Administration Of D-galactosamine Research Articles

Mesenchymal Stem Cells Alleviate Acute Liver Failure through Regulating Hepatocyte Apoptosis and Macrophage Polarization.

Acute liver failure (ALF) is a life-threatening clinical problem with limited treatment options. Administration of human umbilical cord mesenchymal stem cells (hUC-MSCs) may be a promising approach for ALF. This study aimed to explore the role of hUC-MSCs in the treatment of ALF and the underlying mechanisms. A mouse model of ALF was induced by lipopolysaccharide and d-galactosamine administration. The therapeutic effects of hUC-MSCs were evaluated by assessing serum enzyme activity, histological appearance, and cell apoptosis in liver tissues. The apoptosis rate was analyzed in AML12 cells. The levels of inflammatory cytokines and the phenotype of RAW264.7 cells co-cultured with hUC-MSCs were detected. The C-Jun N-terminal kinase/nuclear factor-kappa B signaling pathway was studied. The hUC-MSCs treatment decreased the levels of serum alanine aminotransferase and aspartate aminotransferase, reduced pathological damage, alleviated hepatocyte apoptosis, and reduced mortality in vivo. The hUC-MSCs co-culture reduced the apoptosis rate of AML12 cells in vitro. Moreover, lipopolysaccharide-stimulated RAW264.7 cells had higher levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β and showed more CD86-positive cells, whereas the hUC-MSCs co-culture reduced the levels of the three inflammatory cytokines and increased the ratio of CD206-positive cells. The hUC-MSCs treatment inhibited the activation of phosphorylated (p)-C-Jun N-terminal kinase and p-nuclear factor-kappa B not only in liver tissues but also in AML12 and RAW264.7 cells co-cultured with hUC-MSCs. hUC-MSCs could alleviate ALF by regulating hepatocyte apoptosis and macrophage polarization, thus hUC-MSC-based cell therapy may be an alternative option for patients with ALF.

Influence of Placenta Cryoextract on the Liver Metabolic and Functional State in Case of D-Galactosamine Hepatitis

Background. Viral hepatitis is one of the most common and dangerous diseases in the world and is the third most common infectious disease. The development of new, more effective and safer hepatoprotective drugs is an urgent task of biomedicine. A wide range of proven biological properties in cryoextract of human placenta, in particular the presence of antioxidant, immunomodulating and anti-inflammatory effects, suggests that it has a hepatoprotective effect. A model of D-galactosamine toxic hepatitis, which is similar to human viral hepatitis in terms of morphological and biochemical changes in the liver, was chosen for the study. Objective. We are aimed to study the effect of the therapeutic and preventive administration of cryopreserved placenta extract on the metabolic and functional state of the liver in the model of D-galactosamine hepatitis in rats. Methods. The study was conducted on 28 male rats weighing 200–220 g. Hepatitis was simulated by a single intraperitoneal injection of a 20% aqueous solution of D-galactosamine at a dose of 400 mg/kg. The cryoextract was administered in the treatment-prophylactic mode – 1 time per day for 3 days before the administration of D-galactosamine and another 2 days after the administration of the aminosugar (5 administrations in total). Results. The development of experimental D-galactosamine hepatitis in rats leads to the formation of functional and metabolic disorders in the form of the activation of lipid peroxidation processes, a violation of pigment metabolism, a decrease in the protein-synthesizing function and the development of cytolytic syndrome, which were indicated by an increase (p < 0.001) in the level of reactants with thiobarbituric acid in liver homogenates by 2.2 times, an increase (p < 0.001) in the level of total bilirubin by 2.5 times, a decrease (p < 0.001) in the albumin-globulin ratio by 46.8% and an increase (p < 0.001) in the level of alanine-aminotransferases by 2.2 times and the level of aspartate-aminotransferases by 70.3% compared to the values ​​of intact animals. Against the background of the administration of placenta cryoextract in experimental hepatitis, the level of reactants with thiobarbituric acid decreased (p < 0.001) by 43.8%, the level of alanine-aminotransferases decreased (p < 0.001) by 2.4 times, and the level of aspartate-aminotransferases decreased (p < 0.001) by 45.3%; the level of total protein increased (p < 0.01) by 17.4%, and the level of total bilirubin decreased (p < 0.001) by 53.5% compared to the indicators of untreated animals. Conclusions. Administration of cryopreserved placenta extract normalized metabolic processes in the liver and restored its functional state due to antioxidant and membrane-stabilizing effects, which weakened the cytolytic syndrome caused by the administration of D-galactosamine and restored the protein-synthesizing function of the liver. In addition, administration of the specified cryoextract neutralized D-galactosamine-induced hyperbilirubinemia.

TRAPS mutations in Tnfrsf1a decrease the responsiveness to TNFα via reduced cell surface expression of TNFR1.

Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autoinflammatory periodic fever syndrome associated with heterozygous mutations in TNFRSF1A, which encodes TNF receptor type I (TNFR1). Although possible proinflammatory mechanisms have been proposed, most previous studies were performed using in vitro overexpression models, which could lead to undesirable inflammatory responses due to artificial overexpression. It is crucial to reproduce heterozygous mutations at physiological expression levels; however, such studies remain limited. In this study, we generated TRAPS mutant mice and analyzed their phenotypes. Three Tnfrsf1a mutant strains were generated by introducing T79M, G87V, or T90I mutation. T79M is a known mutation responsible for TRAPS, whereas G87V is a TRAPS mutation that we have reported, and T90I is a variant of unknown significance. Using these murine models, we investigated whether TRAPS mutations could affect the inflammatory responses in vivo and in vitro. We found that none of the mutant mice exhibited detectable inflammatory phenotypes under standard housing conditions for 1 year. Interestingly, TRAPS mutant (T79M and G87V) mice had reduced mortality rates after the administration of lipopolysaccharide (LPS) and D-galactosamine, which induce TNFα-dependent lethal hepatitis. Moreover, TRAPS mutations strongly suppressed the development of TNFα-mediated arthritis when crossed with human TNFα transgenic mice. In in vitro primary bone marrow-derived macrophage cultures, the T79M and G87V mutations attenuated the inflammatory responses to TNFα compared with the wild-type, whereas these mutations did not alter the responsiveness of these cells to LPS. The T90I mutant macrophages behaved similarly to wild type in response to LPS and TNFα. The TNFR1 levels were increased in whole-cell lysates of TRAPS mutant macrophages, whereas the cell surface expression of TNFR1 was significantly decreased in TRAPS mutant macrophages. Taken together, TRAPS mutations did not augment the inflammatory responses to TNFα and LPS; instead, they suppressed the response to TNFα via decreased cell surface expression of TNFR1. The stimulation of lymphotoxin-α, adenosine triphosphate, and norepinephrine in primary macrophages or various stimuli in murine splenocytes did not induce detectable inflammatory responses. In conclusion, TRAPS mutations suppressed responsiveness to TNFα, and TRAPS-associated inflammation is likely induced by unconfirmed disease-specific proinflammatory factors.

Protective activity of purpurin against d-galactosamine and lipopolysaccharide-induced hepatorenal injury by upregulation of heme oxygenase-1 in the RBC degradation cycle.

Acute liver failure, associated with oxidative stress and sustained inflammation is the major clinical manifestation of liver diseases with a high mortality rate due to limited therapeutic options. Purpurin is a bioactive compound of Rubia cordifolia that has been used in textile staining, as a food additive, and as a treatment of multiple chronic and metabolic diseases associated with inflammation and oxidative stress. The present work aimed to investigate the protective efficacy of purpurin against hepatorenal damage. Thirty-six female albino rats were equally assigned into six groups. Purpurin was administered orally once a day for 6 days at doses of 05, 10, and 20 mg/kg, respectively. Intraperitoneal injection of lipopolysaccharide (50 μg/kg) was administered to the animals on 6th day evening, 1 h after d-galactosamine (300 mg/kg) administration to induce hepatorenal injury. The results revealed that purpurin alleviated alterations in serological and hematological parameters as well as restored histoarchitectural and cellular integrity of the liver and kidney. Purpurin restored superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and glutathione content in hepatorenal tissues. Accompanied by the diminution of increased bilirubin and biliverdin, purpurin also diminished total cholesterol, triglyceride, and lipid peroxidation in hepatorenal tissues. Purpurin markedly attenuated the elevation of CYP2E1, restored glutathione-S-transferase, and prevented DNA damage in hepatorenal tissues. Purpurin reduced iron overload by reducing heme depletion and recycling of ferritin and hemosiderin. It also reinforced biliverdin reductase, heme oxygenase-1 to employ hepatorenal protection by regulating antioxidant enzymes and other pathways that produced NADPH. Thus, it may be concluded that purpurin has protective potential against acute hepatorenal injury.

Albumin protects the liver from tumor necrosis factor α-induced immunopathology.

Besides its oncotic power, albumin exerts pleiotropic actions, including binding, transport, and detoxification of endogenous and exogenous molecules, antioxidant activity, and modulation of immune and inflammatory responses. In particular, recent studies have demonstrated that albumin reduces leukocyte cytokine production. Here, we investigated whether albumin also has the ability to protect tissues from the damaging actions of these inflammatory mediators. We circumscribed our investigation to tumor necrosis factor (TNF) α, which exemplifies the connection between immunity and tissue injury. In vivo experiments in analbuminemic mice showed that these mice exhibit a more pronounced response to a model of TNFα-mediated liver injury induced by the administration of lipopolysaccharide (LPS) and D-galactosamine (D-gal). A tissue protective action against LPS/D-gal liver injury was also observed during the administration of human albumin to humanized mice expressing the human genes for albumin and neonatal Fc receptor (hAlb+/+ /hFcRn+/+ ) with preestablished carbon tetrachloride (CCl4 )-induced early cirrhosis. The cytoprotective actions of albumin against TNFα-induced injury were confirmed ex vivo, in precision-cut liver slices, and in vitro, in primary hepatocytes in culture. Albumin protective actions were independent of its scavenging properties and were reproduced by recombinant human albumin expressed in Oryza sativa. Albumin cytoprotection against TNFα injury was related to inhibition of lysosomal cathepsin B leakage accompanied by reductions in mitochondrial cytochrome c release and caspase-3 activity. These data provide evidence that in addition to reducing cytokines, the albumin molecule also has the ability to protect tissues against inflammatory injury.

Complement-5 Inhibition Deters Progression of Fulminant Hepatitis to Acute Liver Failure in Murine Models

Background & AimsAcute liver failure (ALF) is a life-threatening condition with limited treatment alternatives. ALF pathogenesis seemingly involves the complement system. However, no complement-targeted intervention has been clinically applied. In this study, we aimed to investigate the potential of Complement-5 (C5)-targeted ALF treatment.MethodsALF was induced in C5-knockout (KO, B10D2/oSn) mice and their wild-type (WT) counterparts (B10D2/nSn) through intraperitoneal lipopolysaccharide (LPS) and d-galactosamine (D-GalN) administration. Thereafter, monoclonal anti-C5 antibody (Ab) or control immunoglobulin was administered intravenously. Furthermore, a selective C5a-receptor (C5aR) antagonist was administered to WT mice to compare its efficacy with that of anti-C5-Ab-mediated total C5 inhibition. We clarified the therapeutic effect of delayed anti-C5-Ab administration after LPS/D-GalN challenge. We also assessed the efficacy of anti-C5-Ab in another ALF model, using concanavalin-A.ResultsLiver injury was evident 6 hours after LPS/D-GalN administration. C5-KO and anti-C5-Ab treatment significantly improved overall animal survival and significantly reduced serum transaminase and high-mobility group box-1 release with decreased histological tissue damage. This improvement was characterized by significantly reduced CD41+ platelet aggregation, maintained F4/80+ cells, and less infiltration of CD11+/Ly6-G+ cells with lower cytokine/chemokine expression. Furthermore, C5-KO and anti-C5-Ab downregulated tumor necrosis factor-α production by macrophages before inducing marked liver injury. Moreover, single-stranded-DNA cells and caspase activation were reduced, indicating significant attenuation of apoptosis. Anti-C5-Ab treatment protected the liver more effectively than the C5aR antagonist, and its delayed doses were hepatoprotective. In addition, anti-C5-Ab treatment was effective against concanavalin-A–induced ALF.ConclusionsC5 inhibition effectively suppresses progression to ALF in mice models of fulminant hepatitis, serving as a new potential treatment strategy for ALF.

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Liver disease is among the most serious ailments and can be classified as acute or chronic hepatitis, hepatosis, and cirrhosis. Besides expensive and ineffectual modern therapeutic agents like steroids and chemotherapy, South East Asian countries like India and China have an edge in treating hepatic disorders employing their native botanicals. The present study was designed to evaluate the protective effects of Arogyavardhani on D-galactosamine (d-GalN)-induced fulminant hepatic failure. Rats were given an intraperitoneal injection of d-GalN (270 mg/kg). Arogyavardhani (10 mg/kg and 50 mg/kg) was administered orally for 14 days continuously, and on the last day at 1hour before the d-GalN injection. Rats were sacrificed 24 hours after the d-GalN. Silymarin (100 mg/kg body weight) was given orally as a standard hepatoprotective drug. The liver injury was assessed biochemically, investigating biochemical parameters like ALT, AST, ALP, Bilirubin, Total Protein, and Albumin. The survival rates after the application of Arogyavardhani at 24 h were also observed. D-galactosamine administration induced a significant increase (P≤0.01) in total bilirubin associated with a marked elevation in the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) as compared to control rats. The pretreatment of Arogyavardhani attenuated these changes in a dose-dependent manner. The survival rate of the OC groups was significantly higher than that of the d-GalN group. Our results suggest that Arogyavardhani has hepatoprotective effects against D-galactosamine induced hepatic damage. Therefore, Arogyavardhani may be used as a hepatoprotective agent against various liver diseases including toxic liver injury.

EVALUATION OF ANTIOXIDANT PROPERTIES OF MEDGERM IN ACUTE SIMULATED HEPATITIS

Disruption in redox processes plays a significant role in the pathogenesis of hepatitis. A balance between peroxidation, on the one hand, and the antioxidant system, on the other, is a prerequisite for maintaining normal cell activity. The modern arsenal of effective hepatoprotective drugs is not so wide therefore, the development of new hepatoprotectors is one of the topical issues in pharmacology. The purpose of the work is study the antioxidant properties of medgerma in acute experimental hepatitis. Material and methods. The experiments were performed on male rats weighing 180-220 g. Acute toxic hepatitis in rats was induced by intraperitoneal administration of D-galactosamine in a dosage of 400 mg / kg (LD50). The experimental animals were divided into 4 groups: group I (n = 10) included intact animals that were injected 0.9% sodium chloride solution (control group) intraperitoneally, II group (n = 40) involved the animals receiving only D-galactosamine; group III (n = 40) included rats, which received were administered Medmerg intraperitoneally in a dose of 0.4 mg / kg 7 days before D-galactosamine administration and 7 days after D-galactosamine administration. Group IV (n = 40) included rats, which received the comparison medicine, Essentiale® N, in at a dosage of 5 mg / kg in the same mode. The assessment of lipid peroxidation indices and the antioxidant system indices were carried out on days 1, 3, and 7 after the administration of hepatotoxin in the blood serum and the liver tissue homogenate supernatant. The condition of lipid peroxidation processes in the animals with galactosamine hepatitis was assessed by the content of lipid peroxidation end products in the blood and liver tissue that react with 2-thiobarbituric acid (TBA reactants). The condition of the main components of the antioxidant system was evaluated by the level of reduced glutathione and by the activity of catalase and superoxide dismutase. Results and discussion. It has been established that medgerm enhances the liver resistance to hepatotoxicant, as evidenced by a decrease in the level of lipid peroxidation products and the consumption of main components of the antioxidant system on the first day of hepatitis. Moreover, in the rats, which received Medgerm according to prevention scheme under acute toxic hepatitis, the course of the disease was milder than in the animals that did not receive this therapy. Conclusions and prospects for further research. Medgerm possesses hepatoprotective properties, which underlay positive effects on the state of lipid peroxidation and the main components of the antioxidant system.

Protective Role of Shiitake Mushroom-Derived Exosome-Like Nanoparticles in D-Galactosamine and Lipopolysaccharide-Induced Acute Liver Injury in Mice.

Fulminant hepatic failure (FHF) is a rare, life-threatening liver disease with a poor prognosis. Administration of D-galactosamine (GalN) and lipopolysaccharide (LPS) triggers acute liver injury in mice, simulating many clinical features of FHF in humans; therefore, this disease model is often used to investigate potential therapeutic interventions to treat FHF. Recently, suppression of the nucleotide-binding domain and leucine-rich repeat related (NLR) family, pyrin domain containing 3 (NLRP3) inflammasome, was shown to alleviate the severity of GalN/LPS-induced liver damage in mice. Therefore, the goal of this study was to find dietary exosome-like nanoparticles (ELNs) with therapeutic potential in curbing FHF by suppressing the NLRP3 inflammasome. Seven commonly consumed mushrooms were used to extract ELNs. These mushrooms were found to contain ELNs composed of RNAs, proteins, and lipids. Among these mushroom-derived ELNs, only shiitake mushroom-derived ELNs (S-ELNs) substantially inhibited NLRP3 inflammasome activation by preventing inflammasome formation in primary macrophages. S-ELNs also suppressed the secretion of interleukin (IL)-6, as well as both protein and mRNA levels of the Il1b gene. Remarkably, pre-treatment with S-ELNs protected mice from GalN/LPS-induced acute liver injury. Therefore, S-ELNs, identified as potent new inhibitors of the NLRP3 inflammasome, represent a promising class of agents with the potential to combat FHF.

Jie-Du-Hua-Yu Granules Promote Liver Regeneration in Rat Models of Acute Liver Failure: miRNA-mRNA Expression Analysis.

Purpose Jie-Du-Hua-Yu (JDHY) granules are a traditional Chinese medicine with known therapeutic effects for the treatment of acute liver failure (ALF). This study explored the potential molecular mechanism(s) of JDHY granules in promoting liver regeneration and preventing ALF. Methods Rat models of ALF were constructed through administration of D-galactosamine (D-GalN) (600 mg/kg) and lipopolysaccharides (LPS) (20 μg/kg). Rats were gavaged with JDHY granules, and serum and liver samples were collected at 12 h post-D-GalN/LPS administration. The degree of liver injury was evaluated through hepatic pathology and alanine/aspartate aminotransferase (ALT/AST) activity. miRNA chips were used to detect the miRNA expression profiles of rat models. Bioinformatics analysis was used to identify the biological processes and cell signaling pathways mediating the therapeutic effects of JDHY. Real-time PCR (RT-PCR) and western blotting were used to validate the data. Results JDHY granules could effectively decrease the levels of ALT and AST, relieve D-GalN/LPS-induced liver injury, and improve hepatic function. JDHY granules were found to regulate the expression of 20 miRNAs and 19 mRNAs, which influenced 21 biological processes and 9 signaling pathways. Upon analysis of the therapeutic mechanism(s) governing the effects of JDHY granules on liver regeneration, enhanced DNA replication and an improved cholesterol metabolic ratio were identified. JDHY granules were also found to increase the expression of MCM3, CDK4, and TC, confirming the involvement of these pathways. Moreover, JDHY granules were found to promote hepatocyte mitosis and inhibit the progression of ALF. Conclusion JDHY granules protect against D-GalN/LPS-induced ALF in rats by promoting liver regeneration through enhanced DNA replication and an improved cholesterol metabolic ratio.

PROTECTIVE EFFECT OF ROTENONE AGAINST LIPOPOLYSACCHARIDE AND D-GALACTOSAMINE-INDUCED HEMATOTOXICITY

Objective: The aim of the present study was to investigate the protective efficacy of rotenone against lipopolysaccharide (LPS) and D-galactosamine (D-GalN)-induced altered hematology.
 Methods: Hematotoxicity was induced by coinjection of LPS (50 μg/kg i.p.) and D-GalN (300 mg/kg i.p.). Rotenone (5, 10 and 20 mg/kg p.o.) was administered for 6 days as a pre-treatment. Blood was collected through puncturing the retro-orbital sinus to analyze blood parameter. Serum was separated to analyze glucose, triglyceride, and cholesterol.
 Results: The present study revealed decreased in red blood cells, platelets, hemoglobin, and hematocrit value while a significant increase in white blood cells, lymphocyte, and monocytes were observed in LPS and D-GalN treated rats. LPS and D-GalN administration significantly decrease glucose level while serum lipid profile (triglycerides and cholesterol level) were increased significantly at 5% level of significance. LPS and D-GalN-induced altered hematological and serological variables were restored toward control by rotenone pretreatment for 6 days in dose-dependent manner.
 Conclusion: It can be said that LPS and D-GalN administration resulted in alteration of various hematological parameters and rotenone at 20 mg/kg dose restored significant alteration toward control due to the presence of antioxidant activity of rotenone.

Anti-TNF effect of combined pravastatin and cilostazol treatment in an in vivo mouse model

Objectives: Pravastatin and cilostazol are used as lipid-lowering and antiplatelet agents, respectively. Regarding their well-known anti-inflammatory effects, the additive effect of the two drugs on anti-TNF functions has not yet been investigated. In the present investigation, the beneficial effect of combined pravastatin and cilostazol on their anti-TNF activities was assessed using an in vivo mouse model.Methods: Mice were pretreated with pravastatin and/or cilostazol (40 mg/kg of each), orally once two hour prior to an LPS (5 mg/kg, i.p.) challenge. One hour post challenge, blood and descending aorta were collected for serum TNF levels and immune cell infiltration analyses. For survival analysis, pravastatin and/or cilostazol (40 mg/kg of each) were administered 30 minutes prior to d-galactosamine administration (700 mg/kg, i.p.) and TNF (10 µg/kg, i.p.) challenge and mice survival was monitored. We also examined the effect of either drug or the combination of drugs on TNF-mediated MAPK and NF-κB signaling, using Western blot analysis.Results: Combined treatment of pravastatin and cilostazol significantly decreased serum TNF release and immune cell infiltration in the descending aorta following LPS administration, compared to each single treatment. Additionally, the combined drugs significantly decreased TNF-mediated mouse mortality and downregulated TNF-induced MAPK and NF-κB activation.Conclusions: These findings suggest that combined pravastatin and cilostazol is more effective for reducing TNF-driven inflammation through their anti-TNF activity than monotherapy.

Hepatoprotective effects and structure-activity relationship of five flavonoids against lipopolysaccharide/d-galactosamine induced acute liver failure in mice

Acute liver failure (ALF) is a distinct clinical syndrome with high mortality and characterized by metabolic derangements, neurological complication, and multiple failures. Flavonoids exert great biological properties on anti-oxidation, anti-inflammation, and anti-apoptosis. After lipopolysaccharide (LPS)/d-galactosamine (d-GalN) administration, five flavonoids inhibited oxidative activities with reducing nitric oxide synthase (iNOS), malondialdehyde (MDA), and improving catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). They reduced the serum levels of alanine and aspartate aminotransferase (ALT, AST) and pro-inflammatory cytokines, prevented the phosphorylation of IKK, IκBα, and NF-κB/p65 in the NF-κB signaling pathway. Additionally five flavonoids inhibited hepatocyte apoptosis through increasing Bcl-2/Bax ratio and suppressing the Caspase family proteins. Chrysin, luteolin, apigenin, hesperetin and 3′, 4′-dimethoxy hesperetin have apparently hepato-protective effects against ALF induced by LPS/d-GalN. The study found, the C2C3 double bond at A ring, and the hydroxyl group of C3′ or C4′ at B ring increased the protective activities, however, the effect of hydroxymethylation at C3′ and C4′ was reversed. In addition, apigenin has good hepatoprotective effects and potential as a promising therapeutic agent for ALF in clinical application.

Therapeutic Efficacy of Adipose-Derived Stromal Vascular Fraction Cells is associated with CD34 Positivity in Acute-on-Chronic Liver Failure

Background Adipose-derived stromal vascular fraction (SVF) cells, a rich source of primary stem/stromal cells, are promising for administering cell therapy for patients with acute-on-chronic liver failure (ACLF). We evaluated the therapeutic effects of CD34+/CD34− SVF cells in hepatocyte cotransplantation in a rat model of ACLF. Method ACLF was induced in Sprague–Dawley rats by temporary bile duct ligation (chronic) and D-galactosamine administration (acute). Donor hepatocytes and SVF cells were isolated from Tg(UBC–emGFP) rats and human adipose tissues, respectively. Sorted CD34+ and CD34− SVF cells were separated using a magnetic bead system. Rat hepatocytes and SVF cells (labeled with the PKH26 fluorescent dye) were intraportally transplanted into ACLF rats. The surviving animals were sacrificed at 1 and 2 weeks after the transplantation. Results ACLF was evidenced by the onset of acute coagulopathy and hepatocyte necrosis in fibrotic livers. Compared with the control group, the unsorted group showed less biliary ductular proliferation and fibrosis at 1 week after the transplantation. Furthermore, prominent biliary ductular proliferation and significantly progression of fibrosis (P = .005) were observed in the CD34− group than in the CD34+ group. At 2 weeks, the serum level of alkaline phosphatase was significantly higher in the CD34− group than in the CD34+ group (P = .007). The transplanted SVF cells were found in the periportal regions at 1 week, whereas donor hepatocytes were not detected and proliferation of endogenous hepatocytes was observed at times. Conclusion Compared with CD34− SVF cells, cotransplantation of CD34+ SVF cells resulted in the early amelioration of liver fibrosis and biliary ductular proliferation. Heterogeneous therapeutic effects of SVF cells were associated with CD34 positivity, which poses translational significance in future cell therapy.

Novel D-galactosamine-induced cynomolgus monkey model of acute liver failure.

AIMTo establish a simplified, reproducible D-galactosamine-induced cynomolgus monkey model of acute liver failure having an appropriate treatment window.METHODSSixteen cynomolgus monkeys were randomly divided into four groups (A, B, C and D) after intracranial pressure (ICP) sensor implantation. D-galactosamine at 0.3, 0.25, 0.20 + 0.05 (24 h interval), and 0.20 g/kg body weight, respectively, was injected via the small saphenous vein. Vital signs, ICP, biochemical indices, and inflammatory factors were recorded at 0, 12, 24, 36, 48, 72, 96, and 120 h after D-galactosamine administration. Progression of clinical manifestations, survival times, and results of H&E staining, TUNEL, and Masson staining were recorded.RESULTSCynomolgus monkeys developed different degrees of debilitation, loss of appetite, and jaundice after D-galactosamine administration. Survival times of groups A, B, and C were 56 ± 8.7 h, 95 ± 5.5 h, and 99 ± 2.2 h, respectively, and in group D all monkeys survived the 144-h observation period except for one, which died at 136 h. Blood levels of ALT, AST, CK, LDH, TBiL, Cr, BUN, and ammonia, prothrombin time, ICP, endotoxin, and inflammatory markers [(tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6)] significantly increased compared with baseline values in different groups (P < 0.05). Pathological results showed obvious liver cell necrosis that was positively correlated with the dose of D-galactosamine.CONCLUSIONWe successfully established a simplified, reproducible D-galactosamine-induced cynomolgus monkey model of acute liver failure, and the single or divided dosage of 0.25 g/kg is optimal for creating this model.

Genistein Alleviates Neuroinflammation and Restores Cognitive Function in Rat Model of Hepatic Encephalopathy: Underlying Mechanisms.

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome resulting from acute liver failure. Previously, we demonstrated hepatoprotective effects of genistein in D-galactosamine (D-GalN)-induced fulminant hepatic failure (FHF). In this study, we evaluated behavioural and neuroprotective effects of genistein in rat model of HE. HE was induced by intraperitonial administration of D-GalN (250mg/kg BW) twice a week for 30days Genistein was given as co-treatment through oral gavage daily at dose of 5mg/kg BW. D-GalN administration significantly resulted in acute liver failure which was further associated with hyperammonemia, neurological dysfunction, as evident from behavioural and functional impairment and reduced learning ability in Morris water maze. Genistein significantly alleviated behavioural and functional impairment and restored learning ability in Morris water maze. Considerable histopathological changes, including portal inflammation, sinusoidal dilation, necrotic lesions and swelled astrocytes with pale nuclei, were seen in the liver and brain sections of D-GalN-challenged rats while genistein co-treated rats revealed normal cellular and morphological architecture as no pathological features were seen. Furthermore, pro-inflammatory markers (interleukin (IL)-10, IL-4, IL-1β and TNF-α) and membrane expression of subunits α1 of GABAA receptor and GluR2 of AMPA marked significant increase, while subunits GluR1 of AMPA receptors showed reduced expression in D-GalN-challenged rats leading to neuroinflammation and dysregulated neurotransmission. Genistein significantly normalized altered expression of pro-inflammatory cytokines and membrane receptor of GABA and GluR. Our study suggests strong therapeutic potential of genistein in animal model of HE. Genistein can be used a strong anti-oxidant to attenuate neurotoxic effects of xenobiotics.

Glycyrrhetinic acid attenuates lipopolysaccharide-induced fulminant hepatic failure in d-galactosamine-sensitized mice by up-regulating expression of interleukin-1 receptor-associated kinase-M

Glycyrrhetinic acid (GA), the main active ingredient of licorice, reportedly has anti-inflammatory and hepatoprotective properties, but its molecular mechanisms remain be elusive. In the present study, Balb/c mice were pretreated with GA (10, 30, or 100mg/kg) 1h before lipopolysaccharide (LPS)/d-galactosamine (D-GalN) administration. In other in vitro experiment, RAW264.7 macrophages were pretreated with GA before LPS exposure. The mortality, hepatic tissue histology, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed. Toll like receptor 4 (TLR4), interleukin-1 receptor-associated kinases (IRAKs), activation of mitogen-activated protein kinases (MAPKs) and NF-κB, and production of TNF-α were assessed by flow cytometry, western blotting, and enzyme-linked immunosorbent assay (ELISA), respectively. Our results showed that pretreatment with GA protected mice against LPS/D-GalN-induced fulminant hepatic failure (FHF), including a dose-dependent alleviation of mortality and ALT/AST elevation, ameliorating hepatic pathological damage, and decreasing TNF-α release. Moreover, GA inhibited LPS-induced activation of MAPKs and NF-κB in response to LPS, but the expression of TLR4 was not affected in vivo and in vitro. Notably, GA pretreatment in vivo suppressed IRAK-1 activity while inducing IRAK-M expression. Silencing of IRAK-M expression with siRNA blocked these beneficial effects of GA on the activation of MAPKs and NF-κB as well as TNF-α production in LPS-primed macrophages. Taken together, we conclude that GA could prevent LPS/D-GalN-induced FHF. The underlying mechanisms may be related to up-regulation of IRAK-M, which in turn caused deactivation of IRAK-1 and subsequent MAPKs and NF-κB, resulting in inhibiting TNF-α production.

Regression of fibrosis/cirrhosis by Glycine propionyl-l-carnitine treatment in d-Galactosamine induced chronic liver damage

Background and purposeLiver fibrosis and cirrhosis are leading causes of morbidity and mortality, with majority of preventable cases attributed to excessive alcohol consumption, viral hepatitis, or non-alcoholic fatty liver disease. We previously reported the hepatoprotective effect of Glycine propionyl-l-carnitine (GPLC) against the fulminant hepatic failure (FHF) induced by d-Galactosamine (D-GalN). In this study we evaluated the protective effect of GPLC against D-GalN induced chronic liver damage. Experimental approachAnimals received D-GalN twice a week for 12 weeks at a dose of 250 mg/kg body weight (BW). GPLC was given daily for 12 weeks as co-treatment along with D-GalN at a dose of 35 mg/kg BW. Key resultsD-GalN injection resulted in a considerable decrease in body weight, hepatocellular disintegration, necrosis and lipid peroxidation as evident from altered levels of SOD, CAT and MDA while GPLC significantly restored the reduced body weight and ameliorated hepatocellular damage and lipid peroxidation. D-GalN administration resulted in DNA damage as evident from TUNEL positive cells in disease control rats while; GPLC significantly alleviated the genotoxic effects of D-GalN. Further histopathological analysis revealed significant tissue and cellular damage, and increased collagen content in D-GalN challenged rats. GPLC however ameliorated the damage as evident from normal cellular and morphological architecture in GPLC co-treated rats. Hydroxyproline and nitrotyrosine (NTY) levels marked a significant decrease in GPLC co-treated rats relative to disease control. GPLC significantly blocked D-GalN induced pro-inflammatory cytokine (TNF-α, IL-6) production and at the same time inhibited the expression of α-smooth muscle actin (α-SMA), collagen-I (COL-I) and transforming growth factor-β (TGF-β) significantly. Conclusion and implicationsOur results demonstrate significant protective activity of GPLC in chronic liver damage and other complications related to it. This study is a novel study to demonstrate the hepatoprotective effect of GPLC in chronic liver damage.

Hepatoprotective Activity of Nigella sativa Extract in D-Galactosamine Induced Hepatic Injury in Rats

Background: Chronic liver disease (such as hepatitis) is a major health problem worldwide, especially in developing countries. Despite of the tremendous advancement in modern medicine, there is no effective drug available that stimulates liver function. The search of new drugs to protect hepatic injury has been of recent interest. Nigella sativa is used as analgesic, anti-inflammatory, anti-diarrhoeal, antimicrobial, anticancer, immunomodulator, bronchodilator, and also said to be hepatoprotective. Objective: The aim of this work is to evaluate the hepatoprotective activity of Nigella sativa in Wistar rats and to bring about scientific justification for the use of this drug as a hepatoprotective. Method: Healthy Male Wistar Rats Were Treated With The Extract Of Nigella Sativa For 14 Days And On Day 14 Hepatotoxicity were induced by the administration of D-galactosamine through Intraperitoneal (IP) route, blood was collected and analyzed for various biochemical parameters, animals were sacrificed for histopathology, and were compared with the effect of Silymarine as a standard drug. Result: The substantially elevated levels of aspartate amino transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and albumin were restored significantly by the extract of Nigella sativa. Conclusion: Administration of N. sativa extract showed recovery against toxic effects of D-galactosamine in rats. The hepatoprotective effect of the drug was further concluded by the histopathological examination of the liver sections, which reveals that the normal liver shape was disturbed by D-galactosamine. The normal cellular shape was retained as compared to silymarine, thereby confirming the protective effect of the N sativa extract. Keywords: D-Galactosamine, hepatoprotective, Nigella sativa.

No transmission of porcine endogenous retrovirus in an acute liver failure model treated by a novel hybrid bioartificial liver containing porcine hepatocytes

A novel hybrid bioartificial liver (HBAL) was constructed using an anionic resin adsorption column and a multi-layer flat-plate bioreactor containing porcine hepatocytes co-cultured with bone marrow mesenchymal stem cells (MSCs). This study aimed to evaluate the microbiological safety of the HBAL by detecting the transmission of porcine endogenous retroviruses (PERVs) into canines with acute liver failure (ALF) undergoing HBAL. Eight dogs with ALF received a 6-hour HBAL treatment on the first day after the modeling by D-galactosamine administration. The plasma in the HBAL and the whole blood in the dogs were collected for PERV detection at regular intervals until one year later when the dogs were sacrificed to retrieve the tissues of several organs for immunohistochemistry and Western blotting for the investigation of PERV capsid protein gag p30 in the tissue. Furthermore, HEK293 cells were incubated to determine the in vitro infectivity. PERV RNA and reverse transcriptase activity were observed in the plasma of circuit 3, suggesting that PERV particles released in circuit 3. No positive PERV RNA and reverse transcriptase activity were detected in other plasma. No HEK293 cells were infected by the plasma in vitro. In addition, all PERV-related analyses in peripheral blood mononuclear cells and tissues were negative. No transmission of PERVs into ALF canines suggested a reliable microbiological safety of HBAL based on porcine hepatocytes.