Hepatic Ischemia-reperfusion Injury Research Articles

Hepatoprotective effects of baicalein against liver ischemia-reperfusion injury and partial hepatectomy in a rat model.

Baicalein is the main active flavonoid in Scutellariae Radix and is included in shosaikoto, a Kampo formula used for treating hepatitis and jaundice. However, little is known about its hepatoprotective effects against hepatic ischemia-reperfusion injury (HIRI), a severe clinical condition directly caused by interventional procedures. We aimed to investigate the hepatoprotective effects of baicalein against HIRI and partial hepatectomy (HIRI + PH) and its potential underlying mechanisms. Male Sprague-Dawley rats received either baicalein (5mg/kg) or saline intraperitoneally and underwent a 70% hepatectomy 15min after hepatic ischemia. After reperfusion, liver and blood samples were collected. Survival was monitored 30min after hepatic ischemia and hepatectomy. In interleukin 1β (IL-1β)-treated primary cultured rat hepatocytes, the influence of baicalein on inflammatory mediator production and the associated signaling pathway was analyzed. Baicalein suppressed apoptosis and neutrophil infiltration, which are the features of HIRI + PH treatment-induced histological injury. Baicalein also reduced the mRNA expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α). In addition, HIRI + PH treatment induced liver enzyme deviations in the serum and hypertrophy of the remnant liver, which were suppressed by baicalein. In the lethal HIRI + PH treatment group, baicalein significantly reduced mortality. In IL-1β-treated rat hepatocytes, baicalein suppressed TNF-α and chemokine mRNA expression as well as the activation of nuclear factor-kappa B (NF-κB) and Akt. Baicalein treatment attenuates HIRI + PH-induced liver injury and may promote survival. This potential hepatoprotection may be partly related to suppressing inflammatory gene induction through the inhibition of NF-κB activity and Akt signaling in hepatocytes.

Novel IL-4/HB-EGF-dependent crosstalk between eosinophils and macrophages controls liver regeneration after ischaemia and reperfusion injury

ObjectivePrevious studies indicate that eosinophils are recruited into the allograft following orthotopic liver transplantation and protect from ischaemia reperfusion (IR) injury. In the current studies, we aim to explore whether...

Quercetin, a natural flavonoid, protects against hepatic ischemia–reperfusion injury via inhibiting Caspase-8/ASC dependent macrophage pyroptosis

IntroductionHepatic ischemia–reperfusion injury (IRI) is an inevitable adverse event following liver surgery, leading to liver damage and potential organ failure. Despite advancements, effective interventions for hepatic IRI remain elusive, posing a significant clinical challenge. The innate immune response significantly contributes to the pathogenesis of hepatic IRI by promoting an inflammatory cytotoxic cycle. We have reported that blocking GSDMD-induced pyroptosis in innate immunity cells protected hepatic IRI from inflammatory injury. However, the search for effective pyroptosis inhibitors continues. ObjectivesThis study aims to evaluate whether quercetin, a natural flavonoid, can inhibit GSDMD-induced pyroptosis and mitigate hepatic IRI. MethodsWe established the hepatic IRI murine model and cellular pyroptosis model to evaluate the efficacy of quercetin. ResultsQuercetin effectively alleviated hepatic IRI-induced tissue necrosis and inflammation. We found that during hepatic IRI, the cleavage of GSDMD occurred in hepatic macrophages, but not in other non-parenchymal cells. Quercetin inhibited the cleavage of GSDMD in macrophages. Moreover, we found that quercetin blocked the ASC assembly to inhibit the formation of NLRP3 inflammasomes and AIM2 inflammasomes, suppressing macrophage pyroptosis. Co-immunoprecipitation experiments confirmed that quercetin inhibited the interaction between ASC and Caspase-8, which is the mechanism of ASC complex and inflammasome formation. Overexpression of Caspase-8 abolished the anti-pyroptosis effect of quercetin in NLRP3 and AIM2 inflammasome signaling. Furthermore, we found that the hepatoprotective activity of quercetin was reduced in myelocytic GSDMD-deficient mice. ConclusionOur findings suggest that quercetin has beneficial effects on hepatic IRI. Quercetin could attenuate hepatic IRI and target inhibition of macrophage pyroptosis via blocking Caspase-8/ASC interaction. We recommend that quercetin might serve as a targeted approach for the prevention and personalized treatment of hepatic IRI in perioperative patients.

Terf2ip deficiency accelerates non-alcoholic steatohepatitis through regulating lipophagy and fatty acid oxidation via Sirt1/AMPK pathway

Background & aimsOur previous study has demonstrated that Telomeric repeat-binding factor 2-interacting protein 1(Terf2ip), played an important role in hepatic ischemia reperfusion injury. This study is aimed to explore the function and mechanism of Terf2ip in non-alcoholic steatohepatitis (NASH). MethodsThe expression of Terf2ip was detected in liver tissue samples obtained from patients diagnosed with NASH. Mice NASH models were constructed by fed with high-fat diet (HFD) or methionine/choline deficient diet (MCD) in Terf2ip knockout and wild type (WT) mice. To further investigate the role of Terf2ip in NASH, adeno-associated viruses (AAV)-Terf2ip was administrated to mice. ResultsWe observed a significant down-regulation of Terf2ip levels in the livers of NASH patients and mice NASH models. Terf2ip deficiency was associated with an exacerbation of hepatic steatosis in mice under HFD or MCD. Additionally, Terf2ip deficiency impaired lipophagy and fatty acid oxidation (FAO) in NASH models. Mechanically, we discovered that Terf2ip bound to the promoter region of Sirt1 to regulate Sirt1/AMPK pathway activation. As a result, Terf2ip deficiency was shown to inhibit lipophagy through the AMPK pathway, while the activation of Sirt1 alleviated steatohepatitis in the livers of mice. Finally, re-expression of Terf2ip in hepatocyes alleviated liver steatosis, inflammation, and restored lipophagy. ConclusionsThese results revealed that Terf2ip played a protective role in the progression of NASH through regulating lipophagy and FAO by binding to Sirt1 promoter. Our findings provided a potential therapeutic target for the treatment of NASH.

Differential Effects of HDAC6 Inhibition Versus Knockout During Hepatic Ischemia-reperfusion Injury Highlight Importance of HDAC6 C-terminal Zinc-finger Ubiquitin-binding Domain.

Ischemia-reperfusion injury (IRI) causes significant morbidity in liver transplantation among other medical conditions. IRI following liver transplantation contributes to poor outcomes and early graft loss. Histone/protein deacetylases (HDACs) regulate diverse cellular processes, play a role in mediating tissue responses to IRI, and may represent a novel therapeutic target in preventing IRI in liver transplantation. Using a previously described standardized model of murine liver warm IRI, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were assessed at 24 and 48 h after reperfusion to determine the effect of different HDAC inhibitors. Broad HDAC inhibition with trichostatin-A (TSA) was protective against hepatocellular damage (P < 0.01 for AST and P < 0.05 for ALT). Although HDAC class I inhibition with MS-275 provided statistically insignificant benefit, tubastatin-A (TubA), an HDAC6 inhibitor with additional activity against HDAC10, provided significant protection against liver IRI (P < 0.01 for AST and P < 0.001 for ALT). Surprisingly genetic deletion of HDAC6 or -10 did not replicate the protective effects of HDAC6 inhibition with TubA, whereas treatment with an HDAC6 BUZ-domain inhibitor, LakZnFD, eliminated the protective effect of TubA treatment in liver ischemia (P < 0.01 for AST and P < 0.01 for ALT). Our findings suggest TubA, a class IIb HDAC inhibitor, can mitigate hepatic IRI in a manner distinct from previously described class I HDAC inhibition and requires the HDAC6 BUZ-domain activity. Our data corroborate previous findings that HDAC targets for therapeutic intervention of IRI may be tissue-specific, and identify HDAC6 inhibition as a possible target in the treatment of liver IRI.

Loureirin B improves H/R-induced hepatic ischemia-reperfusion injury by downregulating ALOX5 to regulate mitochondrial homeostasis.

This study was conceived to explore the role and the mechanism of Loureirin B (LB) in hepatic IRI. The viability of LB-treated AML-12 cells was assessed using CCK-8 assay and inflammatory cytokines were detected using ELISA. The activities of ROS and oxidative stress markers MDA, SOD, and GSH-Px were detected using DCFH-DA and corresponding assay kits. The cell apoptosis and caspase3 activity were estimated with flow cytometry and caspase3 assay kits. The expressions of arachidonate 5-lipoxygenase (ALOX5) and apoptosis- and mitochondrial dynamics-related proteins were detected using western blot. The interaction between LB and ALOX5 was analyzed with molecular docking. The transfection efficacy of oe-ALOX5 was examined with RT-qPCR and western blot. Mitochondrial membrane potential was detected with JC-1 staining and immunofluorescence (IF) assay was employed to estimate mitochondrial fusion and fission. The present work found that LB revived the viability, inhibited inflammatory response, suppressed oxidative stress, repressed the apoptosis, and maintained mitochondrial homeostasis in H/R-induced AML-12 cells, which were all reversed by ALOX5 overexpression. Collectively,LB regulated mitochondrial homeostasis by downregulating ALOX5, thereby improving hepatic IRI.

A single-chain antibody construct with specificity of a natural IgM antibody reduces hepatic ischemia reperfusion injury in mice.

Natural immunoglobulin M (IgM) antibodies have been shown to recognize post-ischemic neoepitopes following reperfusion of tissues and to activate complement. Specifically, IgM antibodies and complement have been shown to drive hepatic ischemia reperfusion injury (IRI). Herein, we investigate the therapeutic effect of C2 scFv (single-chain antibody construct with specificity of a natural IgM antibody) on hepatic IRI in C57BL/6 mice. Compared with PBS-treated mice, C2 scFv-treated mice displayed almost no necrotic areas, significant reduction in serum ALT, AST and LDH levels, and significantly reduced in the number of TUNEL positive cells. Moreover, C2 scFv-treated mice exhibited a notable reduction in inflammatory cells after hepatic IRI than PBS-treated mice. The serum IL-6, IL-1β, TNF-α and MPC-1 levels were also severely suppressed by C2 scFv. Interestingly, C2 scFv reconstituted hepatic inflammation and IRI in Rag1-/- mice. We found that C2 scFv promoted hepatic cell death and increased inflammatory cytokines and infiltration of inflammatory cells after hepatic IRI in Rag1-/- mice. In addition, IgM and complement 3d (C3d) were deposited in WT mice and in Rag1-/- mice reconstituted with C2 scFv, indicating that C2 scFv can affect IgM binding and complement activation and reconstitute hepatic IRI. C3d expression was significantly lower in C57BL/6 mice treated with C2 scFv compared to PBS, indicating that excessive exogenous C2 scFv inhibited complement activation. These data suggest that C2 scFv alleviates hepatic IRI by blocking complement activation, and treatment with C2 scFv may be a promising therapy for hepatic IRI.

Comparing the protective effects of local and remote ischemic preconditioning against ischemia-reperfusion injury in hepatectomy: a systematic review and network meta-analysis.

Local ischemic preconditioning (LIPC) has been proven to be a protective strategy against hepatic ischemia-reperfusion injury (HIRI) during hepatectomy. Growing evidence suggests remote ischemic preconditioning (RIPC) has the potential to reduce liver injury in hepatectomy. Few studies have directly compared the protective effects of these two mechanical preconditioning strategies. Therefore, we performed a network meta-analysis to compare the efficacy of LIPC and RIPC for hepatic injury during liver resection. We searched Cochrane, PubMed, Embase, and China National Knowledge Infrastructure (CNKI) from the database inception to January 2023. We included studies directly comparing the effectiveness of LIPC and RIPC and those comparing LIPC or RIPC with no-preconditioning in liver resection. Postoperative liver function and surgical events were analyzed. Data were expressed as standardized mean differences (SMDs) or odds ratios (ORs) and analyzed using network meta-analysis with random effects model. Following the screening of 268 citations, we identified 26 eligible randomized clinical trials (RCTs) involving 1,476 participants (LIPC arm: 789, RIPC arm: 859, no-preconditioning arm: 1,072). LIPC and RIPC were superior to no-preconditioning in reducing postoperative serum transaminase levels [aspartate aminotransferase (AST): SMD RIPC versus no-preconditioning: -2.05, 95% confidence interval (CI): -3.39, -0.71; SMD LIPC versus no-preconditioning: -1.10, 95% CI: -2.07, -0.12; alanine aminotransferase (ALT): SMD RIPC versus no-preconditioning: -2.24, 95% CI: -4.15, -0.32; SMD LIPC versus no-preconditioning: -1.32, 95% CI: -2.63, -0.01]. No significant difference was observed between RIPC and LIPC in postoperative liver function and surgical outcomes (AST: SMD RIPC versus LIPC: -0.95, 95% CI: -2.52, 0.62; ALT: SMD RIPC versus LIPC: -0.91, 95% CI: -3.11, 1.28). In addition, the subgroup analysis revealed the potential benefits of RIPC in improving liver function, especially in patients who diagnosed with cirrhosis or underwent major resection. RIPC and LIPC could serve as effective strategies in relieving HIRI during hepatectomy. No significant differences were observed between LIPC and RIPC, however, RIPC may be an easily applicable strategy to relieve liver injury in hepatectomy.

Ceramide kinase-mediated C1P metabolism attenuates acute liver injury by inhibiting the interaction between KEAP1 and NRF2

Acute liver injury is the basis of the pathogenesis of diverse liver diseases. However, the mechanism underlying liver injury is complex and not completely understood. In our study, we revealed that CERK, which phosphorylates ceramide to produce ceramide-1-phosphate (C1P), was the sphingolipid pathway-related protein that had the most significantly upregulated expression during acute liver injury. A functional study confirmed that CERK and C1P attenuate hepatic injury both in vitro and in vivo through antioxidant effects. Mechanistic studies have shown that CERK and C1P positively regulate the protein expression of NRF2, which is a crucial protein that helps maintain redox homeostasis. Furthermore, our results indicated that C1P disrupted the interaction between NRF2 and KEAP1 by competitively binding to KEAP1, which allowed for the nuclear translocation of NRF2. In addition, pull-down assays and molecular docking analyses revealed that C1P binds to the DGR domain of KEAP1, which allows it to maintain its interaction with NRF2. Importantly, these findings were verified in human primary hepatocytes and a mouse model of hepatic ischemia‒reperfusion injury. Taken together, our findings demonstrated that CERK-mediated C1P metabolism attenuates acute liver injury via the binding of C1P to the DGR domain of KEAP1 and subsequently the release and nuclear translocation of NRF2, which activates the transcription of cytoprotective and antioxidant genes. Our study suggested that the upregulation of CERK and C1P expression may serve as a potential antioxidant strategy to alleviate acute liver injury.

Protective Effect of Dexmedetomidine Combined with Remote Ischemic Preconditioning on Hepatic Ischemia-reperfusion Injury: A Report on 16 Cases

Protective Effect of Dexmedetomidine Combined with Remote Ischemic Preconditioning on Hepatic Ischemia-reperfusion Injury: A Report on 16 Cases

Analysis and experimental validation of IL-17 pathway and key genes as central roles associated with inflammation in hepatic ischemia-reperfusion injury.

Hepatic ischemia-reperfusion injury (HIRI) elicits an immune-inflammatory response that may result in hepatocyte necrosis and apoptosis, ultimately culminating in postoperative hepatic dysfunction and hepatic failure. The precise mechanisms governing the pathophysiology of HIRI remain incompletely understood, necessitating further investigation into key molecules and pathways implicated in disease progression to guide drug discovery and potential therapeutic interventions. Gene microarray data was downloaded from the GEO expression profile database. Integrated bioinformatic analyses were performed to identify HIRI signature genes, which were subsequently validated for expression levels and diagnostic efficacy. Finally, the gene expression was verified in an experimental HIRI model and the effect of anti-IL17A antibody intervention in three time points (including pre-ischemic, post-ischemic, and at 1h of reperfusion) on HIRI and the expression of these genes was investigated. Bioinformatic analyses of the screened characterized genes revealed that inflammation, immune response, and cell death modulation were significantly associated with HIRI pathophysiology. CCL2, BTG2, GADD45A, FOS, CXCL10, TNFRSF12A, and IL-17 pathway were identified as key components involved in the HIRI. Serum and liver IL-17A expression were significantly upregulated during the initial phase of HIRI. Pretreatment with anti-IL-17A antibody effectively alleviated the damage of liver tissue, suppressed inflammatory factors, and serum transaminase levels, and downregulated the mRNA expression of CCL2, GADD45A, FOS, CXCL10, and TNFRSF12A. Injection of anti-IL17A antibody after ischemia and at 1h of reperfusion failed to demonstrate anti-inflammatory and attenuating HIRI benefits relative to earlier intervention. Our study reveals that the IL-17 pathway and related genes may be involved in the proinflammatory mechanism of HIRI, which may provide a new perspective and theoretical basis for the prevention and treatment of HIRI.

Lactobacillus reuteri mitigates hepatic ischemia/reperfusion injury by modulating gut microbiota and metabolism through the Nrf2/HO-1 signaling.

This study seeks to investigate the impacts of Lactobacillus reuteri (L. reuteri) on hepatic ischemia-reperfusion (I/R) injury and uncover the mechanisms involved. Mice in the I/R groups were orally administered low and high doses of L.reuteri (L.reuteri-low and L. reuteri-hi; 1 × 1010 CFU/d and 1 × 1011 CFU/d), for 4 weeks prior to surgery. Following this, mice in the model group were treated with an Nrf2 inhibitor (ML-385), palmitoylcarnitine, or a combination of both. After treatment with L. reuteri, mice exhibited reduced levels of serum aminotransferase (ALT), aspartate aminotransferase (AST), and myeloperoxidase (MPO) activity, as well as a lower Suzuki score and apoptosis rate. L. reuteri effectively reversed the I/R-induced decrease in Bcl2 expression, and the significant increases in the levels of Bax, cleaved-Caspase3, p-p65/p65, p-IκB/IκB, p-p38/p38, p-JNK/JNK, and p-ERK/ERK. Furthermore, the administration of L. reuteri markedly reduced the inflammatory response and oxidative stress triggered by I/R. This treatment also facilitated the activation of the Nrf2/HO-1 pathway. L. reuteri effectively counteracted the decrease in levels of beneficial gut microbiota species (such as Blautia, Lachnospiraceae NK4A136, and Muribaculum) and metabolites (including palmitoylcarnitine) induced by I/R. Likewise, the introduction of exogenous palmitoylcarnitine demonstrated a beneficial impact in mitigating hepatic injury induced by I/R. However, when ML-385 was administered prior to palmitoylcarnitine treatment, the previously observed effects were reversed. L. reuteri exerts protective effects against I/R-induced hepatic injury, and its mechanism may be related to the promotion of probiotic enrichment, differential metabolite homeostasis, and the Nrf2/HO-1 pathway, laying the foundation for future clinical applications.

A “defend–attack and capture flag” strategy for cascade management of hepatic ischemia reperfusion injury

Hepatic ischemia reperfusion injury (HIRI) following transplantation or resection can lead to irreversible liver dysfunction and even failure, yet current therapeutic interventions are limited. Herein, we introduce a “defend–attack and capture flag” nanotheranostic strategy for the cascade management of HIRI. Firstly, the NIR-II photothermal pretreatment with the developed nanosystem provides preprotection for the liver, acting as “defend”. Subsequently, the pharmacotherapy furnishes direct cure for HIRI via the delivery and controlled release of wedelolactone from the nanosystem, serving as “attack”. Meanwhile, the photoacoustic (PA) imaging ability of the nanosystem enables the real-time monitoring of the inflammatory hypoxia, which is the “flag” to be captured in HIRI treatment. The photothermal preprotection can activate heat shock factor 1 (HSF-1), while the pharmaceutical therapy directly attacks NF-κB, both of which eventually inhibits NLRP3 inflammasome, thus reducing the activity of proinflammatory cytokine IL-1β, ending up with the alleviation of necrosis and apoptosis in HIRI. Such treatment effect can be reported by the real-time PA imaging. By the organic combination of the above three-step mechanism, significantly enhanced therapeutic efficacy was achieved in the animal model of HIRI, providing an effective solution for overcoming the challenge of HIRI.

Bulk and single-cell RNA sequencing analysis with 101 machine learning combinations reveal neutrophil extracellular trap involvement in hepatic ischemia-reperfusion injury and early allograft dysfunction

BackgroundHepatic ischaemia–reperfusion injury (HIRI) is a major clinical concern during the perioperative period and is closely associated with early allograft dysfunction (EAD), acute rejection (AR) and long-term graft survival. Neutrophil extracellular traps (NETs) are extracellular structures formed by the release of decondensed chromatin and granular proteins following neutrophil stimulation. There is growing evidence that NETs are involved in the progression of various liver transplantation complications, including ischaemia–reperfusion injury (IRI). This study aimed to comprehensively analyse the expression patterns of NET-related genes (NRGs) in HIRI, identify HIRI subtypes with distinct characteristics, and develop a reliable EAD prediction model. MethodsMicroarray, bulk RNA-seq, and single-cell sequencing datasets were obtained from the GEO database. Initially, differentially expressed NRGs (DE-NRGs) were identified using differential gene expression analyses. We then utilised a non-negative matrix factorisation (NMF) algorithm to classify HIRI samples. Subsequently, we employed machine learning algorithms to screen the hub NRGs related to EAD and developed an EAD prediction model based on these hub NRGs. Concurrently, we assessed the expression patterns of hub NRGs at the single-cell level using the HIRI. Additionally, we validated C5AR1 expression and its effect on HIRI and NETs formation in a rat orthotopic liver transplantation (OLT) model. ResultsIn this study, we identified 11 DE-NRGs in the HIRI context. Based on these 11 DE-NRGs, HIRI samples were classified into two distinct clusters. Cluster1 exhibited a low expression of DE-NRGs, minimal neutrophil infiltration, mild inflammation, and a low incidence of EAD. Conversely, Cluster2 displayed the opposite phenotype, with an activated inflammatory subtype and a higher incidence of EAD. Furthermore, an EAD prediction model was developed using the four hub NRGs associated with EAD. Based on risk scores, HIRI samples were classified into high- and low-risk groups. The OLT model confirmed substantial upregulation of C5AR1 expression in the liver tissue, accompanied by increased formation of NETs. Treatment with a C5AR1 antagonist improved liver function, reduced tissue inflammation, and decreased NETs formation. ConclusionsThis study distinguished two apparent HIRI subtypes, established a predictive model for EAD, and validated the effect of C5AR1 on HIRI. These findings provide novel perspectives for the development of advanced clinical strategies to enhance the outcomes of liver transplant recipients.

Gut microbiota-derived gamma-aminobutyric acid from metformin treatment reduces hepatic ischemia/reperfusion injury through inhibiting ferroptosis.

Hepatic ischemia/reperfusion injury (HIRI) is a common and inevitable factor leading to poor prognosis in various liver diseases, making the outcomes of current treatments in clinic unsatisfactory. Metformin has been demonstrated to be beneficial to alleviate HIRI in recent studies, however, the underpinning mechanism remains unclear. In this study, we found metformin mitigates HIRI-induced ferroptosis through reshaped gut microbiota in mice, which was confirmed by the results of fecal microbiota transplantation treatment but showed the elimination of the beneficial effects when gut bacteria were depleted using antibiotics. Detailedly, through 16S rRNA and metagenomic sequencing, we identified that the metformin-reshaped microbiota was characterized by the increase of gamma-aminobutyric acid (GABA) producing bacteria. This increase was further confirmed by the elevation of GABA synthesis key enzymes, glutamic acid decarboxylase and putrescine aminotransferase, in gut microbes of metformin-treated mice and healthy volunteers. Furthermore, the benefit of GABA against HIRI-induced ferroptosis was demonstrated in GABA-treated mice. Collectively, our data indicate that metformin can mitigate HIRI-induced ferroptosis by reshaped gut microbiota, with GABA identified as a key metabolite.

Gut microbiota-derived gamma-aminobutyric acid from metformin treatment reduces hepatic ischemia/reperfusion injury through inhibiting ferroptosis

Hepatic ischemia/reperfusion injury (HIRI) is a common and inevitable factor leading to poor prognosis in various liver diseases, making the outcomes of current treatments in clinic unsatisfactory. Metformin has been demonstrated to be beneficial to alleviate HIRI in recent studies, however, the underpinning mechanism remains unclear. In this study, we found metformin mitigates HIRI-induced ferroptosis through reshaped gut microbiota in mice, which was confirmed by the results of fecal microbiota transplantation treatment but showed the elimination of the beneficial effects when gut bacteria were depleted using antibiotics. Detailedly, through 16S rRNA and metagenomic sequencing, we identified that the metformin-reshaped microbiota was characterized by the increase of gamma-aminobutyric acid (GABA) producing bacteria. This increase was further confirmed by the elevation of GABA synthesis key enzymes, glutamic acid decarboxylase and putrescine aminotransferase, in gut microbes of metformin-treated mice and healthy volunteers. Furthermore, the benefit of GABA against HIRI-induced ferroptosis was demonstrated in GABA-treated mice. Collectively, our data indicate that metformin can mitigate HIRI-induced ferroptosis by reshaped gut microbiota, with GABA identified as a key metabolite.

Piperine attenuates hepatic ischemia/reperfusion injury via suppressing the TLR4 signaling cascade in mice

Piperine, the major active substance in black pepper, has been shown to have anti-inflammatory and antioxidant effects in several ischemic diseases. However, the role of piperine in hepatic ischemia/reperfusion injury (HIRI) and its underlying mechanisms remain unclear. In this study, the mice were administered piperine (30 mg/kg) intragastric administration before surgery. After 24 h of hepatic ischemia-reperfusion, liver histopathological evaluation, serum transaminase measurements, and TUNEL analysis were performed. The infiltration of inflammatory cells and production of inflammatory mediators in the liver tissue were determined by immunofluorescence and immunohistochemical staining. The protein levels of toll-like receptor 4 (TLR4) and related proteins such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interleukin-1 receptor-associated kinase 1 (IRAK1), p65, and p38 were detected by western blotting. The results showed that plasma aminotransferase (ALT), aspartate aminotransferase (AST), hepatocyte apoptosis, oxidative stress, and inflammatory cell infiltration significantly increased in HIRI mice. Piperine pretreatment notably repaired liver function, improved the histopathology and apoptosis of liver cells, alleviated oxidative stress injury, and reduced inflammatory cell infiltration. Further analysis showed that piperine attenuated tumor necrosis factor-a (TNF-α) and interleukin 6 (IL-6) production and reduced TLR4 activation and phosphorylation of IRAK1, p38, and NF-κB in HIRI. Piperine has a protective effect against HIRI through the TLR4/IRAK1/NF-κB signaling pathway and may be a safer option for future clinical treatment and prevention of ischemia-related diseases.

Inhibition of 7-dehydrocholesterol reductase prevents hepatic ferroptosis under an active state of sterol synthesis

Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl β-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.

O-sialoglycoprotein Endopeptidase (OSGEP) Suppresses Hepatic Ischemia-Reperfusion Injury-Induced Ferroptosis Through Modulating the MEK/ERK Signaling Pathway.

Hepatic ischemia-reperfusion injury (HIRI) was widely accepted as a critical complication of liver resection and transplantation. A growing body of evidence suggested that O-sialoglycoprotein endopeptidase (OSGEP) was involved in cell proliferation and mitochondrial metabolism. However, whether OSGEP could mediate the pathogenesis of HIRI has still remained unclarified. This study investigated whether OSGEP could be protective against HIRI and elucidated the potential mechanisms. The OSGEP expression level was detected in cases undergoing ischemia-related hepatectomy and a stable oxygen-glucose deprivation/reoxygenation (OGD/R) condition in hepG2 cells. Additionally, it was attempted to establish a mouse model of HIRI, thus, the function and mechanism of OSGEP could be analyzed. At one day after hepatectomy, the negative association of OSGEP expression level with the elevated serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was noted. Moreover, it was attempted to carry out gain- and loss-of-function analyses of OSGEP in hepG2 cells to reveal its influences on OGD/R-induced injury and relevant signaling pathways. The findings suggested that OSGEP overexpression significantly protected hepG2 cells against ferroptotic cell death, while OSGEP consumption had opposite effects. Consistent with in vitro studies, OSGEP deficiency exacerbated liver functions and ferroptoticcell death in a mouse model of HIRI. The results also revealed that OSGEP mediated the progression of HIRI by regulating the MEK/ERK signaling pathway. Rescue experiments indicated that ERK1/2 knockdown or overexpression reversed the effects of OSGEP overexpression or knockdown on hepG2 cells under OGD/R condition. Taken together, the findings demonstrated that OSGEP could contribute to alleviate HIRI by mediating the MEK-ERK signaling pathway, which may serve as a potential prognostic marker and a therapeutic target for HIRI.

Genetic and pharmacological targeting of XBP1 alleviates hepatic ischemia reperfusion injury by enhancing FoxO1-dependent mitophagy

Hepatic ischemia reperfusion (I/R) injury is a common clinical complication. X-box binding protein 1 (XBP1), as a critical regulator of the endoplasmic reticulum stress, has been implicated in a variety of diseases. In this study, we aimed to investigate the effects and the underlying mechanism of XBP1 in the progression of hepatic I/R injury. Hepatocyte-specific XBP1 knockout mice, multiple viral delivery systems and specific pharmacological inhibitors were applied in vivo in a partial hepatic I/R injury mouse model and in vitro in a cell model of hypoxia-reoxygenation (H/R) injury. Mitophagy and autophagic flux were evaluated and fluorescence resonance energy transfer (FRET) as well as immunoprecipitation were performed. The results demonstrated that reperfusion for 6 h represented a critical timepoint in hepatic I/R injury and resulted in significant intracellular mitochondrial dysfunction; led to the breakdown of hepatocytes accompanied by the highest expression levels of XBP1. Hepatocyte-specific XBP1 knockout alleviated hepatic I/R injury via enhanced mitophagy, as demonstrated by the reduction in hepatocellular damage/necrosis and increased expression of mitophagy markers. Mechanistically, XBP1 interacted with FoxO1 directly and catalyzed the ubiquitination of FoxO1 for proteasomal degradation. Targeting XBP1 by genetic or pharmacological techniques potentiated the protein levels of FoxO1, further promoting the activity of the PINK1/Parkin signaling pathway, thus augmenting mitophagy and exerting hepatoprotective effects upon I/R injury. In conclusion, the inhibition of XBP1 potentiated FoxO1-mediated mitophagy in hepatic I/R injury. Specific genetic and pharmacological treatment targeting XBP1 in the perioperative 6 h prior to reperfusion exerted beneficial effects, thus providing a novel therapeutic approach.