AST Release Research Articles

Microvesicles from quiescent and TGF-β1 stimulated hepatic stellate cells: Divergent impact on hepatic vascular injury.

This study evaluated the effect of microvesicles(MVs) from quiescent and TGF-β1 stimulated hepatic stellate cells (HSC-MVs, TGF-β1HSC-MVs) on H2O2-induced human umbilical vein endothelial cells (HUVECs) injury and CCl4-induced rat hepatic vascular injury. HUVECs were exposed to hydrogen peroxide (H2O2) to establish a model for vascular endothelial cell injury. HSC-MVs or TGF-β1HSC-MVs were co-cultured with H2O2-treated HUVECs, respectively. Indicators including cell survival rate, apoptosis rate, oxidative stress, migration, invasion, and angiogenesis were measured. Simultaneously, the expression of proteins such as PI3K, AKT, MEK1+MEK2, ERK1+ERK2, VEGF, eNOS, and CXCR4 was assessed, along with activated caspase-3. SD rats were intraperitoneally injected with CCl4 twice a week for 10 weeks to induce liver injury models. HSC-MVs or TGF-β1HSC-MVs were injected into the tail vein of rats. Liver and hepatic vascular damage were also detected. In H2O2-treated HUVECs, HSC-MVs increased cell viability, reduced cytotoxicity and apoptosis, improved oxidative stress, migration, and angiogenesis, and upregulated protein expression of PI3K, AKT, MEK1/2, ERK1/2, VEGF, eNOS, and CXCR4. Conversely, TGF-β1HSC-MVs exhibited opposite effects. CCl4- induced rat hepatic injury model, HSC-MVs reduced the release of ALT and AST, hepatic inflammation, fatty deformation, and liver fibrosis. HSC-MVs also downregulated the protein expression of CD31 and CD34. Conversely, TGF-β1HSC-MVs demonstrated opposite effects. HSC-MVs demonstrated a protective effect on H2O2-treated HUVECs and CCl4-induced rat hepatic injury, while TGF-β1HSC-MVs had an aggravating effect. The effects of MVs involve PI3K/AKT/VEGF, CXCR4, and MEK/ERK/eNOS pathways.

Targeting antioxidant factor Nrf2 by raffinose ameliorates lipid dysmetabolism-induced pyroptosis, inflammation and fibrosis in NAFLD

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a persistent liver condition that affects both human health and animal productive efficiency on a global scale. A number of naturally occurring compounds activate nuclear factor erythroid 2-related factor 2 (Nrf2) as a transcription factor with important protective effects against many liver diseases, including NAFLD. Raffinose (Ra), an oligosaccharide extracted from several plants, exhibits diverse biological functions. However, the uncertainty lies in determining whether the activation of Nrf2 by Ra can provide a preventive effect on liver lipotoxicity. PurposeThe aim of this study was to shed light on the molecular pathways by which Ra possesses its protective benefits against NAFLD. MethodsExperimental protocols were established using WT and Nrf2-null (Nrf2−/−) mice. Liver samples from each group were collected for Western blot, RT-qPCR, H & E, Sirius red and Oil red O staining. Additionally, serums were processed for ELISA. ALM12 cells were gathered for Western blot and immunofluorescence. Moreover, to elucidate the molecular mechanism of Ra, molecular docking was performed. ResultsOur results indicated that Ra remarkably alleviated liver lipotoxic in vivo and in vitro. Ra treatment effectively corrected hepatic steatosis, the release of AST, ALT, TG, and TC, as well as the depletion of HDL and LDL. Meanwhile, Ra efficiently prevented inflammation by inhibiting the TLR4-MyD88-NF-κB pathway and pyroptosis. Additionally, these findings implied that Ra reduced the production of fibrosis-related proteins, which enhanced collagen deposition. Molecular docking revealed that Ra possessed the ability to bind specific regions of Nrf2, resulting in the enhancement of Nrf2 activation and nuclear translocation. Ra treatment restored serum redox factors and antioxidant enzymes to normal levels; however, these alterations were clearly reversed in Nrf2−/− mice. ConclusionThis study reveals novel information on Ra's protective benefits against liver injury caused by abnormal lipid metabolism; these effects are mostly mediated by Nrf2 activation, suggesting a potential new medicine or treatment strategy for NAFLD.

Microenvironment-responsive smart hydrogels with antibacterial activity and immune regulation for accelerating chronic wound healing

Current therapeutic strategies for chronic refractory wounds remain challenge owing to their unfavorable wound microenvironment and poor skin regeneration ability. Thus far, a regimen for effective chronic refractory wounds management involves bacterial elimination, alleviation of oxidative stress, inhibition of inflammatory response, and promotion of angiogenesis. In this work, an injectable glycopeptide hydrogel based on phenylboronic acid-grafted ϵ-polylysine (EPBA) and poly (vinyl alcohol) (PVA) with pH/reactive oxygen species (ROS) dual-responsive properties was prepared, which exerted intrinsic antibacterial and antioxidant properties. ROS-responsive micelles (MIC) loaded with herb-derived Astragaloside IV (AST) are introduced into the hydrogel before gelation. Attributed to the acidic condition and oxidative stress microenvironment of wound bed, the hydrogel gradually disintegrates, and the released EPBA could help to eliminate bacterial. Meanwhile, the subsequential release of AST could help to achieve anti-oxidation, anti-inflammatory, proangiogenic effects, and regulation of macrophage polarization to accelerate chronic wound healing. In addition, the wound repair mechanism of composite hydrogel accelerating skin regeneration was assessed by RNA-sequencing, exploring a range of potential targets and pathway for further study. Collectively, this multifunctional hydrogel dressing, matching different healing stages of tissue remodeling, holds a great potential for the treatment of chronic refractory wounds.

Splenectomy ameliorates liver cirrhosis by restoring the gut microbiota balance.

Dysbiosis of gut microbiota is frequent in liver cirrhosis (LC) patients, and splenectomy (SP) has been reported to improve LC. Herein, we report the effects of SP on gut microbiota, especially on Veillonella parvula, a Gram-negative coccus of the gastrointestinal tract, in LC mice, and the underlying mechanism. LC mice models were induced by tail vein injection of concanavalin A (ConA), followed by SP. 16s rRNA sequencing was conducted to analyze the effects of ConA induction and SP on mouse gut microbiota and the gene expression affected by gut microbiota. LC mice receiving SP were gavaged with Veillonella parvula. Likewise, hepatic stellate cells (HSC) and hepatocytes (HC) were induced with conditioned medium (CM) of Veillonella parvula. SP alleviated LC in mice by restoring gut barrier function and maintaining gut microbiota balance, with Veillonella as the key genus. The Veillonella parvula gavage on LC mice reversed the ameliorative effect of SP. The CM of Veillonella parvula promoted the activation of HSC and the release of IL-6, IL-1β, and TNF-α. Also, the CM of Veillonella parvula induced HC pyroptosis and the release of ALT and AST. Veillonella parvula represented an imbalance in the gut microbiota, thus enhancing gut-derived endotoxins in the liver with the main target being Tlr4/Nlrp3. Inhibition of Tlr4 blocked Veillonella parvula-induced HC damage, HSC activation, and subsequent LC progression. SP-mediated gut microbiota regulation ameliorates ConA-related LC progression by inhibiting Tlr4/Nlrp3 in the liver.

Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC.

Rosmarinic acid (RA) possesses promising anticancer potential, but further development of chemotherapeutic agents is hindered by their toxicity to off-target tissue. In particular, chemotherapy-related anemia is a major obstacle in cancer therapy, which may be aggravated by hemolysis and eryptosis. This work presents a toxicity assessment of RA in human RBCs and explores associated molecular mechanisms. RBCs isolated from healthy donors were treated with anticancer concentrations of RA (10-800 μM) for 24 h at 37 °C, and hemolysis and related markers were photometrically measured. Flow cytometry was used to detect canonical markers of eryptosis, including phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, cell size by FSC, and oxidative stress by H2DCFDA. Ions and pH were assessed by an ion-selective electrode, while B12 was detected by chemiluminescence. RA elicited concentration-dependent hemolysis with AST and LDH release but rescued the cells from hypotonic lysis at sub-hemolytic concentrations. RA also significantly increased annexin-V-positive cells, which was ameliorated by extracellular Ca2+ removal and isosmotic sucrose. Furthermore, a significant increase in Fluo4-positive cells and B12 content and a decrease in FSC and extracellular pH with KCl efflux were noted upon RA treatment. Hemolysis was augmented by blocking KCl efflux and was blunted by ATP, SB203580, staurosporin, D4476, isosmotic urea, and PEG 8000. RA stimulates Ca2+-dependent and sucrose-sensitive hemolysis and eryptosis characterized by PS exposure, Ca2+ accumulation, loss of ionic regulation, and cell shrinkage. These toxic effects were mediated through energy deprivation, p38 MAPK, protein kinase C, and casein kinase 1α.

Analysis of antioxidant enzymes and oxidative stress markers in the liver of naturally infected Indian water buffalo (Bubalus bubalis) with cystic echinococcosis.

The present study assessed the antioxidants and oxidative stress markers in the liver of buffalo naturally infected with cystic echinococcosis. Infected and non-infected livers were collected from the abattoir, and processed to determine the markers of oxidative stress and antioxidants. In addition, samples were also analyzed for liver tissue injury markers. A significantly higher level of glutathione-s-transferase (GST) and glutathione peroxidase (GPx) were observed in the infected liver compared to a healthy liver. On the other hand, the levels of glutathione reductase (GR) and thioredoxin reductase (TR) were significantly reduced in the infected liver compared to a healthy liver. Reduced glutathione (GSH), a key non-enzymatic antioxidant, was also decreased in the infected than in the non-infected liver. The cystic echinococcosis is accompanied by the enhanced production of ROS with subsequent elevation of lipid peroxidation and protein oxidation, as evident from increased malondialdehyde (MDA) and protein carbonyl (PC), respectively. Enhanced MDA disrupts the cell membrane leading to the release of liver injury markers AST, ALT, ACP, and ALP, which suggest liver damage. This could result from the mechanical pressure and the space-occupying affect of cystic echinococcosis cysts. In summary, our findings suggest that alteration in the level of antioxidants and oxidative stress markers may potentially serve as evidence for the oxidative stress in the liver of infected buffalo.

Dexamethasone mitigates remdesivir-induced liver toxicity in human primary hepatocytes and COVID-19 patients.

Coronavirus disease 2019 (COVID-19) is a global pandemic that has caused more than 600 million cases and over six million deaths worldwide. Despite the availability of vaccination, COVID-19 cases continue to grow making pharmacological interventions essential. Remdesivir (RDV) is an FDA-approved antiviral drug for treatment of both hospitalized and non-hospitalized COVID-19 patients, albeit with potential for hepatotoxicity. This study characterizes the hepatotoxicity of RDV and its interaction with dexamethasone (DEX), a corticosteroid often co-administered with RDV for inpatient treatment of COVID-19. Human primary hepatocytes and HepG2 cells were used as in vitro models for toxicity and drug-drug interaction studies. Real-world data from hospitalized COVID-19 patients were analyzed for drug-induced elevation of serum ALT and AST. In cultured hepatocytes, RDV markedly reduced the hepatocyte viability and albumin synthesis, while it increased the cleavage of caspase-8 and caspase-3, phosphorylation of histone H2AX, and release of ALT and AST in a concentration-dependent manner. Importantly, co-treatment with DEX partially reversed RDV-induced cytotoxic responses in human hepatocytes. Moreover, data from COVID-19 patients treated with RDV with and without DEX co-treatment suggested that among 1037 patients matched by propensity score, receiving the drug combination was less likely to result in elevation of serum AST and ALT levels (≥ 3 × ULN) compared to the RDV alone treated patients (OR = 0.44, 95% CI = 0.22-0.92, p = 0.03). Our findings obtained from in vitro cell-based experiments and patient data analysis provide evidence suggesting combination of DEX and RDV holds the potential to reduce the likelihood of RDV-induced liver injury in hospitalized COVID-19 patients.

Treprostinil Supplementation Ameliorates Hepatic Ischemia Reperfusion Injury and Regulates Expression of Hepatic Drug Transporters: An Isolated Perfused Rat Liver (IPRL) Study

PurposeIR injury is an unavoidable consequence in deceased donor liver transplantation. Cold preservation and warm reperfusion may change the expression and function of drug transporters in the liver due to vasoconstriction, infiltration of neutrophils and release of cytokines. We hypothesize that vasodilation, anti-platelet aggregation and proinflammatory downregulation activities of treprostinil will diminish the IR injury and its associated effects.MethodsLivers obtained from male SD rats (n = 20) were divided into 1) Control, 2) IR, 3) Treprostinil-1 (preservation only), and 4) Treprostinil-2 (preservation and reperfusion) groups. Control livers were procured and immediately reperfused. Livers in the other groups underwent preservation for 24 h and were reperfused. All the livers were perfused using an Isolated Perfused Rat Liver (IPRL) system. Periodic perfusate, cumulative bile samples and liver tissue at the end of perfusion were collected. Liver injury markers, bile flow rates, m-RNA levels for uptake and efflux transporters (qRT-PCR) were measured.ResultsCold preservation and warm reperfusion significantly increased the release of AST and ALT in untreated livers. Treprostinil supplementation substantially reduced liver injury. Bile flow rate was significantly improved in treprostinil-2 group. m-RNA levels of Slc10a1, Slc22a1, and Slc22a7 in liver were increased and m-RNA levels of Mdr1a were decreased by IR. Treprostinil treatment increased Abcb11 and Abcg2 m-RNA levels and maintained Slc22a1m-RNA similar to control livers.ConclusionsTreprostinil treatment significantly reduced liver injury. IR injury changed expression of both uptake and efflux transporters in rat livers. Treprostinil significantly altered the IR injury mediated changes in m-RNA expression of transporters.

Livogrit Prevents Methionine-Cystine Deficiency Induced Nonalcoholic Steatohepatitis by Modulation of Steatosis and Oxidative Stress in Human Hepatocyte-Derived Spheroid and in Primary Rat Hepatocytes

ABSTRACT The prevalence of nonalcoholic steatohepatitis (NASH), characterized by fatty liver, oxidative injury, and inflammation, has considerably increased in the recent years. Due to the complexity of NASH pathogenesis, compounds which can target different mechanisms and stages of NASH development are required. A robust screening model with translational capability is also required to develop therapies targeting NASH. In this study, we used HepG2 spheroids and rat primary hepatocytes to evaluate the potency of Livogrit, a tri-herbal Ayurvedic prescription medicine, as a hepatoprotective agent. NASH was developed in the cells via methionine and cystine-deficient cell culture media. Livogrit at concentration of 30 µg/mL was able to prevent NASH development by decreasing lipid accumulation, ROS production, AST release, NFκB activation and increasing lipolysis, GSH (reduced glutathione), and mitochondrial membrane potential. This study suggests that Livogrit might reduce the lipotoxicity-mediated ROS generation and subsequent production of inflammatory mediators as evident from the increased gene expression of FXR, FGF21, CHOP, CXCL5, and their normalization due to Livogrit treatment. Taken together, Livogrit showed the potential as a multimodal therapeutic formulation capable of attenuating the development of NASH. Our study highlights the potential of Livogrit as a hepatoprotective agent with translational possibilities.

Activity of Hepatic Enzymes of Isolated Hepatocytes under the Influence of Copper Acetate

The article describes a biochemical method for characterizing the hepatocyte culture under the action of CA. Isolated primary hepatocyte culture was exposed to CA in different concentrations, and then the activity of ALT, AST, ALP, and the amount of glucose, in the culture medium was determined. The result showed that the level of cell membrane damage and the release of ALT and AST into the extracellular space depend on the concentration of the acting substance, and, in addition, AST was found to be more sensitive to the toxic effects of CA. However, the activity of ALP proved to be an indicator of the copper ions' action on the enzyme but not of destructive processes in the hepatocyte culture. At the same time, a decrease in the glucose level may be used as a characteristic of the cell metabolic activity. Based on the results obtained, we can conclude that proposed analysis methods allow us to comprehensively characterize the processes occurring with cultured hepatocytes under the influence of CA.

Porcine Liver Normothermic Machine Perfusion: Methodological Framework and Potential Pitfalls.

Porcine models of liver normothermic machine perfusion (NMP) are increasingly used in transplant research, although known to be challenging because of their complex methodology and their scarcely documented operational aspects. Here, we aimed to provide a methodological framework for researchers looking to adopt NMP technology in research setting by giving an in-detail account of the implementation of a previously validated porcine liver NMP model. We subjected groups of 3–5 porcine livers to 24 h NMP and, using a trial-and-error principle, introduced stepwise changes in the NMP setting with the objective to obtain stable preservation of liver function and histology for 24 h. Female porcine livers were procured, and packed red-blood-cell perfusate was prepared. Perfusate oxygenation, hemodynamics, markers of hepatic injury (aspartate transaminase [AST]), function (lactate, perfusate pH, bile production), and histology were analyzed. Intermediate analysis was performed within groups and a minimum of 3 (out of 5) failed experiments prompted methodological reevaluation. Overall, 13 liver NMP experiments were needed in 3 phases. In phase 1, loss of oxygenator performance occurred from 6 h onward in 3 consecutive experiments because of perfusate leakage. In phase 2, a plasma-tight hollow fiber oxygenator ensured adequate perfusate oxygenation in 5 experiments. However, portal vein resistance increased during all liver NMP, associated with high perfusate AST levels (range, 106–322 IU/L/100 g) and pan-lobular sinusoidal dilation and hemorrhage, suggesting liver outflow impairment. In phase 3, an improved inferior vena cava cannulation technique avoided liver outflow impairment, resulting in lower AST release (range, 29–101 IU/L/100 g), improved lactate clearance, preserved biliary excretion, and normal histology in 5 experiments. This study underscores the critical importance of auditing all equipment and operational components of NMP circuits to obtain successful and reproducible perfusion setup and advocates for in-detail reporting of methodological aspects and potential pitfalls.

In vivo treatment with varespladib, a phospholipase A2 inhibitor, prevents the peripheral neurotoxicity and systemic disorders induced by Micrurus corallinus (coral snake) venom in rats

In this study, we investigated the action of varespladib (VPL) alone or in combination with a coral snake antivenom (CAV) on the local and systemic effects induced by Micrurus corallinus venom in rats. Adult male Wistar rats were exposed to venom (1.5 mg/kg – i.m.) and immediately treated with CAV (antivenom:venom ratio 1:1.5 ‘v/w’ – i.p.), VPL (0.5 mg/kg – i.p.), or both of these treatments. The animals were monitored for 120 min and then anesthetized to collect blood samples used for haematological and serum biochemical analysis; after euthanasia, skeletal muscle, renal and hepatic tissue samples were collected for histopathological analysis. M. corallinus venom caused local oedema without subcutaneous haemorrhage or apparent necrosis formation, although there was accentuated muscle morphological damage; none of the treatments prevented oedema formation but the combination of CAV and VPL reduced venom-induced myonecrosis. Venom caused neuromuscular paralysis and respiratory impairment in approximately 60 min following envenomation; CAV alone did not prevent the neurotoxic action, whereas VPL alone prevented neurotoxic symptoms developing as did the combination of CAV and VPL. Venom induced significant increase of serum CK and AST release, mostly due to local and systemic myotoxicity, which was partially prevented by the combination of CAV and VPL. The release of hepatotoxic serum biomarkers (LDH and ALP) induced by M. corallinus venom was not prevented by CAV and VPL when individually administered; their combination effectively prevented ALP release. The venom-induced nephrotoxicity (increase in serum creatinine concentration) was prevented by all the treatments. VPL alone or in combination with CAV significantly prevented the venom-induced lymphocytosis. In conclusion, VPL shows to be effective at preventing the neurotoxic, nephrotoxic, and inflammatory activities of M. corallinus venom. In addition, VPL acts synergistically with antivenom to prevent a number of systemic effects caused by M. corallinus venom.

Antioxidants improve the semen quality following cryopreservation in Indian yak bulls

The present study was conducted to elucidate the effect of different antioxidants on the semen quality of cryopreserved yak semen. The ejaculates found suitable were extended (1:10) with 4 different Tris extenders by split sample technique containing different antioxidants in each of 3 extenders, viz. Taurine @ 50 mM, Trehalose @ 100 mM or vitamin E @ 2 mM and one control (without additive). The sperm motility, live sperm, HOSTreacted sperm, total incidence of acrosomal changes and extracellular release of ALT and AST differed significantly between antioxidants. Total sperm motility, live sperm and HOST-reacted sperm were significantly higher and total incidence of acrosomal changes and extracellular release of AST were significantly lower in extender containing antioxidants than that in control. The difference between taurine, trehalose and vitamin E was not significant for parameters like sperm motility, HOST-reacted sperm and extracellular release of ALT. The per cent live sperm was significantly higher and total incidence of acrosomal changes was significantly lower for taurine than for trehalose and vitamin E. The extracellular release of ALT was significantly lower in extender containing taurine or trehalose than in control while the extracellular release of AST was significantly lower in the extender containing taurine than that in containing trehalose, vitamin E and control. The findings of the present study revealed that addition of taurine, trehalose and vitamin E in Tris extender significantly improved the post thaw quality of yak semen. Among the antioxidants evaluated, 50 mM of taurine in Tris extender could be effectively used to obtain better quality of frozen thawed yak semen.

Sources and clinical significance of aspartate aminotransferase increases in COVID-19

BackgroundAspartate aminotransferase (AST) is often increased in COVID-19 and, in some studies, AST abnormalities were associated with mortality risk. Methods2054 hospitalized COVID-19 patients were studied. To identify sources of AST release, correlations between AST peak values and other biomarkers of tissue damage, i.e., alanine aminotransferase (ALT) for hepatocellular damage, creatine kinase (CK) for muscle damage, lactate dehydrogenase for multiorgan involvement, alkaline phosphatase and γ-glutamyltransferase for cholestatic injury, and C-reactive protein (CRP) for systemic inflammation, were performed and coefficients of determination estimated. The role of AST to predict death and intensive care unit admission during hospitalization was also evaluated. All measurements were performed using standardized assays. ResultsAST was increased in 69% of patients. Increases could be fully explained by summing the effects of hepatocellular injury [AST dependence from ALT, 66.8% [95% confidence interval (CI): 64.5–69.1)] and muscle damage [AST dependence from CK, 42.6% (CI: 39.3–45.8)]. We were unable to demonstrate an independent association of AST increases with worse outcomes. ConclusionThe mechanisms for abnormal AST in COVID-19 are likely multifactorial and a status related to tissue suffering could play a significant role. The clinical significance of AST elevations remains unclear.

Assessment of cadmium toxicity and its possible effects on goldfish (Carassius auratus), employing microscopy and biochemical techniques.

The present study aimed to evaluate and compare the toxicity level of dietary and waterborne Cadmium (Cd) source on the Goldfish Carassius auratus (C. auratus) using various biochemical and microscopic shreds of evidence. C. auratus were divided into two groups based on Cadmium supplementation methods subjected to the same concentrations in the diet and water; moreover, control (Ctrl) group was set up for comparison. Dietary supplemented group (DSG) group and (Aqueous supplemented groups) ASG were supplied with 0.5% of the Cadmium. Successful completion of the study resulted in the lucrative outcomes, and we noticed significantly high concentrations of Cadmium in the gills of the ASG fish while considerably low levels in the gills of DSG. Histopathological evidence showed clear and distinct changes in the liver cells, dilated sinusoids and unusual occurrence of Kupffer cells were noticed in the AS group fish. At the same time, there were also noticeable changes in the DS group compared to CTRL. Visible changes can be seen on the outer morphology of gills, lamella of ASG and DSG both were shrunk and broken, similar changes were noticed in the muscles myoepithelium. The stress parameters were differentially expressed in both the groups; waterborne Cadmium caused a pronounced release of AST, ALT, cortisol, and glucose concentrations. Comparative mortality showed the highest death rate in the ASG fish compared to DSG fish. The present study depicts the highest toxicity of Cadmium in the soluble Addendum form moreover, administered route and time length are crucial factors.

Study on the role and possible mechanism of hemeoxygenase-1/carbon monoxide system in protection of quercetin against ethanol-induced hepatocytes oxidative injury

Objective: To study the protective effect and potential mechanism of heme oxygenase (HO-1)/carbon monoxide (CO)-mediated quercetin on alcoholic oxidative damage of primary rat hepatocytes. Methods: Primary rat hepatocytes were isolated and cultured by two-step collagenase technique. Ethanol exposed primary rat hepatocytes were simultaneously added with quercetin (100 μmol/L) and/or hemoglobin (100 μmol/L) or different doses of CO-releasing molecules (CORM-2, 5-50 μmol/L) for their combined action. After polling, LDH, AST activities and MDA and GSH levels were measured in the supernatant of cell culture. The alone or combined effects of quercetin, CORM-2, hemoglobin and zinc protoporphyrin IX exposed to ethanol were detected by the activity of CYP2E1 in liver microsomes. Statistical analysis of data was performed by analysis of variance (ANOVA) and intergroup comparison was done by SNK-test. Results: Simultaneous addition of 100 μmol/L quercetin had significantly reduced ethanol-induced AST and LDH release, and GSH consumption and MDA elevation extent. Moreover, quercetin had not only lost the hemoglobin (CO blocker) protective effect but also had further exacerbated ethanol-induced lipid peroxidation. CORM-2 had reduced ethanol-induced AST and LDH release, and GSH consumption and MDA production in liver cells, and thus had dose-dependent protective effect. Ethanol had increased significantly CYP2E1 activity. Quercetin or CORM-2 had inhibited CYP2E1 activity, while hemoglobin or protoporphyrin IX had eliminated quercetin inhibitory effect and had increased the CYP2E1 activity. Quercetin, and CYP2E1 activity was constant as compared to ethanol group when CORM-2, zinc protoporphyrin IX and ethanol were incubated with hepatocytes, but the CYP2E1 activity was significantly decreased (P < 0.05), and the differences were statistically significant. Conclusion: CO/HO-1 metabolite mediates the protective effect of quercetin on alcoholic oxidative damage of hepatocytes, which may be related to the inhibition of CYP2E1 activity.

1080-P: Addressing the Safety Challenges of Aldose Reductase Inhibition: Development of AT-001 for Diabetic Cardiomyopathy, a Potent and Selective Next Generation Aldose Reductase Inhibitor

Diabetic Cardiomyopathy (DbCM) leading to overt heart failure is a common sequelae of both type 1 and type 2 diabetes. Prior attempts to develop treatments for DbCM via inhibition of Aldose Reductase (AR) were unsuccessful, due to low AR binding affinity and off-target binding with Aldehyde Reductase (AldR), an enzyme critical for detoxification of aldehydes in the liver and normal hepatocyte function. This resulted in liver-related safety and tolerability issues with first generation ARIs. We report on the selectivity and specificity of AT-001, a novel small molecule ARI with optimized affinity and specificity for AR and minimal to zero off-target AldR activity. Methods and Results: AT-001 was evaluated for AR binding affinity vs. zopolrestat, a prior best in class ARI, which had been challenged with liver toxicity issues in clinical development. AT-001 demonstrated logarithmically improved AR inhibitory activity (IC50 of 30 pmol for AT-001 and 10 nmol for zopolrestat). Liver enzyme evaluations demonstrated that AT-001 showed no inhibition of AldR at 50x and 100x EC50 levels in assay medium, while zopolrestat inhibited AldR by 50% and 60% respectively (spec activity of 2.3 and 1.9 mmol NADPH/min/mg prot.). Additionally no elevations of ALT/AST’s were observed with AT-001 at doses up to 50x EC50, in contrast to zopolrestat which showed dose dependent release of ALT and AST (consistent with hepatocyte damage and eventual cell death). Finally, AT-001 was evaluated in a standard off-target receptor binding analysis of 87 substrates (13 enzymes, 74 binding assays); with no significant results (&amp;gt;50% inhibitory activity) observed. Conclusion: The unique structure and activity of AT-001 provide potent selectivity for Aldose Reductase and lack of off-target effects. The in-vitro safety of this agent together with the positive safety data from the phase 1/2 program, supports the ongoing pivotal study in DbCM. Disclosure R. Perfetti: Stock/Shareholder; Self; Applied Therapeutics. G. Yepuri: None. N.A. Quadri: None. A.F. Ghannam: Employee; Self; Applied Therapeutics Inc. Stock/Shareholder; Self; Sanofi US. R. Ramasamy: Consultant; Self; Applied Therapeutics. S. Shendelman: Employee; Self; Applied Therapeutics. Stock/Shareholder; Self; Applied Therapeutics. Funding Applied Therapeutics (NCT04083339)

Synergistic hepatoprotective effects of CGplus on CCl4-induced acute injury.

Traditional Chinese/Korean medicine suggests "blood stasis ()", "stagnation of vital energy ()" and "dampness and Phlegm ()" as the main etiologies of liver disorders, and multiherbal formulas are generally believed to exert synergistic action. The present study aimed to investigate the synergistic hepatoprotective effects of CGplus (a mixture of Salviae miltiorrhiza, Artemisia iwayomogi and Ammomum xanthioides) compared to those of the individual herbs. A total of fifty-six male Balb/C mice were randomly divided into eight groups and were administered water (normal and CCl4 groups), 100mg/kg S. miltiorrhiza, A. iwayomogi, or A. xanthioides, 50 or 100mg/kg CGPlus or dimethyl dimethoxybiphenyl dicarboxylate (DDB) as a positive control for 4 consecutive days. After a single CCl4 injection (i.p., 10mL/kg of 0.2% CCl4 in olive oil), blood and liver tissues were collected after 18h of fasting for serum biochemistry, histopathological examination and molecular analyses. CCl4 injection induced drastic hepatic injury characterized by a more than 30-fold increase in the release of AST and ALT into the serum. These alterations were significantly attenuated by pretreatment with each of the three herbs, while the effects of the individual herbs were synergistically augmented by CGPlus pretreatment. The synergistic hepatoprotective actions of CGPlus were demonstrated consistently by analyses of oxidative stress (oxidative stressors, oxidation products and antioxidant enzymes), pro-/anti-inflammatory cytokines (TNF-ɑ, IL-1β, IL-6, IL-10), and apoptosis (caspase-3, p53 and BAX) and histopathology. These data suggest that CGPlus exerts its hepatoprotective effects in a synergistic manner, and further studies are required for clinical application using other chronic models.

Hypothermic oxygenated machine perfusion alleviates liver injury in donation after circulatory death through activating autophagy in mice.

Hypothermic oxygenated machine perfusion (HOPE) is a safe and reliable method that could alleviate liver injury in donation after circulatory death (DCD). This study focuses on the role of autophagy in HOPE's protective effect on DCD liver injury. A 30-minute warm ischemic liver model was established in mice. After 4 hours of cold storage (CS), 1 hour of hypothermic machine perfusion (HMP) with 100% O2 or 100% N2 was employed. During 2 hours of reperfusion, liver tissue and perfusate were collected to evaluate liver function, oxidative stress level, apoptosis, and necrosis. Western blotting was used to explore the level of autophagy. When the liver experienced warm ischemic injury, LC3B-II expression was significantly enhanced. Compared with the CS, HOPE induced lower release of AST and ALT, as well as lower oxidative stress levels, apoptosis, and necrosis cell numbers, and led to higher tissue ATP content. Meanwhile, expression of autophagy-related proteins, such as ULK1, Atg5, and LC3B-II, increased. When oxygen was completely replaced by nitrogen, the washout effect of HMP did not activate autophagy and did not relieve DCD liver injury. When the autophagy inhibitor 3-methyladenine was used in HOPE, the protective effect of HOPE was attenuated. In conclusion, DCD liver injury activated autophagy compared with healthy liver, while HOPE alleviated DCD liver injury by increasing autophagy levels further in this mouse model. However, HMP with 100% of N2 had no beneficial effect on DCD liver injury or on autophagy levels compared with CS. The research on autophagy may provide a new strategy for alleviating DCD liver injury in clinical practice.

The Synthesis Of Coagulation Factors During Normothermic Machine Perfusion Of Livers Is Impaired By Ischemia In Pigs And Might Predict Graft Viability

Normothermic Machine Perfusion (NMP) of livers is a promising tool to test graft viability during preservation. Coagulation factors are synthetized by the liver, hence are candidate markers of function. We evaluated the production of coagulation factors during NMP, tested if ischemia alters their synthesis, and correlated their levels with markers of hepatocyte damage (AST) and sinusoidal endothelial injury {Hyaluronic acid (HAc)}. Method Porcine livers exposed to 60 min Warm Ischemia (WI60, n=5) or not (WI0, n=5) were NMP perfused for 6 h. The concentration of Factors V (FV), VIII (FVIII), and X (FX) in the perfusate was measured at 15, 30min, 1, 2, 3, and 6h of NMP and compared within and between groups (between-within ANOVA). Levels of AST and HAc were correlated to FV and FVIII. Mean ±SD is given. Results FV increased significantly over time in both groups, although with a different kinetics, as a plateau was reached at 2h of NMP in WI0 only. FV concentration was inferior in WI60 vs. WI0 (p<0.0001) (Figure 1A). Similarly, FVIII raised over time significantly in both WI0 and WI60, reaching a plateau at 3h of NMP only in WI0. The production of FVIII was inferior in WI60 vs. WI0 (p=0.006) (Figure 1B). FX raised steadily without a plateau in WI0, while no significant increase was observed in WI60, in which the concentration of FX was inferior (p=0.001) (Figure 1C). The area under the curve of AST was greater in WI60 (3077) vs WI0 (405.3, p=0.0006). FV was inversely correlated to AST release at all time points except 6h. HAc levels increased over time [11.3 ±5.3 ng/mL at 15min to 101.8 ±75.5 ng/mL at 6h in WI0 (p=0.045), and from 51.5 ±6.9 ng/mL at 15min to 303.2 ±107.7 ng/mL at 6h in WI60 (p=0.01)] and were higher in WI60 vs WI0 (p=0.004). FVIII was inversely correlated to HAc levels at 1h (r:-0.58, p=0.02) and 2h (r:-0.59, p=0.03) of NMP. Discussion Coagulation factors are actively produced during NMP and their synthesis was impaired during NMP of severely WI damaged porcine livers. In particular, FV and FVIII, produced by hepatocytes and sinusoidal cells, respectively, can be used as markers of function of the hepatocellular and endothelial compartment, since they were inversely related to markers of hepatocellular injury (AST) and sinusoidal cell damage (HAc). FX synthesis during NMP should be further investigated, since its production is dependent on vitamin K, which was not supplemented in the perfusion system. The correlation of our findings to post-transplant outcomes warrants further investigations. Conclusion The production of coagulation factors during NMP of human livers and their role in the assessment of viability deserve to be explored.