Research Article

Helium preconditioning protects mouse liver against ischemia and reperfusion injury through the PI3K/Akt pathway Rongjia Zhang1, , Ling Zhang2, , Anatol Manaenko3, Zhouheng Ye1, Wenwu Liu1,⇑, Xuejun Sun1,⇑ 1 Department of Diving Medicine, Second Military Medical University, Shanghai, China; 2Department of Medical Genetics, Second Military Medical University, Shanghai, China; 3Department of Physiology and Pharmacology, Loma Linda University Medical Center, Loma Linda, CA, USA

Background & Aims: Hepatic ischemia and reperfusion (I/R) injury is a major complication of liver transplantation, hepatic resection and trauma. Helium preconditioning (HPC) exerts protection against ischemic stress. We investigated potential beneficial effects of HPC on I/R-induced liver injury and investigated mechanisms underlying HPC-induced protection. Methods: We employed a model of segmental warm hepatic I/R on BALB/c mice. Serum ALT was measured and livers were analysed by histology, RT-PCR and western blot. HPC was induced by inhalation of a 70% helium/30% oxygen mixture for three 5-min periods, interspersed with three 5-min washout periods by room air. We tested which component of HPC (the helium/air mixture inhalation, the air room gap, or the interaction between these two factors) is protective. Results: We found that HPC caused a significant increase in Akt phosphorylation in hepatocytes. The HPC-induced Akt phosphorylation resulted in decreased hepatocellular injury and improved survival rate of the treated animals. PI3K inhibitors abolished HPC induced effects. HPC-induced Akt phosphorylation affected expression of its downstream molecules. The effects of HPC on the PI3K/Akt pathway were attenuated by adenosine A2A receptor blockade, but could be re-established by PTEN inhibition. We demonstrated that the interaction of helium/air breathing and air gaps is responsible for the observed effects of HPC.

Keywords: Liver I/R injury; Helium preconditioning; PI3K/Akt; Air gap. Received 30 October 2013; received in revised form 24 May 2014; accepted 14 June 2014; available online 24 June 2014 ⇑ Corresponding authors. Address: Department of Diving Medicine, Second Military Medical University, 800 Xiangyin Road, Shanghai, China. Tel.: +86 21 81871144 809; fax: +86 21 65492382 (W. Liu). Tel.: +86 21 81871143; fax: +86 21 65492382 (X. Sun). E-mail addresses: [email protected] (W. Liu), [email protected] (X. Sun).   These authors contributed equally to this work. Abbreviations: ALT, alanine aminotransferase; I/R, ischemia reperfusion; HPC, helium preconditioning; HEX, helium exposure; NPC, neon preconditioning; NEX, neon exposure; bpv (HOpic), dipotassium bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate (V); PTEN, phosphatase and tensin homolog; PI3K, phosphatidylinositol-3-kinase; GSK-3b, glycogen synthase kinase-3b; BAD, Bcl-2-associated death promoter; JNK, c-Jun N-terminal kinase; p38 MAPK, p38 mitogen-activated protein kinase; NF-jB, nuclear factor jB; TUNEL, terminal dUTP nick end labeling.

Conclusions: HPC may be a promising strategy leading to a decrease in I/R induced liver injury in clinical settings. Additionally, the PI3K/Akt pathway plays an essential role in the protective effects of HPC in hepatic I/R injury. Ó 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction Ischemia/reperfusion (I/R) injury in the liver is a major complication of liver transplantation, liver resection and trauma [1]. The pathophysiology of liver I/R injury includes both, direct cellular damage as the result of the ischemic insult, and delayed dysfunction, which is a consequence of inflammatory pathways activation. Histopathological changes in the liver after I/R injury include: cellular swelling, vacuolization, endothelial cell disruption, neutrophil infiltration, hepatocellular necrosis and apoptosis [2,3]. Despite improved preservation and surgical techniques, I/R injury resulting from donor organ retrieval, cold storage and warm ischemia during surgery often leads to liver dysfunction, a predisposition to chronic rejection, and ultimately contributes to the acute shortage of donor organs available for transplantation [1,2]. New therapeutic strategies, preventing or attenuating the harmful processes, triggered by reperfused ischemic tissues, are therefore urgently needed. Helium is safe for use in clinical practice and diving because of its favourable characteristics and the lack of hemodynamic side effects. Recent experimental research has convincingly shown the protective properties of helium against ischemia in the heart [4–10] and the brain [11–13]. These organs can be protected against I/R injury by subjecting them to several short helium episodes according to a specific protocol, known as helium preconditioning (HPC) [4–10,13]. However, the specific mechanisms and signaling pathways responsible for HPC-induced protections remain poorly understood [14]. The phosphatidylinositol-3-kinase PI3K/Akt pathway is a survival pathway, involved in protection against various stressors. Activation of Akt promotes cell survival by modulation of various downstream elements, including glycogen synthase kinase-3b (GSK-3b) and Bcl-2-associated death promoter (BAD) [15–17]. Phosphorylation of GSK-3b and BAD by Akt can suppress caspase-3 activity and inhibit cell apoptosis. Additionally, Akt inhibits

Journal of Hepatology 2014 vol. 61 j 1048–1055

JOURNAL OF HEPATOLOGY activation of NF-jB, which results in the suppression of hepatic inflammation [18–20]. Activation of Akt also inhibits the activity of c-Jun N-terminal kinase (JNK) and ameliorates cell necrosis [21,22]. PI3K acts as an upstream trigger for p38 mitogen-activated protein kinase (p38 MAPK) activation, which can interfere with the processes leading to cell necrosis [23]. Thus, activation of the PI3K/ Akt pathway plays an important protective role during hepatic I/R [23–25]. In the present study, we used a mouse model of segmental warm hepatic I/R to evaluate the protective effect of HPC on I/R-induced liver injury and to investigate the role of PI3K/Akt pathway in the protection. In this model we also tested, which component of HPC (the mixture of helium/air inhalation, the air room gap, or the interaction between these two factors) is responsible for HPC-induced activation of the PI3K/Akt pathway leading to liver protection. To the best of our knowledge this is the first study that investigates the possible mechanisms of HPC on the experimental validation of hepatic warm I/R injury.

GSK-3b (#11301), total GSK-3b (#21301)); Cell Signaling Technology (phosphorylated-p38 MAPK (#4511), total p38 MAPK (#2387), cleaved caspase-3 (#9661), total caspase-3 (#9662), Bcl-2 (#2876), b-actin (#4967)). Relative protein quantities were determined by an Odyssey infrared scanner (Li-COR Biosciences; Lincoln, NE, USA). Quantitative RT-PCR analysis Real-time PCR was performed with a standard SYBR-Green PCR kit protocol on a StepOnePlus system (Applied Biosystems, Foster City, CA, USA). Target gene expressions were calculated by their ratios to the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT). Primers used to amplify specific gene fragments have been published elsewhere [27–29]. Statistical analysis Data were expressed as the mean ± SD. Differences between experimental groups were analysed by unpaired Student’s t test using SPSS 13.0 (SPSS, Inc., IL, USA). Recipient survival was plotted using the Kaplan-Meier method, and differences between groups were analysed using the log-rank test. A p value of less than 0.05 was considered statistically significant.

Materials and methods Animals

Results

299 male BALB/c mice with a body weight of 25–30 g were obtained from Shanghai Slac Laboratory Animal Co. Ltd. and used for the experiments. The study was approved by the local Ethics Committee.

HPC reduced severity of I/R induced liver injury. The protective effects of HPC were blocked by PI3K/Akt inhibitors

Model of liver I/R A model of segmental (70%) warm hepatic I/R was employed with minor modifications [26]. Briefly, after a midline laparotomy, the structures to the left of the porta hepatis were occluded for 90 min. Reperfusion was initiated via clamp removal. The animal’s body temperature was controlled. For the survival study, 30% of normal liver lobes were removed before starting the reperfusion. Experimental protocol and settings Please see Supplementary materials and methods and Supplementary Fig. 1. Evaluation of hepatocyte and renal cell injuries The degree of hepatic and renal injuries was assessed by measurement of serum alanine aminotransferase (ALT) levels and serum creatinine levels. ALT serum levels and creatinine levels were determined by an automated procedure in the Department of Inspection, Eastern Hepatobiliary Surgery Hospital. Histology and immunohistochemistry Liver sections (5 lm) were stained with hematoxylin and eosin (H&E). Necrotic areas were quantified by image analysis of 10 randomly selected fields per liver, in slides stained with H&E in a blinded manner. Phospho-specific Akt (#11054; Signalway Antibody), CD11b (#ab62817; Abcam), CD68 (#ab125212) and MPO (#ab9535; Abcam) antibody staining of sections was performed according to the manufacturer’s instructions. The data were evaluated by blinded investigators. TUNEL staining TUNEL staining was performed according to the manufacturer’s recommendations. TUNEL-positive hepatocytes were expressed as a percentage of total hepatocytes excluding the surrounding massive necrosis lesion [22]. Antibodies and Western Blotting Western blots were performed according to the manufacturer’s recommendations. The following antibodies were used: Signalway Antibodies (phosphorylated-Akt (#11054), total Akt (#21054), phosphorylated-JNK (#11504-1), total JNK (#21241-1), phosphorylated-BAD (#11068), total BAD (#32330), phosphorylated

I/R caused significant release of ALT six hours after reperfusion compared with sham-operated animals (8539 ± 2151 vs. 44.8 ± 22.0 U/L; ⁄p

Akt pathway.

Hepatic ischemia and reperfusion (I/R) injury is a major complication of liver transplantation, hepatic resection and trauma. Helium preconditioning (...
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