GCN2 Protein Kinase Is Required to Activate Amino Acid Deprivation Responses in Mice Treated with the Anti-cancer Agent l-Asparaginase

Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein β homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1.     

Genetic Activation of Nrf2 Protects against Fasting-Induced Oxidative Stress in Livers of Mice

Acute fasting causes elevated oxidative stress. The current study investigated the effects of the nuclear factor erythoid 2-related factor 2 (Nrf2), the sensor of oxidative stress in cells, on energy homeostasis and liver pathophysiology during fasting. Feed was removed from mice possessing none (Nrf2-null), normal (wild-type, WT), enhanced (Keap1-knockdown, K1-KD), and maximum (hepatocyte-specific Keap1-knockout, K1-HKO) Nrf2 activity in liver for 24 h. Body weight, blood glucose, and blood lipid profiles were similar among mice with graded Nrf2 activity under either fed or fasted conditions. Fasting reduced liver size in mice expressing Nrf2, but not in Nrf2-null mice. Nrf2-null mice accumulated more non-esterified free fatty acids and triglycerides in liver after fasting than the other genotypes of mice. Fatty acids are mainly catabolized in mitochondria, and Nrf2-null mice had lower mitochondrial content in liver under control feeding conditions, which was further reduced by fasting. In contrast, mitochondrial contents in mice with enhanced Nrf2 activity were not affected by fasting. Oxidative stress, determined by staining of free radicals and quantification of malondialdehyde equivalents, was highest in Nrf2-null and lowest in K1-HKO mice after fasting. The exacerbated oxidative stress in livers of Nrf2-null mice is predicted to lead to damages to mitochondria, and therefore diminished oxidation and increased accumulation of lipids in livers of Nrf2-null mice. In summary, the Nrf2-regulated signaling pathway is critical in protecting mitochondria from oxidative stress during feed deprivation, which ensures efficient utilization of fatty acids in livers of mice.     

Total flavonoids,extracted from Polygonum knotweed L,exert beneficial hepatoprotection against liver injury

Hepatic function is of great concern in metabolic and immunological homeostasis. Traditionally, medical management to liver damage may benefit from phytomedicine, such as Chinese herbs. In southern China, Polygonum perfoliatum L can contribute to alleviating pathological symptoms of liver disease, such as hepatitis. However, bioactive compounds of hepatoprotection in this herb are still less to be investigated. In this study, clinical data of patients with drug-induced liver injury were collected on the basis of serological analyses. In addition, we extracted and identified total flavonoids from Polygonum perfoliatum L (TFPPL) before implementing biochemical experiments in vivo. In human data, the blood contents of liver function enzymes were visibly elevated, and the percentage of immune cells were abnormally changed. The data from the animal study showed that TFPPL-treated carbon tetrachloride-exposed mice resulted in reduced absolute liver mass and lowered blood levels of liver functional enzymes (alanine transaminase and aspartate transaminase). In enzyme-linked immunosorbent assay, the comparable data indicated that serological tumor necrosis factor α (TNF-α), interleukin 6, and heat shock protein 90 (Hsp90) contents were reduced in TFPPL-treated mice. In histopathological observations, TFPPL-treated mice exhibited reduced hepatocellular Hsp90, TNF-α, nuclear factor κ-light-chain-enhancer of activated B cells-p65 positive cells, and lowered Bax and caspase-3-labeled cells in the livers. Further, intrasplenic integrin β1, 5′-nucleotidase, and antigen KI-67 positive cells were increased after TFPPL treatments. Taken together, our present findings demonstrate that herb-extracted TFPPL exert potential hepatoprotective activities against chemical-induced liver damage in mice, possibly through the pharmacological mechanisms of inhibiting inflammatory stress and apoptosis, inactivating Hsp90 bioactivity in the liver, and improving splenic immunocompetence.     

Possible involvement of reactive oxygen species in D-galactosamine-induced sensitization against tumor necrosis factor-alpha-induced hepatocyte apoptosis

Intravenous administration of tumor necrosis factor-alpha (TNF-alpha) (0.5 microg/mouse) caused hepatocyte apoptosis in BALB/c mice when they were sensitized with D-galactosamine (GalN, 20 mg/mouse). Activation of nuclear factor kappa B (NF-kappa B) and expression of apoptotic Bcl-2 family members were not significantly different between livers of mice treated with TNF-alpha alone and GalN + TNF-alpha, indicating that neither activation of NF-kappa B nor expression of Bcl-2 family is involved in the sensitization by GalN against TNF-alpha-induced hepatocyte apoptosis. To identify differentially expressed genes implicated in GalN-induced hepatocyte sensitization, we adopted mRNA fingerprinting using an arbitrarily primed polymerase chain reaction. The present analysis revealed that mRNA expression of extracellular antioxidant, selenoprotein P, was up-regulated in the livers after GalN administration. GalN-induced increase in its protein level was confirmed by Western blotting. Increased expression of this gene was also observed in the liver of mice treated with concanavalin A, but not anti-Fas antibody. mRNA of another antioxidant, glutathione peroxidase-1, was also up-regulated, and lipid peroxides were produced in the liver after GalN administration. Selenoprotein P mRNA level also increased in Huh-7 human hepatoma cells incubated with GalN (5 or 10 mM). Accordingly, formation of reactive oxygen species (ROS) was observed in GalN-treated Huh-7 cells. H(2)O(2) induced up-regulation of selenoprotein P mRNA and sensitized Huh-7 cells to TNF-alpha-induced apoptosis. These results suggest that ROS produced by GalN may play a pivotal role in hepatocyte sensitization toward TNF-alpha-induced apoptosis.     

Antihepatotoxic benefits of Poria cocos polysaccharides on acetaminophen-lesioned livers in vivo and in vitro

In our previous study, preliminary data indicates that Poria cocos polysaccharides (PCP) shows beneficial hepatoprotection against acetaminophen (APAP)-induced liver injury in mice. However, biological molecular mechanism warrants to be further discussed. In current study, a number of biochemical tests and immunoassays were subjected to respective PCP-dosed mice in vivo and liver cells in vitro. As a result, PCP-treated mice showed reduced contents of inflammatory cytokines (tumor necrosis factor [TNF]-β and TNFsR-I), enzymological molecules (alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase [LDL]), and heat shock protein 90 (Hsp90) after APAP exposure. Additionally, immunostaining assays exhibited that lowered-positive cells of cleaved-caspase-3, cleaved-poly ADP ribose polymerase, and Hsp90-labeled cells in PCP-treated livers were observed, and increased cluster of differentiation 29 (CD29), CD73-positive cells in the spleen were detected. Further, PCP-treated mouse liver cells resulted in increased cell growth, reduced LDL level. Increased proliferating cell nuclear antigen (PCNA), P38 mitogen-activated protein kinase (MAPK)-labeled cells and decreased Hsp90-positive cells in APAP-exposed liver cells were observed dose-dependently after PCP cotreatments. Collectively, our present experimental findings elucidate that PCP beneficially play hepatoprotective effects against APAP-lesioned liver cells in vivo and in vitro, potentially through the molecular mechanisms of suppressing cell death, reducing hepatocellular inflammatory stress and Hsp90 bioactivity.     

Lipopolysaccharide-induced liver apoptosis is increased in interleukin-10 knockout mice

Although IL-10 down-regulates pro-inflammatory cytokine secretion by hepatic Kupffer cells, the mechanisms underlying its hepatoprotective effects are not fully clear. This study tested the hypothesis that IL-10 protects the liver against pro-inflammatory cytokines by counteracting their pro-apoptotic effects. Wild type and IL-10 knockout mice were treated with bacterial lipopolysaccharide and sacrificed 1, 4, 8, and 12 h later. Plasma ALT activity was measured as a marker of liver injury. Liver pathology and TUNEL response were assessed by histology. Plasma levels and whole liver mRNA levels were measured for TNF-alpha, IL-1 beta, TGF-beta1, IL-10, and their respective receptors. Hepatic mRNA levels were measured for several pro-apoptotic adaptors/regulators, including FasL, Fas receptor, FADD, TRADD, Bad, Bak, Bax, and Bcl-X(S), and anti-apoptotic regulators, including Bcl-w, Bcl-X(L), Bcl-2, and Bfl-1. Caspase-3 activity in the liver was determined as well as immunohistochemistry for IL-1RII, TGF-betaRII and Fas receptor. At all time points the livers from IL-10 knockout mice displayed a significantly increased number of apoptotic nuclei compared to wild type mice. Changes in plasma cytokine levels and their liver mRNA levels were consistent with suppression by IL-10 of pro-inflammatory cytokine secretion. In addition, pro-inflammatory cytokine receptor mRNA levels (TNF-alpha, TGF-beta, and IL-1 beta) were markedly up-regulated by LPS at all time points in IL-10 knockout mice as compared to wild type mice. Expression of the pro-inflammatory cytokine receptor IL-1RII was similarly increased as shown by immunostaining. The mRNA levels of a typical pro-apoptotic cytokine, TRAIL, were increased and LPS also up-regulated the mRNA expression of other apoptotic factors to a larger extent in IL-10 knockout mice than in their wild type counterparts, suggestive of an IL-10 anti-apoptotic effect. In the livers of knockout mice, markedly increased caspase-3 activity was already evident at the 1-h time point following LPS administration, while in the wild type animals this increase was delayed. Immunostaining also indicated that LPS increased hepatic expression of the pro-apoptotic receptors Fas and TGF-betaRII in IL-10 knockout mice. The data presented in this study show that: (i) IL-10 modulates not only the secretion of pro-inflammatory cytokines, but also the receptors of these cytokines, and ii) IL-10 protects the liver against LPS-induced injury at least in part by counteracting pro-inflammatory cytokine-induced liver apoptosis.     

Protective Effects of Polydatin from Polygonum cuspidatum against Carbon Tetrachloride-Induced Liver Injury in Mice

Polydatin is one of main compounds in Polygonum cuspidatum, a plant with both medicinal and nutritional value. The possible hepatoprotective effects of polydatin on acute liver injury mice induced by carbon tetrachloride (CCl₄) and the mechanisms involved were investigated. Intraperitoneal injection of CCl₄ (50 µl/kg) resulted in a significant increase in the levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and hepatic malondialdehyde (MDA), also a marked enhancement in the expression of hepatic tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and nuclearfactor-kappa B (NF-κB). On the other hand, decreased glutathione (GSH) content and activities of glutathione transferase (GST), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were observed following CCl₄ exposure. Nevertheless, all of these phenotypes were evidently reversed by preadministration of polydatin for 5 continuous days. The mRNA and protein expression levels of hepatic growth factor-beta1 (TGF-β₁) were enhanced further by polydatin. These results suggest that polydatin protects mice against CCl₄-induced liver injury through antioxidant stress and antiinflammatory effects. Polydatin may be an effective hepatoprotective agent and a promising candidate for the treatment of oxidative stress- and inflammation-related diseases.     

Constitutive activation of nuclear factor-E2-related factor 2 induces biotransformation enzyme and transporter expression in livers of mice with hepatocyte-specific deletion of Kelch-like ECH-associated protein 1

Chemicals that activate nuclear factor-E2-related factor 2 (Nrf2) often increase multidrug-resistance-associated protein (Mrp) expression in liver. Hepatocyte-specific deletion of Kelch-like ECH-associated protein 1 (Keap1) activates Nrf2. Use of hepatocyte-specific Keap1 deletion represents a nonpharmacological method to determine whether constitutive Nrf2 activation upregulates liver transporter expression in vivo. The mRNA, protein expression, and localization of several biotransformation and transporters were determined in livers of wild-type and hepatocyte-specific Keap1-null mice. Sulfotransferase 2a1/2, NADP(H):quinone oxidoreductase 1, cytochrome P450 2b10, 3a11, and glutamate-cysteine ligase catalytic subunit expression were increased in livers of Keap1-null mice. Organic anion-transporting polypeptide 1a1 expression was nearly abolished, as compared to that detected in livers of wild-type mice. By contrast, Mrp 1-5 mRNA and protein levels were increased in Keap1-null mouse livers, with Mrp4 expression being more than 15-fold higher than wild types. In summary, Nrf2 has a significant role in affecting Oatp and Mrp expressions.     

Hepatoprotective effect of pyrroloquinoline quinone against alcoholic liver injury through activating Nrf2-mediated antioxidant and inhibiting TLR4-mediated inflammation responses

The present study was aimed at investigating the hepatoprotective effect of pyrroloquinoline quinone (PQQ) against acute alcoholic liver injury in mice. Acute alcoholic liver injury model was established in mice, and they were administrated with PQQ to investigate its hepatoprotective effect. Our results shows that PQQ can significantly ameliorate acute alcoholic liver injury by decreasing the hepatic marker enzymes, including serum alanine transaminase (ALT) and aspartate transaminase (AST), and increasing the levels of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in the liver. And PQQ can also significantly reduce the content of hepatic triglyceride (TG) and malondialdehyde (MDA). Moreover, PQQ attenuated alcohol-induced oxidative damage by activating NF-E2-related factor 2 (Nrf2)-mediated signaling pathway, and inhibiting Toll-like receptor 4 (TLR4)-mediated nuclear factor-kappa B (NF-κB) signaling pathway. Our findings have elucidated the liver protection mechanism of PQQ, which would encourage the further exploitation of PQQ as a hepatoprotective functional food.     

The structural analysis and its hepatoprotective activity of melanin isolated from Lachnum sp

In the present study, an intracellular melanin (LIM) isolated from Lachnum YM30 was obtained by submerged fermentation, extraction and purification. The possible structural formula of LIM was concluded, based on Ultraviolet (UV) analysis, IR analysis, elemental analysis, Nuclear Magnetic Resonance (NMR) and ESI-MS analysis, which showed the typical feature of fungal eumelanin. The hepatoprotective effect of LIM was evaluated in an acute LPS/D-GalN induced liver injury mouse model that was proved by the declining of alanine aminotransferase (ALT) and aspartate transaminase (AST), enhancing the content of anti-inflammatory factors interleukin-4 (IL-4) and interleukin-10 (IL-10). Concurrently, LIM could alleviate LPS/D-GalN toxicity through the effects of NF-kappa B (NF-κB) signaling pathway and its related cytokines interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8). The mechanisms underlying the protective effect might be related to inhibition of nuclear factor-kappa B activation and the functional groups of LIM. And compared with the single positive drug treatment or the melanin, the results showed that the combined treatment group could further reduce the inflammatory reaction of the model mice.     

Intereukin-10 and Kupffer cells protect steatotic mice livers from ischemia-reperfusion injury

Steatotic livers are more sensitive to ischemia/reperfusion (I/R) and are thus routinely rejected for transplantation because of their increased rate of primary nonfunction (PNF). Lean livers have less I/R-induced damage and inflammation due toKupffer cells (KC), which are protective after total, warm, hepatic I/R with associated bowel congestion. This protection has been linked to KC-dependent expression of the potent anti-inflammatory cytokine interleukin-10 (IL-10).We hypothesized that pretreatment with exogenous IL-10would protect the steatotic livers of genetically obese (ob/ob) mice from inflammation and injury induced by I/R. Lean and ob/ob mice were pretreated with either IL-10 or liposomally-encapsulated bisphosphonate clodronate (shown to deplete KC) prior to total, warm, hepatic I/R. IL-10 pretreatment increased survival of ob/ob animals at 24 hrs post-I/R from 30% to 100%, and significantly decreased serum ALT levels. At six hrs post-I/R, IL-10 pretreatment increased IL-10 mRNA expression, but suppressed up-regulation of the pro-inflammatory cytokine IL-1β mRNA. However, ALT levels were elevated at six hrs post-I/R in KC-depleted animals. These data reveal that pretreatment with IL-10 protects steatotic livers undergoing I/R, and that phagocytically active KC retain a hepatoprotective role in the steatotic environment.     

Induction of CYP1A by a diterpene quinone tanshinone IIA isolated from a medicinal herb Salvia miltiorrhiza in C57BL/6J but not in DBA/2J mice

Effects of tanshinone IIA, an active diterpene quinone of the herbal medicine Salvia miltiorrhiza (Danshen), on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in the arylhydrocarbon (Ah)-responsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice. Oral treatment of tanshinone IIA caused a dose-dependent increase of liver microsomal 7-methoxyresorufin O-demethylation (MROD) activity in B6 but not in D2 mice. In B6 mice, tanshinone IIA increased hepatic benzo(a)pyrene hydroxylation (AHH), 7-ethoxyresorufin O-deethylation, MROD, and 7-ethoxycoumarin O-deethylation activities. The levels of Cyp1A2 protein and mRNA were elevated. On the contrary, in D2 mice, tanshinone IIA decreased hepatic AHH and nifedipine oxidation activities and the CYP3A protein level without affecting other activities determined. Cyp1A2 protein and mRNA levels were not affected by tanshinone IIA in D2 mice. Tanshinone IIA had no effects on UGT and GST activities in both B6 and D2 mice. These results demonstrated that induction of CYP1A2 by tanshinone IIA depended on the Ah-responsiveness and occurred at pre-translational level.     

Dietary cocoa ameliorates non-alcoholic fatty liver disease and increases markers of antioxidant response and mitochondrial biogenesis in high fat-fed mice

Cocoa powder, derived Theobroma cacao, is a popular food ingredient that is commonly consumed in chocolate. Epidemiological and human intervention studies have reported that chocolate consumption is associated with reduced risk of cardiometabolic diseases. Laboratory studies have reported the dietary supplementation with cocoa or cocoa polyphenols can improve obesity and obesity-related comorbidities in preclinical models. Non-alcoholic fatty liver disease (NAFLD), one such comorbidity, is a risk factor for cirrhosis and hepatocellular carcinoma. Limited studies have examined the effect of cocoa/chocolate on NAFLD and underlying hepatoprotective mechanisms. Here, we examined the hepatoprotective effects of dietary supplementation with 80 mg/g cocoa powder for 10 wks in high fat (HF)-fed obese male C57BL/6J mice. We found that cocoa-supplemented mice had lower rate of body weight gain (22%), hepatic triacylglycerols (28%), lipid peroxides (57%), and mitochondrial DNA damage (75%) than HF-fed controls. These changes were associated with higher hepatic superoxide dismutase and glutathione peroxidase enzyme activity and increased expression of markers of hepatic mitochondrial biogenesis. We also found that the hepatic protein expression of sirtuin 3 (SIRT3), and mRNA expression of peroxisome proliferator activated receptor g coactivator (PGC) 1a, nuclear respiratory factor 1, and forkhead box O3 were higher in cocoa-treated mice compared to HF-fed controls. These factors play a role in coordinating mitochondrial biogenesis and expression of mitochondrial antioxidant response factors. Our results indicate that cocoa supplementation can mitigate the severity of NAFLD in obese mice and that these effects are related to SIRT3/PGC1a-mediated increases in antioxidant response and mitochondrial biogenesis.