Domino hepatocyte transplantation using explanted human livers with metabolic defects attenuates D-GalN/LPS-induced acute liver failure.

BACKGROUND: Explanted livers from patients with inherited metabolic liver diseases possess the potential to be a cell source of good-quality hepatocytes for hepatocyte transplantation (HT). This study evaluated the therapeutic effects of domino HT using hepatocytes isolated from explanted human livers for acute liver failure (ALF). METHODS: Isolated hepatocytes were evaluated for viability and function and then transplanted into D-galactosamine/lipopolysaccharide-induced ALF mice via splenic injection. The survival rate was analyzed by the Kaplan-Meier method and log-rank test. Liver function was evaluated by serum biochemical parameters, and inflammatory cytokine levels were measured by ELISA. The pathological changes in the liver tissues were assessed by hematoxylin-eosin staining. Hepatocyte apoptosis was investigated by TUNEL, and hepatocyte apoptosis-related proteins were detected by western blot. The localization of human hepatocytes in the injured mouse livers was detected by immunohistochemical analyses. RESULTS: Hepatocytes were successfully isolated from explanted livers of 10 pediatric patients with various liver-based metabolic disorders, with an average viability of 85.3% ± 13.0% and average yield of 9.2 × 106 ± 3.4 × 106 cells/g. Isolated hepatocytes had an excellent ability to secret albumin, produce urea, uptake indocyanine green, storage glycogen, and express alpha 1 antitrypsin, albumin, cytokeratin 18, and CYP3A4. Domino HT significantly reduced mortality, decreased serum levels of alanine aminotransferase and aspartate aminotransferase, and improved the pathological damage. Moreover, transplanted hepatocytes inhibited interleukin-6 and tumor necrosis factor-α levels. Domino HT also ameliorates hepatocyte apoptosis, as evidenced by decreased TUNEL positive cells. Positive staining for human albumin suggested the localization of human hepatocytes in ALF mice livers. CONCLUSION: Explanted livers from patients with inheritable metabolic disorders can serve as a viable cell source for cell-based therapies. Domino HT using hepatocytes with certain metabolic defects has the potential to be a novel therapeutic strategy for ALF.

Orientin Attenuated d-GalN/LPS-Induced Liver Injury through the Inhibition of Oxidative Stress via Nrf2/Keap1 Pathway.

Oxidative stress is involved in the pathogenesis of liver diseases, including liver injury, a serious health problem worldwide. Natural polyphenols have attracted increasing attention as potential agents for the prevention and treatment of liver diseases. Orientin, a flavonoid component with antioxidant capacity, has been regarded as a promising nutraceutical for patients with liver damage. This study aimed to investigate the amelioration effect of orientin on d-galactosamine and lipopolysaccharides (d-GalN/LPS) induced liver injury in mice, with a focus on its underlying mechanisms by using the H2O2-induced oxidative damage model of HepG2 cells. Results indicated that orientin alleviated d-GalN/LPS-induced liver damage by improving the hepatic histological changes and reducing the levels of hepatic and serum alanine aminotransferase and aspartic acid aminotransferase. Additionally, supplementation of orientin improved the antioxidant ability in mice by decreasing the levels of hepatic malondialdehyde, protein carbonyl, myeloperoxidase, nitric oxide, glutathione, glutathione peroxidase, gluathione reductase, and superoxide dismutase. Orientin treatment significantly elevated both the protein and mRNA expressions of nuclear factor erythroid 2-related factor 2, Kelch-like ECH-associated protein-1, heme oxygenase-1, and nicotinamide quinone oxidoreductase 1 in liver and HepG2 cells. The management of orientin also elevated the protein expression of glutathione S-transferase and Maf in HepG2 cells. Taken together, it suggested that orientin played an amelioration effect on liver injury by suppressing oxidative stress, which might be strongly related to the activation of Nrf2/ARE through PI3K/Akt and P38/MAPK signal pathways.

The polysaccharides from the Grifola frondosa fruiting body prevent lipopolysaccharide/D-galactosamine-induced acute liver injury via the miR-122-Nrf2/ARE pathways.

Polysaccharides can be used as a potential hepatoprotective agent in the treatment of acute liver injury. However, the underlying mechanism governing how polysaccharides protect against acute liver injury induced by lipopolysaccharide/d-galactosamine (LPS/d-GalN) remains unclear. To investigate the mechanism, the anti-oxidative and anti-inflammatory action and pathways were determined. In this study, we investigated the hepatoprotective effects of Grifola frondosa polysaccharides (GFP), which are obtained from the fruiting body of Grifola frondosa, on (LPS/d-GalN)-induced liver injury in mice. Histopathological analyses showed that GFP protected against LPS/d-GalN-induced lung inflammation. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the levels of the inflammatory mediators tumor necrosis factor-α (TNF-α), interleukin (IL)-2, IL-6, and monocyte chemoattractant protein-1 (MCP-1) were inhibited by GFP. The LPS/d-GalN-induced myeloperoxidase (MPO) activity and malondialdehyde (MDA) content were inhibited by GFP. The levels of superoxide dismutase (SOD) and glutathione (GSH) were upregulated by GFP. The GFP-treated group showed reduced expression levels of miR-122 in liver tissue. Nrf2 has been identified as a potential target of miR-122. The western blotting results showed that GFP attenuates LPS/d-GalN-induced acute liver injury via upregulating transcription factors Nrf2, Nqo-1, and HO-1 and downregulating transcription factor Keap-1 in the Nrf2/ARE signaling pathway. In conclusion, these results indicated that GFP was highly effective in inhibiting liver injury and may be a promising potential therapeutic reagent for liver injury treatment. GFP exerts protective effects against LPS/d-GalN-induced liver injury in mice, which may be related to the regulation of the miR-122-Nrf2/ARE pathways.

Efficacy of decoction from Jieduan Niwan formula on rat model of acute-on-chronic liver failure induced by porcine serum.

OBJECTIVE: To dynamically observe the efficacy of Jieduan Niwan formula (JDNW) on a rat model of acute-on-chronic liver failure (ACLF). METHODS: Seventy Wistar rats were divided into control group (6 rats), model group (22 rats), JDNW group (21 rats), and SP600125 group (21 rats). 13 weeks' porcine serum injection followed with D-galactosamine and lipopolysaccharide joint acute attack was used to establish ACLF model. Rats in JDNW group were orally given JDNW formula for 3 days before acute attack; rats in SP600125 group were injected with SP600125 30 min ahead of acute attack. Rats were sacrificed respectively at 4, 8 and 12 h after model established. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), Creatinine (CR), blood urea nitrogen (BUN), prothrombin activity (PTA) were examined by biochemical process, Tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-10 (IL-10), transformed growth factor-beta 1 (TGF-ß1), High mobility group box-1 (HMGB-1), CD3, CD4, CD8 were analyzed by enzyme-linked immunosorbent assay, apoptotic index (AI) was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling staining, expression of Bad, phosphorylated Jun N-terminal kinases (p-JNK) and Cytochrome C (Cyt C) were detected by immunohistochemical analysis, Bax and Bid were detected by Western blot analysis. RESULTS: In model group, the levels of ALT, AST, TBIL, CR, BUN, IL-1ß, IL-6, IL-10, TGF-ß1 and HMGB-1 remarkably increased and PTA decreased compared with control group (P < 0.05), as time goes on, ALT, AST, TBIL, CR, BUN, continued to grow, while IL-1ß, IL-6, IL-10, HMGB-1, TGF-ß1 and PTA gradually decreased; massive necrosis could be seen; the levels of TNF-a, CD3, CD4, CD8, AI, p-JNK, Bax, Bad, Bid and Cyt C increased at 4 h and peaked at 8 h, but decreased at 12 h (P < 0.05). JDNW group, by contrast, showed less pathological injury, increased PTA level, and reduced ALT, AST, TBIL, TNF-α, IL-1ß, IL-6, IL-10, TGF-ß1, HMGB-1, CD3, CD4 and CD8 levels (P < 0.05), moreover, the AI and expression of p-JNK, Bax, Bad, Bid and Cyt C were lower than model group at 4 and 8 h but were higher at 12 h (P < 0.05). Similar results were observed in SP600125 group. CONCLUSION: An ACLF rat model with low mortality can be established by porcine serum joint with D-galactosamine + lipopolysaccharide induction; JDNW decoction can effectively suppress the inflammatory reaction, improve the immune system, and protect the liver of ACLF rats, the mechanism might involve the inhibition of the JNK-induced mitochondrial apoptotic pathway.

Therapeutic benefits of apocynin in mice with lipopolysaccharide/D-galactosamine-induced acute liver injury via suppression of the late stage pro-apoptotic AMPK/JNK pathway.

The excessive generation of reactive oxygen species (ROS) plays crucial roles in the development of acute liver injury. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is responsible for the robust production of ROS under inflammatory circumstance, but the pathological roles of NOX and the pharmacological significance of NOX inhibitor in acute liver injury remains unclear. In the present study, the potential roles of NOX in acute liver injury were investigated in a mouse model with lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced acute liver injury. The results indicated that LPS/D-Gal exposure time-dependently increased the level of ROS in liver tissue. Pretreatment with the NOX inhibitor apocynin suppressed LPS/D-Gal induced upregulation of ROS, 8-hydroxy-2-deoxyguanosine (8-OH-dG), protein carbonyl content and thiobarbituric acid reactive substances (TBARS). Pretreatment with apocynin also suppressed LPS/D-Gal-induced elevation of aminotransferase, alleviated histological abnormalities, inhibited the production of pro-inflammatory cytokine tumor necrosis factor α (TNF-α), blocked the activation of caspase cascade, reduced the count of TUNEL-positive cells and prevented LPS/D-Gal-induced mortality. Interestingly, post insult treatment with apocynin also suppressed LPS/D-Gal-induced oxidative stress, hepatocyte apoptosis, liver damage but improved the survival rate. Mechanistically, posttreatment with apocynin prohibited LPS/D-Gal-induced activation of the late stage pro-apoptotic AMP-activated protein kinase (AMPK)/c-Jun N-terminal kinase (JNK) pathway. Post-insult treatment with the antioxidant N-acetylcysteine also resulted in suppressed activation of AMPK/JNK, mitigated apoptosis and alleviated liver injury. These data suggest that NOX-derived ROS might be a crucial late stage detrimental factor in LPS/D-Gal-induced acute liver injury via promoting the activation of the pro-apoptotic AMPK/JNK pathway, and the NOX inhibitor might have important value in the pharmacological intervention of inflammation-base liver damage.

Taurine Enhances the Protective Actions of Fish Oil Against D-Galactosamine-Induced Metabolic Changes and Hepatic Lipid Accumulation and Injury in the Rat.

This study has evaluated the effects of a supplementation with taurine (TAU) on the actions of fish oil (FO) against the hypoglycemia, hypoproteinemia, and hepatic accumulation of lipids and liver damage caused by D-galactosamine (GAL) in the rat. To this end, male Sprague-Dawley rats (200-225 g), in groups of 6, were orally treated with physiological saline (2.5 mL, control group), FO (60 mg/kg), TAU (2.4 mmol/kg) or FO-TAU for three consecutive days and before a single oral dose of GAL (400 mg/kg) given on day 3. In parallel, rats receiving only GAL on day 3 or N-acetylcysteine (NAC, 2.4 mmol/kg) for 3 days before GAL served as controls. On day 4 blood samples were collected by cardiac puncture and used to either measure glucose (GLC) or to obtain plasma fractions. Immediately thereafter, the livers were excised, made into a homogenate in phosphate buffered saline pH 7.4, and centrifuged to obtain clear supernatant. Plasma samples were assayed for their total protein (TP), triglycerides (TG), cholesterol (CHOL), phospholipids (PLP), free fatty acids (FFA) and total bilirubin (TB) and direct bilirubin (DB) contents, and for the activities of alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP). The liver homogenates were used to measure TG, CHOL, PLP and total lipids (TL) contents. Without exceptions, GAL was found to markedly affect (p < 0.001) all of the experimental parameters examined, with increases occurring in all instances except for the values of the plasma GLC, TP and PLP which were decreased. A pretreatment with either FO or TAU led to significant attenuation of the effects of GAL and which, in most cases, were of similar magnitude. On the other hand, a combined pretreatment with FO plus TAU usually resulted in a greater protection than with either agent alone (p ≤ 0.05). NAC, serving as a reference treatment, was, in most instances, equipotent with FO alone and. in addition, was the only agent that significantly attenuated the increases in both liver weight and liver weight to body weight ratio caused by GAL.

Protective effects of alpinetin on lipopolysaccharide/d-Galactosamine-induced liver injury through inhibiting inflammatory and oxidative responses.

Alpinetin, a type of novel plant flavonoid derived from Alpinia katsumadai Hayata, has been reported to have anti-inflammatory effects. The aim of this investigation was designed to reveal the protective effects of alpinetin on Lipopolysaccharide (LPS)/d-galactosamine (D-Gal)-induced liver injury in mice. Alpinetin (12.5, 25, 50 mg/kg) were given 1 h before LPS and D-Gal treatment. 12 h after LPS and D-Gal treatment, the liver tissues and serum were collected. Our results showed that alpinetin treatment improved liver histology, indicating a marked decrease of inflammatory cell infiltration and restore hepatic lobular architecture. Alpinetin also inhibited liver myeloperoxidase (MPO) activity and malondialdehyde (MDA) level. Furthermore, LPS/D-Gal-induced tumor necrosis factor-α (TNF-α) and Interleukin-1ß (IL-1ß) production were dose-dependently inhibited by alpinetin. Alpinetin also attenuated LPS/D-Gal-induced expression of phospho-NF-κB p65 and phospho-IκBα. In addition, alpinetin was found to increase the expression of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). In conclusion, these findings suggested that alpinetin inhibited liver injury through inhibiting NF-κB and activating the Nrf2 signaling pathway.

Geraniol protects against lipopolysaccharide and D-galactosamine-induced fulminant hepatic failure by activating PPARγ.

Geraniol (GOH), a natural component of plant essential oils, exhibits potent antioxidant and anti-inflammatory properties. The aim of this study was to assess the protective effects and mechanisms of GOH on lipopolysaccharide (LPS)/d-galactosamine (D-GalN)-induced fulminant hepatic failure (FHF). Mice were treated with GOH (12.5, 25, and 50 µg/kg) 1 h before challenging LPS (60 mg/kg) and D-GalN (800 mg/kg). 8 h later LPS/D-GlaN treatment, mice were sacrificed and the serum and the liver tissues were collected for testing. The liver pathological changes were assessed by H & E staining. MPO activity, MDA level in liver tissues, and AST, ALT levels in serum were detected by specific detection kits. The levels of TNF-α and IL-1ß were detected by ELISA. The expression of NF-κB and PPARγ were detected by western blot analysis and qRT-PCR. The results showed that GOH had a protective effect on LPS/D-GalN-induced FHF, as evidence by the attenuation of liver pathological injury, MPO activity, MDA level, and serum AST and ALT levels. GOH reduced liver TNF-α and IL-1ß levels through inhibiting NF-κB signaling pathway activation. Furthermore, GOH increased PPARγ expression in FHF induced by LPS/D-GalN. In conclusion, the present study proved that GOH protects against LPS/D-GalN-induced FHF through inhibiting inflammatory response and increasing PPARγ expression.

Hepatoprotective effect of chiisanoside from Acanthopanax sessiliflorus against LPS/D-GalN-induced acute liver injury by inhibiting NF-κB and activating Nrf2/HO-1 signaling pathways.

BACKGROUND: In China, Acanthopanax sessiliflorus is a delicious wild vegetable. It is also used to treat inflammation and pain. Chiisanoside (CSS) is the main constituent of the leaf of A. sessiliflorus. Combined use of lipopolysaccharide and d-galactosamine (LPS/D-GalN) can induce acute liver failure in human beings, and there are no reports on the protective effect of CSS against LPS/D-GalN-induced acute liver injury in mice. RESULTS: Chiisanoside pretreatment evidently reduced the activities of alanine transaminase (ALT) and aspartate transaminase (AST) in the changes induced by LPS/D-GalN, and these histopathological changes induced by LPS/GalN were significantly weakened. Catalase (CAT), glutathione (GSH), and superoxide dismutase (SOD) activities increased, and malondialdehyde (MDA) activity decreased after CSS treatment compared with LPS/D-GalN treatment. Pretreatment with CSS also inhibited the expression levels of inflammatory factors. The administration of CSS prevented the phosphorylated expression of inhibitor kappa B (IκB) kinase, and led to a significant increase in heme oxygenase-1 (HO-1) expression and nuclear factor erythroid 2-related factor2 (Nrf2) nuclear translocation. CONCLUSION: The protective effects of CSS are attributed to its antioxidative effect and inflammatory suppression in Nuclear factor kappa beta (NF-κB) and Nrf2/HO-1 signaling pathways. Chiisanoside might therefore be a potential ingredient for drug and food development against acute liver injury in the future. © 2018 Society of Chemical Industry.

Humulus japonicus Extracts Protect Against Lipopolysaccharide/d-Galactosamine-Induced Acute Liver Injury in Rats.

It has been described that Humulus japonicus has potential antituberculosis and anti-inflammatory effects. The antiaging activity of H. japonicus extract (HJE) was also examined not only in yeast but also in human fibroblast cells. We evaluated the protective effect of HJE on hepatotoxicity in this study. We demonstrated the expression of antioxidative enzyme and cytokines in plasma. The vehicle control group received orally administered normal saline. Acute hepatotoxicity rat model was induced by lipopolysaccharide (LPS) (30 µg/kg) and d-galactosamine (D-GalN) (500 mg/kg) by intraperitoneal injection. The positive control group received orally administered silymarin (10 mg/kg). Three HJE groups received 8, 16, and 32 mg/kg. The blood samples were prepared to evaluate malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) level to examine oxidative stress, and hepatic tissue was assayed for myeloperoxidase (MPO). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and tumor necrosis factor-α (TNF-α) activities were also assayed to examine liver cell viability. Pretreatment with HJE decreased levels of AST, ALT, MDA, MPO, and TNF-α and increased levels of SOD and CAT compared with the LPS/D-GalN-treated group. These results suggested that HJE has the potential to reduce oxidative damage and inflammatory reactions in LPS/D-GalN-induced liver injury in the rat model. It can also increase survival rate in LPS/D-GalN-induced hepatotoxicity rat models.

AMSC-derived exosomes alleviate lipopolysaccharide/d-galactosamine-induced acute liver failure by miR-17-mediated reduction of TXNIP/NLRP3 inflammasome activation in macrophages.

BACKGROUND: Mesenchymal stem cell (MSC)-derived exosome administration has been considered as a novel cell-free therapy for liver diseases through cell-cell communication. This study was aimed to determine the effects and mechanisms of AMSC-derived exosomes (AMSC-Exo) for acute liver failure (ALF) treatment. METHODS: AMSC-Exo were intravenously administrated into the mice immediately after lipopolysaccharide and D-galactosamine (LPS/GalN)-exposure and their effects were evaluated by liver histological and serum biochemical analysis. To elucidate its mechanisms in ALF therapy, the expression levels of miRNAs and inflammasome-related genes in macrophages were evaluated by qPCR and Western blot analysis, respectively. The exosomes from miR-17-knockdowned AMSCs (AMSC-ExomiR-17-KD) were used for further determine the role of miR-17 in AMSC-Exo-based therapy. FINDINGS: AMSC-Exo administration significantly ameliorated ALF as determined by reduced serum alanine aminotransferase and aspartate aminotransferase levels and hepatic inflammasome activation. Further experiments revealed that AMSC-Exo were colocalized with hepatic macrophages and could reduce inflammatory factor secretion by suppressing inflammasome activation in macrophages. Moreover, miR-17, which can suppress NLRP3 inflammasome activation by targeting TXNIP, was abundant in AMSC-Exo cargo. While, the therapeutic effects of AMSC-ExomiR-17-KD on ALF were significantly abolished as they could not effectively suppress TXNIP expression and consequent inflammasome activation in vitro and in vivo. INTERPRETATION: Exosome-shuttled miR-17 plays an essential role in AMSC-Exo therapy for ALF by targeting TXNIP and suppressing inflammasome activation in hepatic macrophages. AMSC-Exo-based therapy may present as a promising approach for TXNIP/NLRP3 inflammasome-related inflammatory liver diseases. FUND: Key R&D projects of Zhejiang province (2018C03019) and National Natural Science Fund (81470851 and 81500616).

Daphnetin alleviates lipopolysaccharide/d-galactosamine-induced acute liver failure via the inhibition of NLRP3, MAPK and NF-κB, and the induction of autophagy.

Acute liver failure (ALF), an inflammation-mediated hepatocellular injury process, is a life-threatening and fatal clinical syndrome. Daphnetin (Daph) has been reported to exhibit various pharmacological activities, particularly its antiinflammatory property. The present study was designed to evaluate the protective effects and underlying mechanisms of Daph against lipopolysaccharide and d-galactosamine (LPS/GalN)-induced ALF in mice. Our findings suggested that Daph treatment efficiently protected against LPS/GalN-induced ALF by lessening the lethality, decreasing the alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 secretion, malondialdehyde (MDA) formation and myeloperoxidase (MPO) level, inhibiting nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) protein expression, and increasing the glutathione (GSH) and superoxide dismutase (SOD) contents. Moreover, Daph treatment effectively inhibited LPS/GalN-induced nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3), mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathway activation, and significantly induced autophagy activation by enhancing the level of pro-autophagy proteins expression. Taken together, these findings suggested that Daph plays a pivotal role in liver protection by suppressing inflammatory responses which may be closely associated with the inhibition of NLRP3 inflammasome, MAPK and NF-κB activation, as well as the induction of autophagy.

Protective Effects of Sesquiterpenoids from the Root of Panax ginseng on Fulminant Liver Injury Induced by Lipopolysaccharide/d-Galactosamine.

It is reported that sesquiterpenoids from Panax ginseng (SPG) possess various pharmacological activities, for example, antidepressant, antioxidative, and anti-inflammatory activities. The purpose of this study was to examine the hepatoprotective effects of SPG (2.5 and 10 mg/kg, i.g.) on fulminant liver injury induced by d-galactosamine (d-GalN) and lipopolysaccharide (LPS) and discuss its mechanisms of action. After 24 h of d-GalN (400 mg/kg, i.p.) and LPS (25 µg/kg, i.p.) exposure, the serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), hepatic malondialdehyde (MDA) level, hepatic activities of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), and hepatic tissue histology were measured. Expression levels of tumor necrosis factor α (TNF-α) and interleukin-1ß (IL-1ß) were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Moreover, the nuclear factor κ-light-chain-enhancer of activated B cell (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), sirtuin type 1 (Sirt 1), and heme oxygenase 1 (HO-1) were determined by western blotting. The results indicated that SPG evidently restrained the increase of serum ALT and AST levels induced by d-GalN/LPS. SPG obviously downregulated TNF-α and IL-1ß levels and their mRNA expression in liver. In addition, d-GalN/LPS injection induced severe oxidative stress in liver by boosting the MDA level as well as decreasing CAT, GSH, and SOD capacities, and SPG reversed these changes. Meanwhile, SPG inhibited NF-κB activation induced by d-GalN/LPS and upregulated Sirt 1, Nrf2, and HO-1 expression levels. Therefore, SPG might protect against the fulminant liver injury induced by d-GalN/LPS via inhibiting inflammation and oxidative stress. The protective effect of SPG on fulminant liver injury induced by d-GalN/LPS might be mediated by the Sirt 1/Nrf2/NF-κB signaling pathway. All of these results implied that SPG might be a promising food additive and therapeutic agent for fulminant liver injury.

Isovitexin alleviates liver injury induced by lipopolysaccharide/d-galactosamine by activating Nrf2 and inhibiting NF-κB activation.

The aim of this study was to investigate the protective effects and mechanism of isovitexin, a glycosylflavonoid isolated from rice hulls of Oryza sativa, on Lipopolysaccharide (LPS)/d-galactosamine (D-Gal)-induced acute liver injury. The mice were randomly divided into five groups: control group, LPS/D-Gal group, and LPS/D-Gal + isovitexin groups. The mice of LPS/D-Gal group were received of LPS (50 µg/kg) and D-gal (800 mg/kg) intraperitoneal. The mice of LPS/D-Gal + isovitexin groups were received isovitexin (25, 50, 100 mg/kg) 1 h before LPS/D-Gal treatment. The results showed that the severity of liver injury was attenuated by treatment of isovitexin, as confirmed by the decreased liver histopathologic changes, as well as serum AST and ALT levels. Furthermore, the levels of TNF-α in serum and liver tissues, MPO activity and MDA content were significantly inhibited by isovitexin. In addition, isovitexin significantly attenuated NF-κB phosphorylation induced by LPS/D-Gal. The expression of Nrf2 and HO-1 were significantly up-regulated by isovitexin. In conclusion, isovitexin could protect against LPS/D-Gal-induced liver injury by inhibiting inflammatory and oxidative responses. Isovitexin also had protective effects against carbon tetrachloride (CCl4)-induced liver injury. Isovitexin may used as a potential agent for the treatment of liver injury.

Maslinic acid protects against lipopolysaccharide/d-galactosamine-induced acute liver injury in mice.

Acute liver injury is a life-threatening syndrome that often caused by hepatocyte damage. In this study, we investigated the protective effects of maslinic acid (MA) on lipopolysaccharide (LPS)/d-galactosamine (D-gal)-induced acute liver injury and clarified its mechanism. Mice acute liver injury model was induced by given LPS and D-gal and MA was given intraperitoneally 1 h before LPS and D-gal. Our results showed that MA protected against liver injury by attenuating liver histopathologic changes, serum AST and ALT levels. The increased inflammatory cytokines TNF-α and IL-6 in serum and liver tissues were also inhibited by MA. The level of MDA and the activity of MPO in liver tissues were up-regulated by LPS/D-gal and dose-dependently inhibited by MA. Furthermore, MA attenuated hepatic NF-κB protein expression and increased hepatic Nrf2 and HO-1 protein expression. Taken together, MA offers a protective role against LPS/D-gal-induced liver injury through suppressing NF-κB and activating Nrf2 signaling pathways.

Protective effects of tenuigenin on lipopolysaccharide and d-galactosamine-induced acute liver injury.

Tenuigenin (TEN), a major active component of polygala tenuifolia root, has been reported to have a number of biological properties, such as anti-oxidative and anti-inflammatory activities. However, the protective effect of TEN on acute liver injury has not yet been reported. This research aims to detect the protective effect of TEN on lipopolysaccharide (LPS) and d-galactosamine (D-GalN)-induced acute liver injury in mice and to investigate the molecular mechanisms. TEN was administered intraperitoneally 1 h before LPS/D-GalN treatment. The levels of TNF-α, IL-1ß, ALT, and AST were measured. The expression of NF-κB, ASK1, MAPKs, Nrf2, and HO-1 were detected by western blot analysis. The results showed that TEN significantly inhibited LPS/D-GalN-induced serum ALT and AST levels. TEN also inhibited LPS/D-GalN-induced TNF-α and IL-1ß production. Furthermore, LPS/D-GalN-induced hepatic MDA and MPO activities were also inhibited by TEN. In addition, TEN was found to inhibit LPS/D-GalN-induced ASK1 expression, NF-κB and MAPKs activation and up-regulate the expression of Nrf2 and HO-1. In conclusion, TEN protected against LPS/GalN-induced acute liver injury by suppressing inflammatory and oxidative responses.

Quantitative Determination of Stilbenoids and Dihydroisocoumarins in Shorea roxburghii and Evaluation of Their Hepatoprotective Activity.

A simultaneous quantitative analytical method for 13 stilbenoids including (-)-hopeaphenol (1), (+)-isohopeaphenol (2), hemsleyanol D (3), (-)-ampelopsin H (4), vaticanols A (5), E (6), and G (7), (+)-α-viniferin (8), pauciflorol A (9), hopeafuran (10), (-)-balanocarpol (11), (-)-ampelopsin A (12), and trans-resveratrol 10-C-ß-d-glucopyranoside (13), and two dihydroisocoumarins, phayomphenols A1 (14) and A2 (15) in the extract of Shorea roxburghii (dipterocarpaceae) was developed. According to the established protocol, distributions of these 15 polyphenols (1-15) in the bark and wood parts of S. roxburghii and a related plant Cotylelobium melanoxylon were evaluated. In addition, the principal polyphenols (1, 2, 8, 13-15) exhibited hepatoprotective effects against d-galactosamine (d-galN)/lipopolysaccharide (LPS)-induced liver injury in mice at a dose of 100 or 200 mg/kg, p.o. To characterize the mechanisms of action, the isolates were examined in in vitro studies assessing their effects on (i) d-GalN-induced cytotoxicity in primary cultured mouse hepatocytes; (ii) LPS-induced nitric oxide (NO) production in mouse peritoneal macrophages; and (iii) tumor necrosis factor-α (TNF-α)-induced cytotoxicity in L929 cells. The mechanisms of action of these polyphenols (1, 2, and 8) were suggested to be dependent on the inhibition of LPS-induced macrophage activation and reduction of sensitivity of hepatocytes to TNF-α. However, none of the isolates reduced the cytotoxicity caused by d-GalN.

Nobiletin attenuates lipopolysaccharide/D­galactosamine­induced liver injury in mice by activating the Nrf2 antioxidant pathway and subsequently inhibiting NF­κB­mediated cytokine production.

Inflammation and oxidative stress serve an important role in the development of lipopolysaccharide/D-galactosamine (LPS/GalN)­induced acute liver injury. Nobiletin, which is found in high quantities in the peel of citrus fruits, is able to modulate immune responses, including inflammatory response and oxidative stress. The present study aimed to evaluate the protective effects of nobiletin on LPS/GalN­induced acute liver injury. Male C57BL/6 mice were intraperitoneally treated with nobiletin (50, 100 and 200 mg/kg) 2 h prior to LPS/GalN injection. Liver injury was observed in the LPS/GalN group, as demonstrated by increased levels of serum hepatic enzymes and hepatic inflammatory mediators, as well as by histopathological alterations. Treatment with nobiletin reduced serum alanine aminotransferase and aspartate aminotransferase levels, improved hepatic structure, and suppressed hepatic interleukin (IL)­1ß, IL­6 and tumor necrosis factor­α production 24 h after LPS/GalN exposure. Western blot analysis revealed that nobiletin treatment inhibited inducible nitric oxide synthase and cyclooxygenase­2 liver expression. In addition, nobiletin suppressed LPS/GalN­induced phosphorylation and degradation of inhibitor of nuclear factor (NF)­κB (IκB)α, as well as NF­κB p65 translocation into the nucleus. Nobiletin also upregulated the expression of nuclear NF­E2­related factor 2 (Nrf2) and cytoplasmic heme oxygenase­1. In conclusion, these results indicate that nobiletin serves a protective role in LPS/GalN­induced acute liver injury via activation of the Nrf2 antioxidant pathway and subsequent inhibition of NF­κB­mediated cytokine production. These findings support the potential for nobiletin as a therapeutic agent for the treatment of acute liver injury.

[Role of neutrophils in treatment of rats with D-galactosamine-induced acute liver failure with bone marrow mesenchymal stem cells].

Objective: To investigate the therapeutic effect of bone marrow mesenchymal stromal cell (BMSC) transplantation on D-galactosamine-induced acute liver failure in Sprague-Dawley (SD) rats, as well as the mechanism of neutrophils in this process. Methods: A total of 39 male SD rats were divided into control group (8 rats, intraperitoneal injection of isotonic saline), model group (10 rats, intraperitoneal injection of D-galactosamine), solvent group (9 rats, tail vein injection of isotonic saline at 2 hours after intraperitoneal injection of D-galactosamine), and treatment group (12 rats, tail vein injection of MSCs at 2 hours after intraperitoneal injection of D-galactosamine). The rats were sacrificed at 24 hours after the model of D-galactosamine-induced acute liver failure was established, and the blood and liver tissue were harvested. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and total bilirubin (TBil) were measured, and blood analysis was performed to measure the number and percentage of neutrophils in peripheral blood. Immunofluorescence assay was used to measure the expression of the neutrophil marker Ly6g in the liver, the myeloperoxidase (MPO) kit was used to measure the activity of MPO in liver, and RT-PCR was used to measure the mRNA expression of inflammatory cytokines and chemokines in the liver, i.e., tumor necrosis factor-α(TNF-α), interleukin-1ß(IL-1ß), interferon-γ(IFN-γ), interleukin-10 (IL-10), CXC chemokine ligand 1 (CXCL1), and CXC chemokine ligand 2 (CXCL2). Another 64 male SD rats were randomly divided into groups, and the survival rates of rats in each group were observed for 7 days. The independent samples t-test was used for comparison between any two groups (Levene homogeneity test of variance, and the corrected t-test was used for a P value of < 0.05), and the log-rank test was used for comparison of survival rates between any two groups. Results: At 24 hours after acute liver failure was induced by D-galactosamine in the SD rats, there were significant increases in the liver function parameters (ALT: 2884.1±541.0 U/L vs 45.4±11.0 U/L,P< 0.001; AST: 3634.9±755.9 U/L vs 143.9±23.7 U/L,P< 0.001; TBil: 44.4±8.4µmmol/L vs 0.9±0.2µmmol/L,P< 0.001) and the number and percentage of peripheral blood neutrophils [number: (4.7±1.1)×109 vs (1.4±0.4)×109,P< 0.001; percentage: 44.9%±8.0% vs 18.3%±4.4%,P< 0.001]. A large number of neutrophils aggregated in the liver tissue, and there were significant increases in the MPO activity (4.72±1.09 U/g vs 1.13±0.24 U/g,P< 0.001), inflammatory cytokines, and chemokines. Compared with the model group, the treatment group showed significant improvements in liver function (ALT: 1 823.9±389.2 U/L vs 2 884.1±541.0 U/L,P< 0.001; AST: 2173.0±567.3 U/L vs 3634.9±755.9 U/L,P< 0.001; TBil: 30.9±6.5µmmol/L vs 44.4±8.4µmmol/L,P< 0.001) and survival rate (50% vs 12.5%,P= 0.023). Meanwhile, the treatment group also showed significant reductions in the number and percentage of peripheral blood neutrophils [number: (3.5±1.0)×109 vs (4.7±1.1)×109,P= 0.012; percentage: 35.9%±8.9% vs 44.9%±8.0%,P= 0.021], number of neutrophils in the liver, and MPO activity (3.52±1.03 U/g vs 4.72±1.09 U/g,P= 0.040), as well as significantly inhibited expression of inflammatory cytokines and chemokines (TNF-α: 2.458±0.762 vs 3.778±1.046, P = 0.005; IL-1ß: 2.498±0.547 vs 4.065 ± 0.953,P= 0.002; IFN-γ: 3.977±1.039 vs 5.418±1.255, P = 0.025; IL-10: 6.056±1.542 vs 3.368±0.952,P= 0.001; CXCL1: 7.988±1.911 vs 10.366±1.239,P= 0.010; CXCL2: 3.441±1.005 vs 4.847±1.113,P= 0.019). Conclusion: BMSC transplantation has a therapeutic effect on D-galactosamine-induced acute liver failure in rats, and this process is accompanied by reduced aggregation and activity of neutrophils in peripheral blood and liver. Inflammatory cytokines and chemokines may be involved in the mechanism of regulation of these two aspects.

Caspase Inhibition Prevents Tumor Necrosis Factor-α-Induced Apoptosis and Promotes Necrotic Cell Death in Mouse Hepatocytes in Vivo and in Vitro.

How different cell death modes and cell survival pathways cross talk remains elusive. We determined the interrelation of apoptosis, necrosis, and autophagy in tumor necrosis factor (TNF)-α/actinomycin D (ActD) and lipopolysaccharide/D-galactosamine (GalN)-induced hepatotoxicity in vitro and in vivo. We found that TNF-α/ActD-induced apoptosis was completely blocked by a general caspase inhibitor ZVAD-fmk at 24 hours but hepatocytes still died by necrosis at 48 hours. Inhibition of caspases also protected mice against lipopolysaccharide/GalN-induced apoptosis and liver injury at the early time point, but this protection was diminished after prolonged treatment by switching apoptosis to necrosis. Inhibition of receptor-interacting protein kinase (RIP)1 by necrostatin 1 partially inhibited TNF-α/ZVAD-induced necrosis in primary hepatocytes. Pharmacologic inhibition of autophagy or genetic deletion of Atg5 in hepatocytes did not protect against TNF-α/ActD/ZVAD-induced necrosis. Moreover, pharmacologic inhibition of RIP1 or genetic deletion of RIP3 failed to protect and even exacerbated liver injury after mice were treated with lipopolysaccharide/GalN and a pan-caspase inhibitor. In conclusion, our results suggest that different cell death mode and cell survival pathways are closely integrated during TNF-α-induced liver injury when both caspases and NF-κB are blocked. Moreover, results from our study also raised concerns about the safety of currently ongoing clinical trials that use caspase inhibitors.