Mohamed Bejaoui

AMPK involvement in endoplasmic reticulum stress and autophagy modulation after fatty liver graft preservation: a role for melatonin and trimetazidine cocktail

Ischemia/reperfusion injury (IRI) associated with liver transplantation plays an important role in the induction of graft injury. Prolonged cold storage remains a risk factor for liver graft outcome, especially when steatosis is present. Steatotic livers exhibit exacerbated endoplasmic reticulum (ER) stress that occurs in response to cold IRI. In addition, a defective liver autophagy correlates well with liver damage. Here, we evaluated the combined effect of melatonin and trimetazidine as additives to IGL‐1 solution in the modulation of ER stress and autophagy in steatotic liver grafts through activation of AMPK. Steatotic livers were preserved for 24 hr (4°C) in UW or IGL‐1 solutions with or without MEL + TMZ and subjected to 2‐hr reperfusion (37°C). We assessed hepatic injury (ALT and AST) and function (bile production). We evaluated ER stress (GRP78, PERK, and CHOP) and autophagy (beclin‐1, ATG7, LC3B, and P62). Steatotic livers preserved in IGL‐1 + MEL + TMZ showed lower injury and better function as compared to those preserved in IGL‐1 alone. IGL‐1 + MEL + TMZ induced a significant decrease in GRP78, pPERK, and CHOP activation after reperfusion. This was consistent with a major activation of autophagic parameters (beclin‐1, ATG7, and LC3B) and AMPK phosphorylation. The inhibition of AMPK induced an increase in ER stress and a significant reduction in autophagy. These data confirm the close relationship between AMPK activation and ER stress and autophagy after cold IRI. The addition of melatonin and TMZ to IGL‐1 solution improved steatotic liver graft preservation through AMPK activation, which reduces ER stress and increases autophagy.

Proteasome inhibitors protect the steatotic and non-steatotic liver graft against cold ischemia reperfusion injury.

BACKGROUND The dramatic shortage of organs leads to consider the steatotic livers for transplantation although their poor tolerance against ischemia reperfusion injury (IRI). Ubiquitin proteasome system (UPS) inhibition during hypothermia prolongs myocardial graft preservation. The role of UPS in the liver IRI is not fully understood. Bortezomib (BRZ) treatment at non-toxic doses of rats fed alcohol chronically has shown protective effects by increasing liver antioxidant enzymes. We evaluated and compared both proteasome inhibitors BRZ and MG132 in addition to University of Wisconsin preservation solution (UW) at low and non-toxic dose for fatty liver graft protection against cold IRI. EXPERIMENTAL Steatotic and non-steatotic livers have been stored in UW enriched with BRZ (100 nM) or MG132 (25 μM), for 24h at 4°C and then subjected to 2-h normothermic reperfusion (37 °C). Liver injury (AST/ALT), hepatic function (bile output; vascular resistance), mitochondrial damage (GLDH), oxidative stress (MDA), nitric oxide (NO) (e-NOS activity; nitrates/nitrites), proteasome chymotrypsin-like activity (ChT), and UPS (19S and 20S5 beta) protein levels have been measured. RESULTS ChT was inhibited when BRZ and MG132 were added to UW. Both inhibitors prevented liver injury (AST/ALT), when compared to UW alone. BRZ increased bile production more efficiently than MG132. Only BRZ decreased vascular resistance in fatty livers, which correlated with an increase in NO generation (through e-NOS activation) and AMPK phosphorylation. GLDH and MDA were also prevented by BRZ. In addition, BRZ inhibited adiponectin, IL-1, and TNF alpha, only in steatotic livers. CONCLUSION MG132 and BRZ, administrated at low and non toxic doses, are very efficient to protect fatty liver grafts against cold IRI. The benefits of BRZ are more effective than those of MG132. This evidenced for the first time the potential use of UPS inhibitors for the preservation of marginal liver grafts and for future applications in the prevention of IRI.

Bortezomib enhances fatty liver preservation in Institut George Lopez-1 solution through adenosine monophosphate activated protein kinase and Akt/mTOR pathways

The aim of this study is to investigate the protective mechanisms induced by bortezomib added to Institut George Lopez (IGL)‐1 preservation solution to protect steatotic livers against cold ischaemia reperfusion injury and to examine whether these mechanisms occur through the activation of adenosine monophosphate activated protein kinase (AMPK), Akt/mTOR pathways.

Role of sirtuins in ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) remains an unresolved and complicated situation in clinical practice, especially in the case of organ transplantation. Several factors contribute to its complexity; the depletion of energy during ischemia and the induction of oxidative stress during reperfusion initiate a cascade of pathways that lead to cell death and finally to severe organ injury. Recently, the sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylases has gained increasing attention from researchers, due to their involvement in the modulation of a wide variety of cellular functions. There are seven mammalian sirtuins and, among them, the nuclear/cytoplasmic sirtuin 1 (SIRT1) and the mitochondrial sirtuin 3 (SIRT3) are ubiquitously expressed in many tissue types. Sirtuins are known to play major roles in protecting against cellular stress and in controlling metabolic pathways, which are key processes during IRI. In this review, we mainly focus on SIRT1 and SIRT3 and examine their role in modulating pathways against energy depletion during ischemia and their involvement in oxidative stress, apoptosis, microcirculatory stress and inflammation during reperfusion. We present evidence of the beneficial effects of sirtuins against IRI and emphasize the importance of developing new strategies by enhancing their action.

Targeting the Microbiome in Inflammatory Bowel Disease: Critical Evaluation of Current Concepts and Moving to New Horizons.

Microorganisms present in the intestine possess proinflammatory or anti-inflammatory activities which may modulate inflammatory bowel disease (IBD). The concepts followed by researchers in trying to target the microbiota in IBD were to decrease pathogens or pathobionts, or only the microbial load, and more recently, to favor growth and persistence of favorable microorganisms. We review, here, those concepts and critically analyze the clinical data (especially randomized controlled trials) obtained using antibiotics and probiotics. We eventually present and criticize the rational and data obtained so far following new research strategies including the use of new probiotics, genetically modified organisms and fecal transplantation.

Polyethylene glycol rinse solution: An effective way to prevent ischemia-reperfusion injury

AIM To test whether a new rinse solution containing polyethylene glycol 35 (PEG-35) could prevent ischemia-reperfusion injury (IRI) in liver grafts. METHODS Sprague-Dawley rat livers were stored in University of Wisconsin preservation solution and then washed with different rinse solutions (Ringers lactate solution and a new rinse solution enriched with PEG-35 at either 1 or 5 g/L) before ex vivo perfusion with Krebs-Heinseleit buffer solution. We assessed the following: liver injury (transaminase levels), mitochondrial damage (glutamate dehydrogenase activity), liver function (bile output and vascular resistance), oxidative stress (malondialdehyde), nitric oxide, liver autophagy (Beclin-1 and LCB3) and cytoskeleton integrity (filament and globular actin fraction); as well as levels of metalloproteinases (MMP2 and MMP9), adenosine monophosphate-activated protein kinase (AMPK), heat shock protein 70 (HSP70) and heme oxygenase 1 (HO-1). RESULTS When we used the PEG-35 rinse solution, reduced hepatic injury and improved liver function were noted after reperfusion. The PEG-35 rinse solution prevented oxidative stress, mitochondrial damage, and liver autophagy. Further, it increased the expression of cytoprotective heat shock proteins such as HO-1 and HSP70, activated AMPK, and contributed to the restoration of cytoskeleton integrity after IRI. CONCLUSION Using the rinse solution containing PEG-35 was effective for decreasing liver graft vulnerability to IRI.

Silent information regulator 1 protects the liver against ischemia-reperfusion injury: Implications in steatotic liver ischemic preconditioning

Ischemia–reperfusion (IR) injury is an important problem in liver surgery especially when steatosis is present. Ischemic preconditioning (PC) is the only surgical strategy that has been applied in patients with steatotic livers undergoing warm ischemia. Silent information regulator 1 (SIRT1) is a histone deacetylase that regulates various cellular processes. This study evaluates the SIRT1 implication in PC in fatty livers. Homozygous (Ob) Zucker rats were subjected to IR and IR + PC. An additional group treated with sirtinol or EX527 (SIRT1 inhibitors) before PC was also realized. Liver injury and oxidative stress were evaluated. SIRT1 protein levels and activity, as well as other parameters involved in PC protective mechanisms (adenosine monophosphate protein kinase, eNOS, HSP70, MAP kinases, apoptosis), were also measured. We demonstrated that the protective effect of PC was due in part to SIRT1 induction, as SIRT1 inhibition resulted in increased liver injury and abolished the beneficial mechanisms of PC. In this study, we report for the first time that SIRT1 is involved in the protective mechanisms induced by hepatic PC in steatotic livers.

Acetazolamide Protects Steatotic Liver Grafts against Cold Ischemia Reperfusion Injury

Ischemia reperfusion injury (IRI) is a primary concern in liver transplantation, especially when steatosis is present. Acetazolamide (AZ), a specific carbonic anhydrase (CA) inhibitor, has been suggested to protect against hypoxia. Here, we hypothesized that AZ administration could be efficient to protect fatty livers against cold IRI. Obese Zucker rat livers were preserved in Institut Georges Lopez-1 storage solution for 24 hours at 4°C and ex vivo perfused for 2 hours at 37°C. Alternatively, rats were also treated with intravenous injection of AZ (30 mg/kg) before liver recovery. Liver injury, hepatic function, and vascular resistance were determined. CA II protein levels and CA hydratase activity were assessed as well as other parameters involved in IRI (endothelial nitric oxide synthase, mitogen activated protein kinase family, hypoxic inducible factor 1 alpha, and erythropoietin). We demonstrated that AZ administration efficiently protects the steatotic liver against cold IRI. AZ protection was associated with better function, decreased vascular resistance, and activation of endothelial nitric oxide synthase. This was consistent with an effective mitogen activated protein kinase inactivation. Finally, no effect on the hypoxic inductible factor 1 alpha/erythropoietin pathway was observed. The present study demonstrated that AZ administration is a suitable pharmacological strategy for preserving fatty liver grafts against cold IRI.

Advances in treatment strategies for ischemia reperfusion injury.

ABSTRACT Introduction: Ischemia-reperfusion injury (IRI) involves a complex sequence of events and limits the outcome of various surgical interventions. Clinical trials, based on the data of experimental models, aim to prove whether a pharmacological or technical approach could be suitable to provide a beneficial effect in humans. Due to the complexity of IRI, few pharmacological treatments have been investigated in clinical Phase III. Areas covered: In this review we report clinical trials that test specific drugs in clinical trials of organ transplantation. These studies form part of Phase II trials and examine the administration of caspase inhibitors, P-selectin antagonist or an antioxidant component in order to attenuate cold IRI during transplantation. Moreover, we provide a brief description of drugs tested on trials of different clinical situations associated to IRI, such as the coronary artery bypass graft surgery and percutaneous coronary intervention. Expert opinion: Future clinical trials could be centered on the application of techniques suitable for organs with increased vulnerability toward IRI. Furthermore, the standardization of reliable biomarkers and a careful estimation of the impact of high risk factors may be the key in order to achieve a more critical evaluation of the obtained results.

Sirtuin 1 in rat orthotopic liver transplantation: An IGL-1 preservation solution approach

AIM To investigate the possible involvement of Sirtuin 1 (SIRT1) in rat orthotopic liver transplantation (OLT), when Institute Georges Lopez 1 (IGL-1) preservation solution is enriched with trimetazidine (TMZ). METHODS Male Sprague-Dawley rats were used as donors and recipients. Livers were stored in IGL-1 preservation solution for 8h at 4 °C, and then underwent OLT according to Kamadas cuff technique without arterialization. In another group, livers were stored in IGL-1 preservation solution supplemented with TMZ, at 10(-6) mol/L, for 8 h at 4 °C and then underwent OLT. Rats were sacrificed 24 h after reperfusion, and liver and plasma samples were collected. Liver injury (transaminase levels), mitochondrial damage (glutamate dehydrogenase activity) oxidative stress (malondialdehyde levels), and nicotinamide adenine dinucleotide (NAD(+)), the co-factor necessary for SIRT1 activity, were determined by biochemical methods. SIRT1 and its substrates (ac-FoxO1, ac-p53), the precursor of NAD(+), nicotinamide phosphoribosyltransferase (NAMPT), as well as the phosphorylation of adenosine monophosphate activated protein kinase (AMPK), p-mTOR, p-p70S6K (direct substrate of mTOR), autophagy parameters (beclin-1, LC3B) and MAP kinases (p-p38 and p-ERK) were determined by Western blot. RESULTS Liver grafts preserved in IGL-1 solution enriched with TMZ presented reduced liver injury and mitochondrial damage compared with those preserved in IGL-1 solution alone. In addition, livers preserved in IGL-1 + TMZ presented reduced levels of oxidative stress. This was consistent with enhanced SIRT1 protein expression and elevated SIRT1 activity, as indicated by decreased acetylation of p53 and FoxO1. The elevated SIRT1 activity in presence of TMZ can be attributed to the enhanced NAMPT protein and NAD(+)/NADH levels. Up-regulation of SIRT1 was consistent with activation of AMPK and inhibition of phosphorylation of mTOR and its direct substrate (p-p70S6K). As a consequence, autophagy mediators (beclin-1 and LC3B) were over-expressed. Furthermore, MAP kinases were regulated in livers preserved with IGL-1 + TMZ, as they were characterized by enhanced p-ERK and decreased p-p38 protein expression. CONCLUSION Our study shows that IGL-1 preservation solution enriched with TMZ protects liver grafts from the IRI associated with OLT, through SIRT1 up-regulation.