Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging.

Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/ß-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD(+) and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD(+) levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/ß-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.

Downregulation of Sirtuin 1 Does Not Account for the Impaired Long-Term Potentiation in the Prefrontal Cortex of Female APPswe/PS1dE9 Mice Modelling Alzheimer's Disease.

Alzheimer's disease (AD), which predominantly affects women, involves at its onset a metabolic deregulation associated with a synaptic failure. Here, we performed a behavioral, neurophysiological and neurochemical characterization of 9-month-old female APPswe/PS1dE9 (APP/PS1) mice as a model of early AD. These animals showed learning and memory deficits in the Morris water maze, increased thigmotaxis and anxiety-like behavior and showed signs of fear generalization. Long-term potentiation (LTP) was decreased in the prefrontal cortex (PFC), but not in the CA1 hippocampus or amygdala. This was associated with a decreased density of sirtuin-1 in cerebrocortical synaptosomes and a decreased density of sirtuin-1 and sestrin-2 in total cerebrocortical extracts, without alterations of sirtuin-3 levels or of synaptic markers (syntaxin, synaptophysin, SNAP25, PSD95). However, activation of sirtuin-1 did not affect or recover PFC-LTP deficit in APP/PS1 female mice; instead, inhibition of sirtuin-1 increased PFC-LTP magnitude. It is concluded that mood and memory dysfunction in 9-month-old female APP/PS1 mice is associated with a parallel decrease in synaptic plasticity and in synaptic sirtuin-1 levels in the prefrontal cortex, although sirtiun1 activation failed to restore abnormal cortical plasticity.

Liver Graft Hypothermic Static and Oxygenated Perfusion (HOPE) Strategies: A Mitochondrial Crossroads.

Marginal liver grafts, such as steatotic livers and those from cardiac death donors, are highly vulnerable to ischemia-reperfusion injury that occurs in the complex route of the graft from "harvest to revascularization". Recently, several preservation methods have been developed to preserve liver grafts based on hypothermic static preservation and hypothermic oxygenated perfusion (HOPE) strategies, either combined or alone. However, their effects on mitochondrial functions and their relevance have not yet been fully investigated, especially if different preservation solutions/effluents are used. Ischemic liver graft damage is caused by oxygen deprivation conditions during cold storage that provoke alterations in mitochondrial integrity and function and energy metabolism breakdown. This review deals with the relevance of mitochondrial machinery in cold static preservation and how the mitochondrial respiration function through the accumulation of succinate at the end of cold ischemia is modulated by different preservation solutions such as IGL-2, HTK, and UW (gold-standard reference). IGL-2 increases mitochondrial integrity and function (ALDH2) when compared to UW and HTK. This mitochondrial protection by IGL-2 also extends to protective HOPE strategies when used as an effluent instead of Belzer MP. The transient oxygenation in HOPE sustains the mitochondrial machinery at basal levels and prevents, in part, the accumulation of energy metabolites such as succinate in contrast to those that occur in cold static preservation conditions. Additionally, several additives for combating oxygen deprivation and graft energy metabolism breakdown during hypothermic static preservation such as oxygen carriers, ozone, AMPK inducers, and mitochondrial UCP2 inhibitors, and whether they are or not to be combined with HOPE, are presented and discussed. Finally, we affirm that IGL-2 solution is suitable for protecting graft mitochondrial machinery and simplifying the complex logistics in clinical transplantation where traditional (static preservation) and innovative (HOPE) strategies may be combined. New mitochondrial markers are presented and discussed. The final goal is to take advantage of marginal livers to increase the pool of suitable organs and thereby shorten patient waiting lists at transplantation clinics.

PEG35 as a Preconditioning Agent against Hypoxia/Reoxygenation Injury.

Pharmacological conditioning is a protective strategy against ischemia/reperfusion injury, which occurs during liver resection and transplantation. Polyethylene glycols have shown multiple benefits in cell and organ preservation, including antioxidant capacity, edema prevention and membrane stabilization. Recently, polyethylene glycol 35 kDa (PEG35) preconditioning resulted in decreased hepatic injury and protected the mitochondria in a rat model of cold ischemia. Thus, the study aimed to decipher the mechanisms underlying PEG35 preconditioning-induced protection against ischemia/reperfusion injury. A hypoxia/reoxygenation model using HepG2 cells was established to evaluate the effects of PEG35 preconditioning. Several parameters were assessed, including cell viability, mitochondrial membrane potential, ROS production, ATP levels, protein content and gene expression to investigate autophagy, mitochondrial biogenesis and dynamics. PEG35 preconditioning preserved the mitochondrial function by decreasing the excessive production of ROS and subsequent ATP depletion, as well as by recovering the membrane potential. Furthermore, PEG35 increased levels of autophagy-related proteins and the expression of genes involved in mitochondrial biogenesis and fusion. In conclusion, PEG35 preconditioning effectively ameliorates hepatic hypoxia/reoxygenation injury through the enhancement of autophagy and mitochondrial quality control. Therefore, PEG35 could be useful as a potential pharmacological tool for attenuating hepatic ischemia/reperfusion injury in clinical practice.

miR-378a: a new emerging microRNA in metabolism.

Metabolic diseases, such as type 2 diabetes or obesity, are the consequence of the disruption of the organism's metabolic pathways. The discovery of small non-coding RNAs-microRNAs (miRNAs)-as post-transcriptional gene regulators opened new doors for the development of novel strategies to combat said diseases. The two strands of miR-378a, miR-378a-3p, and miR-378a-5p are encoded in the Ppargc1b gene and have an active role in the regulation of several metabolic pathways such as mitochondrial metabolism and autophagy. Recent studies recognized miR-378a as an important regulator of energy and glucose homeostasis, highlighting it as a potential target for the improvement of metabolic dysregulation. In the present review, the current knowledge on miR-378a will be discussed with a particular emphasis on its biological functions and mechanisms of action in metabolism, mitochondria, and autophagy.

miR-378a-3p Participates in Metformin's Mechanism of Action on C2C12 Cells under Hyperglycemia.

Metformin is the most used biguanide drug for the treatment of type 2 diabetes mellitus. Despite being mostly known for its hepatic anti-gluconeogenic effect, it is also known to modulate microRNAs (miRNAs, miRs) associated with metabolic diseases. The latter mechanism could be relevant for better understanding metformin's mechanisms underlying its biological effects. In the current work, we found that metformin increases miR-378a-3p expression (p < 0.002) in C2C12 myoblasts previously exposed to hyperglycemic conditions. While the inhibition of miR-378a-3p was shown to impair metformin's effect in ATP production, PEPCK activity and the expression of Tfam. Finally, mitophagy, an autophagic process responsible for the selective degradation of mitochondria, was found to be induced by miR-378a-3p (p < 0.04). miR-378a-3p stimulated mitophagy through a process independent of sestrin-2 (SESN2), a stress-responsible protein that has been recently demonstrated to positively modulate mitophagy. Our findings provide novel insights into an alternative mechanism of action of metformin involving miR-378a-3, which can be used in the future for the development of improved therapeutic strategies against metabolic diseases.

Chenodeoxycholic Acid Has Non-Thermogenic, Mitodynamic Anti-Obesity Effects in an In Vitro CRISPR/Cas9 Model of Bile Acid Receptor TGR5 Knockdown.

Bile acids (BA) have shown promising effects in animal models of obesity. However, the said effects are thought to rely on a thermogenic effect, which is questionably present in humans. A previous work has shown that the BA chenodeoxycholic acid (CDCA) can revert obesity and accelerate metabolism in animal and cell culture models. Thus, the aim of this study was to understand if this obesity reduction is indeed thermogenically-dependent. A CRISPR/Cas9 model of TGR5 (BA receptor) knockdown in 3T3-L1 adipocytes was developed to diminish thermogenic effects. Various parameters were assessed, including mitochondrial bioenergetics by Seahorse flux analysis, oxidative stress and membrane potential by fluorometry, intermediary metabolism by NMR, protein content assessment by Western Blot, gene expression by qPCR, and confocal microscopy evaluation of mitophagy. CDCA was still capable, for the most part, of reversing the harmful effects of cellular obesity, elevating mitophagy and leading to the reduction of harmed mitochondria within the cells, boosting mitochondrial activity, and thus energy consumption. In summary, CDCA has a non-thermogenic, obesity reducing capacity that hinges on a healthy mitochondrial population, explaining at least some of these effects and opening avenues of human treatment for metabolic diseases.

Role of PEG35, Mitochondrial ALDH2, and Glutathione in Cold Fatty Liver Graft Preservation: An IGL-2 Approach.

The total damage inflicted on the liver before transplantation is associated with several surgical manipulations, such as organ recovery, washout of the graft, cold conservation in organ preservation solutions (UW, Celsior, HTK, IGL-1), and rinsing of the organ before implantation. Polyethylene glycol 35 (PEG35) is the oncotic agent present in the IGL-1 solution, which is an alternative to UW and Celsior solutions in liver clinical transplantation. In a model of cold preservation in rats (4 °C; 24 h), we evaluated the effects induced by PEG35 on detoxifying enzymes and nitric oxide, comparing IGL-1 to IGL-0 (which is the same as IGL-1 without PEG). The benefits were also assessed in a new IGL-2 solution characterized by increased concentrations of PEG35 (from 1 g/L to 5 g/L) and glutathione (from 3 mmol/L to 9 mmol/L) compared to IGL-1. We demonstrated that PEG35 promoted the mitochondrial enzyme ALDH2, and in combination with glutathione, prevented the formation of toxic aldehyde adducts (measured as 4-hydroxynonenal) and oxidized proteins (AOPP). In addition, PEG35 promoted the vasodilator factor nitric oxide, which may improve the microcirculatory disturbances in steatotic grafts during preservation and revascularization. All of these results lead to a reduction in damage inflicted on the fatty liver graft during the cold storage preservation. In this communication, we report on the benefits of IGL-2 in hypothermic static preservation, which has already been proved to confer benefits in hypothermic oxygenated dynamic preservation. Hence, the data reported here reinforce the fact that IGL-2 is a suitable alternative to be used as a unique solution/perfusate when hypothermic static and preservation strategies are used, either separately or combined, easing the logistics and avoiding the mixture of different solutions/perfusates, especially when fatty liver grafts are used. Further research regarding new therapeutic and pharmacological insights is needed to explore the underlying mitochondrial mechanisms exerted by PEG35 in static and dynamic graft preservation strategies for clinical liver transplantation purposes.

Target Score-A Proteomics Data Selection Tool Applied to Esophageal Cancer Identifies GLUT1-Sialyl Tn Glycoforms as Biomarkers of Cancer Aggressiveness.

Esophageal cancer (EC) is a life-threatening disease, demanding the discovery of new biomarkers and molecular targets for precision oncology. Aberrantly glycosylated proteins hold tremendous potential towards this objective. In the current study, a series of esophageal squamous cell carcinomas (ESCC) and EC-derived circulating tumor cells (CTCs) were screened by immunoassays for the sialyl-Tn (STn) antigen, a glycan rarely expressed in healthy tissues and widely observed in aggressive gastrointestinal cancers. An ESCC cell model was glycoengineered to express STn and characterized in relation to cell proliferation and invasion in vitro. STn was found to be widely present in ESCC (70% of tumors) and in CTCs in 20% of patients, being associated with general recurrence and reduced survival. Furthermore, STn expression in ESCC cells increased invasion in vitro, while reducing cancer cells proliferation. In parallel, an ESCC mass spectrometry-based proteomics dataset, obtained from the PRIDE database, was comprehensively interrogated for abnormally glycosylated proteins. Data integration with the Target Score, an algorithm developed in-house, pinpointed the glucose transporter type 1 (GLUT1) as a biomarker of poor prognosis. GLUT1-STn glycoproteoforms were latter identified in tumor tissues in patients facing worst prognosis. Furthermore, healthy human tissues analysis suggested that STn glycosylation provided cancer specificity to GLUT1. In conclusion, STn is a biomarker of worst prognosis in EC and GLUT1-STn glycoforms may be used to increase its specificity on the stratification and targeting of aggressive ESCC forms.

Elucidating the mechanisms of action of parecoxib in the MG-63 osteosarcoma cell line.

Different types of tumors often present an overexpression of cyclooxygenase-2. The aim of this study was to evaluate the effects of parecoxib (NSAID, cyclooxygenase-2 selective inhibitor) in the behavior of the human osteosarcoma MG-63 cell line, concerning several biological features. Cells were exposed to several concentrations of parecoxib for 48 hours. Cell viability/proliferation, cyclooxygenase-2 expression, morphologic alterations, membrane integrity, cell cycle evaluation, cell death and genotoxicity were evaluated. When compared with untreated cells, parecoxib led to a marked decrease in cell viability/proliferation, in COX-2 expression and changes in cell morphology, in a concentration-dependent manner. Cell recuperation was observed after incubation with drug-free medium. Parecoxib exposure increased lactate dehydrogenase release, an arrest of the cell cycle at S-phase and G2/M-phase, as well as growth of the sub-G0/G1-fraction and increased DNA damage. Parecoxib led to a slight increase of necrosis regulated cell death in treated cells, and an increase of autophagic vacuoles, in a concentration-dependent manner. In this study, parecoxib showed antitumor effects in the MG-63 human osteosarcoma cells. The potential mechanism was inhibiting cell proliferation and promoting necrosis. These results further suggested that parecoxib might be a potential candidate for in-vivo studies.

Polyethylene Glycol 35 as a Perfusate Additive for Mitochondrial and Glycocalyx Protection in HOPE Liver Preservation.

Organ transplantation is a multifactorial process in which proper graft preservation is a mandatory step for the success of the transplantation. Hypothermic preservation of abdominal organs is mostly based on the use of several commercial solutions, including UW, Celsior, HTK and IGL-1. The presence of the oncotic agents HES (in UW) and PEG35 (in IGL-1) characterize both solution compositions, while HTK and Celsior do not contain any type of oncotic agent. Polyethylene glycols (PEGs) are non-immunogenic, non-toxic and water-soluble polymers, which present a combination of properties of particular interest in the clinical context of ischemia-reperfusion injury (IRI): they limit edema and nitric oxide induction and modulate immunogenicity. Besides static cold storage (SCS), there are other strategies to preserve the organ, such as the use of machine perfusion (MP) in dynamic preservation strategies, which increase graft function and survival as compared to the conventional static hypothermic preservation. Here we report some considerations about using PEG35 as a component of perfusates for MP strategies (such as hypothermic oxygenated perfusion, HOPE) and its benefits for liver graft preservation. Improved liver preservation is closely related to mitochondria integrity, making this organelle a good target to increase graft viability, especially in marginal organs (e.g., steatotic livers). The final goal is to increase the pool of suitable organs, and thereby shorten patient waiting lists, a crucial problem in liver transplantation.

The Soluble Adenylyl Cyclase Inhibitor LRE1 Prevents Hepatic Ischemia/Reperfusion Damage Through Improvement of Mitochondrial Function.

Hepatic ischemia/reperfusion (I/R) injury is a leading cause of organ dysfunction and failure in numerous pathological and surgical settings. At the core of this issue lies mitochondrial dysfunction. Hence, strategies that prime mitochondria towards damage resilience might prove applicable in a clinical setting. A promising approach has been to induce a mitohormetic response, removing less capable organelles, and replacing them with more competent ones, in preparation for an insult. Recently, a soluble form of adenylyl cyclase (sAC) has been shown to exist within mitochondria, the activation of which improved mitochondrial function. Here, we sought to understand if inhibiting mitochondrial sAC would elicit mitohormesis and protect the liver from I/R injury. Wistar male rats were pretreated with LRE1, a specific sAC inhibitor, prior to the induction of hepatic I/R injury, after which mitochondria were collected and their metabolic function was assessed. We find LRE1 to be an effective inducer of a mitohormetic response based on all parameters tested, a phenomenon that appears to require the activity of the NAD+-dependent sirtuin deacylase (SirT3) and the subsequent deacetylation of mitochondrial proteins. We conclude that LRE1 pretreatment leads to a mitohormetic response that protects mitochondrial function during I/R injury.

Plasma Extracellular Vesicle-Derived TIMP-1 mRNA as a Prognostic Biomarker in Clear Cell Renal Cell Carcinoma: A Pilot Study.

The tumor microenvironment has gained a lot of attention from the scientific community since it has a proven impact in the development of tumor progression and metastasis. Extracellular vesicles (EVs) are now considered one of the key players of tumor microenvironment modulation. Clear cell renal cell carcinoma (ccRCC) is the most lethal urological neoplasia and presents a high metastatic potential, which reinforces the need for the development of more effective predictive biomarkers. Our goal was to evaluate the applicability of EV-derived matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) as prognostic biomarkers for ccRCC. To do so, we studied the plasma EV content of 32 patients with localized ccRCC and 29 patients with metastatic ccRCC. We observed that patients with localized disease and tumors larger than 7 cm presented higher levels of plasma EV-derived TIMP-1 mRNA when compared with patients presenting smaller tumors (p = 0.020). Moreover, patients with metastatic disease presented higher levels of EV-derived TIMP-1 mRNA when compared with patients with localized disease (p = 0.002) and when we stratified those patients in high and low levels of TIMP-1 EV-derived mRNA, the ones presenting higher levels had a lower overall survival (p = 0.030). EV-derived TIMP-1 mRNA may be a good prognostic biomarker candidate for ccRCC.

Exploration of the cellular effects of the high-dose, long-term exposure to coffee roasting product furan and its by-product cis-2-butene-1,4-dial on human and rat hepatocytes.

Coffee is the most popular hot beverage and caffeine is the most used psychoactive drug in the world. Roasting of coffee beans leads to the generation of minute quantities of undesirable compounds, such as furan. It is now thought that the toxicity of furan derives from its processing by CYP450 family of detoxifying enzymes, leading to the formation of cis-2-butene-1,4-dial (BDA). BDA has known cytotoxicity capacities, binding to proteins, nucleic acids, and glutathione (GSH). BDA also appears to mediate furan's toxic effects, since the inhibition of CYP450 family impedes the aforementioned toxicological effects of furan. There are some studies performed on furan's toxicity, but very few on BDA. Furthermore, the doses used in these studies appear to be fairly high when compared with the expected dosage one could be exposed to in a standard day. As such, to understand if furan and BDA could have toxic effects using more realistic doses and longer time frames, human and rat hepatocytes were exposed to furan or BDA for up to 96 h, and several biochemical parameters were assessed. We report here that human hepatocytes were more sensitive than rat's, in particular to furan, for we show a decrease in MTT reduction, ATP levels and increase in carbonyl formation and 8-OHdG accumulation in the longer time points. BDA was mostly ineffective, which we attribute to a low import rate into the cells. In conclusion, we show that there is potential for harm from furan in high doses, which should be carefully addressed.

Mild hypothermia during the reperfusion phase protects mitochondrial bioenergetics against ischemia-reperfusion injury in an animal model of ex-vivo liver transplantation-an experimental study.

The organ preservation paradigm has changed following the development of new ways to preserve organs. The use of machine perfusion to preserve organs appears to have several advantages compared with conventional static cold storage. For liver transplants, the temperature control provided by machine perfusion improves organ preservation. In this experimental study, we measured the effects of different temperatures on mitochondrial bioenergetics during the reperfusion phase. An experimental model of ex-vivo liver transplantation was developed in Wistar rats (Rattus norvegicus). After total hepatectomy, cold static preservation occurred at 4ºC and reperfusion was performed at 37ºC and 32ºC using a Langendorff system. We measured parameters associated with mitochondrial bioenergetics in the livers. Compared with the livers that underwent normothermic reperfusion, mild hypothermia during reperfusion caused significant increases in the mitochondrial membrane potential, the adenosine triphosphate content, and mitochondrial respiration, and a significant reduction in the lag phase (all P < 0.001). Mild hypothermia during reperfusion reduced the effect of ischemia-reperfusion injury on mitochondrial activity in liver tissue and promoted an increase in bioenergetic availability compared with normothermic reperfusion.

Indirubin and NAD+ prevent mitochondrial ischaemia/reperfusion damage in fatty livers.

BACKGROUND: Fatty livers are considerably more susceptible to acute stressors, such as ischaemia/reperfusion (I/R). As the incidence of I/R is high due to surgical events and some pathologies, there is an urgent need to find strategies against I/R injury (I/RI) in fatty livers. We postulate that an acute pretreatment with indirubin-3'-oxime (Ind) or NAD+ prevents mitochondrial dysfunction associated with warm I/RI in fatty livers. MATERIALS AND METHODS: Zucker fatty rats were subjected to warm ischaemia and 12 hours of reperfusion. Ind or NAD+ was administered in the hepatic artery 30 minutes before ischaemia. Hepatic mitochondrial isolation was performed, and functional assays as well as molecular analysis were performed. RESULTS: Pretreatment decreased markers of liver injury while preserving mitochondrial cytochrome c content, which is related to the prevention of calcium-induced mitochondrial permeability transition (mPT), the decline in mitochondrial respiratory state 3 and ATP content. The generation of reactive oxygen species (ROS) was also diminished. Inhibition of GSK-3ß by Ind resulted in the prevention of cyclophilin-D (CypD) phosphorylation, unabling it to bind to the adenine nucleotide translocator (ANT), thus, preventing mPT induction. Furthermore, deacetylation of CypD at Lys residue by sirtuin 3 (SIRT3) caused its dissociation from ANT, contributing to an increase in mPT threshold in NAD+ -pretreated animals. CONCLUSIONS: Pretreatment with Ind or NAD+ protects fatty livers by maintaining mitochondrial calcium homoeostasis, thus, preserving mitochondrial function and energetic balance. As such, CypD might be a new protective target against I/RI in fatty livers.

Recent insights into mitochondrial targeting strategies in liver transplantation.

Ischemia/reperfusion (I/R) injury in liver transplantation can disrupt the normal activity of mitochondria in the hepatic parenchyma. This potential dysfunction of mitochondria after I/R injury could be responsible for the initial poor graft function or primary nonfunction observed after liver transplantation. Thus, determining the mechanisms that lead to human hepatic mitochondrial dysfunction might contribute to improving the outcome of liver transplantation. Furthermore, early identification of novel prognostic factors involved in I/R injury could serve as a key endpoint to predict the outcome of liver grafts and also to promote the early adoption of novel strategies that protect against I/R injury. Here, we briefly review recent advances in the study of mitochondrial dysfunction and I/R injury, particularly in relation to liver transplantation. Next, we highlight various pharmacological therapeutic strategies that could be applied, and discuss their relationship to relevant mitochondrion-related processes and targets. Lastly, we note that although considerable progress has been made in our understanding of I/R injury and mitochondrial dysfunction, further investigation is required to elucidate the cellular and molecular mechanisms underlying these processes, thereby identifying biomarkers that can help in evaluating donor organs.

Addition of Berberine to Preservation Solution in an Animal Model of Ex Vivo Liver Transplant Preserves Mitochondrial Function and Bioenergetics from the Damage Induced by Ischemia/Reperfusion.

Liver transplantation is a therapeutic regimen to treat patients with non-malignant end-stage liver diseases and malignant tumors of hepatic origin. The ischemia/reperfusion (I/R) injury in liver transplantation is associated with disruption of mitochondrial function in the hepatic parenchyma. Several studies have been conducted in animal models to identify pharmacological therapeutic strategies to minimize the injury induced by the cold/warm I/R in liver transplantation. Most of these studies were conducted in unrealistic conditions without the potential to be translated to clinical usage. Berberine (BBR) is a pharmacological compound with a potential protective effect of the mitochondrial function in the context of I/R. For the future clinical application of these pharmacological strategies, it is essential that a close resemblance exists between the methodology used in the animals models and real life. In this study, we have demonstrated that the addition of BBR to the preservation solution in an I/R setting preserves mitochondrial function and bioenergetics, protecting the liver from the deleterious effects caused by I/R. As such, BBR has the potential to be used as a pharmacological therapeutic strategy.