Melatonin and its protective role in attenuating warm or cold hepatic ischaemia/reperfusion injury

Although the liver is the only organ with regenerative capacity, various injury factors induce irreversible liver dysfunction and end‐stage liver disease. Liver resection and liver transplantation (LT) are effective treatments for individuals with liver failure, liver cirrhosis and liver cancers. The remnant or transplanted liver tissues will undergo hepatic ischaemia/reperfusion (IR), which leads to oxidative stress, inflammation, immune injury and liver damage. Moreover, systemic ischaemia induced by trauma, stroke, myocardial ischaemia, haemorrhagic shock and other injury factors also induces liver ischaemia/reperfusion injury (IRI) in individuals. Hepatic IRI can be divided into warm IRI, which is induced by liver surgery and systemic ischaemia, and cold IRI, which is induced by LT. Multiple studies have shown that melatonin (MT) acts as an endogenous free radical scavenger with antioxidant capacity and is also able to attenuate hepatic IRI via its anti‐inflammatory and antiapoptotic capacities. In this review, we discuss the potential mechanisms and current strategies of MT administration in liver surgery for protecting against warm or cold hepatic IRI. We highlight strategies to improve the efficacy and safety of MT for attenuating hepatic IRI in different conditions. After the potential mechanisms underlying the interactions between MT and other important cellular processes during hepatic IR are clarified, more opportunities will be available to use MT to treat liver diseases in the future.     

Dual role of chloroquine in liver ischemia reperfusion injury: reduction of liver damage in early phase,but aggravation in late phase

The anti-malaria drug chloroquine is well known as autophagy inhibitor. Chloroquine has also been used as anti-inflammatory drugs to treat inflammatory diseases. We hypothesized that chloroquine could have a dual effect in liver ischemia/reperfusion (I/R) injury: chloroquine on the one hand could protect the liver against I/R injury via inhibition of inflammatory response, but on the other hand could aggravate liver I/R injury through inhibition of autophagy. Rats (n=6 per group) were pre-treated with chloroquine (60 mg/kg, i.p.) 1 h before warm ischemia, and they were continuously subjected to a daily chloroquine injection for up to 2 days. Rats were killed 0.5, 6, 24 and 48 h after reperfusion. At the early phase (i.e., 0–6 h after reperfusion), chloroquine treatment ameliorated liver I/R injury, as indicated by lower serum aminotransferase levels, lower hepatic inflammatory cytokines and fewer histopathologic changes. In contrast, chloroquine worsened liver injury at the late phase of reperfusion (i.e., 24–48 h after reperfusion). The mechanism of protective action of chloroquine appeared to involve its ability to modulate mitogen-activated protein kinase activation, reduce high-mobility group box 1 release and inflammatory cytokines production, whereas chloroquine worsened liver injury via inhibition of autophagy and induction of hepatic apoptosis at the late phase. In conclusion, chloroquine prevents ischemic liver damage at the early phase, but aggravates liver damage at the late phase in liver I/R injury. This dual role of chloroquine should be considered when using chloroquine as an inhibitor of inflammation or autophagy in I/R injury.     

Early release of macrophage migration inhibitory factor after liver ischemia and reperfusion injury in rats

Macrophage migration inhibitory factor (MIF) is an important mediator of ischemia/reperfusion (I/R) injury in heart, brain and intestine. We previously demonstrated that MIF was released during warm/cold ischemia in vitro. However, the role of MIF in liver I/R injury remains unclear. We aimed to test the hypothesis that MIF acts as an early proinflammatory cytokine and could mediate the inflammatory injury in liver I/R. Rats (n = 6 per group) were subjected to 90 min warm ischemia followed by 0.5 h, 6 h and 24 h reperfusion, respectively to liver transplantation (LTx) after 6 h of cold ischemia followed by 24 h of reperfusion. The expression of MIF, its receptor (cluster of differentiation 74 (CD74)) and the downstream inflammatory cytokines (tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)) were analyzed. Peritoneal macrophages were cultured for 6 h alone or in the presence of effluent from cold-preserved livers or effluent depleted of MIF. Warm I/R increased hepatic MIF-mRNA and protein expression. MIF-protein was released into peripheral circulation in vivo with a maximum at 0.5 h after reperfusion. Induction of MIF-expression was associated with the expression of proinflammatory cytokines and its receptor in both models. MIF released by isolated cold preserved livers, induced TNF-α and IL-1β production by cultured peritoneal macrophages. Intrahepatic upregulation of MIF, release into systemic circulation and the associated upregulation of the proinflammatory mediators suggest a role of MIF in mediating the inflammatory response to I/R injury. Blocking experiments will help to elucidate its role as potential molecular target for preventing hepatic I/R injury.     

Inhibition of mitochondrial autophagy protects donor lungs for lung transplantation against ischaemia‐reperfusion injury in rats via the mTOR pathway

Impaired mitochondrial function is a key factor attributing to lung ischaemia‐reperfusion (IR) injury, which contributes to major post‐transplant complications. Thus, the current study was performed to investigate the role of mitochondrial autophagy in lung I/R injury and the involvement of the mTOR pathway. We established rat models of orthotopic left lung transplantation to investigate the role of mitochondrial autophagy in I/R injury following lung transplantation. Next, we treated the donor lungs with 3‐MA and Rapamycin to evaluate mitochondrial autophagy, lung function and cell apoptosis with different time intervals of cold ischaemia preservation and reperfusion. In addition, mitochondrial autophagy, and cell proliferation and apoptosis of pulmonary microvascular endothelial cells (PMVECs) exposed to hypoxia‐reoxygenation (H/R) were monitored after 3‐MA administration or Rapamycin treatment. The cell apoptosis could be inhibited by mitochondrial autophagy at the beginning of lung ischaemia, but was rendered out of control when mitochondrial autophagy reached normal levels. After I/R of donor lung, the mitochondrial autophagy was increased until 6 hours after reperfusion and then gradually decreased. The elevation of mitochondrial autophagy was accompanied by promoted apoptosis, aggravated lung injury and deteriorated lung function. Moreover, the suppression of mitochondrial autophagy by 3‐MA inhibited cell apoptosis of donor lung to alleviate I/R‐induced lung injury as well as inhibited H/R‐induced PMVEC apoptosis, and enhanced its proliferation. Finally, mTOR pathway participated in I/R‐ and H/R‐mediated mitochondrial autophagy in regulation of cell apoptosis. Inhibition of I/R‐induced mitochondrial autophagy alleviated lung injury via the mTOR pathway, suggesting a potential therapeutic strategy for lung I/R injury.     

细胞自噬在大鼠肺缺血/再灌注引发肝脏损伤中的作用*

目的: 探讨肺缺血/再灌注(LI/R)时肝脏损伤的影响,并初步探索细胞自噬(Autophagy)在其中发挥的作用。方法: 构建大鼠缺血/再灌注肺损伤(LI/RI)模型,模型制备方法为大鼠麻醉后切开气管进行机械通气,使用动脉夹将肺门夹闭模拟缺血过程,30 min后松开动脉夹,恢复灌注3 h。24只大鼠随机分为伪手术组(Sham组)、缺血/再灌注组(I/R组)、溶剂组(DMSO组)和自噬抑制剂组(3-MA组),每组均6只,后2组大鼠术前分别腹腔注射DMSO和3-MA,造模结束后使用肺湿/干重比判断造模是否成功;抽取静脉血测定肝脏转氨酶指标ALT与AST;取肝脏组织,光镜下观察肝脏形态改变,以及电镜下观察肝细胞超微结构;使用RT-qPCR和Western blot实验分别检测肝脏组织细胞中自噬相关蛋白的基因mRNA表达水平和蛋白表达水平。结果: 与Sham组相比,其余各组肺湿/干重比均升高;血AST和ALT均有大幅升高且肝脏组织损伤明显,其中以I/R组升高最为明显,光镜下组织形态学及电镜下细胞微细结构均有不同程度的破坏;肝脏中自噬相关蛋白的基因表达水平与蛋白表达水平均有明显不同,表现为自噬上升 (P＜0.01或P＜0.05)。I/R组和DMSO组肝脏组织均有较重损伤,肝细胞结构破坏严重,自噬小体形成,而AST、ALT、自噬相关蛋白转录和表达水平等各项指标均无统计学差异(P＞0.05)。而相较于DMSO组,3-MA组肝脏组织损伤有所减轻,肝细胞微细结构损伤程度低,且无自噬小体形成,血中AST和ALT下降,肝脏组织内自噬水平均下降 (P＜0.05)。结论: 肺缺血/再灌注可引起大鼠肝损伤;细胞自噬可介导大鼠肺缺血/再灌注引起的肝损伤,抑制细胞自噬可以有效减轻大鼠LI/R引起的肝损伤。     

Loss of sirtuin 1 and mitofusin 2 contributes to enhanced ischemia/reperfusion injury in aged livers

Ischemia/reperfusion (I/R) injury is a causative factor contributing to morbidity and mortality during liver resection and transplantation. Livers from elderly patients have a poorer recovery from these surgeries, indicating reduced reparative capacity with aging. Mechanisms underlying this age‐mediated hypersensitivity to I/R injury remain poorly understood. Here, we investigated how sirtuin 1 (SIRT1) and mitofusin 2 (MFN2) are affected by I/R in aged livers. Young (3 months) and old (23–26 months) male C57/BL6 mice were subjected to hepatic I/R in vivo. Primary hepatocytes isolated from each age group were also exposed to simulated in vitro I/R. Biochemical, genetic, and imaging analyses were performed to assess cell death, autophagy flux, mitophagy, and mitochondrial function. Compared to young mice, old livers showed accelerated liver injury following mild I/R. Reperfusion of old hepatocytes also showed necrosis, accompanied with defective autophagy, onset of the mitochondrial permeability transition, and mitochondrial dysfunction. Biochemical analysis indicated a near‐complete loss of both SIRT1 and MFN2 after I/R in old hepatocytes, which did not occur in young cells. Overexpression of either SIRT1 or MFN2 alone in old hepatocytes failed to mitigate I/R injury, while co‐overexpression of both proteins promoted autophagy and prevented mitochondrial dysfunction and cell death after reperfusion. Genetic approaches with deletion and point mutants revealed that SIRT1 deacetylated K655 and K662 residues in the C‐terminus of MFN2, leading to autophagy activation. The SIRT1‐MFN2 axis is pivotal during I/R recovery and may be a novel therapeutic target to reduce I/R injury in aged livers.     

The protective mechanisms of defibrotide on liver ischaemia-reperfusion injury

During some surgical interventions, temporary occlusion of the hepatic blood supply may cause ischaemia-reperfusion (I/R) injury and hepatic dysfunction. In this study the protective effect of defibrotide (DEF) was evaluated in a rat model of liver I/R injury. Four groups of rats were subjected to the following protocols: saline infusion without ischaemia, DEF infusion without ischaemia, DEF infusion with hepatic I/R, and saline infusion with hepatic I/R. After a midline laporatomy, liver ischaemia was induced by 45 min of portal occlusion. DEF 175 mg/kg(-1) was infused before ischaemia in 10 ml of saline. The same volume of saline was infused into the control animals. At the end of the 45-min reperfusion interval, the animals were sacrified. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzyme activities were determined in haemolysates, and malondialdehyde (MDA) level in the liver tissue was measured. Tissue MDA levels were significantly higher in the I/R plus saline group compared to the sham operation control groups (p < 0.01 and p < 0.05, respectively). Tissue MDA levels decreased in the DEF plus I/R group compared to the I/R plus saline group (p < 0.05), but DEF could not reduce tissue lipid peroxidation to the levels of the control sham operation groups. SOD and GSH-Px enzyme activities were significantly higher in DEF-treated animals than in the other groups (p < 0.05). These results suggest that DEF protects liver against I/R injury by increasing the antioxidant enzyme levels.     

Pre‐conditions for eliminating mitochondrial dysfunction and maintaining liver function after hepatic ischaemia reperfusion

The liver, the largest organ with multiple synthesis and secretion functions in mammals, consists of hepatocytes and Kupffer, stem, endothelial, stellate and other parenchymal cells. Because of early and extensive contact with the external environment, hepatic ischaemia reperfusion (IR) may result in mitochondrial dysfunction, autophagy and apoptosis of cells and tissues under various pathological conditions. Because the liver requires a high oxygen supply to maintain normal detoxification and synthesis functions, it is extremely susceptible to ischaemia and subsequent reperfusion with blood. Consequently, hepatic IR leads to acute or chronic liver failure and significantly increases the total rate of morbidity and mortality through multiple regulatory mechanisms. An increasing number of studies indicate that mitochondrial structure and function are impaired after hepatic IR, but that the health of liver tissues or liver grafts can be effectively rescued by attenuation of mitochondrial dysfunction. In this review, we mainly focus on the subsequent therapeutic interventions related to the conservation of mitochondrial function involved in mitigating hepatic IR injury and the potential mechanisms of protection. Because mitochondria are abundant in liver tissue, clarification of the regulatory mechanisms between mitochondrial dysfunction and hepatic IR should shed light on clinical therapies for alleviating hepatic IR‐induced injury.     

Involvement of kindlin‐2 in irisin’s protection against ischaemia reperfusion‐induced liver injury in high‐fat diet‐fed mice

Liver steatosis is associated with increased ischaemia reperfusion (I/R) injury. Our previous studies have shown that irisin, an exercise‐induced hormone, mitigates I/R injury via binding to αVβ5 integrin. However, the effect of irisin on I/R injury in steatotic liver remains unknown. Kindlin‐2 directly interacts with β integrin. We therefore suggest that irisin protects against I/R injury in steatotic liver via a kindlin‐2 dependent mechanism. To study this, hepatic steatosis was induced in male adult mice by feeding them with a 60% high‐fat diet (HFD). At 12 weeks after HFD feeding, the mice were subjected to liver ischaemia by occluding partial (70%) hepatic arterial/portal venous blood for 60 minutes, which was followed by 24 hours reperfusion. Our results showed HFD exaggerated I/R‐induced liver injury. Irisin (250 μg/kg) administration at the beginning of reperfusion attenuated liver injury, improved mitochondrial function, and reduced oxidative and endoplasmic reticulum stress in HFD‐fed mice. However, kindlin‐2 inhibition by RNAi eliminated irisin''s direct effects on cultured hepatocytes. In conclusion, irisin attenuates I/R injury in steatotic liver via a kindlin‐2 dependent mechanism.     

Identification and preliminary validation of novel biomarkers of acute hepatic ischaemia/reperfusion injury using dual-platform proteomic/degradomic approaches

Hepatic ischaemia/reperfusion (I/R), a major cause of liver damage associated with multiple trauma, haemorrhagic and septic shock, and liver transplantation, contributes significantly to multiple organ failure. Development of novel sensitive biomarkers that detect early stages of liver damage is vital for effective management and treatment of ischaemic liver injury. By using high-throughput immunoblotting and cation-anion exchange chromatography/reversed-phase liquid chromatography-tandem mass-spectrometry, we identified several hepatic proteins, including argininosuccinate synthase (ASS) and estrogen sulfotransferase (EST-1), which were degraded in the liver and rapidly released into circulation during I/R injury. ASS accumulated in serum within 10 min, reached a steady state at 30 min, and persisted up until 3 h after reperfusion following 30 min of total hepatic ischaemia. EST-1 appeared rapidly in blood and attained maximum within 1 hour followed by a decline at 3 h of reperfusion. No ASS or EST-1 protein was detected in serum of control or sham operated rats. ASS and EST-1 exhibited greater sensitivity and specificity toward I/R liver injury as compared with alanine aminotransferase (ALT), an established marker of hepatocellular necrosis. In contrast, serum ASS and EST-1 were undetectable in rats with chronic alcoholic liver disease, while the levels of ALT protein were significantly increased. In addition, ASS, but not EST-1 or ALT accumulated in blood only 6 h after treatment with hepatotoxic combination of lipopolysaccharide and D-galactosamine. These data demonstrate the utility of ASS and EST-1 as novel sensitive and specific biomarkers of acute liver ischaemic injury for prospective clinical studies.     

Identification and preliminary validation of novel biomarkers of acute hepatic ischaemia/reperfusion injury using dual-platform proteomic/degradomic approaches

Hepatic ischaemia/reperfusion (I/R), a major cause of liver damage associated with multiple trauma, haemorrhagic and septic shock, and liver transplantation, contributes significantly to multiple organ failure. Development of novel sensitive biomarkers that detect early stages of liver damage is vital for effective management and treatment of ischaemic liver injury. By using high-throughput immunoblotting and cation–anion exchange chromatography/reversed-phase liquid chromatography-tandem mass-spectrometry, we identified several hepatic proteins, including argininosuccinate synthase (ASS) and estrogen sulfotransferase (EST-1), which were degraded in the liver and rapidly released into circulation during I/R injury. ASS accumulated in serum within 10 min, reached a steady state at 30 min, and persisted up until 3 h after reperfusion following 30 min of total hepatic ischaemia. EST-1 appeared rapidly in blood and attained maximum within 1 hour followed by a decline at 3 h of reperfusion. No ASS or EST-1 protein was detected in serum of control or sham operated rats. ASS and EST-1 exhibited greater sensitivity and specificity toward I/R liver injury as compared with alanine aminotransferase (ALT), an established marker of hepatocellular necrosis. In contrast, serum ASS and EST-1 were undetectable in rats with chronic alcoholic liver disease, while the levels of ALT protein were significantly increased. In addition, ASS, but not EST-1 or ALT accumulated in blood only 6 h after treatment with hepatotoxic combination of lipopolysaccharide and D-galactosamine. These data demonstrate the utility of ASS and EST-1 as novel sensitive and specific biomarkers of acute liver ischaemic injury for prospective clinical studies.     

Ischaemic preconditioning modulates the activity of Kupffer cells during in vivo reperfusion injury of rat liver

This work was performed to elucidate further the main cellular events underlying the protective effect of ischaemic preconditioning in an in vivo rat liver model of 90 min ischaemia followed by 30 min reperfusion. A significant attenuation of the various aspects of post-ischaemic injury, namely necrosis and the levels of hydrogen peroxide and 5- and 15-hydroperoxyeicosatetraenoic acids, was afforded by the prior application of a short cycle of ischaemia/reperfusion (10 + 10 min) or when rats were previously treated with gadolinium chloride. However, when preconditioning was applied on Kupffer cell-depleted livers, no additional level of ischaemic tolerance was obtained. In terms of cellular pathology, this result could be suggestive of Kupffer cells as the target of the preconditioning phenomenon during the warm ischaemia/reperfusion injury. Accordingly, modulation of Kupffer cell activity was associated with a well-preserved hepatocyte integrity, together with low levels of pro-oxidant generation during reperfusion. As activated Kupffer cells can generate and release potentially toxic substances, their modulation by ischaemic preconditioning could help to provide new surgical and/or pharmacological strategies to protect the liver against reperfusion damage.     

MicroRNA-449b-5p targets HMGB1 to attenuate hepatocyte injury in liver ischemia and reperfusion

MicroRNAs (miRNAs) participate in the pathological process of liver ischemia/reperfusion (I/R) injury. MiR-449b-5p is the target miRNA of high mobility group box 1 (HMGB1). Its role and molecular mechanism in liver I/R injury remain unidentified. In this study, we found a protective effect of miR-449b-5p against hepatic I/R injury. HMGB1 expression significantly increased, whereas miR-449b-5p dramatically decreased in patients after liver transplant and in L02 cells exposed to hypoxia/reoxygenation (H/R). A dual-luciferase reporter assay confirmed the direct interaction between miR-449b-5p and the 3′ untranslated region of HMGB1 messenger RNA. We also found that overexpression of miR-449b-5p significantly promoted cell viability and inhibited cell apoptosis of L02 cells exposed to H/R. Moreover, miR-449b-5p repressed HMGB1 protein expression and nuclear factor-κB (NF-κB) pathway activation in these L02 cells. In an in vivo rat model of hepatic I/R injury, overexpression of miR-449b-5p significantly decreased alanine aminotransferase and aspartate aminotransferase and inhibited the HMGB1/NF-κB pathway. Our study thus suggests that miR-449b-5p alleviated hepatic I/R injury by targeting HMGB1 and deactivating the NF-κB pathway, which may provide a novel and promising therapeutic target for hepatic I/R injury.     

The damage-associated molecular pattern HMGB1 is released early after clinical hepatic ischemia/reperfusion

Objective and backgroundActivation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice.MethodsPlasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury.ResultsIn patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects.ConclusionHMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage.     

Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling

The traditional Chinese medicine Danshensu (DSS) has a protective effect on cardiac ischaemia/reperfusion (I/R) injury. However, the molecular mechanisms underlying the DSS action remain undefined. We investigated the potential role of DSS in autophagy and apoptosis using cardiac I/R injury models of cardiomyocytes and isolated rat hearts. Cultured neonatal rat cardiomyocytes were subjected to 6 hrs of hypoxia followed by 18 hrs of reoxygenation to induce cell damage. The isolated rat hearts were used to perform global ischaemia for 30 min., followed by 60 min. reperfusion. Ischaemia/reperfusion injury decreased the haemodynamic parameters on cardiac function, damaged cardiomyocytes or even caused cell death. Pre‐treatment of DSS significantly improved cell survival and protected against I/R‐induced deterioration of cardiac function. The improved cell survival upon DSS treatment was associated with activation of mammalian target of rapamycin (mTOR) (as manifested by increased phosphorylation of S6K and S6), which was accompanied with attenuated autophagy flux and decreased expression of autophagy‐ and apoptosis‐related proteins (including p62, LC3‐II, Beclin‐1, Bax, and Caspase‐3) at both protein and mRNA levels. These results suggest that alleviation of cardiac I/R injury by pre‐treatment with DSS may be attributable to inhibiting excessive autophagy and apoptosis through mTOR activation.     

New therapeutic strategy of hinokitiol in haemorrhagic shock‐induced liver injury

Haemorrhagic shock and resuscitation (HS/R) may cause global ischaemia‐reperfusion injury, which can result in systemic inflammation, multiorgan failure (particularly liver failure) and high mortality. Hinokitiol, a bioactive tropolone‐related compound, exhibits antiplatelet and anti‐inflammatory activities. Targeting inflammatory responses is a potential strategy for ameliorating hepatic injury during HS/R. Whether hinokitiol prevents hepatic injury during HS/R remains unclear. In the present study, we determined the role of hinokitiol following HS/R. The in vivo assays revealed that hinokitiol markedly attenuated HS/R‐induced hepatic injury. Hinokitiol could inhibited NF‐κB activation and IL‐6 and TNF‐α upregulation in liver tissues. Moreover, hinokitiol reduced caspase‐3 activation, upregulated Bax and downregulated Bcl‐2. These findings suggest that hinokitiol can ameliorate liver injury following HS/R, partly through suppression of inflammation and apoptosis. Furthermore, the in vitro data revealed that hinokitiol significantly reversed hypoxia/reoxygenation (H/R)‐induced cell death and apoptosis in the primary hepatocytes. Hinokitiol prevented H/R‐induced caspase‐3 activation, PPAR cleavage, Bax overexpression and Bcl‐2 downregulation. Moreover, hinokitiol attenuated H/R‐stimulated NF‐κB activation and reduced the levels of IL‐6 and TNF‐α mRNAs, suggesting that hinokitiol can protect hepatocytes from H/R injury. Collectively, our data suggest that hinokitiol attenuates liver injury following HS/R, partly through the inhibition of NF‐κB activation.     

ARRB1 ameliorates liver ischaemia/reperfusion injury via antagonizing TRAF6‐mediated Lysine 6‐linked polyubiquitination of ASK1 in hepatocytes

Hepatic ischaemia/reperfusion (I/R) injury is a major clinical problem during liver surgical procedures, which usually lead to early transplantation failure and higher organ rejection rate, and current effective therapeutic strategies are still limited. Therefore, in‐depth exploring of the molecular mechanisms underlying liver I/R injury is key to the development of new therapeutic methods. β‐arrestins are multifunctional proteins serving as important signalling scaffolds in numerous physiopathological processes, including liver‐specific diseases. However, the role and underlying mechanism of β‐arrestins in hepatic I/R injury remain largely unknown. Here, we showed that only ARRB1, but not ARRB2, was down‐regulated during liver I/R injury. Hepatocyte‐specific overexpression of ARRB1 significantly ameliorated liver damage, as demonstrated by decreases in serum aminotransferases, hepatocellular necrosis and apoptosis, infiltrating inflammatory cells and secretion of pro‐inflammatory cytokines relative to control mice, whereas experiments with ARRB1 knockout mice gotten opposite effects. Mechanistically, ARRB1 directly interacts with ASK1 in hepatocytes and inhibits its TRAF6‐mediated Lysine 6‐linked polyubiquitination, which then prevents the activation of ASK1 and its downstream signalling pathway during hepatic I/R injury. In addition, inhibition of ASK1 remarkably abolished the disruptive effect result from ARRB1 deficiency in liver I/R injury in vivo, indicating that ASK1 was required for ARRB1 function in hepatic I/R injury. In conclusion, we proposed that ARRB1 is a novel protective regulator during liver I/R injury, and modulation of the regulatory axis between ARRB1 and ASK1 could be a novel therapeutic strategy to prevent this pathological process.     

HMGB1 in ischemic and non-ischemic liver after selective warm ischemia/reperfusion in rat

High mobility group box 1 (HMGB1) acts as an early mediator in inflammation and organ injury. Ischemia reperfusion (I/R) injury induces HMGB1 translocation and expression in ischemic areas. However, it is unknown whether selective warm liver I/R injury also induces the expression of HMGB1 in non-ischemic lobes. The present study aimed to test the hypothesis that selective liver I/R injury also causes HMGB1 translocation and up-regulates its expression in non-ischemic liver areas. In the present study, selective I/R injury was induced by clamping the median and left lateral liver lobes for 90 min followed by 0.5, 6 and 24 h reperfusion. We used male inbred Lewis rats; six animals for each point in time and six animals for the normal control group. Selective hepatic I/R injury induced morphological changes not only in ischemic lobes but also in non-ischemic lobes. HMGB1 translocation and expression was increased in a time-dependent manner in the ischemic lobes, and increased in with delayed onset in the non-ischemic lobes. Serum HMGB1 levels were increased after reperfusion. Furthermore, liver I/R injury up-regulated the expression of HMGB1 receptors (Toll-like receptor 4 and receptor for advanced glycation end products and pro-inflammatory cytokines (Tumor necrosis factor-alpha and interleukin-6) in both ischemic lobes, however, the up-regulation of these cytokines was more prominent in the ischemic lobes. In conclusion, selective warm I/R induces a substantial “sympathetic/bystander” effect on the non-ischemic lobes in terms of HMGB1 translocation and local cytokine production.     

Protective effect of ulinastatin on hepatic ischemia reperfusion injury through autophagy activation in Chang liver cells

This study aimed to investigate the protective effect of ulinastatin in hepatic ischemia-reperfusion progress, involving its association with the role of autophagy during hypoxia-induced hypoxia-reoxygenation injury in vitro. The model of hepatic hypoxia/reoxygenation (H/R) injury in Chang liver cells was established. After treatment with ulinastatin at the doses of 10, 100, and 1000 U/mL in H/R liver cells, the cell proliferation was significantly increased, morphological damage was reduced, and the cell apoptosis rate was decreased. The protein levels of antiapoptotic myeloid cell leukemia-1 (Mcl-1) and caspase-3 were upregulated, and C-PARP protein was downregulated. Meanwhile, ulinastatin led to an increase in the messenger RNA and protein levels of autophagy maker Unc-like kinase 1 (ULK1), Beclin-1, and microtubule-associated protein 1 light chain 3 (LC-3) and a decrease in p62. Then, 3-methyladenine (3-MA), an inhibitor of autophagy, made morphological damage and cell apoptosis worsen in ulinastatin-treated H/R liver cells. And the expression levels of caspase-3, C-PARP, p62, Beclin-1, and LC-3, proteins were also reversed by 3-MA. Taken together, our results demonstrate that ulinastatin inhibited the hepatic H/R injury in Chang liver cells, which was, to some extent, related to the autophagy activation.