Design and biological evaluation of novel long-acting adalimumab Fab conjugated with the albumin binding domain.

Antigen-binding fragments (Fabs) are preferred alternatives to antibodies for medical application, whereas their short half-lives limit therapeutic effectiveness. Albumin binding domain (ABD) with high affinity for albumin possesses a great potential in enhancing in vivo performance of biotherapeutics. In this study, to mitigate the poor pharmacokinetics of adalimumab Fab targeting tumor necrosis factor-α (TNFα), an ABD fusion strategy was applied innovatively using GA3, ABD035, ABD094 and ABDCon with high affinities for albumin. The prokaryotic expression, bioactivities and half-lives of those novel Fab-ABD fusions were investigated in vitro and in vivo. All Fab-ABD fusions were successfully purified, and they retained similar TNFα-binding activities with the unmodified Fab control, also presented high affinities for human/mouse serum albumin (HSA/MSA). Additionally, the simultaneous binding of the difunctional Fab-ABD fusions to TNFα and albumin was verified, and ABD fused to Fab neither interfered with Fab-TNFα binding nor impaired the association between Fc fragment of IgG receptor and transporter (FcRn) and albumin. Based on the highest binding affinity for HSA and maximal yield, Fab-ABDCon was selected for further evaluation. Fab-ABDCon showed similar thermostability with the Fab control and robust stability in human and mouse plasma. Most notably, the pharmacokinetics of Fab-ABDCon in mice was significantly improved with a 22-fold longer plasma half-life (28.2 h) compared with that of Fab control (1.31 h), which have contributed to its satisfactory therapeutic efficacy in murine TNFα-induced hepatonecrosis model. Thus, Fab-ABDCon could be a promising long-acting candidate suitable for drug development targeting TNFα-mediated inflammatory disease.

Galactosamine reduces sandfly gut protease activity through TOR downregulation and increases Lutzomyia susceptibility to Leishmania.

In sandflies, males and females feed on carbohydrates but females must get a blood meal for egg maturation. Using artificial blood meals, this study aimed to understand how galactosamine interferes with sandfly digestive physiology. We also used galactosamine to manipulate the digestive physiology of Lutzomyia longipalpis to investigate its influence on sandfly digestion and Leishmania development within their insect vectors. Galactosamine was capable to reduce Lu. longipalpis trypsinolytic activity in a dose-dependent manner. This effect was specific to galactosamine as other similar sugars were not able to affect sandfly trypsin production. An excess of amino acids supplemented with the blood meal and 15 mM galactosamine was able to abrogate the reduction of the trypsinolytic activity caused by galactosamine, suggesting this phenomenon may be related to an impairment of amino acid detection by sandfly enterocytes. The TOR inhibitor rapamycin reduces trypsin activity in the L. longipalpis midgut. Galactosamine reduces the phosphorylation of the TOR pathway repressor 4EBP, downregulating TOR activity in the gut of L. longipalpis. Galactosamine reduces sandfly oviposition, causes an impact on sandfly longevity and specifically reduces sandfly gut proteases whereas increasing α-glycosidase activity. The administration of 15 and 30 mM galactosamine increased the number of promastigote forms of Le. mexicana and Le. infantum in galactosamine-treated L. longipalpis. Our results showed that galactosamine influences amino acid sensing, reduces sandfly gut protease activity through TOR downregulation, and benefits Leishmania growth within the Lu. longipalpis gut.

Blockade of Dopamine D2 Receptors as a Novel Approach to Stimulation of Notch1+ Endothelial Progenitor Cells and Angiogenesis in C57BL/6 Mice with Pulmonary Emphysema Induced by Proteases and Deficiency of α1-Antitrypsin.

We studied the effects of spiperone, a selective blocker of dopamine D2 receptors, on the model of pulmonary emphysema provoked by administration of elastase and D-galactosamine hydrochloride to female C57BL/6 mice and characterized by activation of proteases in the lungs and systemic deficiency of its inhibitor α1-antitrypsin. In this model, spiperone prevented the development of inflammatory reaction and reduced the area of emphysematous expanded alveolar tissue. The expression of angiogenic marker CD31 in the lungs increased under these conditions. Regeneration of the damaged microvascular bed under the action of spiperone resulted from recruiting of Notch1+ endothelial progenitor cells (CD45-CD31+CD34+) into the lungs and blockade of the inhibitory effect of dopamine on phosphorylation of VEGF-2 receptors in endothelial cells of different maturity. In addition, spiperone produced a protective effect on hepatocytes and restored the production and secretion of α1-antitrypsin by these cells.

Panaxynol from Saposhnikovia diviaricata exhibits a hepatoprotective effect against lipopolysaccharide + D-Gal N induced acute liver injury by inhibiting Nf-κB/IκB-α and activating Nrf2/HO-1 signaling pathways.

We investigated the mechanism of action of panaxynol (PAL) extract from the root of Saposhnikovia diviaricata (Turcz.) Schischk for treating acute liver injury caused by lipopolysaccharide (LPS) and D-galactosamine (D-Gal N) in mice. A mouse model of acute liver failure induced by LPS/D-Gal N was established. Mice were divided randomly into three equal groups: control group, LPS/D-Gal N group and PAL group. After seven days of continuous PAL administration, all animals except controls were injected with 50 µg/kg LPS and 800 mg/kg D-Gal N; blood and liver samples were collected after 8 h. Compared to the LPS/D-Gal N group, the levels of catalase, glutathione and superoxide dismutase were increased in the liver of the PAL group. The inflammatory response index indicated that PAL attenuated LPS/D Gal N-induced liver pathological injury and decreased levels of hepatic malondialdehyde, serum alanine aminotransferase, aspartate transaminase, tumor necrosis factor-α, and interleukins 1ß and 6. PAL also inhibited LPS/D-Gal N induced nuclear factor-kappa B (Nf-κB), inhibitor kappa B-α (IκB-α) activation, and up-regulated Nrf2 and heme oxygenase-1 (HO-1) expression. PAL can prevent LPS/D-Gal N induced acute liver injury by activating Nrf2/HO-1 to stimulate antioxidant defense and inhibit the IkB-α/NF-κB signaling pathway.

Lactobacillus helveticus R0052 alleviates liver injury by modulating gut microbiome and metabolome in D-galactosamine-treated rats.

The liver is an important digestive gland, and acute liver failure results in high mortality. Probiotics are considered potential adjuvant therapies for liver disease. This study aimed to investigate the beneficial effects of Lactobacillus helveticus R0052 on acute liver injury and the underlying mechanisms. Sprague-Dawley rats were gavaged with L. helveticus R0052 suspensions (3 × 109 CFU) for 1 week. Subsequently, acute liver injury was induced by intraperitoneal D-galactosamine injection on the eighth day. After 24 h, samples (blood, liver, ileum, faeces) were collected and assessed for histological injury, inflammation, intestinal barrier, gut microbiome and metabolome. L. helveticus R0052 alleviated aminotransferase, bilirubin and total bile acid elevation and histological hepatic injuries. Additionally, L. helveticus R0052 exhibited anti-inflammatory properties by downregulating Toll-like receptors, tumour necrosis factor-α and nuclear factor-κb transcription in liver samples and decreasing proinflammatory cytokine plasma concentrations. Additionally, L. helveticus R0052 ameliorated intestinal abnormalities and regulated Toll-like receptors, claudin2 and mucin3 gene transcription in the intestine. These effects were associated with gut microbiome and metabolome modulation by L. helveticus R0052. Probiotic pretreatment enriched Lactobacillus and Bacteroides and depleted Flavonifractor and Acetatifactor in the gut microbiome. Meanwhile, L. helveticus R0052 improved carbohydrate and fatty acid metabolism and reduced lithocholic acid levels. These results indicate that L. helveticus R0052 is promising for alleviating acute liver injury and provide new insights regarding the correlations among the microbiome, the metabolome, the intestinal barrier and liver disease.

Effective Increase of Serum Vitamin D3 by IV Application of a Cholecalciferol-N-Acetyl-Galactosamine-Stabilized Dimer: a Clinical Murine Trial Study.

BACKGROUND: Pre-clinical toxicology studies of human Gc-protein (vitamin D binding protein) are of special interest as to the transport of vitamin D and its biological activities. We have demonstrated that the oral application of a special dimeric vitamin D complex reduces oxidative stress and increases the quality of life in autistic children. Therefore, safety and toxic effects of two dimeric cholecalciferol-N-acetyl-galactosamine-albumin complexes were evaluated in increasing intravenous (iv.) vitamin D levels administered in a pre-clinical trial in mice over a 5-week period. METHODS: Over a period of 5 weeks, two times a week, mice received iv. administration of one of the following: (a) 1.2 IE of vitamin D-N-acetyl-galactosamine-albumin (Vitamin D3 NAGA, ImmunoD® group), (b) 1.2 IE of vitamin-D-poly-N-acetyl-galactosamine-albumin (Poly-Nac group), or (c) isotonic saline solution (sham group). Before and after the trial, red and white blood cell panels (RBS, WBC and platelets) were determined. Furthermore, vitamin D levels, electrolytes, and C-reactive protein levels were measured directly before sacrificing. RESULTS: No toxic effects were observed during iv. injection with dimeric vitamin D complexes, neither in the sham group, nor in the two treatment groups. Vitamin D levels increased significantly within 5 weeks in the Poly-Nac group (26.6 ± 8.8 ng/mL; p = 0.001) compared to the sham group (3.1 ± 0.9 ng/mL), and the Poly-Nac group to the ImmunoD group (7.0 ± 3.6 ng/mL; p = 0.003). A significant increase of vitamin D was also obtained in favor of the ImmunoD group compared to the sham (p = 0.03). Electrolytes (K, Na, Cl, Mg, Ca) and C-reactive protein showed no significant differences after administration in all three mice groups. Also, no significant differences were observed between these three groups in the WBC and RBC blood panels. CONCLUSIONS: The two dimeric vitamin D complexes used in this pre-clinical study showed no side or toxic effects after iv. administration in mice, but a sole increase in vitamin D levels without any change in electrolytes or blood cells. Therefore, we assume this newly developed composition to be safe in oral or iv.-administration and further pre-clinical studies can be conducted to evaluate the value in treatment of various diseases related to vitamin D deficiencies.

The effects of vitamins and selenium mixture against brain tissue induced by d-galactosamine.

Brain damage is a major complication of fulminant hepatic failure. d-Galactosamine (d-GalN)-induced liver toxicity causes damage to brain. The effects of vitamins and selenium mixture against d-GalN stimulated brain injury were investigated in this study. Sprague-Dawley female rats aged 2.0-2.5 months were used for the study. The rats were divided into four categories. A 0.9% NaCl solution was intraperitoneally given to the experimental rats in the first group. Using gavage technique, the second group of animals were subjected to a formulation consisting of 100 mg·kg-1 ·day-1 vitamin C, 15 mg·kg-1 ·day-1 of ß-carotene, 100 mg·kg-1 ·day-1 of α-tocopherol in addition to 0.2 mg·kg-1 ·day-1 of sodium selenate for 3 days. The third group was given a single dose of d-GalN hydrochloride at the concentration of 500 mg·kg-1 through a saline injection. The final group was given similar concentrations of both the antioxidant combination and d-GalN. Tissue samples were collected under ether anesthesia. The rats treated with d-GalN showed brain damage; increased myeloperoxidase, catalase, glutathione peroxidase, glutathione-S-transferase, lactate dehydrogenase, and superoxide dismutase activities; and decreased glutathione levels. Treatment with vitamins and selenium combination resulted in alleviation of these alterations in the rats. These findings suggest that administration of the vitamins and selenium combination suppresses oxidative stress and protects brain cells from injury induced by d-GalN.

Carbon monoxide-induced TFEB nuclear translocation enhances mitophagy/mitochondrial biogenesis in hepatocytes and ameliorates inflammatory liver injury.

Carbon monoxide (CO) can confer protection against cellular stress, whereas the potential involvement of autophagy and lysosomal biogenesis remains incompletely understood. We demonstrate here that the activation of protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK) with CO increased the nuclear translocation of transcription factor EB (TFEB). PERK activation by CO increased intracellular Ca2+ concentration and the phosphatase activity of calcineurin against TFEB. Moreover, we found that in the deficiency of TFEB, CO not only failed to recruit Parkin to the mitochondria but also failed to increase expression of lysosomal genes such as Lamp1, CathB, and TPP1. Therefore, we suggest that CO increases mitophagy through TFEB nuclear translocation by PERK-calcinuerin activation. In addition, the inhibition of TFEB with siRNA against TFEB abrogated the increase of mtDNA with CO, markers of mitochondrial biogenesis such as PGC1α, NRF1, and TFAM, and the mitochondrial proteins COX II, COX IV, and cytochrome c. To investigate the effects of CO on mitochondrial homeostasis in vivo, mice were treated with lipopolysaccharide (LPS)/D-galactosamine (D-GalN). CO inhalation reduced liver injury after challenge with LPS/GalN. Furthermore, CO inhalation increased TFEB activation, mitophagy and mitochondrial biogenesis in mice treated with LPS/GalN. Our findings describe novel mechanisms underlying CO-dependent cytoprotection in hepatocytes and liver tissue via activation of TFEB-dependent mitophagy and associated induction of both lysosomal and mitochondrial biogenesis.

Macrophage-targeting and reactive oxygen species (ROS)-responsive nanopolyplexes mediate anti-inflammatory siRNA delivery against acute liver failure (ALF).

As one of the intractable challenges in the clinic, the treatment of acute liver failure (ALF) is limited due to high mortality and resource cost. RNA interference (RNAi) provides a new modality for the anti-inflammatory therapy of ALF, while its therapeutic efficacy is greatly hampered by the lack of effective carriers to cooperatively overcome the various systemic barriers. Herein, we developed macrophage-targeting and reactive oxygen species (ROS)-responsive polyplexes to enable efficient systemic delivery of TNF-α siRNA (siTNF-α) to attenuate hepatic inflammation in mice bearing ALF. Se-PEI, obtained from the cross-linking of 600 Da polyethylenimine (PEI) via the ROS-responsive diselenide bond, was developed to condense siTNF-α, and the obtained polyplexes were further coated with carboxylated mannan (Man-COOH). Man-COOH coating allowed active targeting of polyplexes to macrophages with over-expressed mannose receptors (MRs), and it shielded the surface positive charges to enhance the serum stability of polyplexes. Se-PEI could be degraded by ROS in inflammatory macrophages to promote intracellular siRNA release to potentiate the gene knockdown efficiency, and in the meantime reduce the material cytotoxicity associated with high molecular weight. As such, i.v. injected Man-COOH/Se-PEI/siTNF-α polyplexes afforded notable TNF-α silencing by ∼80% in inflamed liver tissues at 500 µg siRNA per kg, and notably reduced serum TNF-α levels to achieve potent anti-inflammatory performance against ALF.

Reduction in activating transcription factor 4 promotes carbon tetrachloride and lipopolysaccharide/D­galactosamine­mediated liver injury in mice.

Although activating transcription factor 4 (ATF4) is involved in the regulation of numerous biological functions, whether ATF4 has a direct role in liver injury is unknown. The aim of the present study was to investigate the role of ATF4 in liver injury using mouse models. The results revealed that ATF4 protein is expressed markedly higher in the mouse liver when in comparison with other tissues. Notably, tunicamycin treatment, an endoplasmic reticulum (ER) stress inducer, induced the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), but decreased ATF4 protein levels in the mouse liver. This suggested an unconventional regulation pattern of ATF4 protein not associated with ER stress or eIF2α. In addition, it was also observed that the liver levels of ATF4 protein were significantly reduced upon chronic liver injury induced by carbon tetrachloride (CCl4). ATF4 protein was also decreased in acute liver injury induced by lipopolysaccharide (LPS) plus D­galactosamine (D­GalN). Furthermore, the results revealed that knockdown of ATF4 by injecting ATF4­targeting Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)­CRISPR associated protein 9 plasmids exacerbated CCl4 and LPS/D­GalN­induced liver injury as demonstrated by elevated serum aspartate transaminase and alanine aminotransferase levels. ATF4 suppression also enhanced CCl4 and LPS/D­GalN mediated c­Jun N­terminal kinase activation. By contrast, ATF4 overexpression alleviated CCl4 and LPS/D­GalN­induced liver injury. Taken together, these observations suggested that ATF4 may serve a protective role in the mouse liver.

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.

Histone deacetylase 6 inhibitor ACY-1215 protects against experimental acute liver failure by regulating the TLR4-MAPK/NF-κB pathway.

Histone deacetylase 6 (HDAC6) is considered a new target for anticancer, anti-inflammatory, and neurodegenerative treatment. ACY-1215 is a selective histone deacetylase 6 inhibitor, and it has been recognized as a potential anticancer and anti-inflammation drug. The aim of our study was to investigate whether ACY-1215 has protective effects on acute liver failure (ALF) in mice and explore its potential mechanism. Male C57/BL6 mice were divided into normal, model, and ACY-1215 groups. ACY-1215 (25mg/kg) and same amounts of saline were given to mice. After 2h, the ALF models were induced by lipopolysaccharide (LPS, 100µg/kg) combined with D-galactosamine (D-gal, 400mg/kg). All animals were killed after 24h. The expressions of HDAC6 were determined by western blotting and RT-PCR assay. The expression levels of inflammatory cytokines were detected by ELISA and RT-PCR. The protein expression of Toll-like receptor 4 (TLR4), mitogen-activated protein kinase (MAPK), and nuclear factor κB (NF-κB) species were determined by western blot. The mortality of mice with ALF induced by LPS and D-gal was significantly decreased by ACY-1215 pretreatment. Procedures to manage ALF caused adversely affected liver histology and function; this damage was repaired by pretreatment of ACY-1215. ACY-1215 treatment also attenuated the serum and messenger RNA levels of the proinflammatory cytokines. Pretreatment of ACY-1215 significantly decreased the protein expression of TLR4 and the activation of MAPK and NF-κB signalling pathways. ACY-1215 has potential therapeutic value in mice with ALF by directly inhibiting inflammatory response via regulation of the TLR4-MAPK/NF-kB pathway.

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

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

Hepatoprotective effect of quercetin against LPS/d-GalN induced acute liver injury in mice by inhibiting the IKK/NF-κB and MAPK signal pathways.

Quercetin is regarded as a potential hepatoprotective agent in the treatment of acute liver injury. However, the underlying mechanism of how quercetin to protect against lipopolysaccharides/d-galactosamine (LPS/d-GalN) induced acute liver injury remains unclear. To investigate the mechanism, the antioxidative, anti-inflammatory and antiapoptotic responses were performed. The results showed that quercetin pretreatment improved the survival rate and substantially reduced the liver histopathological changes in mice. It also alleviated the hepatic damage and reduced the productions of oxidative markers induced by LPS/d-GalN. In addition, quercetin pretreatment significantly diminished the production of inflammatory cytokines, including TNF-α, IL-6 and IL-1ß, and inhibited the activation of the NF-κB and MAPK signaling pathways as well as the expression of apoptotic-related proteins induced by LPS/d-GalN. We found that the potential mechanism of this quercetin-induced protection is mainly mediated through its powerful antioxidative capacity, inhibition of hepatocyte apoptosis and suppression of inflammatory cytokines through the IKK/NF-κB and MAPK signaling pathways. Thus, quercetin shows a promising therapeutic effect on acute liver injury in mice.

Prostacyclin mimetics afford protection against lipopolysaccharide/d-galactosamine-induced acute liver injury in mice.

Prostacyclin (PGI2) serves as a protective, anti-inflammatory mediator and PGI2 mimetics may be useful as a hepatoprotective agent. We examined whether two PGI2 mimetics, ONO-1301 and beraprost, are beneficial in acute liver injury and attempted to delineate the possible mechanism underlying the hepatoprotective effect. Acute liver injury was induced by lipopolysaccharide/d-galactosamine (LPS/GalN) in mice. Mice were given an intraperitoneal injection of PGI2 mimetics 1h before LPS/GalN challenge. Both ONO-1301 and beraprost significantly declined the LPS/GalN-induced increase in serum aminotransferase activity. ONO-1301 and, to a lesser extent, beraprost inhibited hepatic gene expression levels of pro-inflammatory cytokines, which were sharply elevated by LPS/GalN. The hepatoprotective effects of ONO-1301, to a lesser extent, of beraprost were also supported by liver histopathological examinations. The PGI2 receptor antagonist CAY10441 abrogated their hepatoprotective effects. The mechanisms behind the benefit of PGI2 mimetics in reducing LPS/GalN-induced liver injury involved, in part, their suppressive effects on increased generation of reactive oxygen species (ROS), since their ability to prevent LPS/GalN-induced hepatic apoptosis was mimicked by the antioxidant N-acetyl-l-cysteine. They significantly diminished LPS/GalN-induced activation of signal transducers and activators of transcription 3 (STAT3) in liver tissues, an effect which was highly associated with their hepatoprotective effects. We indicate that IP receptor activation with PGI2 mimetics can rescue the damage in the liver induced by LPS/GalN by undermining activation of STAT3 and leading to a lower production of ROS. Our findings point to PGI2 mimetics, especially ONO-1301, as a potential novel therapeutic modality for the treatment of acute liver injury.

The role of MAP2 kinases and p38 kinase in acute murine liver injury models.

c-Jun N-terminal kinase (JNK) mediates hepatotoxicity through interaction of its phospho-activated form with a mitochondrial outer membrane protein, Sh3bp5 or Sab, leading to dephosphorylation of intermembrane Src and consequent impaired mitochondrial respiration and enhanced ROS release. ROS production from mitochondria activates MAP3 kinases, such as MLK3 and ASK1, which continue to activate a pathway to sustain JNK activation, and amplifies the toxic effect of acetaminophen (APAP) and TNF/galactosamine (TNF/GalN). Downstream of MAP3K, in various contexts MKK4 activates both JNK and p38 kinases and MKK7 activates only JNK. The relative role of MKK4 versus 7 in liver injury is largely unexplored, as is the potential role of p38 kinase, which might be a key mediator of toxicity in addition to JNK. Antisense oligonucleotides (ASO) to MKK4, MKK7 and p38 (versus scrambled control) were used for in vivo knockdown, and in some experiments PMH were used after in vivo knockdown. Mice were treated with APAP or TNF/GalN and injury assessed. MKK4 and MKK7 were expressed in liver and each was efficiently knocked down with two different ASOs. Massive liver injury and ALT elevation were abrogated by MKK4 but not MKK7 ASO pretreatment in both injury models. The protection was confirmed in PMH. Knockdown of MKK4 completely inhibited basal P-p38 in both cytoplasm and mitochondria. However, ALT levels and histologic injury in APAP-treated mice were not altered with p38 knockdown versus scrambled control. p38 knockdown significantly increased P-JNK levels in cytoplasm but not mitochondria after APAP treatment. In conclusion, MKK4 is the major MAP2K, which activates JNK in acute liver injury. p38, the other downstream target of MKK4, does not contribute to liver injury from APAP or TNF/galactosamine.

Absence of DCIR1 reduces the mortality rate of endotoxemic hepatitis in mice.

Dendritic cell immunoreceptor (DCIR) is a C-type lectin with an immunoreceptor tyrosine-based inhibitory motif (ITIM). Mice lacking DCIR1 (Dcir1-/- mice) show higher susceptibility to chronic arthritis with increasing age, suggesting that DCIR1 is involved in immune modulation via its ITIM. However, the role of DCIR1 in acute immune responses is not clear. In this study, we explored its role in acute experimental hepatitis. Upon injection of d-galactosamine and lipopolysaccharide, Dcir1-/- mice showed decreased mortality rates and serum levels of alanine aminotransferase. In early onset hepatitis, serum levels of TNF-α, which primarily cause inflammation and hepatocyte apoptosis, were significantly lower in Dcir1-/- mice than in WT mice. In the liver of Dcir1-/- mice, influx of neutrophils and other leukocytes decreased. Consistently, the levels of neutrophil-chemoattractant chemokine CXCL1/KC, but not CXCL2/MIP-2, were lower in Dcir1-/- mice than in WT mice. However, chemotaxis of Dcir1-/- neutrophils to CXCL1/KC appeared normal. Pervanadate treatment induced binding of DCIR1 and Src homology region 2 domain-containing phosphatase (SHP)-2, possibly leading to CXCL1/KC expression. These results suggest that DCIR1 is involved in exacerbation of endotoxemic hepatitis, providing a new therapeutic target for lethal hepatitis.

The protective effect of the natural compound hesperetin against fulminant hepatitis in vivo and in vitro.

BACKGROUND AND PURPOSE: Liver diseases are mostly accompanied by inflammation and hepatocyte death. Therapeutic approaches targeting both hepatocyte injury and inflammation are not available. Natural compounds are considered as potential treatment for inflammatory liver diseases. Hesperetin, a flavonoid component of citrus fruits, has been reported to have anti-inflammatory properties. The aim of this study was to evaluate the cytoprotective and anti-inflammatory properties of hesperetin both in vitro and in models of fulminant hepatitis. EXPERIMENTAL APPROACH: Apoptotic cell death and inflammation were induced in primary cultures of rat hepatocytes by bile acids and cytokine mixture respectively. Apoptosis was quantified by caspase-3 activity and necrosis by LDH release. The concanavalin A (ConA) and D-galactosamine/LPS (D-GalN/LPS) were used as models of fulminant hepatitis. Liver injury was assessed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, liver histology and TUNEL assay and inflammation by inducible NOS (iNOS) expression. KEY RESULTS: Hesperetin blocked bile acid-induced apoptosis and cytokine-induced inflammation in rat hepatocytes. Moreover, hesperetin improved liver histology and protected against hepatocyte injury in ConA- and D-GalN/LPS-induced fulminant hepatitis, as assessed by TUNEL assay and serum AST and ALT levels. Hesperetin also reduced expression of the inflammatory marker iNOS and the expression and serum levels of TNFα and IFN-γ, the main mediators of cell toxicity in fulminant hepatitis. CONCLUSION AND IMPLICATIONS: Hesperetin has anti-inflammatory and cytoprotective actions in models of acute liver toxicity. Hesperetin therefore has therapeutic potential for the treatment of inflammatory liver diseases accompanied by extensive hepatocyte injury, such as fulminant hepatitis.

[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.

Protective effect of Xuebijing injection on D-galactosamine- and lipopolysaccharide-induced acute liver injury in rats through the regulation of p38 MAPK, MMP-9 and HO-1 expression by increasing TIPE2 expression.

Xuebijing injection (XBJ) has long been used to treat infectious diseases in China. The therapeutic effect of XBJ is probably associated with anti-inflammatory effects. However, the precise mechanisms responsible for the effects of XBJ remain unknown. The present study was conducted in order to evaluate the protective effects of XBJ in a rat model of D-galactosamine (D-Gal)- and lipopolysaccharide (LPS)­induced acute liver injury. In the present study, the rats were injected with D-Gal and LPS intraperitoneally to induce acute liver injury. Two hours prior to D-Gal and LPS administration, the treatment group was administered XBJ by intravenous infusion. The effects of XBJ on D-Gal- and LPS-induced expression of tumor necrosis factor (TNF)­alpha­induced protein 8-like 2 (TIPE2), nuclear factor-κB (NF-κB), matrix metalloproteinase-9 (MMP-9) and heme oxygenase-1 (HO-1) as well as mitogen-activated protein kinase (MAPK) signaling was examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot analysis, immunofluorescence, as well as by analysing the serum levels of pro-inflammatory cytokines and the transaminases, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Myeloperoxidase (MPO), malondialdehyde (MDA) and superoxide dismutase (SOD) levels in the rat liver tissues were also measured. For histological analysis, hematoxylin and eosin (H&E)-stained liver samples were evaluated. The results showed that XBJ upregulated TIPE2 and HO-1 expression, reduced the expression of NF-κB65 and MMP-9, inhibited the LPS-induced gene expression of c-jun N-terminal kinase (JNK) and p38 MAPK, decreased the generation of pro-inflammatory cytokines [interleukin (IL)-6, IL-13 and TNF-α], inhibited ALT and AST activity, and ameliorated D-Gal- and LPS-induced liver injury. The histological results also demonstrated that XBJ attenuated D-Gal- and LPS-induced liver inflammation. It was found that XBJ may prevent LPS-induced pro-inflammatory gene expression through inhibiting the NF-κB and MAPK signaling pathways by upregulating TIPE2 expression, thereby attenuating LPS-induced liver injury in rats. The marked protective effects of XBJ suggest that it has the potential to be used in the treatment of LPS-induced liver injury.