Fatty Liver Ischemia-reperfusion Injury Research Articles

Targeting S100A9-TLR2 axis controls macrophage NLRP3 inflammasome activation in fatty liver ischemia reperfusion injury

Abstract Liver ischemia reperfusion (IR) injury significantly impacts clinical outcomes by increasing the risk of hepatic dysfunction after liver surgery. Fatty livers are more susceptible to IR stress. Recent studies have demonstrated that S100A9 plays a crucial role in both IR injury and the progression of liver steatosis. Nevertheless, the precise mechanisms underlying these effects remain unclear. In our study, transcriptome analysis of fatty livers subjected to IR insult in mice identified S100A9 as an important mediator. Employing loss-of-function approaches, we investigated the immune regulatory function of S100A9 and its downstream signaling in fatty liver IR injury. As expected, S100A9 emerged as one of the most significantly upregulated genes during the reperfusion stage in fatty livers. Genetic knockdown of S100A9 markedly ameliorated liver pathological damage, evidenced by reduced macrophage/neutrophil infiltration as well as the decreased expression of proinflammatory factors. Transcriptome/functional studies revealed that S100A9 triggered liver inflammatory response via regulating Toll-like receptor 2 (TLR2)/Activating transcription factor 4 (ATF4) signaling. Additionally, TLR2 expression was notably increased in macrophages from ischemic fatty livers. In vitro, recombinant S100A9-stimulated macrophages exhibited the elevated production of proinflammatory factors and TLR2/ATF4 pathway activation. Intriguingly, S100A9 facilitated ATF4 nuclear translocation and enhanced NEK7/NLRP3 inflammasome activation in macrophages. In conclusion, our study identified S100A9 as a key regulator responsible for macrophage NLRP3 inflammasome activation and subsequent inflammatory injury in fatty liver IR process. Targeting TLR2/ATF4 signaling may offer a novel therapeutic strategy for mitigating S100A9-mediated liver injury.

TBHQ mitigates fatty liver ischemia–reperfusion injury by activating Nrf2 to attenuate hepatocyte mitochondrial damage and macrophage STING activation

BackgroundLiver ischemia–reperfusion (IR) injury is an inevitable pathophysiological process in various liver surgeries. Previous studies have found that IR injury is exacerbated in fatty liver due to significant hepatocellular damage and macrophage inflammatory activation, though the underlying mechanisms are not fully understood. In this study, we aim to explore the role and mechanism of Nrf2 (Nuclear factor erythroid 2-related factor 2) signaling in regulating hepatocellular damage and macrophage immune response in fatty liver IR injury. MethodsThe study used high-fat diet-induced fatty liver mice to establish an IR model, alongside an in vitro co-culture system of primary hepatocytes and macrophages. This approach was used to examine mitochondrial dysfunction, oxidative stress, mitochondrial DNA (mtDNA) release, and activation of macrophage STING (Stimulator of interferon genes) signaling. We also conducted recovery verification using H-151 (a STING inhibitor) and tBHQ (an Nrf2 activator). ResultsCompared to the control group, mice on a high-fat diet demonstrated more severe liver IR injury, as evidenced by increased histological damage, elevated liver enzyme levels, and heightened inflammatory markers. The HFD group showed significant oxidative stress and mitochondrial dysfunction and damage post-IR, as indicated by elevated levels of ROS and lipid peroxidation markers, and decreased antioxidant enzyme activity. Elevated mtDNA release from hepatocytes post-IR activated macrophage STING signaling, worsening inflammation and liver damage. However, STING signaling inhibition with H-151 in vivo or employing STING knockout macrophages significantly reduced these injuries. In-depth mechanism studies have found that the transfer of Nrf2 protein into the nucleus of liver cells after IR in fatty liver is reduced. Pre-treatment with tBHQ ameliorated liver oxidative stress, mitochondrial damage and suppressed the macrophage STING signaling activation. ConclusionsOur study reveals a novel mechanism where the interaction between hepatocellular damage and macrophage inflammation intensifies liver IR injury in fatty liver. Enhancing Nrf2 activation to protect mitochondrial from oxidative stress damage and inhibiting macrophage STING signaling activation emerge as promising strategies for clinical intervention in fatty liver IR injury.

Human umbilical cord mesenchymal stem cells alleviate fatty liver ischemia-reperfusion injury by activating autophagy through upregulation of IFNγ.

Liver ischemia-reperfusion injury (IRI) is an important factor affecting the prognosis of liver transplantation, and extended criteria donors (e.g., steatosis donor livers) are considered to be more sensitive to ischemia-reperfusion injury in liver transplantation. Currently, the application of human umbilical cord mesenchymal stem cells (hMSCs) has great promise in the treatment of various injuries in the liver. This study aimed to investigate the therapeutic role and mechanism of hMSCs in fatty liver IRI. After more than 8 weeks of high-fat chow feeding, we constructed a fatty liver mouse model and established ischemic injury of about 70% of the liver. Six hours after IRI, liver injury was significantly alleviated in hMSCs-treated mice, and the expression levels of liver enzyme, inflammatory factor TNF-α, and apoptotic proteins were significantly lower than those of the control group, which were also significant in pathological sections. Transcriptomics analysis showed that IFNγ was significantly upregulated in the hMSCs group. Mechanistically, IFNγ, which activates the MAPK pathway, is a potent agonist that promotes the occurrence of autophagy in hepatocytes to exert a protective function, which was confirmed by in vitro experiments. In summary, hMSCs treatment could slow down IRI in fatty liver by activating autophagy through upregulation of IFNγ, and this effect was partly direct.

Exacerbated ischemia-reperfusion injury in fatty livers is mediated by lipid peroxidation stress and ferroptosis

BackgroundIschemia-reperfusion injury is a common problem in liver surgery and transplantation. Although ischemia-reperfusion injury is known to be more pronounced in fatty livers, the underlying mechanisms for this difference remain poorly understood. We hypothesized that ferroptosis plays a significant role in fatty liver ischemia-reperfusion injury due to increased lipid peroxidation in the presence of stored iron in the fatty liver. To test this hypothesis, the ferroptosis pathway was evaluated in a murine fatty liver ischemia-reperfusion injury model. MethodsC57BL6 mice were fed with a normal diet or a high fat, high sucrose diet for 12 weeks. At 22 weeks of age, liver ischemia-reperfusion injury was induced through partial (70%) hepatic pedicle clamping for 60 minutes, followed by 24 hours of reperfusion before tissue harvest. Acyl–coenzyme A synthetase long-chain family member 4 and 4-hydroxynonenal were quantified in the liver tissues. In separate experiments, liproxstatin-1 or vehicle control was administered for 7 consecutive days before liver ischemia-reperfusion injury. ResultsExacerbated ischemia-reperfusion injury was observed in the livers of high fat, high sucrose diet fed mice. High fat, high sucrose diet + ischemia-reperfusion injury (HDF+IRI) livers had a significantly greater abundance of acyl–coenzyme A synthetase long-chain family member 4 and 4-hydroxynonenal compared with normal diet + ischemia-reperfusion injury (ND+IRI) livers or sham fatty livers, which indicated an increase of ferroptosis. HFD fed animals receiving liproxstatin-1 injections had a significant reduction in serum aspartate transaminase and alanine transaminase after ischemia-reperfusion injury, consistent with attenuation of ischemia-reperfusion injury in the liver. ConclusionFerroptosis plays a significant role in ischemia-reperfusion injury in fatty livers. Inhibiting ferroptotic pathways in the liver may serve as a novel therapeutic strategy to protect the fatty liver in the setting of ischemia-reperfusion injury.

IL-17a exacerbates hepatic ischemia-reperfusion injury in fatty liver by promoting neutrophil infiltration and mitochondria-driven apoptosis.

Hepatic ischemia-reperfusion (IR) injury is a critical issue during liver transplantation (LT). Recent studies have demonstrated that IL-17a contributes to IR injury and steatohepatitis. However, the underlying mechanism is not understood. This study aimed to examine the role of IL-17a on hepatic IR injury in fatty liver and to investigate the underlying mechanisms. The correlation between serum IL-17a levels and liver function was analyzed in LT patients receiving fatty (n=42) and normal grafts (n=44). Rat LT model was applied to validate the clinical findings. IL-17a knockout (KO) and wild-type mice were fed with high-fat diets to induce fatty liver and subjected to hepatic IR injury with major hepatectomy. Frequency of circulating neutrophils and IL-17a expression on PBMCs were analyzed by flow cytometry. Mitochondrial outer membrane permeabilization (MOMP) was examined by a living intravital image system. Serum IL-17a was elevated after human LT, especially with fatty grafts. The aspartate aminotransferase and alanine transaminase levels were increased in recipients with fatty grafts compared with normal grafts. In rat LT model, the intragraft IL-17a expression was significantly higher in fatty grafts than normal ones post-LT. KO of IL-17a in mice notably attenuated liver damage after IR injury in fatty liver, characterized by better-preserved liver architecture, improved liver function, and reduced neutrophil infiltration. MOMP triggered cell death after hepatic IR injury in a caspase-independent way via IL-17a/NF-κB signaling pathway. KO of IL-17a protected the fatty liver against IR injury through the suppression of neutrophil infiltration and mitochondria-driven apoptosis.

Renalase Attenuates Mouse Fatty Liver Ischemia/Reperfusion Injury through Mitigating Oxidative Stress and Mitochondrial Damage via Activating SIRT1.

Liver ischemia/reperfusion (IR) injury is a severe complication of liver surgery. Moreover, nonalcoholic fatty liver disease (NAFLD) patients are particularly vulnerable to IR injury, with higher rates of postoperative morbidity and mortality after liver surgeries. Our previous study found that renalase (RNLS) was highly sensitive and responsive to oxidative stress, which may be a promising biomarker for the evaluation of the severity of liver IR injury. However, the role of RNLS in liver IR injury remains unclear. In the present study, we intensively explored the role and mechanism of RNLS in fatty liver IR injury in vivo and in vitro. C57BL/6 mice were divided into 2 groups feeding with high-fat diet (HFD) and control diet (CD), respectively. After 20 weeks' feeding, they were suffered from portal triad blockage and reflow to induce liver IR injury. Additionally, oleic acid (OA) and tert-butyl hydroperoxide (t-BHP) were used in vitro to induce steatotic hepatocytes and to simulate ROS burst and mimic cellular oxidative stress following portal triad blockage and reflow, respectively. Our data showed that RNLS was downregulated in fatty livers, and RNLS administration effectively attenuated IR injury by reducing ROS production and improving mitochondrial function through activating SIRT1. Additionally, the downregulation of RNLS in the fatty liver was mediated by a decrease of signal transduction and activator of transcription 3 (STAT3) expression under HFD conditions. These findings make RNLS a promising therapeutic strategy for the attenuation of liver IR injury.

Exogenous hydrogen sulfide protects fatty liver against ischemia-reperfusion injury by regulating endoplasmic reticulum stress-induced autophagy in macrophage through mediating the class A scavenger receptor pathway in rats.

Fatty liver disease is a disease manifested with excessive alcohol intake and obese. Importantly, hydrogen sulfide (H2 S) has been revealed to participate in the progression of fatty liver; however, the underlying mechanism has not been clearly elucidated yet. In this study, we aimed to investigate the effects of exogenous H2 S on fatty liver ischemia-reperfusion injury (IRI) through mediating class A scavenger receptor (SRA) pathway in rats. By determining endoplasmic reticulum stress (ERS)-related factors, autophagy markers and apoptosis-related factors in liver tissue and liver function, levels of oxidative stress, inflammatory factors, and hepatocyte apoptosis, the effects of H2 S on IRI-induced autophagy, oxidative stress, and inflammation were all examined in rat model of fatty liver IRI. Results from obtained data showed that H2 S decreased the expression of SRA, Grp78, PERK, CHOP, and Caspase-3, and increased that of LC3-II/LC3-I, in addition to alleviating the pathological changes of liver and reducing the levels of ALT, AST, LDH TBARS, and MDA. Moreover, H2 S decreased the levels of oxidative stress, the expression of pro-inflammatory factors including tumor necrosis factor α, interleukin 1, and interleukin 6, and the apoptosis of hepatocytes. Our findings suggested exogenous H2 S could reduce ERS by mediating the SRA pathway and protect liver function by inducing autophagy, and protect against IRI by reducing oxidative stress and inflammation.

CXCR6 deficiency ameliorates ischemia-reperfusion injury by reducing the recruitment and cytokine production of hepatic NKT cells in a mouse model of non-alcoholic fatty liver disease

Fatty liver is used for transplantation due to organ shortage, but prone to cause complications like ischemia-reperfusion injury (IRI). NKT cells as a bridge between innate and adaptive immunity were reported to infiltrate the liver at the early phase of IRI induced in normal liver. However, the localization mechanism of NKT cells is not precise, and the role of NKT cells in fatty liver IRI is poorly understood. In present murine IRI model of non-alcoholic fatty liver disease, we demonstrated that although the number reduced in fatty liver, NKT cells still activated and accumulated to fatty liver following IRI, and contributed to IRI by producing inflammatory cytokine IFN-γ. We revealed that NKT cells in fatty liver expressed more CXCR6, a vital chemokine receptor; meanwhile, the ligand CXCL16 mRNA expression level in fatty liver was up-regulated. The up-regulation of the CXCR6/CXCL16 axis in fatty liver happened in IRI, which maybe endow NKT cells more chemotaxis. We further found CXCR6 deficiency reduced the recruitment of NKT cells in a tissue-dependent manner, and impaired the IFN-γ producing capacity of hepatic NKT cells. Serum ALT level and hepatic histology were both improved in CXCR6 deficient mice. The results provide evidence of the pathogenic role of NKT cells in fatty liver IRI, and important localization mechanism involving up-regulated CXCR6/CXCL16. Deficiency of CXCR6 protects the fatty liver from IRI by reducing the recruitment and cytokine production of hepatic NKT cells.

Effect of berberine on fatty liver ischemia-reperfusion injury in rats: the relationship with endoplasmic reticulum stress in liver tissues

Objective To evaluate the effect of berberine on fatty liver ischemia-reperfusion(I/R) injury and the relationship with endoplasmic reticulum stress in liver tissues. Methods Thirty-two male Wistar rats, aged 4 weeks, weighing 100-150 g, were divided into 4 groups(n=8 each) using a random number table method: control group(group C), fatty liver group(group FL), I/R group and berberine group(group BBR ). Rats were fed a normal fat diet for 12 weeks, normal saline 3.5 ml was given intragastrically for 4 weeks starting from 9th week, and rats only underwent simple laparotomy in group C. Rats were fed a high-fat diet(45% energy originating from fat) for 12 weeks, and the other treatments were similar to those previously described in group C. Rats were fed a high-fat diet(45% energy originating from fat) for 12 weeks, normal saline 3.5 ml was given intragastrically for 4 weeks starting from 9th week, and then the model of liver I/R injury was established in group I/R. Rats were fed a high-fat diet(45% energy originating from fat) for 12 weeks, berberine solution(300 mg/kg) 3.5 ml was given intragastrically for 4 weeks starting from 9th week, and then the model of liver I/R injury was established in group BBR. Hepatic ischemia was induced by clamping the portal vein, hepatic artery, right gastric vein, and supra- and inferior-hepatic vena cava to perform cold perfusion with 4 ℃ lactated Ringer′s solution lasting for 30 min, followed by reperfusion. The serum triglyceride(TG) concentrations were determined after 4, 8 and 12 weeks of diet. Blood samples were collected at 6 h of reperfusion for measurement of serum aspartate transminase(AST )and alanine transaminase(ALT)concentrations. Livers were removed after blood sampling at 6 h of reperfusion and liver tissues were obtained and stained with oil red O and haematoxylin and eosin for examination of pathological changes and for determination of the expression of Bip, CCAAT/enhancer-binding protein homologous protein(CHOP), protein kinase RNA-like endoplasmic reticulum kinase(PERK) and phosphorylated PERK(p-PERK)(by Western blot). p-PERK/PERK ratio was calculated. Results Compared with group C, the serum TG, ALT and AST concentrations were significantly increased, the expression of Bip and CHOP was up-regulated, p-PERK/PERK ratio was increased(P<0.05), lipid deposition was increased, and liver steatosis was found in group FL. Compared with group FL, the serum AST and ALT concentrations were significantly increased, the expression of Bip and CHOP was up-regulated, p-PERK/PERK ratio was increased(P<0.05), and the pathological changes of liver tissues were accentuated in group I/R. Compared with group I/R, the serum TG, ALT and AST concentrations were significantly decreased, the expression of Bip and CHOP was down-regulated, p-PERK/PERK ratio was decreased(P<0.05), lipid deposition was reduced, and the pathological changes of liver tissues were significantly attenuated in group BBR. Conclusion Berberine can ameliorate fatty liver I/R injury, and the mechanism may be related to inhibiting endoplasmic reticulum stress in liver tissues of rats. Key words: Berberine; Fatty liver; Reperfusion injury; Endoplasmic reticulum; Stress

Influence of intravenous injected sodium hydrosulfide on fatty liver ischemia-reperfusion injury in rats

Objective To investigate whether intravenous injected sodium hydrosulfide (NaHS) could alleviate the fatty liver ischemia-reperfusion injury in rats and the mechanism. Methods 30 healthy male Sprague-Dawley (SD) rats were fed with high fat diet for 4 weeks then divided into five groups randomly, sham operation group (S group), ischemia-reperfusion group (IR group) and different doses of sodium hydrosulfide groups (NaHS 1-3 groups). In S group hepatic portal was only exposed but not ligated. In IR group the left and middle lobes of hepatic portal vein and hepatic artery were clamped for 1 h and then reperfused. NaHS 1-3 groups received intravenous of NaHS 14, 28, 56 μmol/kg 5 min before reperfusion. Blood samples were collected for measuring the level of alanine transaminase (ALT) in serum at 6 h after reperfusion. Liver tissue samples were collected for measurement the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) and pathologic examination. Results The level of ALT in serum of S group, IR group and NaHS 1-3 group were(64±8), (1 393±69), (852±53), (823±42), (807±57) U/L respectively; The content of MDA in liver tissue of S group, IR group and NaHS 1-3 group were (2.15±0.24), (7.97±0.42), (5.26±0.34), (4.95±0.42), (4.77±0.40) nmol/mg prot respectively; the activity of SOD in liver tissue were (421±20), (201±32), (517±27), (601±23), (607±30) U/mg prot respectively. Compared with S group, the level of ALT in serum and MDA content in liver tissue were increased in IR group (t=44.194, P=0.000; t=42.068, P=0.000), and the activity of SOD in liver tissue was decreased in IR group (t=-14.658, P=0.000). Compared with IR group, the level of ALT in serum were decreased in NaHS 1-3 group (t=-11.563, P=0.000; t=-36.415, P=0.000; t=-13.372, P=0.000), and the content MDA in liver tissue were decreased in NaHS 1-3 group (t=-10.375, P=0.000; t=-17.532, P=0.000; t=-12.402, P=0.000), the activity of SOD in liver tissue was increased in NaHS 1-3 group (t=24.087, P=0.000; t=30.451, P=0.000; t=42.542, P=0.000). The pathological damage degree was lighter in NaHS 1-3 group (Suzuki’s score 2.50, 2.33, 2.17) than IR group (Suzuki’s score 3.67) (t=3.873, P=0.012; t=6.325, P=0.001; t=11.608, P=0.000). Conclusion Intravenous injected sodium hydrosulfide can reduce fatty liver ischemia-reperfusion injury in rats through the inhibition of lipid peroxidation. Key words: Sodium hydrosulfide; Liver ischemia; Reperfusion injury; Fatty liver; Lipid peroxidation

Effect of hydrogen sulfide on fatty liver ischemia-reperfusion injury in rats

Objective To investigate the role of hydrogen sulfide in fatty liver ischemia-reperfusion injury in rats. Methods Eighteen healthy male Sprague-Dawley (SD) rats were fed on high fat diet for 4 weeks, and then divided into three groups according to the method of random number table, sham operation group (S group), ischemia-reperfusion group (IR group) and sodium hydrosulfide group (NaHS group). In S group hepatic portal was only exposed but not ligated. In IR group hepatic portal vein and hepatic artery of the left and middle lobes were clamped for 1 h and then reperfused. NaHS group received intravenous injection of NaHS (28 μmol/kg) 5 min before reperfusion. Blood samples were collected for measuring the level of alanine transaminase (ALT) and tumor necrosis factor-α (TNF-α) in serum at 6 h after reperfusion. Liver tissue samples were collected for pathologic examination. Results The serum ALT levels in S group, IR group and NaHS group were (64±8), (1 393±69) and (823±42) U/L, and the serum TNF-α levels in S group, IR group and NaHS group were (55.62±7.24), (288.97±41.42) and (203.65±37.36) ng/L respectively. As compared with S group, the serum levels of ALT and TNF-α were significantly increased in IR group (t=44.194, P=0.000; t=20.586, P=0.000). As compared with IR group, the serum levels of ALT and TNF-α were significantly decreased in NaHS group (t=-36.415, P=0.000; t=-4.556, P=0.006). The pathological damage degree was milder in NaHS group (Suzuki’s score 2.33) than in IR group (Suzuki’s score 3.67; t=6.325, P=0.001). Conclusion Hydrogen sulfide can reduce fatty liver ischemia-reperfusion injury in rats through the inhibition of TNF-α expression. Key words: Hydrogen sulfide; Liver ischemia; Reperfusion injury; Fatty liver; Tumor necrosis factor-α

Ischemia-Reperfusion Injury in Fatty Liver Is Mediated by Activated NADPH Oxidase 2 in Rats.

Liver ischemia-reperfusion (I/R) injury is a severe complication of liver surgery, and steatosis is a risk factor for liver damage. Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate oxidase (NOX) contribute to liver dysfunction. Here we examined the role of NOX in I/R injury of fatty livers. Rats were fed a methionine and choline-deficient diet to induce a fatty liver. Rats then underwent surgically induced partial hepatic ischemia followed by reperfusion. The overall survival rate after I/R was lower in rats with fatty livers than with normal livers (P < 0.01). Necrotic area and the concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG), TNFα, and IL-6 were higher in fatty liver tissue than in normal liver tissue (P < 0.01). The number of p47phox-positive cells was significantly higher in fatty liver tissue than in normal liver tissue after reperfusion and peaked 24 hours after reperfusion. The number of TLR-4 positive cells was significantly higher in fatty liver tissue than in normal liver tissue after reperfusion and peaked 4 and 24 hours after reperfusion coupled with a decreased number of high-mobility group box 1-positive hepatocytes. Apocynin significantly improved the survival rate, necrotic area, and concentrations of 8-hydroxy-2'-deoxyguanosine, TNFα, and IL-6 (P < 0.01). The protective effect of apocynin on fatty livers was greater than on normal livers. Ischemia-reperfusion injury was associated with increased high-mobility group box 1, TLR4, and NOX2. Inhibition of NOX activity improved oxidative stress and may prevent I/R injury in fatty liver.

Intracellular oxygenation and cytochrome oxidase C activity in ischemic preconditioning of steatotic rabbit liver

Background Mild to moderate steatotic livers are used as marginal donors in liver transplantation. Very little is known about the mechanisms of ischemia reperfusion (IR) injury (IRI) in fatty liver. This study aimed to establish whether cytochrome oxidase C (COX) activity is compromised by IRI in fatty liver and whether ischemic preconditioning (IPC) can protect COX activity. Methods New Zealand rabbits were fed on a high-cholesterol diet for 8 weeks to induce moderate hepatic steatosis. Three groups were tested. The IR group underwent 60 minutes of ischemia, followed by 7 hours of reperfusion. The IPC group (IPC + IR) underwent 5 minutes of ischemia, followed by 10 minutes of reperfusion and then 60 minutes of ischemia and 7 hours of reperfusion. The control group (sham) underwent the same surgical procedure, but ischemia was not induced. Deoxyhemoglobin, oxyhemoglobin, and change in the redox state of COX was continuously monitored in vivo by near-infrared spectroscopy. COX and citrate synthase (CS) activity assays were carried out on liver biopsy specimens in vitro. Bile was collected continuously during the procedure and analyzed using proton nuclear magnetic resonance spectroscopy. Results The IR group had decreased COX activity and tissue oxygenation represented by deoxyhemoglobin, oxyhemoglobin, COX, and elevated redox ratios of lactate/pyruvate and β-hydroxybutarate/acetoacetate in vivo and a decrease in COX and CS activity in vitro. The IPC + IR group showed higher levels of all measured parameters in vivo and showed a smaller decrease in COX and CS activity in vitro. Conclusion This study shows that IRI affects COX activity in fatty livers. This is attenuated by IPC.