Liver Ischemia And Reperfusion Research Articles

ER stress promotes mitochondrial calcium overload and activates the ROS/NLRP3 axis to mediate fatty liver ischemic injury.

Fatty livers are widely accepted as marginal donors for liver transplantation but are more susceptible to liver ischemia and reperfusion (IR) injury. Increased macrophage-related inflammation plays an important role in the aggravation of fatty liver IR injury. Here, we investigate the precise mechanism by which endoplasmic reticulum (ER) stress activates macrophage NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) signaling by regulating mitochondrial calcium overload in fatty liver IR. Control- and high-fat diet-fed mice were subjected to a partial liver IR model. The ER stress, mitochondrial calcium levels, and NLRP3 signaling pathway in macrophages were analyzed. Liver steatosis exacerbated liver inflammation and IR injury and enhanced NLRP3 activation in macrophages. Myeloid NLRP3 deficiency attenuated intrahepatic inflammation and fatty liver injury following IR. Mechanistically, increased ER stress and mitochondrial calcium overload were observed in macrophages obtained from mouse fatty livers after IR. Suppression of ER stress by tauroursodeoxycholic acid effectively downregulated mitochondrial calcium accumulation and suppressed NLRP3 activation in macrophages, leading to decreased inflammatory IR injury in fatty livers. Moreover, Xestospongin-C-mediated inhibition of mitochondrial calcium influx decreased reactive oxygen species (ROS) expression in macrophages after IR. Scavenging of mitochondrial ROS by mito-TEMPO suppressed macrophage NLRP3 activation and IR injury in fatty livers, indicating that excessive mitochondrial ROS production was responsible for macrophage NLRP3 activation induced by mitochondrial calcium overload. Patients with fatty liver also exhibited upregulated activation of NLRP3 and the ER stress signaling pathway after IR. Our findings suggest that ER stress promotes mitochondrial calcium overload to activate ROS/NLRP3 signaling pathways within macrophages during IR-stimulated inflammatory responses associated with fatty livers.

Defective efferocytosis by aged macrophages promotes STING signaling mediated inflammatory liver injury

Aged livers have shown aggravated liver ischemia and reperfusion (IR) injury. Timely efferocytosis of apoptotic cells is a key mechanism for avoiding excessive inflammation and tissue injury. Here, we investigated the alteration of efferocytosis by aged macrophages and its role in regulating macrophage STING (stimulator of interferon genes) signaling and liver IR injury. Aged and young mice were subjected to liver partial IR model. Liver injury and inflammation were measured. Efferocytosis by aged macrophages and the underlying regulatory mechanism were analyzed as well. Aged macrophages exhibited impaired efferocytosis with decreased MerTK (c-mer proto-oncogene tyrosine kinase) activation, which was reversed by treatment of the MerTK CRISPR activation plasmid. Increased MerTK cleavage by ADAM17 (a disintegrin and metalloproteinase 17) due to enhanced ROS (reactive oxygen species) levels contributed to defective efferocytosis by aged macrophages. MerTK activation by suppressing ADAM17 or ROS improved aged macrophage efferocytosis, leading to reduced inflammatory liver injury. Moreover, increased apoptotic hepatocytes, DNA accumulation, and macrophage STING activation were observed in aged ischemic livers. Improvement in efferocytosis by aged macrophages via MerTK activation suppressed STING activation and inflammatory liver injury. Our study demonstrates that aging suppresses MerTK- mediated macrophage efferocytosis to promote macrophage STING activation and inflammatory liver IR injury, suggesting a new mechanism and potential therapy to promote inflammation resolution and efferocytosis in aged livers.

MSCs Ameliorate Hepatic IR Injury by Modulating Phenotypic Transformation of Kupffer Cells Through Drp-1 Dependent Mitochondrial Dynamics.

Hepatic ischemia and reperfusion (IR) injury, characterized by reactive oxygen species (ROS) production and immune disorders, leads to exogenous antigen-independent local inflammation and hepatocellular death. Mesenchymal stem cells (MSCs) have been shown to be immunomodulatory, antioxidative and contribute to liver regeneration in fulminant hepatic failure. We aimed to investigate the underlying mechanisms by which MSCs protect against liver IR injury in a mouse model. MSCs suspension was injected 30min prior to hepatic warm IR. Primary kupffer cells (KCs) were isolated. Hepatic injury, inflammatory responses, innate immunity, KCs phenotypic polarization and mitochondrial dynamics were evaluated with or without KCs Drp-1 overexpression RESULTS: MSCs markedly ameliorated liver injury and attenuated inflammatory responses and innate immunity after liver IR injury. MSCs significantly restrained M1 phenotypic polarization but boosted M2 polarization of KCs extracted from ischemic liver, as demonstrated by lowered transcript levels of iNOS and IL-1β but raised transcript levels of Mrc-1 and Arg-1 combined with p-STAT6 up-regulation and p-STAT1 down-regulation. Moreover, MSCs inhibited KCs mitochondrial fission, as evidenced by decreased Drp1 and Dnm2 levels. We overexpressed Drp-1 in KCs which promote mitochondrial fission during IR injury. the regulation of MSCs towards KCs M1/M2 polarization was abrogated by Drp-1 overexpression after IR injury. Ultimately, in vivo Drp-1 overexpression in KCs hampered the therapeutic effects of MSCs against hepatic IR injury CONCLUSIONS: We revealed that MSCs facilitated M1-M2 phenotypic polarization through inhibiting Drp-1 dependent mitochondrial fission and further attenuated liver IR injury. These results add a new insight into regulating mechanisms of mitochondrial dynamics during hepatic IR injury and may offer novel opportunities for developing therapeutic targets to combat hepatic IR injury.

MLKL deficiency attenuated hepatocyte oxidative DNA damage by activating mitophagy to suppress macrophage cGAS-STING signaling during liver ischemia and reperfusion injury

Mixed-lineage kinase domain-like protein (MLKL)-mediated necroptosis has been implicated in aggravating liver ischemia and reperfusion (IR) injury. However, the precise role and mechanism of MLKL in regulating oxidative DNA damage of hepatocytes and subsequent activation of macrophage stimulator of interferon genes (STING) signaling remains unclear. In this study, we investigated the role of MLKL in regulating the interplay between hepatocyte injury and macrophage pro-inflammatory responses during liver IR injury. We found that IR increased MLKL expression in liver tissues of wild type (WT) mice. MLKL knockout (KO) attenuated liver IR injury and suppressed the activation of cGAS-STING signaling in intrahepatic macrophages, which was abrogated by STING activation with its agonist. Mechanistically, IR induced oxidative DNA damage in hepatocytes, leading to cGAS-STING activation in macrophages, which was suppressed by MLKL KO. Moreover, increased PTEN-induced kinase 1 (PINK1)-mediated mitophagy contributed to reduced oxidative DNA damage in hepatocytes and subsequent decreased activation of STING signaling in macrophages in MLKL KO mice. Our findings demonstrated a non-canonical role of MLKL in the pathogenesis of liver IR. MLKL deficiency significantly promoted PINK1-mediated mitophagy activation to inhibit oxidative DNA damage in hepatocytes, which in turn suppressed macrophage cGAS-STING activation and inflammatory liver IR injury.

Ovarian hormones influence immune response to liver ischemia-reperfusion.

Liver injury occurs after ischemia and reperfusion (IR), as seen in transplant settings. Sex hormones have been implicated in many pathophysiological mechanisms in females and this could lead to liver protection under inflammatory reperfusion conditions where an excessive immune response occurs. Despite such assumptions, this fact needs to be further investigated. To address this, female and male C57BL/6J mice (8-12 weeks old) were studied. Bilateral ovariectomy (OVX) was performed in females to decrease estradiol levels. IR was performed, and after two weeks, all animals underwent a sham control operation or IR with euthanasia at the following time points after reperfusion: 6, 12, 24, and 48 h. IR triggered an inflammatory process in the liver with recruitment of neutrophils into the parenchyma of male mice. The female sham mice were protected against liver IR presenting no alteration of aminotransferase (ALT) levels compared to males. OVX caused loss of protection, increasing hepatic injury as represented by increased ALT levels and myeloperoxidase (MPO) activity. Female sham mice showed increased Akt phosphorylation and activation, while males showed reduced Akt activation. Estradiol pretreatment recovered ALT levels after IR injury, which was associated with decreased liver injury.

Increased oxidative stress caused by impaired mitophagy aggravated liver ischemia and reperfusion injury in diabetic mice.

Emerging evidence has suggested the detrimental role of oxidative stress in aggravating ischemia and reperfusion (IR) injury in diabetic livers. Interplay between oxidative stress and mitophagy has been shown. However, the role and mechanism of mitophagy in regulating oxidative stress and IR injury in diabetic livers remain unclear. Wild-type and db/db (DB) mice were subjected to a partial warm liver IR model. Liver injury, oxidative stress, mitophagy and related molecular pathways were analyzed. Here, we found that increased liver IR injury was observed in DB mice, as evidenced by higher levels of serum alanine aminotransferase and serum aspartate, worsened liver architecture damage and more hepatocellular death. DB mice also showed increased mitochondrial oxidative stress. Mitochondrial reactive oxygen species scavenge alleviated liver IR injury in DB mice. Mechanistic analysis showed that 5' adenosine monophosphate-activated protein kinase-mediated mitophagy was suppressed in DB mice post-IR. Pharmacological activation of 5' adenosine monophosphate-activated protein kinase by its agonist effectively restored mitophagy activation, leading to decreased mitochondrial oxidative stress and attenuated liver IR injury in DB mice. Our findings showed that diabetes increased oxidative stress to exacerbate liver IR injury by impairing 5' adenosine monophosphate-activated protein kinase-mediated mitophagy. Strategies targeting oxidative stress and mitophagy might provide a promising approach to ameliorate liver IR injury in diabetes patients.

422.5: CHOP Promotes ROS-Mediated Liver Ischemia and Reperfusion Injury by Inhibiting Mitophagy in Hepatocytes

Background: Liver ischemia and reperfusion (IR) injury represents a major risk factor in both partial hepatectomy and liver transplantation. CCAAT/enhancer-binding protein homologous protein (CHOP) is a key regulator of cell death, its precise molecular basis in regulating hepatocyte death during liver IR has not been delineated. Methods: Hepatocellular CHOP deficient mice were generated by bone marrow chimera models using global CHOP knockout mice. Liver partial warm ischemia model and hypoxia/reoxygenation (H/R) model of primary hepatocytes were applied. Liver injury and mitophagy related signaling pathways were investigated. IR-stressed patient liver tissues and serum samples were analyzed as well. Results: Mice with hepatocellular CHOP deficiency exhibited alleviated cell death, decreased reactive oxygen species (ROS) expression and enhanced mitophagy in hepatocytes after IR, confirmed by in vitro studies of hepatocytes after H/R. Mitochondria ROS scavenge by Mito TEMPO effectively attenuated hepatocyte death and liver IR injury of WT mice, whereas no significant effects were observed in hepatocellular CHOP-deficient mice. CHOP depletion upregulated dynamin-related protein 1 (Drp1) and Beclin-1 activation in the mitochondria of hepatocytes leading to enhanced mitophagy. Following IR, increased CHOP expression and impaired mitophagy activation were observed in the livers of patients undergoing hepatectomy. N-acetylcysteine (NAC) pretreatment significantly improved the liver function of patients after surgery. Conclusion: IR-induced CHOP activation exacerbates ROS-mediated hepatocyte death by inhibiting Drp1-Beclin-1-dependent mitophagy. National Natural Science Foundation of China (82071798, 81901628, 81600450), National Science Foundation of Jiangsu Province (BK20191490), CAMS Innovation Fund for Medical Sciences (No.2019-I2M-5-035).

CCAAT/Enhancer-binding Protein Homologous Protein Promotes ROS-mediated Liver Ischemia and Reperfusion Injury by Inhibiting Mitophagy in Hepatocytes.

Liver ischemia and reperfusion (IR) injury represent a major risk factor in both partial hepatectomy and liver transplantation. CCAAT/enhancer-binding protein homologous protein (CHOP) is a key regulator of cell death, its precise molecular basis in regulating hepatocyte death during liver IR has not been delineated. Hepatocellular CHOP deficient mice were generated by bone marrow chimera models using global CHOP knockout mice. Liver partial warm ischemia model and hypoxia/reoxygenation model of primary hepatocytes were applied. Liver injury and mitophagy-related signaling pathways were investigated. IR-stressed patient liver tissues and serum samples were analyzed as well. Mice with hepatocellular CHOP deficiency exhibited alleviated cell death, decreased reactive oxygen species (ROS) expression, and enhanced mitophagy in hepatocytes after IR, confirmed by in vitro studies of hepatocytes after hypoxia/reoxygenation. Mitochondria ROS scavenge by Mito TEMPO effectively attenuated hepatocyte death and liver IR injury of wild-type mice, whereas no significant effects were observed in hepatocellular CHOP -deficient mice. CHOP depletion upregulated dynamin-related protein 1 and Beclin-1 activation in the mitochondria of hepatocytes leading to enhanced mitophagy. Following IR, increased CHOP expression and impaired mitophagy activation were observed in the livers of patients undergoing hepatectomy. N-acetyl cysteine pretreatment significantly improved the liver function of patients after surgery. IR-induced CHOP activation exacerbates ROS-mediated hepatocyte death by inhibiting dynamin-related protein 1-Beclin-1-dependent mitophagy.

Propionic Acid, Induced in Gut by an Inulin Diet, Suppresses Inflammation and Ameliorates Liver Ischemia and Reperfusion Injury in Mice.

Liver ischemia and reperfusion injury (IRI) is one of the obstacles in liver surgery such as liver resection and transplantation. In this study, we investigated the preventive effect on mouse liver IRI by feeding mice with inulin, which is a heterogeneous blend of indigestible fructose polymer. Mice were fed either a control ordinary diet (CD) or an inulin diet (ID) containing 5% inulin in the CD, for 14 days before the ischemia and reperfusion (IR) maneuver. IR induced-liver damages were significantly ameliorated in the ID group, compared with those in the CD group. Feeding mice with an ID, but not a CD, elevated levels of Bacteroidetes among gut microbiota, and especially increased Bacteroides acidifaciens in mouse feces, which resulted in significant elevation of short-chain fatty acids (SCFAs) in the portal vein of mice. Among SCFAs, propionic acid (PA) was most significantly increased. The microbial gene functions related to PA biosynthesis were much higher in the fecal microbiome of the ID group compared to the CD. However, the action of PA on liver IRI has not been yet clarified. Direct intraperitoneal administration of PA alone prior to the ischemia strongly suppressed liver cell damages as well as inflammatory responses caused by liver IR. Furthermore, PA suppressed the secretion of inflammatory cytokines from peritoneal macrophages stimulated in vitro through TLR-4 with high-mobility group box 1 protein (HMGB-1), known to be released from apoptotic liver cells during the IR insult. The present study shows that PA may play a key role in the inulin-induced amelioration of mouse liver IRI.

TGR5/Cathepsin E signaling regulates macrophage innate immune activation in liver ischemia and reperfusion injury.

The role and underlying mechanism of plasma membrane-bound G protein-coupled bile acid receptor (TGR5) in regulating macrophage innate immune activation during liver ischemia and reperfusion (IR) injury remains largely unclear. Here, we demonstrated that TGR5 depletion in myeloid cells aggravated liver injury with increased macrophage infiltration and enhanced inflammation in livers post-IR. While TGR5 deficiency enhanced mobility and proinflammatory M1 polarization of macrophages, TGR5 agonist enhanced the anti-inflammatory effect of TGR5 both in vivo and in vitro. Microarray profiling revealed that TGR5-deficient macrophages exhibited enhanced proinflammatory characteristics and cathepsin E (Cat E) was the most upregulated gene. Knockdown of Cat E abolished the enhanced mobility and shift of macrophage phenotypes induced by TGR5 depletion. Moreover, Cat E knockdown attenuated liver IR injury and liver inflammation in myeloid TGR5-deficient mice. In patients undergoing partial hepatectomy, IR stress promoted TGR5 activation of CD11b+ cells in peripheral blood mononuclear cells, correlating with the shift in macrophage M2 polarization. Ursodeoxycholic acid administration enhanced TGR5 activation and the trend in macrophage M2 polarization. Our results suggest that TGR5 attenuates proinflammatory immune activation by restraining macrophage migration and facilitating macrophage M2 polarization via suppression of Cat E and thereby protects against liver IR injury.

Aging aggravated liver ischemia and reperfusion injury by promoting hepatocyte necroptosis in an endoplasmic reticulum stress-dependent manner.

Aggravated liver ischemia and reperfusion (IR) injury has been reported in aged mice. Although necroptosis inhibition showed no crucial effect on hepatic IR injury in young mice, whether and how necroptosis affects liver IR injury in aged mice remains unclear. Young and aged mice were subjected to liver IR modeling. Liver injury, hepatocyte necroptosis and endoplasmic reticulum (ER) stress were analyzed in different groups. Significantly increased liver necroptosis was found in aged mice post IR compared with young mice. Necroptosis inhibition by necrostatin-1 (Nec-1) decreased hepatocyte necroptosis and liver injury post IR in aged mice, with no significant effects on young mice. Furthermore, IR induced ER stress in both young and aged mice, and enhanced ER stress was observed in aged mice post IR. Administration of 4-phenylbutyrate (4-PBA), an ER stress antagonist, alleviated liver IR injury in both young and aged mice. However, ER stress inhibition reduced hepatocyte necroptosis in aged mice but not in young mice. Aging increased ER stress in IR-stressed hepatocytes, leading to aggravated necroptosis and liver IR injury. Our study demonstrated a novel mechanism of ER stress in the regulation of hepatocyte necroptosis in aged livers post IR, which would be a potential therapeutic target to reduce liver IR injury in elderly patients.

Dexmedetomidine preconditioning alleviated murine liver ischemia and reperfusion injury by promoting macrophage M2 activation via PPARγ/STAT3 signaling

BackgroundAlthough a protective role of dexmedetomidine in liver ischemia and reperfusion (IR) injury has been reported, the underlying mechanism remains to be determined. The aim of this study is to analyze the effects of dexmedetomidine on the regulation of macrophage innate immune activation during liver IR. MethodsMice were randomly divided into dexmedetomidine preconditioning (DEX) and phosphate buffered saline vehicle control (VEH) groups. A murine 70% warm liver IR model was used, and liver injury and intrahepatic inflammation was compared between groups. Bone marrow-derived macrophages (BMDMs) were stimulated with LPS in the presence or absence of dexmedetomidine. The inflammatory cytokine production was measured, and the macrophage M1/M2 polarization was determined in different groups. The underlying mechanism of dexmedetomidine in regulating macrophage M2 activation was also analyzed. ResultsCompared to mice observed in the control group, mice in the DEX group showed reduced liver injury and diminished proinflammatory immune responses in livers post IR. In vitro, dexmedetomidine pretreatment promoted BMDMs M2 activation, as evidenced by increased Arg1 and Mrc1 gene induction, decreased iNOS gene induction, inhibited phosphorated-signal transducer and activator of transcription 1 (p-STAT1) but enhanced p-STAT6 expression, much lower levels of proinflammatory TNF-α and IL-6, and higher levels of anti-inflammatory IL-10 cytokine secretion. Signaling pathway analysis revealed that peroxisome proliferator-activated receptor-γ (PPARγ)/ STAT3 activation was upregulated in BMDMs with dexmedetomidine pretreatment. Furthermore, PPARγ knockdown by siRNA not only inhibited STAT3 activation but also abrogated the promotion effects of macrophage M2 activation in BMDMs pretreated with dexmedetomidine. Finally, in vivo PPARγ inhibition in macrophages by siRNA significantly increased liver IR injury and intrahepatic inflammation in mice from the Dex group, with no significant effect in the VEH group. ConclusionsOur results indicate that dexmedetomidine preconditioning inhibited intrahepatic proinflammatory innate immune activation by promoting macrophage M2 activation in a PPARγ/STAT3 dependent manner. Our results demonstrate a novel innate immune regulatory mechanism by dexmedetomidine preconditioning during liver IR injury.

Combined ischemic and rapamycin preconditioning alleviated liver ischemia and reperfusion injury by restoring autophagy in aged mice

Old livers are more damaged by hepatic ischemia and reperfusion (IR) injury than young livers. The aim of this study was to investigate the effects of ischemic and rapamycin preconditioning on IR injury in old livers. Young (8-week-old) and aged (60-week-old) mice were subjected to IR or a sham control procedure. The aged mice were randomly divided into six groups: IR (CON), IR with ischemic preconditioning (IPC), IR with rapamycin preconditioning (RAPA), IR with combined ischemic and rapamycin preconditioning (IPC + RAPA), IR with 3-methyladenine (3-MA), IR with combined ischemic and rapamycin preconditioning with 3-MA pretreatment (IPC + RAPA+3-MA). Liver injury was evaluated 6 h after reperfusion. Hepatocellular autophagy induction was also analyzed by western blotting. The results revealed that aged mice had aggravated liver IR injury as compared to young mice. In aged mice following IR, IPC + RAPA but not IPC or RAPA alleviated liver injury, as evidenced by lower levels of serum ALT, improved preservation of liver architecture with lower Suzuki scores, and decreased caspase-3 activity compared with CON. In addition, western blot analysis revealed increased LC3B II but decreased p62 protein expression levels in the IPC + RAPA group, indicating that autophagic flux was restored by combined ischemic and rapamycin preconditioning. Furthermore, autophagy inhibition by the inhibitor 3-MA abrogated the protective role in the IPC + RAPA group, while no significant effects were observed in the CON group. In conclusions, our results demonstrated that combined ischemic and rapamycin preconditioning protected old livers against IR injury, which was likely attributed to restored autophagy activation.

Topical hepatic hypothermia associated with ischemic preconditioning. Histopathological and biochemical analysis of ischemia reperfusion damage in a 24 hour model 1.

To develop a new 24 hour extended liver ischemia and reperfusion (LIR) model analyzing the late biochemical and histopathological results of the isolated and combined application of recognized hepatoprotective mechanisms. In addition, we used a new stratification with zoning to classify the histological lesion. A modified animal model of severe hepatic damage produced through 90 minutes of segmental ischemia (70% of the organ) and posterior observation for 24 hours of reperfusion, submitted to ischemic preconditioning (IPC) and topical hypothermia (TH) at 26ºC, in isolation or in combination, during the procedure. Data from intraoperative biometric parameters, besides of late biochemical markers and histopathological findings, both at 24 hours evolution time, were compared with control (C) and normothermic ischemia (NI) groups. All groups were homogeneous with respect to intraoperative physiological parameters. There were no losses once the model was stablished. Animals subjected to NI and IPC had worse biochemical (gamma-glutamyl transpeptidase, alkaline phosphatase, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, direct bilirubin, and total bilirubin) and histopathological scores (modified Suzuki score) compared to those of control groups and groups with isolated or associated TH (p < 0.05). The new extended model demonstrates liver ischemia and reperfusion at 24 hour of evolution and, in this extreme scenario, only the groups subjected to topical hypothermia, combined with ischemic preconditioning or alone, had better outcomes than those subjected to only ischemic preconditioning and normothermic ischemia, reaching similar biochemical and histopathological scores to those of the control group.

Hyperglycemia Aggravates Hepatic Ischemia and Reperfusion Injury by Inhibiting Liver-Resident Macrophage M2 Polarization via C/EBP Homologous Protein-Mediated Endoplasmic Reticulum Stress.

Aggravated liver ischemia and reperfusion (IR) injury has been observed in hyperglycemic hosts, but its underlying mechanism remains undefined. Liver-resident macrophages (Kupffer cells, KCs) and endoplasmic reticulum (ER) stress play crucial roles in the pathogenesis of liver IR injury. In this study, we evaluated the role of ER stress in regulating KC activation and liver IR injury in a streptozotocin-induced hyperglycemic/diabetic mouse model. Compared to the control group (CON group), hyperglycemic mice exhibited a significant increase in liver injury and intrahepatic inflammation following IR. KCs obtained from hyperglycemic mice secreted higher levels of the pro-inflammatory factors TNF-α and IL-6, while they secreted significantly lower levels of the anti-inflammatory factor IL-10. Furthermore, enhanced ER stress was revealed by increased C/EBP homologous protein (CHOP) activation in both IR-stressed livers and KCs from hyperglycemic mice. Specific CHOP knockdown in KCs by siRNA resulted in a slight decrease in TNF-α and IL-6 secretion but dramatically enhanced anti-inflammatory IL-10 secretion in the hyperglycemic group, while no significant changes in cytokine production were observed in the CON group. We also analyzed the role of hyperglycemia in macrophage M1/M2 polarization. Interestingly, we found that hyperglycemia inhibited IL-10-secreting M2-like macrophage polarization, as revealed by decreased Arg1 and Mrc1 gene induction accompanied by a decrease in STAT3 and STAT6 signaling pathway activation. CHOP knockdown restored Arg1 and Mrc1 gene induction, STAT3 and STAT6 activation, and most importantly, IL-10 secretion in hyperglycemic KCs. Finally, in vivo CHOP knockdown in KCs enhanced intrahepatic anti-inflammatory IL-10 gene induction and protected the liver against IR injury in hyperglycemic mice but had no significant effects in control mice. Our results demonstrate that hyperglycemia induces hyper-inflammatory activation of KCs during liver IR injury. Thus, hyperglycemia-induced CHOP over-activation inhibits IL-10-secreting M2-like macrophage polarization by liver-resident macrophages, thereby leading to excessive inflammation and the exacerbation of liver IR injury in diabetic/hyperglycemic hosts. This study provides novel mechanistic insight into macrophage inflammatory activation under hyperglycemic conditions during liver IR.

Isoflurane Preconditioning Alleviated Murine Liver Ischemia and Reperfusion Injury by Restoring AMPK/mTOR-Mediated Autophagy.

Isoflurane has a pharmacological preconditioning effect against ischemia injury in the heart, kidney, and brain, but whether and how isoflurane preconditioning protects livers against ischemia and reperfusion (IR) injury is unclear. Mice were randomly divided into an isoflurane preconditioning (ISO) group and control group, receiving 1.5% isoflurane or carrier gas for 40 minutes, respectively (n = 8/group). A partial warm liver IR model was used, and liver injury was evaluated. Primary hepatocytes were pretreated with 1.5% isoflurane for 2 hours before the induction of cell death by hydrogen peroxide. Cell death and survival were evaluated with the lactate dehydrogenase and cell counting kit-8 assay. Autophagy and regulatory molecules in stressed livers and hepatocytes were analyzed by Western blot (n = 6/group). An autophagy inhibitor (3-methyladenine [3-MA]) and 5' adenosine monophosphate-activated protein kinase (AMPK) inhibitor (dorsomorphin) were administered in vivo (n = 8/group) and in vitro (n = 6/group). Compared to that observed in the control group, mice in the ISO group showed reduced liver injury (alanine aminotransferase [ALT] levels, control versus ISO group, 8285 ± 769 vs 4896 ± 917 U/L, P < .001) and enhanced hepatocellular antiapoptosis in livers after IR. Furthermore, liver autophagy was restored by ISO as indicated by elevated LC3B II protein levels accompanied with increased p62 degradation. The in vitro study of primary hepatocytes also found that ISO effectively attenuated hepatocyte cell death induced by hydrogen peroxide. In addition, 3-MA pretreatment showed no significant influence in the control group, but abrogated the protective role of ISO both in stressed livers (ALT levels, phosphate-buffered saline + ISO versus 3-MA + ISO group, 5081 ± 294 vs 8663 ± 607 U/L, P < .001) and in hepatocytes. Finally, signaling pathway analysis demonstrated that AMPK was activated by ISO. Pretreatment with an AMPK inhibitor also abrogated liver protection by ISO (ALT levels, phosphate-buffered saline + ISO versus dorsomorphin [DOR] + ISO group, 5081 ± 294 vs 8710 ± 500 U/L, P < .001), with no significant effect in control mice. Our results indicate that isoflurane preconditioning attenuates liver IR injury via AMPK/mTOR-mediated hepatocellular autophagy restoration. Our findings provide a novel potential therapeutic strategy for managing liver IR injury.

Mild hypothermia pretreatment protects against liver ischemia reperfusion injury via the PI3K/AKT/FOXO3a pathway.

Mild hypothermia is known to protect against ischemia and reperfusion (IR) injury. The exact mechanisms of the protection are not fully understood. Forkhead box O3 (FOXO3a) has been defined as a critical mediator in cellular processes, including oxidative stress, apoptosis, inflammation, cell death and DNA repair; however, the protection function in mild hypothermia has not been reported previously. The current study was designed to investigate the function of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/FOXO3a pathway in pretreatment with mild hypothermia during IR injury. Additionally, PI3K/AKT/FOXO3a signaling was inhibited using Ly294002 and the effect on the protective function of mild hypothermia pretreatment was evaluated. Furthermore, the apoptotic and inflammatory response induced by the IR injury was evaluated. Liver IR injury induced a significant increase in the level of apoptosis and inflammatory responses. However, pretreatment with mild hypothermia increased phospho (p)-AKT and p-FOXO3a following IR injury, and significantly reduced apoptosis and inflammatory cytokines release. However, inhibiting p-AKT and p-FOXO3a using Ly294002 suppressed the liver protection produced by mild hypothermia. In conclusion, these findings indicated that mild hypothermia pretreatment exhibited liver protective effects against IR injury associated with suppressing inflammatory cytokine release and apoptosis via the PI3K/AKT/FOXO3a pathway.

MicroRNA-29a/b/c targets iNOS and is involved in protective remote ischemic preconditioning in an ischemia-reperfusion rat model of non-alcoholic fatty liver disease.

Remote ischemic preconditioning (RIPC) protects against the injury that is incurred by ischemia and reperfusion (IR); however, the role of RIPC in liver IR injury in non-alcoholic fatty liver disease (NAFLD) remains unclear. In this study, a NAFLD rat model was utilized in a series of different surgical procedures and molecular experiments. Rats of the IR group and the RIPC+IR group exhibited more severe injury than NAFLD control rats (in which the liver was prodded following a median-incision laparotomy). The liver condition, measured by serum alanine transaminase and aspartate transaminase levels, of the RIPC+IR group was better than that of the IR group. In addition, alanine transaminase and aspartate transaminase levels were lower in the RIPC+IR group compared with the IR group (P<0.001). Flow cytometry revealed that the cell apoptosis ratio was significantly lower in the RIPC+IR group than in the IR group (P<0.001). Reverse transcription-polymerase chain reaction (RT-qPCR) was used to assess miR-29a/b/c levels, revealing that they were significantly reduced in the RIPC and RIPC+IR groups, but did not vary in the IR group compared with the control group. RT-qPCR also revealed that iNOS mRNA levels were not significantly different among any of the NAFLD groups; however, western blot analysis indicated that iNOS protein levels were increased in the RIPC group and the RIPC+IR group compared with the control and IR groups. A luciferase reporter assay demonstrated that transfection with miR-29a/b/c mimics significantly decreased the luciferase activities of plasmids containing the wild-type iNOS 3'-untranslated region (UTR) (relative fluorescence intensity: 0.47±0.06 for miR-29a, 0.36±0.07 for miR-29b, 0.41±0.04 for miR-29c; P<0.001), whereas the activities of plasmids containing the mutant iNOS 3'-UTR sequence were not markedly affected [relative fluorescence intensity: 0.99±0.08 for miR-29a (P=0.1349), 0.99±0.09 for miR-29b (P=0.1607), 0.97±0.07 for miR-29c (P=0.1824)]. This suggested that miR-29a/b/c downregulates iNOS by directly targeting its 3'-UTR. In summary, the results suggest that RIPC has a protective effect in NAFLD liver IR injury, which may be due to reduced miR-29a/b/c levels in the skeletal muscle, leading to increased iNOS and, therefore, nitric oxide.

N-acetyl cysteine improves glycogenesis after segmental liver ischemia and reperfusion injury in pigs

Objective. N-acetylcysteine (NAC) is an antioxidative molecule known to protect liver tissue from oxygen radical species generated during ischemia and reperfusion (IR). Nutritional and toxicology studies have shown that NAC also improves glucose metabolism and glycogen stores. We hypothesized that NAC improves glycogenesis and that impaired glycogenesis is a key element in IR injury. Material and Methods. In an experimental model, 80 min of segmental liver ischemia was induced in 16 pigs and the reperfusion was followed for 360 min. Eight animals received NAC 150 mg/kg as a bolus injection followed by an infusion of NAC 50 mg/kg/h intravenously. Results. AST and leukocyte density were lower in the NAC-treated animals, unrelated to the glutathione levels or apoptosis. Glycogen stores returned to a higher degree in the NAC-treated animals and microdialysis revealed lower levels of lactate during the reperfusion phase. Nitrite/Nitrate levels in the NAC group were lower in both serum and microdialysates, indicating that NAC scavenges radical nitrosative species. Conclusions. NAC treatment improves glycogenesis after liver IR injury and reduces the level of intraparenchymal lactate during reperfusion, possibly due to the scavenging of radical nitrosative species.

Paneth cell-derived interleukin-17A causes multiorgan dysfunction after hepatic ischemia and reperfusion injury

Hepatic ischemia and reperfusion (IR) injury is a major clinical problem that leads to frequent extrahepatic complications including intestinal dysfunction and acute kidney injury (AKI). In this study we aimed to determine the mechanisms of hepatic IR-induced extrahepatic organ dysfunction. Mice subjected to 60 minutes of hepatic IR not only developed severe hepatic injury but also developed significant AKI and small intestinal injury. Hepatic IR induced small intestinal Paneth cell degranulation and increased interleukin-17A (IL-17A) levels in portal vein plasma and small intestine. We also detected increased levels of IL-17A messenger RNA (mRNA) and protein in Paneth cells after hepatic IR with laser capture dissection. IL-17A-neutralizing antibody treatment or genetic deletion of either IL-17A or IL-17A receptors significantly protected against hepatic IR-induced acute liver, kidney, and intestinal injury. Leukocyte IL-17A does not contribute to organ injury, as infusion of wildtype splenocytes failed to exacerbate liver and kidney injury in IL-17A-deficient mice after hepatic IR. Depletion of Paneth cell numbers by pharmacological (with dithizone) or genetic intervention (SOX9 flox/flox Villin cre+/- mice) significantly attenuated intestinal, hepatic, and renal injury following liver IR. Finally, depletion of Paneth cell numbers significantly decreased small intestinal IL-17A release and plasma IL-17A levels after liver IR. Taken together, the results show that Paneth cell-derived IL-17A plays a critical role in hepatic IR injury and extrahepatic organ dysfunction. Modulation of Paneth cell dysregulation may have therapeutic implications by reducing systemic complications arising from hepatic IR.