Liver Ischemia-reperfusion Injury Research Articles

Small extracellular vesicles from HO-1-modified bone marrow-derived mesenchymal stem cells attenuate ischemia-reperfusion injury after steatotic liver transplantation by suppressing ferroptosis via miR-214-3p

Donor shortage is a major problem that limits liver transplantation availability. Steatotic donor liver presents a feasible strategy to solve this problem. However, severe ischemia-reperfusion injury (IRI) is an obstacle to the adoption of steatotic transplanted livers. Evidence from our prior studies indicated that bone marrow mesenchymal stem cells modified with heme oxygenase-1 (HMSCs) can attenuate non-steatotic liver IRI. However, the contribution of HMSCs in transplanted steatotic liver IRI is unclear. Here, HMSCs and their derived small extracellular vesicles (HM-sEVs) alleviated IRI in transplanted steatotic livers. After liver transplantation, there was significant enrichment of the differentially expressed genes in the glutathione metabolism and ferroptosis pathways, accompanied by ferroptosis marker upregulation. The HMSCs and HM-sEVs suppressed ferroptosis and attenuated IRI in the transplanted steatotic livers. MicroRNA (miRNA) microarray and validation experiments indicated that miR-214-3p, which was abundant in the HM-sEVs, suppressed ferroptosis by targeting cyclooxygenase 2 (COX2). In contrast, COX2 overexpression reversed this effect. Knockdown of miR-214-3p in the HM-sEVs diminished its ability to suppress ferroptosis and protect liver tissues/cells. The findings suggested that HM-sEVs suppressed ferroptosis to attenuate transplanted steatotic liver IRI via the miR-214-3p–COX2 axis.

Type 2 innate lymphoid cells are protective against hepatic ischaemia/reperfusion injury

Although type 2 innate lymphoid cells (ILC2s) were originally found to be liver-resident lymphocytes, the role and importance of ILC2 in liver injury remains poorly understood. In the current study, we sought to determine whether ILC2 is an important regulator of hepatic ischaemia/reperfusion injury (IRI). ILC2-deficient mice (ICOS-T or NSG) and genetically modified ILC2s were used to investigate the role of ILC2s in murine hepatic IRI. Interactions between ILC2s and eosinophils or macrophages were studied in coculture. The role of human ILC2s was assessed in an immunocompromised mouse model of hepatic IRI. Administration of IL-33 prevented hepatic IRI in association with reduction of neutrophil infiltration and inflammatory mediators in the liver. IL-33-treated mice had elevated numbers of ILC2s, eosinophils, and regulatory T cells. Eosinophils, but not regulatory T cells, were required for IL-33-mediated hepatoprotection in IRI mice. Depletion of ILC2s substantially abolished the protective effect of IL-33 in hepatic IRI, indicating that ILC2s play critical roles in IL-33-mediated liver protection. Adoptive transfer of exvivo-expanded ILC2s improved liver function and attenuated histologic damage in mice subjected to IRI. Mechanistic studies combining genetic and adoptive transfer approaches identified a protective role of ILC2s through promoting IL-13-dependent induction of anti-inflammatory macrophages and IL-5-dependent elevation of eosinophils in IRI. Furthermore, invivo expansion of human ILC2s by IL-33 or transfer of exvivo-expanded human ILC2s ameliorated hepatic IRI in an immunocompromised mouse model of hepatic IRI. This study provides insight into the mechanisms of ILC2-mediated liver protection that could serve as therapeutic targets to treat acute liver injury. We report that type 2 innate lymphoid cells (ILC2s) are important regulators in a mouse model of liver ischaemia/reperfusion injury (IRI). Through manipulation of macrophage and eosinophil phenotypes, ILC2s mitigate liver inflammation and injury during liver IRI. We propose that ILC2s have the potential to serve as a therapeutic tool for protecting against acute liver injury and lay the foundation for translation of ILC2 therapy to human liver disease.

The Protective Effect of Nutraceuticals on Hepatic Ischemia-Reperfusion Injury in Wistar Rats.

Nutraceuticals are bioactive compounds present in foods, utilized to ameliorate health, prevent diseases, and support the proper functioning of the human body. They have gained attention due to their ability to hit multiple targets and act as antioxidants, anti-inflammatory agents, and modulators of immune response and cell death. Therefore, nutraceuticals are being studied to prevent and treat liver ischemia-reperfusion injury (IRI). This study evaluated the effect of a nutraceutical solution formed by resveratrol, quercetin, omega-3 fatty acid, selenium, ginger, avocado, leucine, and niacin on liver IRI. IRI was performed with 60 min of ischemia and 4 h of reperfusion in male Wistar rats. Afterward, the animals were euthanized to study hepatocellular injury, cytokines, oxidative stress, gene expression of apoptosis-related genes, TNF-α and caspase-3 proteins, and histology. Our results show that the nutraceutical solution was able to decrease apoptosis and histologic injury. The suggested mechanisms of action are a reduction in gene expression and the caspase-3 protein and a reduction in the TNF-α protein in liver tissue. The nutraceutical solution was unable to decrease transaminases and cytokines. These findings suggest that the nutraceuticals used favored the protection of hepatocytes, and their combination represents a promising therapeutic proposal against liver IRI.

FOXO1 regulates Th17 cell-mediated hepatocellular carcinoma recurrence after hepatic ischemia-reperfusion injury

BackgroundHepatic ischemia-reperfusion injury (IRI) is considered as an effecting factor for hepatocellular carcinoma (HCC) recurrence. Th17/Treg cells are a pair of essential components in adaptive immune response in liver IRI, and forkhead box O1 (FOXO1) has the properties of maintaining the function and phenotype of immune cells. Herein, we illuminated the correlation and function between Th17/Treg cell balance and FOXO1 in IRI-induced HCC recurrence.MethodsRNA sequencing was performed on naive CD4+ T cells from normal and IRI model mice to identify relevant transcription factors. Western blotting, qRT-PCR, immunohistochemical staining, and flow cytometry were performed in IRI models to indicate the effect of FOXO1 on the polarization of Th17/Treg cells. Then, transwell assay of HCC cell migration and invasion, clone formation, wound healing assay, and Th17 cells adoptively transfer was utilized to assess the function of Th17 cells in IRI-induced HCC recurrence in vitro and in vivo.ResultsOwning to the application of RNA sequencing, FOXO1 was screened and assumed to perform a significant function in hepatic IRI. The IRI model demonstrated that up-regulation of FOXO1 alleviated IR stress by attenuating inflammatory stress, maintaining microenvironment homeostasis, and reducing the polarization of Th17 cells. Mechanistically, Th17 cells accelerated IRI-induced HCC recurrence by shaping the hepatic pre-metastasis microenvironment, activating the EMT program, promoting cancer stemness and angiogenesis, while the upregulation of FOXO1 can stabilize the liver microenvironment homeostasis and alleviate the negative effects of Th17 cells. Moreover, the adoptive transfer of Th17 cells in vivo revealed its inducing function in IRI-induced HCC recurrence.ConclusionsThese results indicated that FOXO1-Th17/Treg axis exerts a crucial role in IRI-mediated immunologic derangement and HCC recurrence, which could be a promising target for reducing the HCC recurrence after hepatectomy.Liver IRI affects the balance of Th17/Treg cells by inhibiting the expression of FOXO1, and the increase of Th17 cells has the ability to induce HCC recurrence through EMT program, cancer stemness pathway, the formation of premetastatic microenvironment and angiogenesis.

Fucoidan Ameliorates Ferroptosis in Ischemia-reperfusion-induced Liver Injury through Nrf2/HO-1/GPX4 Activation.

Liver ischemia-reperfusion (IR) injury is a common pathological process in liver surgery. Ferroptosis, which is closely related to lipid peroxidation, has recently been confirmed to be involved in the pathogenesis of IR injury. However, the development of drugs that regulate ferroptosis has been slow, and a complete understanding of the mechanisms underlying ferroptosis has not yet been achieved. Fucoidan (Fu) is a sulfated polysaccharide that has attracted research interest due to its advantages of easy access and wide biological activity. In this study, we established models of IR injury using erastin as an activator of ferroptosis, with the ferroptosis inhibitor ferrostatin-1 (Fer-1) as the control. We clarified the molecular mechanism of fucoidan in IR-induced ferroptosis by determining lipid peroxidation levels, mitochondrial morphology, and key pathways in theta were involved. Ferroptosis was closely related to IR-induced hepatocyte injury. The use of fucoidan or Fer-1 inhibited ferroptosis by eliminating reactive oxygen species and inhibiting lipid peroxidation and iron accumulation, while those effects were reversed after treatment with erastin. Iron accumulation, mitochondrial membrane rupture, and active oxygen generation related to ferroptosis also inhibited the entry of nuclear factor erythroid 2-related factor 2 (Nrf2) into the nucleus and reduced downstream heme oxygenase-1 (HO-1) and glutathione peroxidase 4 (GPX4) protein levels. However, fucoidan pretreatment produced adaptive changes that reduced irreversible cell damage induced by IR or erastin. Fucoidan inhibited ferroptosis in liver IR injury via the Nrf2/HO-1/GPX4 axis.

Hepatocyte-derived extracellular vesicles miR-122-5p promotes hepatic ischemia reperfusion injury by regulating Kupffer cell polarization.

Ischemia reperfusion injury remains a major barrier to liver transplantation, especially using grafts from donation after circulatory death, and it is also a pressing issue to be solved in clinical practice. Kupffer cell polarization toward a proinflammatory M1 phenotype is an early trigger of liver ischemia-reperfusion injury. However, the molecular mechanism regulating Kupffer cell polarization has not yet been fully elucidated. We induced liver ischemia reperfusion injury in mice and obtained samples from patients undergoing liver transplantation, serum and hepatocytes-derived extracellular vesicles were isolated by differential ultracentrifugation. Kupffer cell polarization was examined by flow cytometry and immunofluorescence histochemistry. RNA-seq was conducted to detect the differentially expressed miRNAs in extracellular vesicles. The role and mechanism of exosomal miR-122-5p in liver ischemia-reperfusion injury were determined both in vitro and in vivo. We identified ischemia reperfusion induced extracellular vesicles as a major cause of hepatic inflammation and tissue damage using adoptive transfer and release inhibition. The study also demonstrated that hepatocyte-derived exosomal miR-122-5p mediates liver ischemia reperfusion injury by polarizing Kupffer cell via PPARδ down-regulation and NF-κB pathway activation using profiling and functional analysis. Moreover, inhibiting miR-122-5p with antagomir suppressed Kupffer cell M1 polarization and attenuated liver ischemia reperfusion injury. Overall, our study demonstrated that hepatocyte-derived exosomal miR-122-5p played a critical role in promoting hepatic ischemia reperfusion injury through modulating PPARδ signaling and NF-κB pathway to introduce M1 polarization of Kupffer cell. Inhibition of miR-122-5p exhibited a protective effect against liver ischemia reperfusion injury, suggesting a potential therapeutic target for liver transplantation.

Establishment and applicability comparison of four models of acute liver ischemia/reperfusion injury in rat

To clarify the preparation methods of four rat models of liver ischemia/reperfusion injury (IRI) and to determine a liver IRI animal model that is consistent with clinical conditions, has stable pathological and physiological injury, and is easy to operate. A total of 160 male Sprague-Dawley (SD) rats were randomly divided into four groups using an interval grouping method: 70% IRI (group A), 100% IRI (group B), 70% IRI with 30% hepatectomy (group C), and 100% IRI with 30% hepatectomy (group D), with 40 rats in each group. Each model was further divided into sham operation group (S group) and ischemia groups of 30, 60, and 90 minutes, with 10 rats in each group. After surgery, the survival status and awakening time of the rats were observed, and the liver lobectomy weight, bleeding volume, and hemostasis time of groups C and D were recorded. Blood samples were collected by cardiac puncture after 6 hours of reperfusion for determination the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), serum creatinine (SCr), and γ-glutamyl transpeptidase (γ-GT) in the serum to assess liver and kidney function. Hematoxylin-eosin (HE) staining and immunohistochemical staining of macrophages were performed to analyze the liver tissue structure damage from a pathological perspective. Rats in group A exhibited earlier awakening and acceptable mental status, while rats in the other groups showed delayed awakening and poor mental status. The hemostasis time in group D was approximately 1 second longer than that in group C. The mortality of rats subjected to 60 minutes of 70% hepatic ischemia was 0. Compared to the sham operation group, rats in each experimental group showed significant increases in serum levels of AST, ALT, ALP, BUN, SCr, and γ-GT, indicating impaired liver and kidney function in the rat models of liver IRI. In groups A, B, and C, the 90-minute ischemia subgroup exhibited more pronounced elevation in AST, ALT, ALP, BUN, SCr, and γ-GT levels compared to the 30-minute ischemia subgroup [AST (U/L): group A, 834.94±56.73 vs. 258.74±18.33; group B, 547.63±217.40 vs. 277.67±57.92; group C, 930.38±75.48 vs. 640.51±194.20; ALT (U/L): group A, 346.78±25.47 vs. 156.58±13.25; group B, 408.40±138.25 vs. 196.80±58.60; group C, 596.41±193.32 vs. 173.76±72.43; ALP (U/L): group A, 431.21±34.30 vs. 315.95±15.64; group B, 525.88±62.13 vs. 215.63±17.31; group C, 487.53±112.37 vs. 272.46±92.33; BUN (U/L): group A, 18.35±5.63 vs. 14.32±2.30; group B, 30.21±4.55 vs. 17.41±8.14; group C, 20.50±3.64 vs. 15.93±3.22; SCr (U/L): group A, 27.47±8.91 vs. 22.37±5.66; group B, 43.60±15.57 vs. 36.80±7.95; group C, 63.81±20.24 vs. 42.47±7.03; γ-GT (U/L): group A, 15.64±3.57 vs. 6.82±1.48; group B, 9.28±1.91 vs. 5.62±1.21; group C, 10.98±3.18 vs. 5.67±1.10; all P < 0.05]. The 100% IRI 90-minute group and 100% IRI 90-minute group with 30% hepatectomy exhibited more pronounced increases in the above-mentioned indicators compared to the corresponding 70% IRI control group, indicating increased liver and kidney damage in rats subjected to combined blood flow occlusion and hepatectomy. HE staining showed clear liver tissue structure with intact and orderly arranged cells in the sham operation group, while the experimental groups exhibited cell structure damage, including cell rupture or collapse, cell swelling, nuclear pyknosis, deep cytoplasm staining, cell shedding, and necrosis. The interstitium showed infiltration of inflammatory cells. Immunohistochemical staining revealed a higher number of macrophages in the experimental groups compared to the sham operation group. Four models of liver IRI in rat were successfully established. As the duration and severity of hepatic ischemia increased, liver cell ischemia worsened, leading to increased hepatocellular necrosis and exhibiting characteristic features of liver IRI. These models can effectively simulate liver IRI following liver trauma, with the group subjected to 100% ischemia and 30% hepatectomy showing the most severe liver injury. The designed models are reasonable, easy to perform, and exhibit good reproducibility. They can be used for investigating the mechanisms, therapeutic efficacy, and diagnostic methods related to clinical liver IRI.

SAT-378 - The Ninj1/Dusp1 axis contributes to liver ischemia reperfusion injury by regulating macrophage activation and neutrophil infiltration

SAT-378 - The Ninj1/Dusp1 axis contributes to liver ischemia reperfusion injury by regulating macrophage activation and neutrophil infiltration

Recipient TIM4 signaling regulates ischemia reperfusion-induced ER stress and metabolic responses in liver transplantation: from mouse-to-human.

T-cell immunoglobulin and mucin (Tim)4 is expressed on APCs, including macrophages, as one of the main amplifiers in the mechanism of liver ischemia-reperfusion injury (IRI) following orthotopic liver transplantation (OLT). Though donor Tim4 selectively expressed on Kupffer cells serves as a checkpoint regulator of innate immune-driven IRI cascades, its role on cells outside the OLT remains unclear. To dissect the role of donor vs. recipient-specific Tim4 signaling in IR-induced stress and hepatocellular function, we employed a murine OLT model utilizing Tim4-knockout (KO) mice as either donor or recipient (WT → WT, WT → Tim4-KO, Tim4-KO → WT). In the experimental arm, disruption of donor Tim4 attenuated IRI-OLT damage, while recipient Tim4-null mutation aggravated hepatic IRI concomitant with disturbed lipid metabolism, enhanced endoplasmic reticulum stress, and activated pro-apoptotic signaling in the grafts. In the in vitro study, murine hepatocytes co-cultured with Tim4-null adipose tissue showed enhanced C/EBP homologous protein (CHOP) expression pattern and susceptibility to hepatocellular death accompanied by activated caspase cascade in response to TNF-α stimulation. In the clinical arm, liver grafts from forty-one transplant patients with enhanced TIM4 expression showed higher body mass index, augmented hepatic endoplasmic reticulum stress, enhanced pro-apoptotic markers, upregulated innate/adaptive immune responses, exacerbated hepatocellular damage, and inferior graft survival. In conclusion, although TIM4 is considered a principal villain in peri-transplant early tissue injury, recipient TIM4 signaling may serve as a savior of IR-triggered metabolic stress in mouse and human OLT recipients.

Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus d-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia–reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia–reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

Combination of Cold Storage in a Heavy Water-Containing Solution and Post-Reperfusion Hydrogen Gas Treatment Reduces Ischemia-Reperfusion Injury in Rat Livers

We previously reported the efficacy of cold storage (CS) using a heavy water-containing solution (Dsol) and post-reperfusion hydrogen gas treatment separately. This study aimed to clarify the combined effects of these treatments. Rat livers were subjected to 48-hour CS and a subsequent 90-minute reperfusion in an isolated perfused rat liver system. The experimental groups were the immediately reperfused control group (CT), the CS with University of Wisconsin solution (UW) group, the CS with Dsol group, the CS with UW and post-reperfusion H2 treatment group (UW-H2), and the CS with Dsol and post-reperfusion H2 group (Dsol-H2). We first compared the Dsol-H2, UW, and CT groups to evaluate this alternative method to conventional CS. The protective potential of the Dsol-H2 group was superior to that of the UW group, as evidenced by lower portal venous resistance and lactate dehydrogenase leakage, a higher oxygen consumption rate, and increased bile production. Multiple comparison tests among the UW, Dsol, UW-H2, and Dsol-H2 groups revealed that both treatments, during CS and after reperfusion, conferred a similar extent of protection and showed additive effects in combination therapy. Furthermore, the variance in all treatment groups appeared smaller than that in the no-treatment or no-stress groups, with excellent reproducibility. In conclusion, combination therapy with Dsol during CS and hydrogen gas after reperfusion additively protects against graft injury.

Identification of distinct immune infiltration and potential biomarkers in patients with liver ischemia-reperfusion injury

AimsTo identify alterations of specific gene expression, immune infiltration components, and potential biomarkers in liver ischemia-reperfusion injury (IRI) following liver transplantation (LT). Materials and methodsGSE23649 and GSE151648 datasets were obtained from the Gene Expression Omnibus (GEO) database. To determine the differentially expressed genes (DEGs), we utilized the R package “limma”. We also identify the infiltration of different immune cells through single-sample gene-set enrichment analysis (ssGSEA). Furthermore, we utilized LASSO logistic regression to select feature genes and Spearman's rank correlation analysis to determine the correlation between these genes and infiltrating immune cells. Finally, the significance of these feature genes was confirmed using a mouse model of hepatic IRI. Key findingsA total of 17 DEGs were acquired, most of which were associated with inflammation, apoptosis, cell proliferation, immune disorders, and cellular response. 28 immune cell types were determined using ssGSEA. 5 feature genes (ADM, KLF6, SERPINE1, SLC20A1, and HBB) were screened using LASSO analysis, but the HBB gene was ultimately excluded due to the lack of statistical significance in the GSE151648 dataset. These 4 feature genes were predominantly related to immune cells. Finally, 15 significantly distinctive types of immune cells between the control and IRI groups were verified. SignificanceWe unveiled that macrophages, dendritic cells (DCs), neutrophils, CD4 T cells, and other immune cells infiltrated the IRI that occurred after LT. Moreover, we identified ADM, KLF6, SERPINE1, and SLC20A1 as potential biological biomarkers underlying IRI post-transplant, which may improve the diagnosis and prognosis of this condition.

Protective Effects of Lumbricus Extract on the Antioxidant System and Liver in an Experimentally Created Liver Ischemia Reperfusion Injury Model in Rats

Protective Effects of Lumbricus Extract on the Antioxidant System and Liver in an Experimentally Created Liver Ischemia Reperfusion Injury Model in Rats

Responses of hepatic sinusoidal cells to liver ischemia-reperfusion injury.

The liver displays a remarkable regenerative capacity in response to acute liver injury. In addition to the proliferation of hepatocytes during liver regeneration, non-parenchymal cells, including liver macrophages, liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) play critical roles in liver repair and regeneration. Liver ischemia-reperfusion injury (IRI) is a major cause of increased liver damage during liver resection, transplantation, and trauma. Impaired liver repair increases postoperative morbidity and mortality of patients who underwent liver surgery. Successful liver repair and regeneration after liver IRI requires coordinated interplay and synergic actions between hepatic resident cells and recruited cell components. However, the underlying mechanisms of liver repair after liver IRI are not well understood. Recent technological advances have revealed the heterogeneity of each liver cell component in the steady state and diseased livers. In this review, we describe the progress in the biology of liver non-parenchymal cells obtained from novel technological advances. We address the functional role of each cell component in response to liver IRI and the interactions between diverse immune repertoires and non-hematopoietic cell populations during the course of liver repair after liver IRI. We also discuss how these findings can help in the design of novel therapeutic approaches. Growing insights into the cellular interactions during liver IRI would enhance the pathology of liver IRI understanding comprehensively and further develop the strategies for improvement of liver repair.

Lin28 promoting the protective effect of PMSCs on hepatic ischaemia-reperfusion injury by regulating glucose metabolism.

Human placental mesenchymal stem cells (PMSCs) can prevent liver ischaemia-reperfusion injury (LIRI). However, their therapeutic effects are limited. Therefore, additional research is required to elucidate the mechanisms of PMSC-mediated LIRI prevention and enhance the related therapeutic effects. This study aimed to examine the role of the Lin28 protein in the regulation of glucose metabolism in PMSCs. Further, it explored whether Lin28 could enhance the protective effects of PMSCs against LIRI and investigated the underlying mechanisms. Western blotting was performed to examine Lin28 expression in PMSCs under hypoxic conditions. A Lin28 overexpression construct was introduced into PMSCs, and the effect on glucose metabolism was examined using a glucose metabolism kit. Further, the expression of some proteins involved in glucose metabolism and the PI3K-AKT pathway and the levels of microRNA Let-7a-g were examined using western blots and real-time quantitative PCR, respectively. To examine the relationship between Lin28 and the PI3K-Akt pathway, the effects of AKT inhibitor treatment on the changes induced by Lin28 overexpression were examined. Subsequently, AML12 cells were co-cultured with PMSCs to elucidate the mechanisms via which PMSCs prevent hypoxic injury in liver cells in vitro. Finally, C57BL/6J mice were used to establish a partial warm ischaemia-reperfusion model. The mice received intravenous injections containing PMSCs (control and Lin28-overexpressing PMSCs). Finally, their serum transaminase levels and degree of liver injury were assessed using biochemical and histopathological methods, respectively. Lin28 was upregulated under hypoxic conditions in PMSCs. Lin28 exerted protective effects against hypoxia-induced cell proliferation. Moreover, it increased the glycolytic capacity of PMSCs, allowing PMSCs to produce more energy under hypoxic conditions. Lin28 also activated the PI3K-Akt signalling pathway under hypoxic conditions, and its effects were attenuated by AKT inhibition. Lin28 overexpression could protect cells against LIRI-induced liver damage, inflammation and apoptosis and could also attenuate hypoxia-induced hepatocyte injury. Lin28 enhances glucose metabolism under hypoxic conditions in PMSCs, thereby exerting protective effects against LIRI by activating the PI3K-Akt signalling pathway. Our study is the first to report the potential of genetically modified PMSCs for LIRI treatment.

Electroacupuncture Pretreatment at Zusanli (ST36) Ameliorates Hepatic Ischemia/Reperfusion Injury in Mice by Reducing Oxidative Stress via Activating Vagus Nerve-Dependent Nrf2 Pathway.

Current pharmacological approaches to prevent hepatic ischemia/reperfusion injury (IRI) are limited. To mitigate hepatic injury, more research is needed to improve the understanding of hepatic IRI. Depending on traditional Chinese medicine (TCM) theory, acupuncture therapy has been used for the treatment of ischemic diseases with good efficacy. However, the efficacy and mechanism of acupuncture for hepatic IRI are still unclear. Blood provided to the left and middle lobe of mice livers was blocked with a non-invasive clamp and then the clamps were removed for reperfusion to establish a liver IRI model. Quantitative proteomics approach was used to evaluate the impact of EA pretreatment on liver tissue proteome in the IRI group. Serum biochemistry was used to detect liver injury, inflammation, and oxidative stress levels. H&E staining and TUNEL staining were used to detect hepatocyte injury and apoptosis. Immunohistochemistry and ELISA were used to detect the degree of inflammatory cell infiltration and the level of inflammation. The anti-inflammatory and antioxidant capacities were detected by Quantitative RT-PCR and Western blotting. We found that EA at Zusanli (ST36) has a protective effect on hepatic IRI in mice by alleviating oxidative stress, hepatocyte death, and inflammation response. Nuclear factor E2-related factor 2 (Nrf2) as a crucial target was regulated by EA and was then successfully validated. The Nrf2 inhibitor ML385 and cervical vagotomy eliminated the protective effect in the EA treatment group. This study firstly demonstrated that EA pretreatment at ST36 significantly ameliorates hepatic IRI in mice by inhibiting oxidative stress via activating the Nrf2 signal pathway, which was vagus nerve-dependent.

Caspase 6/NR4A1/SOX9 signaling axis regulates hepatic inflammation and pyroptosis in ischemia-stressed fatty liver

The mechanism of nonalcoholic fatty liver susceptibility to ischemia/reperfusion (IR) injury has not been fully clarified. Caspase 6 is a critical regulator in innate immunity and host defense. We aimed to characterize the specific role of Caspase 6 in IR-induced inflammatory responses in fatty livers. Human fatty liver samples were harvested from patients undergoing ischemia-related hepatectomy to evaluate Caspase 6 expression. in mice model, we generated Caspase 6-knockout (Caspase 6KO) mice to investigate cellular and molecular mechanisms of macrophage Caspase 6 in IR-stimulated fatty livers. In human liver biopsies, Caspase 6 expression was upregulated combined with enhanced serum ALT level and severe histopathological injury in ischemic fatty livers. Moreover, Caspase 6 was mainly accumulated in macrophages but not hepatocytes. Unlike in controls, the Caspase 6-deficiency attenuated liver damage and inflammation activation. Activation of macrophage NR4A1 or SOX9 in Caspase 6-deficient livers aggravated liver inflammation. Mechanistically, macrophage NR4A1 co-localized with SOX9 in the nuclear under inflammatory conditions. Specifically, SOX9 acts as a coactivator of NR4A1 to directly target S100A9 transcription. Furthermore, macrophage S100A9 ablation dampened NEK7/NLRP3-driven inflammatory response and pyroptosis in macrophages. In conclusion, our findings identify a novel role of Caspase 6 in regulating NR4A1/SOX9 interaction in response to IR-stimulated fatty liver inflammation, and provide potential therapeutic targets for the prevention of fatty liver IR injury.

Preservation of Mitochondrial Health in Liver Ischemia/Reperfusion Injury.

Liver ischemia-reperfusion injury (LIRI) is a major cause of the development of complications in different clinical settings such as liver resection and liver transplantation. Damage arising from LIRI is a major risk factor for early graft rejection and is associated with higher morbidity and mortality after surgery. Although the mechanisms leading to the injury of parenchymal and non-parenchymal liver cells are not yet fully understood, mitochondrial dysfunction is recognized as a hallmark of LIRI that exacerbates cellular injury. Mitochondria play a major role in glucose metabolism, energy production, reactive oxygen species (ROS) signaling, calcium homeostasis and cell death. The diverse roles of mitochondria make it essential to preserve mitochondrial health in order to maintain cellular activity and liver integrity during liver ischemia/reperfusion (I/R). A growing body of studies suggest that protecting mitochondria by regulating mitochondrial biogenesis, fission/fusion and mitophagy during liver I/R ameliorates LIRI. Targeting mitochondria in conditions that exacerbate mitochondrial dysfunction, such as steatosis and aging, has been successful in decreasing their susceptibility to LIRI. Studying mitochondrial dysfunction will help understand the underlying mechanisms of cellular damage during LIRI which is important for the development of new therapeutic strategies aimed at improving patient outcomes. In this review, we highlight the progress made in recent years regarding the role of mitochondria in liver I/R and discuss the impact of liver conditions on LIRI.

Effect of the Non-steroidal Anti-inflammatory Drug Diclofenac on Ischemia-Reperfusion Injury in Rat Liver: A Nitric Oxide-Dependent Mechanism.

Ischemia/reperfusion injury (IRI) is an inevitable complication of liver surgery and transplantation. The purpose of this study was to examine the beneficial effects of diclofenac on hepatic IRI and the mechanism behind it. Wistar rats' livers were subjected to warm ischemia for 60min followed by 24h of reperfusion. Diclofenac was administered intravenously 15min before ischemia at 10, 20, and 40mg/kg body weight. To determine the mechanism of diclofenac protection, the NOS inhibitor L-Nitro-arginine methyl ester (L-NAME) was administered intravenously 10min after diclofenac injection (40mg/kg). Liver injury was evaluated by aminotransferases (ALT and AST) activities and histopathological analysis. Oxidative stress parameters (SOD, GPX, MPO, GSH, MDA, and PSH) were also determined. Then, eNOS gene transcription and p-eNOS and iNOS protein expressions were evaluated. The transcription factors PPAR-γ and NF-κB in addition to the regulatory protein IκBα were also investigated. Finally, the gene expression levels of inflammatory (COX-2, IL-6, IL-1β, IL-18, TNF-α, HMGB-1, and TLR-4) and apoptosis (Bcl-2 and Bax) markers were measured. Diclofenac, at the optimal dose of 40mg/kg, decreased liver injury and maintained histological integrity. It also reduced oxidative stress, inflammation, and apoptosis. Its mechanism of action essentially depended on eNOS activation rather than COX-2 inhibition, since pre-treatment with L-NAME abolished all the protective effects of diclofenac. To our knowledge, this is the first study demonstrating that diclofenac protects rat liver against warm IRI through the induction of NO-dependent pathway. Diclofenac reduced oxidative balance, attenuated the activation of the subsequent pro-inflammatory response and decreased cellular and tissue damage. Therefore, diclofenac could be a promising molecule for the prevention of liver IRI.

Blackberry-Loaded AgNPs Attenuate Hepatic Ischemia/Reperfusion Injury via PI3K/Akt/mTOR Pathway

To achieve site-specific delivery of blackberry leaves extract (BBE) loaded AgNPs to the hepatocyte in IRI and to verify possible molecular mechanisms. IRI was induced in male Wister rats. Liver injury, hepatic histology, oxidative stress markers, hepatic expression of apoptosis-related proteins were evaluated. Non-targeted metabolomics for chemical characterization of blackberry leaves extract was performed. Pre-treatment with BBE protected against the deterioration caused by I/R, depicted by a significant improvement of liver functions and structure, as well as reduction of oxidative stress with a concomitant increase in antioxidants. Additionally, BBE promoted phosphorylation of antiapoptotic proteins; PI3K, Akt and mTOR, while apoptotic proteins; Bax, Casp-9 and cleaved Casp-3 expressions were decreased. LC-HRMS-based metabolomics identified a range of metabolites, mainly flavonoids and anthocyanins. Upon comprehensive virtual screening and molecular dynamics simulation, the major annotated anthocyanins, cyanidin and pelargonidin glucosides, were suggested to act as PLA2 inhibitors. BBE can ameliorate hepatic IRI augmented by BBE-AgNPs nano-formulation via suppressing, oxidative stress and apoptosis as well as stimulation of PI3K/Akt/mTOR signaling pathway.