TUNEL-positive Hepatocytes Research Articles

Alanyl-Glutamine Protects against Lipopolysaccharide-Induced Liver Injury in Mice via Alleviating Oxidative Stress, Inhibiting Inflammation, and Regulating Autophagy.

Acute liver injury is a worldwide problem with a high rate of morbidity and mortality, and effective pharmacological therapies are still urgently needed. Alanyl-glutamine (Ala-Gln), a dipeptide formed from L-alanine and L-glutamine, is known as a protective compound that is involved in various tissue injuries, but there are limited reports regarding the effects of Ala-Gln in acute liver injury. This present study aimed to investigate the protective effects of Ala-Gln in lipopolysaccharide (LPS)-induced acute liver injury in mice, with a focus on inflammatory responses and oxidative stress. The acute liver injury induced using LPS (50 μg/kg) and D-galactosamine (D-Gal) (400 mg/kg) stimulation in mice was significantly attenuated after Ala-Gln treatment (500 and 1500 mg/kg), as evidenced by reduced plasma alanine transaminase (ALT) (p < 0.01, p < 0.001), aspartate transaminase (AST) (p < 0.05, p < 0.001), and lactate dehydrogenase (LDH) (p < 0.01, p < 0.001) levels, and accompanied by improved histopathological changes. In addition, LPS/D-Gal-induced hepatic apoptosis was also alleviated by Ala-Gln administration, as shown by a greatly decreased ratio of TUNEL-positive hepatocytes, from approximately 10% to 2%, and markedly reduced protein levels of cleaved caspase-3 (p < 0.05, p < 0.001) in liver. Moreover, we found that LPS/D-Gal-triggered oxidative stress was suppressed after Ala-Gln treatment, the effect of which might be dependent on the elevation of SOD and GPX activities, and on GSH levels in liver. Interestingly, we observed that Ala-Gln clearly inhibited LPS/D-Gal exposure-induced macrophage accumulation and the production of proinflammatory factors in the liver. Furthermore, Ala-Gln greatly regulated autophagy in the liver in LPS/D-Gal-treated mice. Using RAW264.7 cells, we confirmed the anti-inflammatory role of Ala-Gln-targeting macrophages.

Flash Irradiation Inhibits Breast Onset and Spares Radiation-Induced Liver Injury

<h3>Purpose/Objective(s)</h3> To clarify the efficacy of FLASH radiotherapy (FLASH-RT) in inhibiting mammary tumorigenesis and normal tissue sparing. <h3>Materials/Methods</h3> We used a spontaneous mammary tumor model of MMTV-PyMT mice for the study. Prior to the formation of mammary tumor, MMTV-PyMT mice (3–6-week-old) were treated with a single fraction dose of 9-16 Gy FLASH-RT or conventional radiotherapy (CONV-RT) using a mice-applicator designed targeting five pair of mammary glands. Tumor growth was detected by thorough palpation of all 10 mammary glands twice a week. Liver function, H&E staining and TUNEL staining of liver tissues were performed to determine the irradiation-induced liver injury. Inflammatory cytokines of TNF-a, IFN-g, IL-6 and IL-10 in the liver tissue and in the serum on Days 6 (D6), 18 (D18) and 31 days (D31) post-irradiation (IR) were tested by ELISA. DHE staining in frozen sections of tumor tissue and liver tissue at 4 weeks post-IR were performed, respectively. <h3>Results</h3> Median days for first tumor occurrence were 65, 62, 58.5 and 52 days of age, while median days for the involvement of 10th mammary gland were 79, 76, 76 and 63 days of age for FLASH (14-16 Gy), FLASH (9-12 Gy), CONV (9-12 Gy) and Control (0 Gy) groups, respectively. Tumor weight at 12 weeks after birth in FLASH and CONV were significantly lower than controls. On 18 days after IR, serum ALT level was significantly increased in the CONV compared with the samples in FLASH. H&E staining of liver in 7 weeks after 9-12 Gy IR revealed that FLASH caused less histological abnormalities in comparison to the samples from CONV, which was similar to the tissue of control. The number of TUNEL-positive hepatocytes in the FLASH was less than that of the CONV but similar to that of the control. In liver tissue, TNF-a, IFN-g and IL-6 significantly increased in CONV compared with FLASH and control; however, IFN-g and IL-6 also significantly increased in FLASH compared with control, while IL-10 significantly decreased in CONV compared with control. Serum IL-6, IFN-g and TNF-a levels in FLASH did not markedly change on D6, D18 and D31 post-IR compared with control. Serum IL-6, IFN-g and TNF-a levels were elevated on D6 and continuously increased on D18 and D31 in CONV, but began to increase on D6, and then recovered to the control level on D18 or D31 in FLASH. Serum IL-10 levels decreased at D6, D18 and D31 in CONV, but began to decrease at D6, and then recovered to the control level at D18 or D31 in FLASH. FLASH induced reactive oxygen species (ROS) accumulation in the tumor tissue but not in normal tissue; while absence of ROS accumulation in either tumor or normal tissue under CONV. Notably, the ROS level of normal liver tissue did not differ under CONV or FLASH. <h3>Conclusion</h3> We found that mammary tumorigenesis was inhibited by FLASH without significant liver injury compared with CONV-RT. The alteration of serum and liver inflammatory cytokines induced by CONV-RT was mildly altered by FLASH. This unique benefit can by partially explained by the accumulatio of ROS in tumor tissue after FLASH.

Diploid hepatocytes promote compensatory liver regeneration following acetaminophen induced acute liver injury

Background Acetaminophen (APAP) is a commonly used analgesic that can be safely used at doses under 4 g/day. When taken in excess, APAP causes acute liver injury, which leads to acute liver failure and death. APAP overdose leads to 56,000 emergency room visits and 26,000 hospitalizations annually and is the leading cause of acute liver failure in the United States. The only available pharmacological treatment, N-acetyl cysteine, is effective within hours of overdose. The only other treatment option is orthotopic liver transplantation, and this is limited by the availability of donor organs. Considering the paucity of therapeutic options, there is an urgent need to understand mechanisms of liver regeneration and repair. Most mammalian somatic cells are diploid and contain pairs of each chromosome, but there are also polyploid cells, such as hepatocytes that contain additional sets of chromosomes. Polyploid hepatocytes are among the best described, and in adult humans and mice comprise more than 25% and 90%, respectively, of the hepatocyte population. The cellular and molecular mechanisms that regulate polyploidy have been well-characterized; however, it is poorly understood if diploid and polyploid hepatocytes play specialized roles in liver injury and repair. Methods Our lab recently found that diploid hepatocytes are inherently more proliferative than polyploid hepatocytes. Additionally, we can specifically study diploid hepatocytes using mice lacking E2f7 and E2f8 in the liver (referred to as “LKO” mice), which are functionally normal, but livers are depleted of polyploid hepatocytes. Considering that liver regeneration affects the outcome of APAP-medicated injury, we asked if LKO mice enriched with highly proliferative diploid hepatocytes would respond differently than control livers. We fasted LKO and control mice for 16 hours, injected intraperitoneally with 300 mg/kg (the LD50 dose) APAP and harvested livers after 0.5, 3, 6, 12, 24, 48, 72 and 96 hours. We used these tissues to investigate differences in liver injury, necrosis, and proliferation. Results Liver injury enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were higher in control mice compared to LKO mice beginning at 3 hours. Necrotic liver tissue was abundant in control mice and was markedly reduced in LKO mice. We also measured more apoptotic, TUNEL positive hepatocytes in control mice. We next assessed the proliferation response after APAP-induced liver injury by staining tissues for PCNA which was induced by 6 hours in LKO mice compared to 24 hours in control mice. We treated wild-type 19-day old hepatocytes with 5 mM APAP for 12, 24, and 36 hours in vitro and found that diploid hepatocytes proliferate more readily compared to polyploids as marked by BrdU incorporation. Finally, we found higher doses of APAP eliminate the advantages seen in LKO mice. Conclusion Based on these data, diploid hepatocytes appear to be the main contributors of early-stage compensatory regeneration following APAP-mediated liver injury. These data suggest that, compared to polyploid hepatocytes, diploids initiate and drive liver healing/regeneration after acute injury.

The hepatotoxicity of altrazine exposure in mice involves the intestinal microbiota

Atrazine (ATR), a bio accumulative herbicide is frequently used in agriculture to control unwanted weeds. Due to continuous application, atrazine persists in the environment and causes deleterious impacts including neurotoxicity, hepatotoxicity, and gut microbiota disorders. Therefore, this study for the first time reports the variation in the gut microbiota, induction of process of apoptosis and autophagy in mice induced by ATR. Results indicated that TUNEL-positive hepatocytes suggestive of apoptosis were increased in livers of different experimental mice. Results on metabolic analysis in liver tissues indicated an overall change in seventy-six metabolites particularly Uridine 5′-diphosphate, Propenoylcarnitine and Chinenoside V resulting in generation of energy-related metabolic disorders and imbalance of oxidation/autoxidation status. Results on gut microbiome inquisition showed that ATR changed the richness and diversity of gut microbiota of mice and number of Firmicutes. Moreover, results also revealed that ATR induced apoptosis via disruption of apoptotic (Bax, Bcl2, and Casp3) and autophagy (LC3/Map1lc3a, Beclin 1/Becn1 and P62/Sqstm1) genes. Results of our experimental study confirmed that changes in gut microbiota play a significant role in process of gut immune regulation and inflammation via different metabolites. In conclusion, the findings of our study provide a new idea for the involvement of mechanisms of detoxification in liver and inquisition of gut microbiota plays crucial role in regulation of physiological activities through liver-gut axis to mitigate toxic effects in animals.

Sleep deprivation aggravated lipopolysaccharide/D-galactosamine-induced acute liver injury by suppressing melatonin production.

Sleep loss is common in patients with liver injury, but the effects of sleep deprivation (SD) on liver injury remain unclear. In the present study, the potential effects of SD on acute liver injury and the underlying mechanisms have been investigated. The sleep of male BALB/c mice has been deprived by using a modified multiple platform water bath for 3 days and acute liver injury was induced by intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (D-Gal). The degree of liver injury was detected by aminotransferase determination, histopathology and survival rate analysis. Inflammatory response and melatonin (MT) were measured by enzyme-linked immunosorbent assay (ELISA). In addition, hepatocyte apoptosis was determined by caspase activity measurement and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. We observed that SD increased plasma aminotransferases, TUNEL-positive hepatocytes, histological abnormalities and mortality rates in mice with LPS/D-Gal treatment. SD also promoted LPS/D-Gal-induced production of TNF-α and upregulated hepatic caspase-8, caspase-9, and caspase-3 activities in LPS/D-Gal-exposed mice. In addition, SD significantly decreased MT contents in plasma of mice with acute liver injury, but supplementation with MT reversed these SD-promoted changes. Our data suggested that SD exacerbated LPS/D-Gal-induced liver injury via decreasing melatonin production.

TCDD promotes liver fibrosis through disordering systemic and hepatic iron homeostasis

2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic environmental pollutant which can cause severe health problems, such as fibrosis. However, the toxic effects and related mechanism of TCDD on the liver remain largely unknown. In this study, we established a liver fibrosis mouse model upon exposure of TCDD, as evidenced by increased collagen I, tumor growth factor β1 (TGFβ1), α-smooth muscle actin (α-SMA), and Masson staining. Meanwhile, there was also a significant increase of inflammatory factors and TUNEL-positive hepatocytes in liver, indicating that liver inflammation and hepatic cell apoptosis occurred. In addition, increased serum and liver iron were concomitant with liver injury induced by TCDD. We further investigated the mechanism underlying TCDD-induced hepatocyte apoptosis through apoptosis polymerase chain reaction array, and found that a crucial apoptosis-related gene, cell death-inducing DFF45-like effector b (Cideb), was significantly increased in primary hepatocytes from TCDD-exposed mice, and accompanied by liver iron deposition in hepcidin knockout mice. Therefore, Cideb depletion could effectively attenuated TCDD or iron induced cell death related genes expression. In conclusion, our results showed that iron-induced Cideb expression played a critical role in promoting TCDD-induced hepatocyte apoptosis and liver fibrosis, which provide a novel mechanism for understanding TCDD-induced liver injury.

Diploid Hepatocytes Resist Acetaminophen Induced Acute Liver Injury and Drive Compensatory Liver Regeneration

BackgroundAcetaminophen (APAP) is a commonly used analgesic that can be safely used at doses under 4 g/day. When taken in excess, APAP causes acute liver injury, which leads to acute liver failure and death. APAP overdose leads to 56,000 emergency room visits and 26,000 hospitalizations annually and is the leading cause of acute liver failure in the United States. The only available pharmacological treatment, N‐acetyl cysteine, is effective within hours of overdose. The only other treatment option is orthotopic liver transplantation, and this is limited by the availability of donor organs. Considering the paucity of therapeutic options, there is an urgent need to understand mechanisms of liver regeneration and repair.Most mammalian somatic cells are diploid and contain pairs of each chromosome, but there are also polyploid cells, such as hepatocytes that contain additional sets of chromosomes. Polyploid hepatocytes are among the best described, and in adult humans and mice comprise more than 25% and 90%, respectively, of the hepatocyte population. The cellular and molecular mechanisms that regulate polyploidy have been well‐characterized; however, it is poorly understood if diploid and polyploid hepatocytes play specialized roles in liver injury and repair.MethodsOur lab recently found that diploid hepatocytes are inherently more proliferative than polyploid hepatocytes. Additionally, we can specifically study diploid hepatocytes using mice lacking E2f7 and E2f8 in the liver (referred to as “LKO” mice), which are functionally normal, but livers are depleted of polyploid hepatocytes. Considering that liver regeneration affects the outcome of APAP‐medicated injury, we asked if LKO mice enriched with highly proliferative diploid hepatocytes would respond differently than control livers. We fasted LKO and control mice for 16 hours, injected intraperitoneally 300 mg/kg (the LD50 dose) APAP and harvested livers after 0.5, 3, 6, 12, 24, and 48 hours. We used these tissues to investigate differences in liver injury, necrosis, and proliferation.ResultsLiver injury enzymes, alanine aminotransferase and aspartate aminotransferase, were higher in control mice compared to LKO mice beginning at 3 hours. Necrotic liver tissue was abundant in control mice and was markedly reduced in LKO mice. We also measured more apoptotic, TUNEL positive hepatocytes in control mice. We next assessed the proliferation response after APAP‐induced liver injury by staining for PCNA which was induced by 6 hours in LKO mice compared to 24 hours in control mice. Finally, we treated wild‐type 19‐day old hepatocytes with 5 mM APAP for 12, 24, and 36 hours in vitro and found that diploid hepatocytes proliferate more readily compared to polyploids as marked by BrdU incorporation.ConclusionBased on these data, diploid hepatocytes appear to be the main contributors of early‐stage compensatory regeneration following APAP‐mediated liver injury. These data suggest that, compared to polyploid hepatocytes, diploids initiate and drive liver healing/regeneration after acute injury.Support or Funding InformationThis work supported by the NIH to AWD (R01DK103645).

Abstract 1728: Overactivation of tumor suppressor P53 in hepatocytes promotes hepatocarcinogenesis

Abstract Aim: p53 is a tumor suppressor and its dysfunction promotes carcinogenesis of several organs including the liver. Meanwhile, p53 is reported to be overactivated in non-tumorous liver tissues of patients with chronic liver diseases, presumably due to several stressful stimuli to hepatocytes, including viral infection, ethanol exposure, fat accumulation. The aim of this study is to clarify the impact of p53 overactivation on hepatocarcinogenesis. Methods: To induce hepatocyte-specific overactivation of p53 in liver cancer models, hepatic Mdm2, a negative regulator of p53, was genetically ablated in diethylnitrosoamine (DEN)-treated mice (Alb-CreMdm2fl/fl) and liver-specific constitutive-active Kras-mutant knock-in mice (Alb-CreKrasLSL-G12DMdm2fl/fl called as KrasG12DΔMdm2 mice). To analyze the significance of p53, hepatic p53 was deleted in Mdm2-wild KrasG12D mice (Alb-CreKrasLSL-G12DMdm2+/+p53fl/fl) and KrasG12DΔMdm2 mice (Alb-CreKrasLSL-G12DMdm2fl/flp53fl/fl). Results: Mdm2 deletion significantly elevated serum ALT levels and increased tumor penetrance in both DEN-treated mice and Kras-mutant mice, suggesting that p53 overactivation accelerated hepatocarcinogenesis. KrasG12DΔMdm2 mice upregulated hepatic expressions of pro-apoptotic molecules (p16, p21, Bax, Noxa), number of TUNEL positive hepatocytes and serum caspase-3/7 levels, indicating accelerated hepatocyte apoptosis. They also increased the number of β-galactosidase positive hepatocytes and upregulated hepatic inflammatory cytokines (Tnf, Ccl2, Il-1β), suggesting senescence-associated secretory phenotype (SASP). Expressions of hepatic progenitor cell (HPC) markers (Afp, Epcam, Ck7, Ck19, Cd90, Cd133, Dlk1) were up-regulated in their liver. HPC marker staining suggested that liver tumors in KrasG12DΔMdm2 mice may originate from HPCs. All the phenotypes including accelerated hepatocyte apoptosis, SASP and emergence of HPCs in KrasG12DΔMdm2 mice were significantly alleviated by p53 deletion. Most importantly, deletion of p53 significantly decelerated hepatocarcinogenesis in those mice, indicating that p53 overactivation was responsible for the accelerated hepatocarcinogenesis. In sharp contrast, p53 deletion significantly accelerated hepatocarcinogenesis in Mdm2-wild Kras-mutant mice, confirming the tumor suppressive role of normal level of p53 in the liver. Conclusion: Whereas p53 deletion accelerated hepatocarcinogenesis, p53 overactivation induced liver injury and activation of HPCs, leading to the acceleration of hepatocarcinogenesis. p53 overactivation in hepatocytes might be associated with liver cancer development in patients with chronic liver diseases. Citation Format: Yuki Makino. Overactivation of tumor suppressor P53 in hepatocytes promotes hepatocarcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1728.

Bolus Administration of Polyamines Boosts Effects on Hepatic Ischemia-Reperfusion Injury and Regeneration in Rats

Background:It was demonstrated that polyamines ameliorate ischemia-reperfusion injury (IRI) and promote regeneration in the liver. An optimal protocol of polyamine treatment remains unknown in the clinical setting. We examined 2 types of administration methods using rat models. Methods: Experiment 1: evaluation of pharmacokinetics of polyamines. Experiment 2: for 3 days preoperatively and 5 days postoperatively, polyamines were given to male Lewis rats in the following three groups: the control group, no polyamine administration; the chow group, 0.05% polyamines mixed in chow; the bolus group, polyamines (200 μmol/kg) given by gastric tube once a day. All rats received 70% hepatectomy after 40 min of warm IRI. Postoperatively, IRI and regeneration were evaluated with assessment of serum levels of hepatic enzymes, histology and immunohistochemistry of liver tissue, and measurement of remnant liver weight. Results: The blood concentrations of polyamines in the portal vein increased at 1 h of bolus administration, while they did not increase without the bolus. The bolus group was significantly associated with lower serum levels of aspartate/alanine aminotransferases (p < 0.05), decreased hepatocyte congestion, vacuolization and necrosis in histopathological scoring (p < 0.05), a lower number of TUNEL-positive hepatocytes (p < 0.05), higher remnant liver weight at 24, 48, and 168 h (p < 0.05), and a higher Ki-67 labeling index (24 h, p < 0.01) compared with the chow group. Conclusion: The bolus administration of polyamines was more effective in ameliorating IRI and promoting regeneration than chow administration. Perioperative bolus administration of polyamines might be an optimal treatment, when clinically applied.

Dietary Iron Overload Abrogates Chemically-Induced Liver Cirrhosis in Rats.

Chronic liver disease is an intractable disease, which can progress to cirrhosis and hepatocellular carcinoma. Hepatic iron overload is considered to be involved in the progression of chronic liver diseases; however, the mechanism remains to be elucidated. Here we investigate the role of dietary iron overload using chemically-induced liver cirrhosis model. Rats were fed a high-iron or standard diet and were injected intraperitoneally with thioacetamide (TAA) or saline twice a week for 20 weeks. Rats with TAA treatment (TAA group) had progressive liver cirrhosis characterized by persistent hepatocellular injury, mononuclear cell inflammation and bridging fibrosis; these lesions were markedly reduced in rats with iron feeding and TAA treatment (Fe-TAA group). Rats with iron feeding alone (Fe group) had no evidence of liver injury. Hepatic expression of cleaved caspase-3, but not phospho-RIP3, was decreased in Fe-TAA group compared with that in TAA group. The number of TUNEL-positive (terminal deoxynucleotidyl transferase dUTP nick end labeling) apoptotic hepatocytes was lower in the Fe-TAA group than in the TAA group. Hepatic xenobiotic metabolism and lipid peroxidation were shown to be less related to the abrogation of liver cirrhosis. Our results suggested that dietary hepatic iron overload abrogates chemically-induced liver cirrhosis in rats, which could partly involve decreased hepatocellular apoptosis.

Prevention of acute liver injury by suppressing plasma kallikrein-dependent activation of latent TGF-β

Acute liver injury (ALI) is highly lethal acute liver failure caused by different etiologies. Transforming growth factor β (TGF-β) is a multifunctional cytokine and a well-recognized inducer of apoptotic and necrotic cell death in hepatocytes. Latent TGF-β is activated partly through proteolytic cleavage by a serine protease plasma kallikrein (PLK) between the R58 and L59 residues of its propeptide region. Recently, we developed a specific monoclonal antibody to detect the N-terminal side LAP degradation products ending at residue R58 (R58 LAP-DPs) that reflect PLK-dependent TGF-β activation. This study aimed to explore the potential roles of PLK-dependent TGF-β activation in the pathogenesis of ALI. We established a mouse ALI model via the injection of anti-Fas antibodies (Jo2) and observed increases in the TGF-β1 mRNA level, Smad3 phosphorylation, TUNEL-positive apoptotic hepatocytes and R58-positive cells in the liver tissues of Jo2-treated mice. The R58 LAP-DPs were observed in/around F4/80-positive macrophages, while macrophage depletion with clodronate liposomes partly alleviated the Jo2-induced liver injury. Blocking PLK-dependent TGF-β activation using either the serine proteinase inhibitor FOY305 or the selective PLK inhibitor PKSI-527 or blocking the TGF-β receptor-mediated signaling pathway using SB431542 significantly prevented Jo2-induced hepatic apoptosis and mortality. Furthermore, similar phenomena were observed in the mouse model of ALI with the administration of acetaminophen (APAP). In summary, R58 LAP-DPs reflecting PLK-dependent TGF-β activation may serve as a biomarker for ALI, and targeting PLK-dependent TGF-β activation has potential as a therapeutic strategy for ALI.

Sunitinib induces hepatocyte mitochondrial damage and apoptosis in mice

Reports concerning hepatic mitochondrial toxicity of sunitinib are conflicting. We therefore decided to conduct a toxicological study in mice. After having determined the highest dose that did not affect nutrient ingestion and body weight, we treated mice orally with sunitinib (7.5 mg/kg/day) for 2 weeks. At the end of treatment, peak sunitinib plasma concentrations were comparable to those achieved in humans and liver concentrations were approximately 25-fold higher than in plasma. Sunitinib did not affect body weight, but increased plasma ALT activity 6-fold. The activity of enzyme complexes of the electron transport chain (ETC) was decreased numerically in freshly isolated and complex III activity significantly in previously frozen liver mitochondria. In previously frozen mitochondria, sunitinib decreased NADH oxidase activity concentration-dependently in both treatment groups. The hepatic mitochondrial reactive oxygen species (ROS) content and superoxide dismutase 2 expression were increased in sunitinib-treated mice. Protein and mRNA expression of several subunits of mitochondrial enzyme complexes were decreased in mitochondria from sunitinib-treated mice. Protein expression of PGC-1α, citrate synthase activity and mtDNA copy number were all decreased in livers of sunitinib-treated mice, indicating impaired mitochondrial proliferation. Caspase 3 activation and TUNEL-positive hepatocytes were increased in livers of sunitinib-treated mice, indicating hepatocyte apoptosis. In conclusion, sunitinib caused concentration-dependent toxicity in isolated mitochondria at concentrations reached in livers in vivo and inhibited hepatic mitochondrial proliferation. Daily mitochondrial insults and impaired mitochondrial proliferation most likely explain hepatocellular injury observed in mice treated with sunitinib.

Dopamine alleviated acute liver injury induced by lipopolysaccharide/d-galactosamine in mice

Dopamine (DA), a crucial neurotransmitter, not only functions in the central nervous system but also plays important roles in the modulation of inflammation. Several studies suggest that DA might suppress the inflammatory response both in vitro and in vivo. In the present study, the potential effects of DA in a mouse model with lipopolysaccharide (LPS)/d-galactosamine (D-Gal)-induced acute liver injury were investigated. The results show that DA-treated LPS/D-Gal-exposed mice had reduced incidence of histologic lesions, lower plasma aminotransferases and improved the survival rates compared to LPS/D-Gal-exposed mice. Treatment with DA also suppressed LPS/D-Gal-induced production of TNF-α, phosphorylation of c-jun-N-terminal kinase (JNK), cleavage of caspase-3, up-regulation of hepatic caspase-3, caspase-8, and caspase-9 activities and reduced the count of TUNEL-positive hepatocytes. These data indicate that DA attenuated LPS/D-Gal-induced fulminant liver injury in mice, which implies that DA might have value for the prevention of inflammatory liver disease.

Differences in Genetic Background Contribute to Pseudomonas Exotoxin A-Induced Hepatotoxicity in Rats

Pseudomonas aeruginosa exotoxin A (PEA) causes severe hepatotoxicity in experimental animals and is useful in investigations of immune-mediated liver injury. However, strain differences in the sensitivity to PEA-induced hepatotoxicity in rats remains be elucidated. In this study, we determined the severity of PEA-induced hepatotoxicity in six genetically different rat strains. Male LE (Long Evans), Wistar, F344, WKY, BN/SsN and LEW rats were administered a single intravenous injection of PEA (20 μg/kg). Significantly elevated serum ALT and AST levels, massive necrosis and hemorrhage, and numerous TUNEL-positive hepatocytes were observed in BN/SsN rats. In contrast, low levels of ALT and AST as well as mild changes in liver histopathology were observed in Wistar and F344 rats. Moderate levels of hepatic injuries were observed in LE, WKY, and LEW rats. Pro-inflammatory cytokines including TNF-α, IL-2 and IL-6 serum levels were markedly increased in BN/SsN rats compared to Wistar and F344 rats. However, the hepatic levels of low density lipoprotein receptor-related protein (LRP), which functions as the PEA receptor, were not significantly different in each strain. Taken together, we suggest that BN/SsN is the most sensitive rat strain, whereas Wistar and F344 were the most resistant rat strains to PEA-induced liver damage. The different genetic background of rat strains plays an important role in the susceptibility to PEA-induced epatotoxicity that may depend on immune-regulation but not LRP receptor levels.

Tamoxifen Attenuates Lipopolysaccharide/Galactosamine-induced Acute Liver Failure by Antagonizing Hepatic Inflammation and Apoptosis

ABSTRACTBacterial lipopolysaccharide (LPS)-induced acute liver failure (ALF) is a common severe clinical syndrome in intensive care unit. No other methods are available for its prevention apart from supportive treatment and liver transplantation. Tamoxifen (TAM) was reported to attenuate ALF induced by excessive acetaminophen, while its effect on LPS-induced ALF remained unknown. For this, in the present study, we comprehensively assessed whether TAM can attenuate ALF induced by LPS/galactosamine (GaIN). Mice were given TAM once a day for three times. Twelve hours after the last treatment, mice were given LPS/GaIN (intraperitoneally [i.p.]). Survival, plasma transaminases, and histopathology were examined. Serum TNF-α and IL-1β were analyzed by ELISA. Hepatic apoptosis was analyzed by TUNEL and caspase-3 Western blotting, respectively. Compared to the model group, ALF induced by LPS/GaIN was alleviated remarkably following TAM administration, as evidenced by the improvement of survival (87.5% vs. 37.5%), hepatic swell, moderate transaminases, slightly increased serum TNF-α, IL-1β (P < 0.05), and moderate histopathology. In respect of apoptosis, severe hepatocellular apoptosis was reduced notably by TAM treatment confirmed by less TUNEL-positive hepatocytes and decreased caspase-3 cleavage. The results demonstrated that TAM could attenuate LPS/GaIN-induced ALF effectively, probably due to hepatic inflammation and apoptosis antagonism. Furthermore, it was the first report about the effect of TAM on LPS/GaIN-induced ALF.

Novel molecular triggers underlie valproate-induced liver injury and its alleviation by the omega-3 fatty acid DHA: role of inflammation and apoptosis.

Background/AimHepatic injury is a hallmark adverse reaction to Valproate (VPA), a common used drug in the management of numerous CNS disorders, including epilepsy. DHA has a myriad of health benefits, including renal- and hepato-protective effects. Unfortunately, however, the underpinnings of such liver-pertinent VPA- and DHA-actions remain largely undefined. Accordingly, this study attempted to unveil the cellular and molecular triggers whereby VPA evokes, while DHA abates, hepatotoxicity.MethodsWe evaluated activity and/or expression of cellular markers of oxidative stress, inflammation, and apoptosis in rat liver, following treatment with VPA (500 mg/kg/day) with and without concurrent treatment with DHA (250 mg/kg/day) for two weeks.Results and conclusionVPA promoted hepatic oxidative stress as evidenced by enhancing activity/expression of NADPH-oxidase and its subunits, a ROS-generator, and by accumulation of lipid-peroxides. Moreover, VPA enhanced hepatic phosphorylation/activation of mitogen-activated protein kinase (MAPK), and expression of cyclooxygenase-2(COX-2), as proinflammatory signals. Besides, VPA promoted hepatocellular apoptosis, as attested by enhanced expression of cleaved caspase-9 and increased number of TUNEL-positive hepatocytes. Lastly, VPA upregulated levels of hypoxia-inducible factor-1-alpha (HIF-1α), a multifaceted modulator of hepatocytic biology, and activity of its downstream antioxidant enzyme heme-oxygenase-1(HO-1). These changes were significantly blunted by co-administration of DHA. Our findings demonstrate that VPA activated NADPH-oxidase and HIF-1α to induce oxidative-stress and hypoxia as initiators of hepatic injury. These changes were further aggravated by up-regulation of inflammatory (MAPK and COX-2) and apoptotic cascades, but could be partly lessened by HO-1 activation. Concurrent administration of DHA mitigated all VPA-induced anomalies.

장수풍뎅이 유충의 간보호 효능 및 항암활성

Beetle larvae have been used as a traditional medicine to treat various human liver diseases. To prove the liver protective function of Allomyrina dichotoma larvae (ADL), we induced liver damage by the intraperitoneal injection of a hepatotoxic reagent, diethylnitrosamine (DEN), to C3H/HeN male mice and orally administered freeze-dried ADL powder. ADL powder lessened DEN-induced hepatotoxicity considering the reduced signs of acute and chronic hepatotoxicities, such as the ALP level in the blood serum, TUNEL-positive hepatocytes, ductural reactions, steatotic hepatocytes, and collagen deposition of the Masson’s trichrome staining. In addition to hepatoprotection, the anti-cancer activity of ADL has been examined. The ADL powder was extracted with ethanol and then fractionated with hexane, ethyl acetate, and water by a solvent partition technique. The ethyl acetate fraction showed cytotoxicity to various cancer cells through induction of apoptosis and necrosis, as well as the perturbed metabolism of the cancer cell to trigger autophagy. Collectively, ADL contains bioactive substances that can protect hepatocytes from toxic chemicals and trigger cell death in cancer cells. Thus, further purification and analyses of ADL fractions could lead to the identification of novel bioactive compounds.

Caloric restriction mimetic 2-deoxyglucose alleviated lethal liver injury induced by lipopolysaccharide/d-galactosamine in mice

The glycolytic inhibitor 2-deoxyglucose (2-DG) is a calorie restriction (CR) mimetic produces CR-like beneficial effects in both acute and chronic pathological processes, but whether 2-DG is also helpful in critical and life-threatening situation is not known. In the present study, the potential benefits of 2-DG in lipopolysaccharide/d-galactosamine (LPS/D-Gal)-induced lethal liver injury were investigated. The results indicated that treatment with 2-DG suppressed the elevation of plasma aminotransferases, alleviated the histopathological abnormalities and improved the survival rate of LPS/D-Gal-exposed mice. Treatment with 2-DG also suppressed the production of pro-apoptotic cytokine TNF-α, the phosphorylation of JNK, the activation of caspase cascade and the count of TUNEL-positive apoptotic hepatocytes. These data suggested that the CR mimetic 2-DG could also provide beneficial effects in lethal pathological process such as LPS/D-Gal-induced fulminant liver injury.

New drug delivery system for liver sinusoidal endothelial cells for ischemia-reperfusion injury.

To investigate the cytoprotective effects in hepatic ischemia-reperfusion injury, we developed a new formulation of hyaluronic acid (HA) and sphingosine 1-phophate. We divided Sprague-Dawley rats into 4 groups: control, HA, sphingosine 1-phosphate (S1P), and HA-S1P. After the administration of each agent, we subjected the rat livers to total ischemia followed by reperfusion. After reperfusion, we performed the following investigations: alanine aminotransferase (ALT), histological findings, TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining, and transmission electron microscopy (TEM). We also investigated the expression of proteins associated with apoptosis, hepatoprotection, and S1P accumulation. S1P accumulated in the HA-S1P group livers more than S1P group livers. Serum ALT levels, TUNEL-positive hepatocytes, and expression of cleaved caspase-3 expression, were significantly decreased in the HA-S1P group. TEM revealed that the liver sinusoidal endothelial cell (LSEC) lining was preserved in the HA-S1P group. Moreover, the HA-S1P group showed a greater increase in the HO-1 protein levels compared to the S1P group. Our results suggest that HA-S1P exhibits cytoprotective effects in the liver through the inhibition of LSEC apoptosis. HA-S1P is an effective agent for hepatic ischemia/reperfusion injury.

Rotenone, a mitochondrial respiratory complex I inhibitor, ameliorates lipopolysaccharide/D-galactosamine-induced fulminant hepatitis in mice.

The syntheses of inflammatory mediators are energy-intensive processes and the mitochondria play pivotal roles in supporting these energy-requiring molecular responses. In the present studies, a mitochondrial respiratory complex I inhibitor rotenone was administrated in mice with lipopolysaccharide/D-galactosamine (LPS/D-Gal)-induced fulminant liver injury and the prophylactic and therapeutic effects on tissue injury were evaluated. We found that pretreatment with rotenone suppressed the elevation of plasma aminotransferases, alleviated the histopathological abnormalities and improved the survival rate of LPS/D-Gal-challenged mice. Pretreatment with rotenone has no obvious effects on hepatic malondialdehyde (MDA) contents but it significantly inhibited the up-regulation of both hepatic mRNA level and plasma protein level of TNF-α and IL-6. In the rotenone-pretreated group, the elevation of hepatic caspase-3, caspase-8 and caspase-9 activities induced by LPS/D-Gal decreased and rotenone reduced the count of TUNEL-positive apoptotic hepatocytes. In addition, posttreatment with rotenone at 1h after LPS/D-Gal challenge also suppressed the elevation of plasma aminotransferases. These data suggest that mitochondrial complex I inhibition might be a potential approach for the control of inflammation.