The mechanisms responsible for the evolution of steatosis towards NASH (non-alcoholic steatohepatitis) and fibrosis are not completely defined. In the present study we evaluated the role of CD4(+) T-helper (Th) cells in this process. We analysed the infiltration of different subsets of CD4(+) Th cells in C57BL/6 mice fed on a MCD (methionine choline-deficient) diet, which is a model reproducing all phases of human NASH progression. There was an increase in Th17 cells at the beginning of NASH development and at the NASH-fibrosis transition, whereas levels of Th22 cells peaked between the first and the second expansion of Th17 cells. An increase in the production of IL (interleukin)-6, TNFα (tumour necrosis factor α), TGFβ (transforming growth factor β) and CCL20 (CC chemokine ligand 20) accompanied the changes in Th17/Th22 cells. Livers of IL-17(-/-) mice were protected from NASH development and characterized by an extensive infiltration of Th22 cells. In vitro, IL-17 exacerbated the JNK (c-Jun N-terminal kinase)-dependent mouse hepatocyte lipotoxicity induced by palmitate. IL-22 prevented lipotoxicity through PI3K (phosphoinositide 3-kinase)-mediated inhibition of JNK, but did not play a protective role in the presence of IL-17, which up-regulated the PI3K/Akt inhibitor PTEN (phosphatase and tensin homologue deleted on chromosome 10). Consistently, livers of IL-17(-/-) mice fed on the MCD diet displayed decreased activation of JNK, reduced expression of PTEN and increased phosphorylation of Akt compared with livers of wild-type mice. Hepatic infiltration of Th17 cells is critical for NASH initiation and development of fibrosis in mice, and reflects an infiltration of Th22 cells. Th22 cells are protective in NASH, but only in the absence of IL-17. These data strongly support the potentiality of clinical applications of IL-17 inhibitors that can prevent NASH by both abolishing the lipotoxic action of IL-17 and allowing IL-22-mediated protection.

Ischemia/reperfusion (I/R) injury still represents an important cause of morbidity following hepatic surgery and limits the use of marginal livers in hepatic transplantation. Transient blood flow interruption followed by reperfusion protects tissues against damage induced by subsequent I/R. This process known as ischemic preconditioning (IP) depends upon intrinsic cytoprotective systems whose activation can inhibit the progression of irreversible tissue damage. Compared to other organs, liver IP has additional features as it reduces inflammation and promotes hepatic regeneration. Our present understanding of the molecular mechanisms involved in liver IP is still largely incomplete. Experimental studies have shown that the protective effects of liver IP are triggered by the release of adenosine and nitric oxide and the subsequent activation of signal networks involving protein kinases such as phosphatidylinositol 3-kinase, protein kinase C δ/ε and p38 MAP kinase, and transcription factors such as signal transducer and activator of transcription 3, nuclear factor-κB and hypoxia-inducible factor 1. This article offers an overview of the molecular events underlying the preconditioning effects in the liver and points to the possibility of developing pharmacological approaches aimed at activating the intrinsic protective systems in patients undergoing liver surgery.

NEFA (non-esterified 'free' fatty acid)-mediated lipotoxicity plays a critical role in the pathogenesis of NASH (non-alcoholic steatohepatitis). In the light of the growing need for new therapeutic options for NASH, we investigated the action of A2aR (adenosine A(2a) receptor) stimulation against lipotoxicity. The effects of the A(2a)R agonist CGS21680 [2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxyamidoadenosine] were evaluated 'in vitro' in liver cells exposed to SA (stearic acid) and 'in vivo' in rats with NASH induced by 8 weeks of feeding with an MCD diet (methionine/choline-deficient diet). In cultured hepatocytes, SA promoted apoptosis by inducing MKK4 (mitogen-activated protein kinase kinase 4)/SEK1 (stress-activated protein kinase/extracellular-signal-regulated kinase kinase-1) and JNK-1/2 (c-Jun N-terminal kinase-1/2) activation. CGS21680 addition prevented JNK-1/2 activation and reduced apoptosis without interfering with lipid accumulation. CGS21680 action required PI3K (phosphoinositide 3-kinase)/Akt-mediated block of MKK4/SEK1. Consistently, PI3K inhibition with wortmannin abolished the cytoprotective action of CGS21680 and reverted MKK4 inhibition. SA lipotoxicity was also prevented by transfecting HTC cells with a specific MKK4/SEK1 siRNA (small interfering RNA). In rats receiving the MCD diet, the development of NASH was associated with MKK4/SEK1 and JNK-1/2 activation. CGS21680 (0.5 mg/kg of body weight, intraperitoneal) administration to MCD-fed rats prevented JNK-1/2 activation by acting on MKK4/SEK1. CGS21680 also effectively reduced NASH-associated ALT (alanine aminotransferase) release, hepatocyte apoptosis, liver inflammation and fibrosis without affecting hepatic steatosis. Taken together, these results demonstrate that, by inhibiting JNK-1/2, A(2a)R stimulation reduces lipotoxicity and ameliorates NASH, giving a rationale to investigate A(2a)R agonists as possible new therapeutic agents in preventing fatty liver progression to NASH.

Postconditioning is a procedure based on the induction of intracellular protective reactions immediately after the onset of reperfusion. Because of the growing need to prevent ischemia/reperfusion (I/R) injury during liver surgery and transplantation, we investigated the possibility of pharmacologically inducing hepatic postconditioning. The effects of the adenosine A2A receptor agonist 2p-(2-carboxyethyl)-phenyl-amino-5 0 -N-ethylcarboxyamido-adenosine (CGS21680; 5 lmol/L) and the phosphatase and tensin homologue deleted from chromosome 10 (PTEN) inhibitor dipotassium bisperoxo-(5-hydroxypyridine-2-carboxyl)-oxovanadate [bpV(HOpic); 250 nmol/L] were investigated in primary rat hepatocytes during reoxygenation after 24 hours of cold storage and in an in vivo model of rat liver warm I/R. The addition of CGS21680 at reoxygenation significantly reduced hepatocyte death through the activation of the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB)/ Akt signal pathway and through the reduction of the intracellular level of PTEN. PTEN lowering was associated with the increased generation of reactive oxygen species after A2A receptor-mediated stimulation of b-nicotinamide adenine dinucleotide phosphate oxidase (NOX). The inhibition of PI3K or NOX with wortmannin or diphenyleneiodonium chloride, respectively, and the addition of the antioxidant N,N 0 -diphenyl-p-phenylenediamine reversed the effects of CGS21680. The PTEN inhibitor bpV(HOpic) mimicked the protection provided by CGS21680 against reoxygenation damage. An in vivo rat treatment with CGS21680 or bpV(HOpic) during reperfusion after 1 hour of partial hepatic ischemia also promoted PKB/Akt activation and ameliorated alanine aminotransferase release and histological lesions induced by 2 hours of reperfusion. We conclude that adenosine A2A receptor agonists and PTEN inhibitors are possibly useful agents for the pharmacological induction of postconditioning in the liver.

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