survival in lethally injured mice Finally, to confirm the therapeutic efficacy of PPARc agonist in hepatic I/R injury, we utilized a lethal model. In this model, after 105 min of 70% liver ischemia, the non-ischemic shunt right lobe was surgically resected at the initiation of reperfusion. In this model, over 80% of mice in control group died within 24 h after reperfusion. In contrast, PPARc agonist treatment significantly improved the survival rate (Fig. 8). Thus, data clearly demonstrated that PPARc agonist had a therapeutic efficacy in hepatic I/R injury.

4. Discussion

Fig. 3. Histological analysis of the liver after ischemia/reperfusion. (a–d) Representative histological appearances of the liver staining by hematoxylin-eosin (H&E) from untreated (a and b) and peroxisome proliferator-activated receptor-c (PPARc) agonist-treated mice (c and d) at 6 h after reperfusion following 60 min of ischemia. (a and b) Massive cellular infiltration and extensive hepatic cellular necrosis were observed in the control liver. (c and d) In contrast, mild cellular infiltration, few necrosis as well as comparatively preserved lobular architecture were seen in PPARc agonist-treated liver. (a) and (c) are 40· magnification; (b) and (d) are 400· magnification.

Recent studies have demonstrated that stimulation of PPARc exhibits the significant functions in tissue protection and repair [18–20]. In this study, we have investigated the functions of PPARc in regulating hepatic I/R injury and the therapeutic potential of PPARc agonist for the protection of hepatic injury. First, we examined the local expression of PPARc and found that PPARc expression was significantly upregulated, suggesting that PPARc may play some roles in the acute hepatic injury induced by I/R. Then, to investigate the pathophysiological function of PPARc, we utilized a pharmacological approach using a highly specific agonist for PPARc. As a result, PPARc agonist significantly inhibited hepatic I/R injury. Taken together, our data suggest that endogenous PPARc may be physiologically important to protect acute hepatic injury. This was consistent with previous report demonstrating that PPARc-deficient

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Fig. 4. Effect of peroxisome proliferator-activated receptor-c (PPARc) agonist on the local expressions of cytokine, chemokine, and adhesion molecule in the ischemic liver at 2 and 6 h after reperfusion following 60 min of ischemia. (a) PPARc agonist treatment significantly inhibited the mRNA expression of tumor necrosis factor (TNF)-a and interleukin (IL)-1b after reperfusion. (b) The treatment inhibited the monocyte chemoattractant protein-1 (MCP-1), interferon-inducible protein-10 (IP-10) and macrophage inflammatory protein-2 (MIP-2) at 2 and 6 h after reperfusion. (c) It also significantly inhibited the E-selectin at 6 h after reperfusion. *P < 0.05.

mice display more severe tissue injury compared to wild type mice in an intestinal I/R injury model [22]. Next, we were intrigued with the therapeutic efficacy of PPARc agonist and tried to reveal the underlying mechanisms for its beneficial effect on hepatic I/R injury. Previous studies have suggested that the diverse function of PPARc agonists might be responsible for its protective effect on I/R injury in several models. Among them, the regulation of cytokine may be a crucial mechanism. Cytokines such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b, interferon (IFN)-c as well as chemokines such as macrophage inflammatory protein-2 (MIP-2) have been proposed to play important roles in the process of hepatic I/R injury [4,5,31–34].

PPARc agonists induce the downregulation of several proinflammatory cytokines probably via inhibition of NF-jB, a key regulator of genes encoding inflammatory cytokines [8,20–26,35,36]. To clarify the in vivo dynamics of cytokines induced by PPARc agonist treatment in this study, we analyzed the local expression of several cytokines. As a result, we found a significant reduction of TNF-a and IL-1b by the treatment. Furthermore, chemokines also play an important role in hepatic I/R injury. Due to their potent chemotactic activity for neutrophils, it is generally assumed that CXC chemokines recruit neutrophils into the post-ischemic liver [6]. In this study, one of CXC chemokines, IP-10, was downregulated by PPARc agonist treatment. In addition,

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Fig. 5. Effect of peroxisome proliferator-activated receptor-c (PPARc) agonist on the expressions of endothelial nitric oxide synthase (eNOS) and inducible NO synthase (iNOS) in the ischemic liver at 2 and 6 h after reperfusion following 60 min of ischemia. (a) PPARc agonist treatment significantly inhibited the mRNA expression of eNOS at 6 h after reperfusion. (b) The treatment inhibited the iNOS expression at 2 and 6 h after reperfusion. *P < 0.05.

our data indicated that MCP-1, MIP-2 as well as IP-10 were inhibited by the treatment. Since both clinical and experimental studies have indicated that hepatic levels of these chemokines are markedly enhanced during various types of liver injury, the reduction of such chemokines may be responsible for the effect of PPARc [30,37–39]. These findings are also consistent with previous in vitro data shown in the protective function of PPARc agonist treatment [29,40]. Furthermore, we confirmed

that a potent adhesion molecule, E-selectin, was also downregulated. The interactions between the expressions of adhesion molecules and PPARc agonist have not been reported so far. The increased expression of adhesion molecules on sinusoidal endothelial cells promotes the neutrophil infiltration into the liver, contributing to the progression of parenchymal injury [41–43]. Taken together, the beneficial effect of PPARc agonist on hepatic I/R injury may, at least in part, depend on

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Fig. 6. Inhibitory effect of peroxisome proliferator-activated receptor-c (PPARc) agonist on the accumulation of neutrophils in the liver at 6 h after reperfusion following 60 min of ischemia. (a and b) Paraffin-embedded sections were stained with a monoclonal antibody against MPO (magnification 200·). (a) A considerable number of neutrophils were observed in the control liver. (b) Relatively less neutrophils were seen in the liver treated with PPARc agonist. (c) Quantitative analysis revealed that the treatment significantly inhibited the neutrophil accumulation (*P < 0.05).

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Fig. 7. Effect of Peroxisome proliferator-activated receptor-c (PPARc) agonist on apoptosis in the liver after reperfusion. (a–c) Terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining was performed on liver sections from mice subjected to 60 min of ischemia and 6 h of reperfusion (magnification 200·). (a) A considerable number of TUNEL-positive cells were observed mainly in hepatocytes of the control liver. (b) Relatively less TUNEL-positive cells were seen in the liver treated with PPARc agonist. The TUNEL-positive inflammatory cells infiltrating into the injured liver were observed around the vessels (c) TUNEL-positive cells were counted in non-necrotic areas in 10 high-power fields (magnification 400·). PPARc agonist treatment significantly inhibited the accumulation of TUNEL-positive cells (*P < 0.05). (d) PPARc agonist treatment significantly downregulated the mRNA expression of caspase 3 at 2 h after reperfusion following 60 min of ischemia (*P < 0.05). [This figure appears in colour on the web].

the restraints of local immune activation in the liver. Such downregulation of local immune activation can lead to less infiltration and accumulation of various pathogenic cells such as neutrophils and macrophages, thereby resulting in the significant protection from hepatic injury. In addition, we found significantly

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Time after reperfusion (hr) Fig. 8. The therapeutic efficacy of peroxisome proliferator-activated receptor-c (PPARc) agonist in mouse survival after severe hepatic ischemia/reperfusion (I/R) injury. The non-ischemic shunt lobe was surgically removed at the time of reperfusion following 105 min of ischemia. PPARc agonist treatment significantly improved the survival (n = 12 of each group) (P < 0.05).

decreased expressions of eNOS and iNOS in the liver by PPARc agonist treatment. These data were partly consistent with previous studies [44]. Although the role of nitric oxide in hepatic I/R injury is still controversial, our data suggested that PPARc agonist might exert the protective effect through the downregulation of both eNOS and iNOS. Although the pathophysiological role of apoptosis and oncotic necrosis in hepatic I/R injury has not been fully elucidated yet, they are undoubtedly prominent features [45]. In addition, several reports have provided evidence for therapeutic efficacy of targeting apoptosis in the inhibition of hepatic I/R injury. To evaluate the dynamics of apoptosis by the PPARc agonist treatment, we analyzed the local expression of caspase 3, a component of enzymatic cascades that cause apoptotic death of cells, by real-time RT-PCR and examined the apoptotic cells by TUNEL assay. As a result, we found a significant downregulation of caspase 3 as well as reduction of the TUNEL-positive cells in the PPARc agonist-treated liver. Data suggested that the inhibition of apoptosis might be responsible for the therapeutic efficacy of PPARc agonist on hepatic I/R injury. On the other hand, it has been recently reported that PPARc agonist induces apoptosis on T cells under some circumstances such as infection [46]. In addition, it has been demonstrated that PPARc could also induce apoptosis of mac-

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rophages in human tissues [47]. Thus, PPARc activation by selective agonist might inhibit hepatic I/R injury through reduction or elimination of these pathogenic cells by inducing apoptosis, while it prevents the induction of apoptosis in hepatocytes. In fact, while comparatively few TUNEL-positive hepatocytes were identified in the PPARc agonist-treated liver, the TUNEL-positive inflammatory cells infiltrating into the injured liver were observed around the vessels. These histopathological findings support our interpretations. In addition, the other mechanisms might be important for the protection of acute hepatic injury induced by PPARc agonist. For instance, PPARc agonists have effects on generation of reactive oxygen species and lipid peroxidation [48–50]. To clarify the other fundamental mechanisms, further studies will be required. Several PPARc agonists have been shown to have diverse biological properties and regulate various physiological as well as pathological disease conditions. As shown in our data indicating pioglitazone significantly improves mouse survival in a lethal hepatic I/R injury model, this study may further support its therapeutic potential in acute hepatic injury such as liver transplantation and surgery. Since PPARc agonists are widely used as common agents to treat diabetic patients, the expansion of clinical indication for them might be relatively easy. Furthermore, PPARc agonists have been recently proven to inhibit the growth and function of hepatic stellate cells (HSC) by the downregulation of proteins such as a1-collagen, a-SMA, and fibronectin [19,29,51–53]. HSCs are the most relevant cell type for liver fibrosis. Therefore, PPARc agonists are anticipated to prevent the development of liver cirrhosis as well as fibrosis that are chronic hepatic diseases. Taken together, PPARc agonists may have significant therapeutic potential for the treatment of both acute and chronic liver disorders through distinct, but not mutually exclusive mechanisms. In conclusion, we have demonstrated for the first time the inhibitory role of PPARc in the process of hepatic I/R injury. Furthermore, selective PPARc agonist had a therapeutic efficacy for the protection of the liver from ischemic injury. Therefore, PPARc may be a potent target for protecting the acute hepatic injury and the clinical application of PPARc selective agonist can be considered in several clinical conditions such as liver transplantation and major hepatic surgery. References [1] Lemasters JJ, Thurman RG. Reperfusion injury after liver preservation for transplantation. Annu Rev Pharmacol Toxicol 1997;37:327–338. [2] Huguet C, Gavelli A, Bona S. Hepatic resection with ischemia of the liver exceeding one hour. J Am Coll Surg 1994;178:454–458. [3] Jaeschke H. Preservation injury: mechanisms, prevention and consequences. J Hepatol 1996;25:774–780.

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