Adhesions formation is a significant postsurgical complication. At present, there is no effective method for preventing adhesions formation 1, although barrier products such as Dextran (Dex) 2 and sodium hyaluronate (SH) 3 have proved the most clinically successful 456, This study is designed to investigate the preventive and therapeutic potential of a novel penicillamine-bound membrane for abdominal adhesions formation.

Rationale: The functions of fibrinogen-like protein 2 (fgl2) have been studied in many inflammatory and neoplastic diseases, but the role of fgl2 in nonalcoholic fatty liver disease has not yet been elucidated. In this study, we sought to investigate the role of fgl2 in the pathogenesis of nonalcoholic steatohepatitis (NASH). Methods: Hepatic fgl2 expression was tested in patients with nonalcoholic fatty liver (NAFL) or NASH and controls. Wild-type and fgl2-/- C57BL/6 mice were subjected to a methionine/choline-deficient (MCD) diet or a high-fat diet (HFD) to establish NASH models. Bone marrow-derived macrophages (BMDMs) stimulated with LPS or free fatty acids were used for the in vitro study. Results: In both humans and mice with NASH, macrophage accumulation was concomitant with significantly increased fgl2 expression in the liver. Fgl2 deficiency attenuated liver steatosis and inflammation in diet-induced murine models of NASH. In both liver tissues and BMDMs from NASH mice, fgl2 deficiency resulted in reduced levels of proinflammatory cytokines and reactive oxygen species (ROS) compared with levels in wild-type controls. Activation of NF-κB, p38-MAPK and NLRP3 inflammasomes was also suppressed upon fgl2 disruption. Moreover, lipogenic genes (Fasn and SREBP-2) were downregulated while lipolytic genes (PPAR and CPT1A) were upregulated in the livers of fgl2-/- NASH mice. Primary hepatocytes incubated with the medium collected from fgl2-/- BMDMs showed less fat deposition than those incubated with WT BMDMs. Furthermore, we discovered that fgl2 combined with TLR4 mediates the activation of the Myd88-dependent signaling pathway, which may contribute to inflammation and lipid metabolism disorders. Conclusions: These data suggest that fgl2 aggravates the progression of NASH through activation of NF-κB, p38-MAPK and NLRP3 inflammasomes in macrophages, which consequently induces overproduction of proinflammatory cytokines and lipid metabolism disorders. An interaction of fgl2 and TLR4 may in part contribute to the activation of inflammatory signaling pathways in macrophages.

Heterogeneous macrophages play an important role in multiple liver diseases, including viral fulminant hepatitis (VFH). Fibrinogen-like protein 2 (FGL2) is expressed on macrophages and regulates VFH pathogenesis; however, the underlying mechanism remains unclear.

Fulminant viral hepatitis (FVH) is a life-threatening disease, but its pathogenesis is not fully understood. Neutrophil extracellular traps (NETs) were an unrecognized link between inflammation and coagulation, which are 2 main features of FVH. Here, we investigated the role and mechanism of NETs in the pathogenesis of FVH.

Single minded 1 (SIM1) is a transcription factor involved in brain patterning and control of energy balance. In humans, haploinsufficiency of SIM1 causes early-onset obesity. Mice deficient in the homologous gene, SIM1, also exhibit early onset obesity and increased sensitivity to a high fat diet. SIM1 is expressed in several areas of the brain implicated in control of energy balance including the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the medial amygdala and nucleus of the lateral olfactory tract. We have previously shown that mice with global Sim1 neuron ablation exhibit obesity with hyperphagia as the primary defect. The PVN has a critical role in feeding and in high-fat appetite, thus, we sought to determine the effect of Sim1 neuron ablation limited to the PVN. We achieved PVN-SIM1 limited ablation through stereotactic injection of diphtheria toxin into the PVN of Sim1Cre-iDTR mice. The specificity of this ablation was confirmed by immunohistochemistry and quantitative real time PCR of the PVN, supraoptic nucleus and the amygdala. Mice with PVN Sim1 neuron ablation, similar to mice with global Sim1 neuron ablation, exhibit early onset obesity with hyperphagia as the primary defect. However, PVN-Sim1 neuron ablated mice have a decreased response to fasting-induced hyperphagia. Consistent with this decrement, PVN-Sim1 neuron ablated mice have a decreased hyperphagic response to PVN injection of agouti-related peptide (AgRP). When PVN-Sim1 neuron ablated mice are placed on a high fat diet, surprisingly, their intake decreases and they actually lose weight. When allowed ad lib access to high fat diet and normal chow simultaneously, PVN-Sim1 neuron ablated mice exhibit overall decreased intake. That is, in PVN-Sim1 neuron ablated mice, access to fat suppresses overall appetite.

Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1.

Fluid accumulation is critical for lung distension and normal development. The multi-subunit γ-amino butyric acid type A receptors (GABAA) mainly act by mediating chloride ion (Cl-) fluxes. Since fetal lung actively secretes Cl--rich fluid, we investigated the role of GABAA receptors in fetal lung development. The physiological ligand, GABA, and its synthesizing enzyme, glutamic acid decarboxylase, were predominantly localized to saccular epithelium. To examine the effect of activating GABAA receptors in fetal lung development in vivo, timed-pregnant rats of day 18 gestation underwent an in utero surgery for the administration of GABAA receptor modulators into the fetuses. The fetal lungs were isolated on day 21 of gestation and analyzed for changes in fetal lung development. Fetuses injected with GABA had a significantly higher body weight and lung weight when compared to phosphate-buffered saline (control)-injected fetuses. GABA-injected fetal lungs had a higher number of saccules than the control. GABA increased the number of alveolar epithelial type II cells as indicated by surfactant protein C-positive cells. However, GABA decreased the number of α-smooth muscle actin-positive myofibroblasts, but did not affect the number of Clara cells or alveolar type I cells. GABA-mediated effects were blocked by the GABAA receptor antagonist, bicuculline. GABA also increased cell proliferation and Cl- efflux in fetal distal lung epithelial cells. In conclusion, our results indicate that GABAA receptors accelerate fetal lung development, likely through an enhanced cell proliferation and/or fluid secretion.

Viral hepatitis remains the most common cause of liver disease and a major public health problem. Here, we focus on the role of CD4 CD8 double negative T (DN T) cells involved in the mechanisms of viral persistence in hepatitis. C3H/HeJ mice infected with murine hepatitis virus strain 3 (MHV-3) were used to display chronic viral hepatitis. DN T cells dramatically increased in MHV-3 infected mice. Adoptive transfer of DN T cells from MHV-3 infected mice led to a significant increase in mice survival. The DN T cells with production of IFN-γ and IL-2 are able to kill virus-specific CD8(+) T cells via the Fas/FasL dependent pathway. The delicate balance of multiple effects of DN T cells may lead to viral persistence in MHV-3 induced hepatitis. In short, our study identified DN T cells contributing to viral persistence in MHV-3 induced hepatitis in C3H/HeJ mice, which provides a rationale for modulating DN T cells for the management of viral hepatitis.

Fibrin deposition was universal in the lungs of SARS patients and fgl2 prothrombinase gene, a novel procoagulant, was demonstrated to express highly in a clinically relevant SARS model. To investigate whether and which structural protein of SARS-CoV induced transcription of hfgl2 prothrombinase gene, three eukaryotic expression plasmids expressing nucleocapsid protein (N), membrane protein (M) and spike protein 2 (S2) of SARS-CoV were co-transfected with hfgl2 promoter luciferase-reporter plasmids and beta-galactosidase plasmid in CHO cells, respectively. M, N and S2 protein of SARS-CoV were detected by western blotting and immunohistochemistry analysis. Further assays demonstrated that expression of hfgl2 gene was related with N protein, but not with M or S2 protein in THP-1 cells and Vero cells. N protein significantly induced functional procoagulant activity in comparison with control group. Luciferase assay showed that N protein of SARS-CoV could activate the transcription of hfgl2 promoter compared with the pcDNA3.1 empty vector. Site-directed mutagenesis and EMSA assay further demonstrated that transcription factor C/EBP alpha band with its cognate cis-element in hfgl2 promoter. The results showed that N protein of SARS-CoV induced hfgl2 gene transcription dependent on the transcription factor C/EBP alpha, which maybe contribute to the development of thrombosis in SARS.

Gold-coated iron oxide nanoparticle Hepatitis B virus (HBV) DNA probes were prepared, and their application for HBV DNA measurement was studied. Gold-coated iron oxide nanoparticles were prepared by the citrate reduction of tetra-chloroauric acid in the presence of iron oxide nanoparticles which were added as seeds. With a fluorescence-based method, the maximal surface coverage of hexaethiol 30-mer oligonucleotides and the maximal percentage of hybridization strands on gold-coated iron oxide nanoparticles were (120 ± 8) oligonucleotides per nanoparticle, and (14 ± 2%), respectively, which were comparable with those of (132 ± 10) and (22 ± 3%) in Au nanoparticle groups. Large network aggregates were formed when gold-coated iron oxide nanoparticle HBV DNA gene probe was applied to detect HBV DNA molecules as evidenced by transmission electron microscopy and the high specificity was verified by blot hybridization. Our results further suggested that detecting DNA with iron oxide nanoparticles and magnetic separator was feasible and might be an alternative effective method.

Sugar-sweetened beverage consumption is a known independent risk factor for nonalcoholic steatohepatitis (NASH). Non-caloric sweeteners (NCS) are food additives providing sweetness without calories and are considered safe and/or not metabolized by the liver. The potential role of newer NCS in the regulation of NASH, however, remain unknown. Our study aimed to determine the impact of newer NCS including Rebaudioside A and sucralose on NASH using high fat diet induced obesity mouse model by substituting fructose and sucrose with NCS in the drinking water. We characterized the phenotype of NCS- treated obesity and investigated the alterations of hepatic function and underlying mechanisms. We found that NCS have no impact on weight gain and energy balance in high fat diet induced obesity. However, in comparison to fructose and sucrose, Rebaudioside A significantly improved liver enzymes, hepatic steatosis and hepatic fibrosis. Additionally, Rebaudioside A improved endoplasmic reticulum (ER) stress related gene expressions, fasting glucose levels, insulin sensitivity and restored pancreatic islet cell mass, neuronal innervation and microbiome composition. We concluded that Rebaudioside A significantly ameliorated murine NASH, while the underlying mechanisms requires further investigation.

The selective involvement of a subset of neurons in many psychiatric disorders, such as gamma-aminobutyric acid (GABA)-ergic interneurons in schizophrenia, creates a significant need for in-depth analysis of these cells. Here we introduce a combination of techniques to examine the relative gene expression of N-methyl-d-aspartic acid (NMDA) receptor subtypes in GABAergic interneurons from the rat prefrontal cortex. Neurons were identified by immunostaining, isolated by laser microdissection and RNA was prepared for reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. These experimental procedures have been described individually; however, we found that this combination of techniques is powerful for the analysis of gene expression in individual identified neurons. This approach provides the means to analyze relevant molecular mechanisms that are involved in the neuropathological process of a devastating brain disorder.

Soluble fibrinogen-like protein 2 (sFGL2), a secretory protein expressed by regulatory T cells (Tregs) with immunosuppressive activity, is highly expressed in both the peripheral blood and tumor tissue of patients with hepatocellular carcinoma (HCC); however, sFGL2 function in HCC remains largely unknown. Here, we elucidated the potential role of sFGL2 in HCC progression.

Fulminant hepatitis (FH) is characterized by rapid liver failure and high mortality. The pathogenesis of viral FH includes virus-induced immune activation, inflammation, and subsequent hepatic apoptosis and necrosis. However, the mechanisms that underlie FH progression are unclear. IL-33 is a member of the IL-1-related cytokines, considered to be an "alarmin" that participates in various diseases, but its precise role in the coagulation of FH is not very clear. In our study, we found that IL-33 is significantly elevated in mice infected with murine hepatitis virus strain 3 (MHV-3). This is accompanied by an increase in pro-coagulant fibrinogen-like protein 2 (FGL2) in the liver. Previous studies have suggested that an increase in FGL2 is diagnostic of FH and liver necrosis, and animals with no FGL2 had better survivorship during FH. Our studies showed that IL-33 administration in a MHV-3 infection promoted survival during FH, with a significant reduction in FGL2 expression and liver inflammation. In vitro IL-33 treatment abrogated MHV-3 and IFN-γ induced FGL2 expression in RAW264.7 and THP-1 cells, respectively. In conclusion, our research suggests that IL-33 protects against viral fulminant hepatitis in mice by antagonizing expression of the pro-coagulant protein FGL2.

The paraventricular nucleus (PVN) is a critical locus of energy balance control. Three sets of neurons in the PVN are involved in regulating energy balance: oxytocin-expressing neurons (OXT-neurons), thyrotropin-releasing hormone-expressing neurons, and corticotrophin-releasing hormone-expressing neurons. To examine the role of OXT-neurons in energy balance, we ablated these neurons in mice by injecting diphtheria toxin into mice possessing both the oxytocin promoter driving cre expression and a cre-inducible diphtheria toxin receptor. Immunohistochemistry and real-time reverse transcriptase polymerase chain reaction confirmed that this injection caused a significant decrease in PVN OXT-neurons and OXT-mRNA abundance. OXT-neuron ablation did not alter food intake, weight, or energy expenditure at room temperature on either chow or a high-fat diet. To further characterize OXT-neuron-ablated mice, we examined their response to 1) intraperitoneal cholecystokinin (CCK) injection and 2) thermogenic stress. OXT-neuron-ablated mice had a blunted decrease in feeding response to CCK. When exposed to the extreme cold (4°C) for 3 hours, OXT-neuron-ablated mice had significant decreases in both rectal and brown adipose tissue temperature relative to controls, which was rescued by OXT treatment. Thermographic imaging revealed that OXT-neuron-ablated mice had increased body surface temperature. Thus, we report that OXT-neuron ablation shows no role for OXT-neurons in energy homeostasis at neutral temperature but reveals a heretofore unappreciated role for OXT-neurons and oxytocin specifically in regulating the thermogenic stress response.