PF Mice Research Articles

Exploring TβRI inhibitors from Arenaria kansuensis based on 3D-QSAR, molecular docking and molecular dynamics simulation methods and its anti-pulmonary fibrosis molecular mechanism validation

Ethnopharmacological relevancePulmonary fibrosis (PF) is a kind of interstitial lung disease that seriously threatens human life and health. Up to now, there is no specifically therapeutic drug. Arenaria kansuensis, a typical Tibetan medicine, has been previously proved to have anti-PF pharmacological activity by our group. However, the specific target and molecular mechanism of pharmacological active ingredients from it are still unknown. Aim of the studyThis study aimed to explore the molecular mechanism and specific target of pharmacological active ingredients from A. kansuensis for treating PF. Materials and methodsVirtual screening including 3D-QSAR, molecular docking and molecular dynamics simulation were used to screen TβRI inhibitor. CETSA experiment was used to verify the interaction between GAK (a β-carboline alkaloid isolated from A. kansuensis) and TβRI. Cell and molecular experiments including observation of cell morphology and Western blot were applied to investigate the molecular mechanism of action of GAK for treating PF. Animal experiments including physiological index, immunohistochemistry and ELISA were used to comprehensively evaluate the anti-PF effect of GAK and explore the corresponding mechanism of action. ResultsResults of 3D-QSAR experiment indicated that GAK is a much stronger potential TβRI inhibitor, molecular mechanism study showed that 30 μM GAK could significantly keep TβRI more stable which indicated that the direct binding interaction between GAK and TβRI, it targetedly inhibited TβRI through forming hydrogen bonds with LYS232, SER280 and ASP351 and the binding energies is −56.05 kcal/mol.In vitro experiment showed GAK could suppress downstream signal pathways of TβRI including MAPK, PI3K/AKT and NF-κB pathways during EMT process. In vivo experiment showed that GAK could improve the survival rate and body weight of PF mice, alleviate the symptoms of histopathological severity, inflammatory cell infiltration and collagen deposition in lung tissue of PF mice through inhibiting EMT process of PF. ConclusionsThis work not only provided evidence to support GAK as a novel TβRI inhibitor for treating PF through multiple pathways, but also reveal the specific target and molecular mechanism of β-carboline alkaloids from A. kansuensis for treating PF.

ShaShen-MaiDong decoction attenuates bleomycin-induced pulmonary fibrosis by inhibiting TGF-β/smad3, AKT/MAPK, and YAP/TAZ pathways

Ethnopharmacological relevancePulmonary fibrosis (PF) is progressive and terminal lung disease, which is also the most common sequelae of Corona Virus Disease (2019) (COVID-19) survivors. Unfortunately, there is currently no cure for PF. ShaShen-MaiDong decoction (SMT), a traditional Chinese medicine, has been employed in treating various lung diseases, which may offer potential therapeutic benefits for PF. Aim of the studyTo investigate the antifibrotic efficacy of SMT and its major active ingredients as well as the underlying mechanisms for treating PF. Materials and methodsFist, we build the UPLC-MS based qualitative and quantitative profiling for the quality control of SMT. Then, the antifibrotic efficacy of SMT was investigated in bleomycin (BLM)-induced PF mice model. Network pharmacology was used to predict the mechanism and active components of SMT for the treatment of PF, which was further verified in vitro and in vivo. ResultsSMT improved the weight loss and attenuated hydroxyproline, inflammatory cytokines, and collagen deposition in BLM-induced PF mice model in a dose-dependent manner. Mechanistically, as predicted by network pharmacology analysis, SMT and its active compounds (kaempferol, quercetin, and isorhamnetin) regulated the mitogen-activated protein kinase (MAPK) signaling pathways, TGF-β/Smad signaling pathway, and YAP/TAZ signaling pathway, which was further verified in the PF mice and TGF-β-induced A549 cell model. Moreover, SMT balanced the proportions of increased CD4+ and decreased CD8+ T cells in the peripheral blood of PF mice model. ConclusionsConsidering the high mortality and complex pathogenesis of fibrotic diseases, our results provide novel evidence that SMT would be beneficial for pulmonary fibrosis therapy by modulating MAPK, TGF-β/Smad, and YAP/TAZ signaling pathways at same time.

Forsythoside A regulates pulmonary fibrosis by inhibiting endothelial-to-mesenchymal transition and lung fibroblast proliferation via the PTPRB signaling

BackgroundPulmonary fibrosis (PF) is an end-stage change in many interstitial lung diseases, whereas no proven effective anti-pulmonary fibrotic treatments. Forsythoside A (FA) derived from Forsythia suspensa (Thunb.) Vahl, has been found to possess lung-protective effect. However, studies on its anti-pulmonary fibrosis effect are limited and its mechanism of action remains unknown. PurposeThis study aimed to explore the underlying mechanism of FA on PF. MethodsMale C57BL/6 mice were randomized into normal (CON), model (BLM), pirfenidone (PFD), low- and high-dose FA (FA-L, FA-H, respectively). Except for the CON group, which was injected with the same dose of saline, the model of PF was established by intratracheal instillation of BLM, during which the survival rate and body weight changes of the mice were measured. The lung histopathology was evaluated by Hematoxylin–eosin, Sirius red, and Masson staining. Transcriptome analysis was performed to screen for the differential genes associated with the role of FA in PF. Differential genes in normal and pulmonary fibrosis patients with the GSE2052 dataset were analyzed in the GEO database. The levels of CTGF, α-SMA, MMP-8 in lung and TNF-α in bronchoalveolar lavage fluid (BALF) were detected by ELISA. The levels of HYP in lungs were detected by digestion. The mRNA and protein levels of MMP-7, E-cadherin, CD31, α-SMA, TGF-β1, IL-6, β-catenin, ZO-1, PTPRB, E-cadherin, and vimentin in lungs were detected by RT-qPCR and Western blot. The expression of CD31, α-SMA, TGF-β1 and ZO-1 were detected by immunofluorescence. TGF-β1-stimulated HFL1 cells and human umbilical vein endothelial cells (HUVECs) were used in an attempt to explore the possible role of protein tyrosine phosphatase receptor type B (PTPRB) involved in FA-induced improvement of PF. ResultsThe results showed that FA could improve the survival rate and body weight of PF mice. FA could alleviate the symptoms of alveolar wall thickening, inflammatory cell infiltration, blue collagen fiber deposition, collagen fiber type Ⅰ and type Ⅲ in mice with PF. In addition, FA could reduce the levels of HYP, CTGF, α-SMA, TGF-β1, TNF-α, β-catenin and MMP8, and regulate the expression levels of CD31, ZO-1, PTPRB and E-cadherin in lung of mice with PF, inhibiting endothelial-to-mesenchymal transition (EndMT) and fibroblasts proliferation. In the GSE2052 dataset, the expression level of PTPRB is reduced in lung tissue from PF patients, and results from transcriptome sequencing indicate that PTPRB expression is also reduced in PF mice. In addition, the effect of FA on TGF-β1-induced HFL1 or HUVECs cells could be attenuated by the inhibitor of PTPRB, suggesting that the effect of FA on PF is related to PTPRB. ConclusionThis study demonstrated that FA could ameliorate PF by inhibiting lung fibroblast proliferation and EndMT, and that PTPRB might be a target of FA to ameliorate PF, which provided evidence to support FA as a candidate phytochemical for PF.

Specneuzhenide improves bleomycin-induced pulmonary fibrosis in mice via AMPK-dependent reduction of PD-L1

BackgroundPulmonary fibrosis (PF) is an escalating global health issue, characterized by rising rates of morbidity and mortality annually. Consequently, further investigation of potential damage mechanisms and potential preventive strategies for PF are warranted. Specnuezhenide (SPN), a prominent secoiridoid compound derived from Ligustrum lucidum Ait, exhibits anti-inflammatory and anti-oxidative capacities, indicating the potential therapeutic actions on PF. However, the underlying mechanisms of SPN on PF remain unclear. PurposeThis work was aimed at investigating the protective actions of SPN on PF and the potential mechanism. MethodsIn vivo, mice were administrated with bleomycin (BLM) to establish PF model. PF mice were treated with SPN (45/90 mg/kg) by gavage. In vitro, we employed TGF-β1 (10 ng/mL)-induced MLE-12 and PLFs cells, which then were treated with SPN (5, 10, 20 µM). DARTS assay, biofilm interference experiment and molecular docking were performed to investigate the molecular target of SPN. ResultsIn vivo, we found SPN treatment improved survival rate, alleviated pathological changes through reducing BLM-induced extracellular matrix (ECM) deposition, as well as BLM-induced epithelial-mesenchymal transition (EMT). In vitro, SPN inhibited EMT and lung fibroblast transdifferentiation. Mechanistically, SPN activated the AMPK protein to decrease the abnormally high level of PD-L1. Furthermore, the compound C, known as an AMPK inhibitor, exhibited a significant hindrance to the inhibition of SPN on TGF-β1-caused fibroblast transdifferentiation and proliferation. This outcome could be attributed to the fact that compound C could eliminate the inhibitory effects of SPN on PD-L1 expression. Interestingly, DARTS assay, biofilm interference experiment and molecular docking results all indicated that SPN could bind to AMPK, which suggested that SPN might be a potential agonist targeting AMPK protein. ConclusionAltogether, the results in our work illustrated that SPN promoted AMPK-dependent reduction of PD-L1 protein, contributing to the inhibition of fibrosis progression. Thus, SPN may represent a potential AMPK agonist for PF treatment.

Vincamine as an agonist of G protein-coupled receptor 40 effectively ameliorates pulmonary fibrosis in mice

BackgroundPulmonary fibrosis (PF) is an irreversible and fatal lung disease with limited therapeutic options. G protein-coupled receptor 40 (GPR40) has been developed as a promising therapeutic target for metabolic disorders and functions potently in varied pathological and physiological processes. Vincamine (Vin) is a monoterpenoid indole alkaloid originated from Madagascar periwinkle and was reported as a GPR40 agonist in our previous work. PurposeHere, we aimed to clarify the role of GPR40 in PF pathogenesis by using the determined GPR40 agonist Vin as a probe and explore the potential of Vin in ameliorating PF in mice. MethodsPulmonary GPR40 expression alterations were assessed in both PF patients and bleomycin-induced PF mice (PF mice). Vin was used to evaluate the therapeutic potential of GPR40 activation for PF and the underlying mechanism was intensively investigated by assays against GPR40 knockout (Ffar1−/−) mice and the cells transfected with si-GPR40 in vitro. ResultsPulmonary GPR40 expression level was highly downregulated in PF patients and PF mice. Pulmonary GPR40 deletion (Ffar1−/−) exacerbated pulmonary fibrosis as evidenced by the increases in mortality, dysfunctional lung index, activated myofibroblasts and extracellular matrix (ECM) deposition in PF mice. Vin-mediated pulmonary GPR40 activation ameliorated PF-like pathology in mice. Mechanistically, Vin suppressed ECM deposition by GPR40/β-arrestin2/SMAD3 pathway, repressed inflammatory response by GPR40/NF-κB/NLRP3 pathway and inhibited angiogenesis by decreasing GPR40-mediated vascular endothelial growth factor (VEGF) expression in the region of interface to normal parenchyma in pulmonary fibrotic tissues of mice. ConclusionPulmonary GPR40 activation shows promise as a therapeutic strategy for PF and Vin exhibits high potential in treating this disease.

Local administration of liposomal-based Plekhf1 gene therapy attenuates pulmonary fibrosis by modulating macrophage polarization.

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with limited therapeutic options. Macrophages, particularly alternatively activated macrophages (M2), have been recognized to contribute to the pathogenesis of pulmonary fibrosis. Therefore, targeting macrophages might be a viable therapeutic strategy for IPF. Herein, we report a potential nanomedicine-based gene therapy for IPF by modulating macrophage M2 activation. In this study, we illustrated that the levels of pleckstrin homology and FYVE domain containing 1 (Plekhf1) were increased in the lungs originating from IPF patients and PF mice. Further functionality studies identified the pivotal role of Plekhf1 in macrophage M2 activation. Mechanistically, Plekhf1 was upregulated by IL-4/IL-13 stimulation, after which Plekhf1 enhanced PI3K/Akt signaling to promote the macrophage M2 program and exacerbate pulmonary fibrosis. Therefore, intratracheal administration of Plekhf1 siRNA-loaded liposomes could effectively suppress the expression of Plekhf1 in the lungs and notably protect mice against BLM-induced lung injury and fibrosis, concomitant with a significant reduction in M2 macrophage accumulation in the lungs. In conclusion, Plekhf1 may play a crucial role in the pathogenesis of pulmonary fibrosis, and Plekhf1 siRNA-loaded liposomes might be a promising therapeutic approach against pulmonary fibrosis.

TMT proteomics analysis reveals the mechanism of bleomycin-induced pulmonary fibrosis and effects of Ginseng honeysuckle superfine powdered tea

BackgroundPulmonary fibrosis (PF) is a chronic and potentially fatal lung disease and disorder. Although the active ingredients of ginseng honeysuckle superfine powdered tea (GHSPT) have been proven to have anti-inflammatory and antioxidant effects, the mechanism of GHSPT on PF remains unclear. The present study was to explore the underlying mechanism of GHSPT in treating PF based on proteomics and network pharmacology analysis and to confirm it in vivo.Materials and methodsWe used intratracheal instillation of bleomycin to induce the PF mouse model and GHSPT (640 mg/kg) intragastrically administrated to PF mice for 21 days. The lung tissues were harvested for TMT-based proteomics. The UPLC-Q-Exactive MS/MS analyze the serum migrant compounds of GHSPT in the PF mice. Moreover, components of GHSPT were harvested from the pharmacology database of the TCMSP system. PF-related targets were retrieved using NCBI and GeneCards databases.ResultsOur results showed that GHSPT significantly alleviated PF mice. Proteomics analysis showed that 525 proteins had significantly changed in the lung of untreated PF mice. Among them, 19 differential proteins were back-regulated to normal levels after GHSPT therapy. Moreover, 25 compounds originating from GHSPT were identified in the serum sample. Network analysis showed 159 active ingredients and 92 drug targets against PF. The signaling pathways include apoptosis, ferroptosis, cytokine-cytokine receptor, P53, and PI3K-Akt signaling pathway.ConclusionThe evidence suggests that GHSPT might play an effective role in the treatment of PF by multi-target interventions against multiple signaling pathways.

Protective effects of Qing-Re-Huo-Xue formula on bleomycin-induced pulmonary fibrosis through the p53/IGFBP3 pathway

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrosing lung disease with high mortality. Inflammation and epithelial mesenchymal transformation (EMT) may play an important role in the occurrence and development of IPF. Qing-Re-Huo-Xue formula (QRHXF) has been used clinically by our team for half a century and has obvious therapeutic effects on lung disease. Nevertheless, the role and mechanism of QRHXF in the treatment of IPF have never been studied.MethodsA mouse pulmonary fibrosis model was established by intratracheal injection of BLM. The effects of QRHXF on the treatment of pulmonary fibrosis were studied by pulmonary function testing, imaging examination, pathological staining, transmission electron microscopy (TEM) observation and mRNA expression. Tandem mass tag (TMT)-based quantitative proteomics was carried out to analyse the lung protein expression profiles between the control (CTL), bleomycin (BLM) and QRHXF (BLM + QRHXF) groups. Immunohistochemistry and qRT-PCR were used to verify the possible existence of drug target proteins and signalling pathways.ResultsThe results of pulmonary function, lung pathology and imaging examinations showed that QRHXF could significantly alleviate BLM-induced pulmonary fibrosis in vivo. Additionally, inflammatory cell infiltration and EMT were markedly reduced in BLM-induced PF mice administered QRHXF. Proteomics detected a total of 35 proteins, of which 17 were upregulated and 18 were downregulated. A total of 19 differentially expressed proteins (DEPs) overlapped between the BLM versus CTL groups and the BLM + QRHXF versus BLM groups. The expression of p53 and IGFBP3 was reversed in the QRHXF intervention group, which was verified by immunohistochemistry and qRT-PCR.ConclusionsQRHXF attenuated BLM-induced pulmonary fibrosis, and regulation of the p53/IGFBP3 pathway might be associated with its efficacy, which holds promise as a novel treatment strategy for pulmonary fibrosis patients.Graphical

The Therapeutic Mechanism of Schisandrol A and Its Metabolites on Pulmonary Fibrosis Based on Plasma Metabonomics and Network Analysis.

Schisandrol A (Sch A) is the main active ingredient of Schisandra chinensis (Turcz.) Baill. Our previous study showed that Sch A has anti-pulmonary fibrosis (PF) activity, but its metabolic-related mechanisms of action are not clear. Here, we explored the therapeutic mechanisms of Sch A on PF by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) metabolomics approach and network analysis. The metabolites of Sch A in mice (bleomycin + Sch A high-dose group) plasma were identified based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). 32 metabolites were detected reversed to normal level after treating bleomycin (BLM)-induced PF mice with Sch A. The 32 biomarkers were enriched in energy metabolism and several amino acid metabolisms, which was the first report on the therapeutic effects of Sch A on PF through rescuing the disordered energy metabolism. The UPLC-Q-TOF/MS analysis identified 17 possible metabolites (including isomers) of Sch A in mice plasma. Network analysis revealed that Sch A and 17 metabolites were related to 269 genes, and 1109 disease genes were related to PF. The construction of the Sch A/metabolites-target-PF network identified a total of 79 intersection genes and the TGF-β signaling pathway was determined to be the main signaling pathway related to the treatment of PF by Sch A. The integrated approach involving metabolomics and network analysis revealed that the TGF-β1-ID3-creatine pathway, TGF-β1-VIM-carnosine pathway were two of the possible pathways Sch A regulated to modulate metabolic disorders, especially energy metabolism, and the metabolite of Sch A M5 was identified as a most likely active metabolite. The results suggested the feasibility of combining metabolomics and network analysis to reflect the effects of Sch A on the biological network and the metabolic state of PF and to evaluate the drug efficacy of Sch A and its related mechanisms.

CFD simulation of porous microsphere particles in the airways of pulmonary fibrosis

Background and objectivePulmonary fibrosis (PF) is a chronic progressive disease with an extremely high mortality rate and is a complication of COVID-19. Inhalable microspheres have been increasingly used in the treatment of lung diseases such as PF in recent years. Compared to the direct inhalation of drugs, a larger particle size is required to ensure the sustained release of microspheres. However, the clinical symptoms of PF may lead to the easier deposition of microspheres in the upper respiratory tract. Therefore, it is necessary to understand the effects of PF on the deposition of microspheres in the respiratory tract. MethodsIn this study, airway models with different degrees of PF in humans and mice were established, and the transport and deposition of microspheres in the airway were simulated using computational fluid dynamics. ResultsThe simulation results showed that PF increases microsphere deposition in the upper respiratory tract and decreases bronchial deposition in both humans and mice. Porous microspheres with low density can ensure deposition in the lower respiratory tract and larger particle size. In healthy and PF humans, porous microspheres of 10 µm with densities of 700 and 400 kg/m³ were deposited most in the bronchi. Unlike in humans, microspheres larger than 4 µm are completely deposited in the upper respiratory tract of mice owing to their high inhalation velocity. For healthy and PF mice, microspheres of 6 µm with densities of and 100 kg/m³ are recommended. ConclusionsThe results showed that with the exacerbation of PF, it is more difficult for microsphere particles to deposit in the subsequent airway. In addition, there were significant differences in the deposition patterns among the different species. Therefore, it is necessary to process specific microspheres from different individuals. Our study can guide the processing of microspheres and achieve differentiated drug delivery in different subjects to maximize therapeutic effects.

Antifibrotic effect of AD-1 on lipopolysaccharide-mediated fibroblast injury in L929 cells and bleomycin-induced pulmonary fibrosis in mice.

20(R)-25-methoxyl-dammarane-3β,12β,20-triol (25-OCH3-PPD, AD-1) is a dammarane ginsenoside that is isolated from Panax notoginseng. The present study aimed to explore its anti-pulmonary fibrosis (PF) effect in vitro and in vivo. L929 cells were treated with 10 μg mL-1 lipopolysaccharide (LPS) to establish a PF model in vitro and mice were administered with 3.5 mg kg-1 bleomycin (BLM) by endotracheal intubation to establish a PF model in vivo for investigating the anti-PF effect and its potential mechanism. The results demonstrated that AD-1 reduced the injury, extracellular matrix (ECM) buildup and α-smooth muscle actin (α-SMA) protein expression levels of L929 induced by LPS. Oral administration of AD-1 downregulated the expression of interleukins (such as IL-1β, IL-6 and IL-18), increased the expression of superoxide dismutase (SOD) and glutathione (GSH), reduced the lung coefficient and the content of hydroxyproline (HYP), and mediated the Bax/Bcl-2 protein ratio and P-p53, β-catenin and SIRT3 expression in the lung tissue of mice. Furthermore, AD-1 inhibited the expression levels of TGF-β1, TIMP-1 and α-SMA and reduced inflammatory cell infiltration and collagen deposition in the lung tissue of PF mice. These results indicated that AD-1 could alleviate PF both in vitro and in vivo, and the underlying mechanism may be related to the decrease in ECM deposition and inflammation, the enhancement of antioxidant capacity, and the mediation of lung cell apoptosis and the TGF-β1/TIMP-1/α-SMA signaling pathway, which provide a theoretical basis for the rehabilitation treatment of PF.

Health Effects of Alternate Day Fasting Versus Pair-Fed Caloric Restriction in Diet-Induced Obese C57Bl/6J Male Mice.

Alternate day fasting (ADF) induces weight loss and improves various markers of health in rodents and humans. However, it is unclear whether the benefits of ADF are derived from the lower caloric intake of ADF or from the 24-h fasting period. Therefore, this study directly compared selected markers for health – such as glucose control, body weight, liver triglycerides, T cell frequencies, and others – in high-fat (60% calories from fat) diet-induced obese mice subjected to either ADF or caloric restriction (CR). Obese mice were randomly assigned to one of four groups: (1) ADF: remained on the high-fat diet, but fed on alternate days (n = 5), (2) PF: remained on the high-fat diet, but pair-fed to the ADF group (n = 5), (3) LF: moved to a chow ad libitum diet (n = 5; 17% calories from fat), and (4) HF: remained on the high-fat ad libitum diet (n = 5). An additional group of non-obese mice maintained on a chow diet since weaning were used as controls (CON: n = 5). After 10 weeks, ADF, PF, and LF mice ate fewer kcals, had a lower body mass, had smaller epididymal fat pads, improved glucose tolerance, and had a lower hepatic triglyceride content relative to HF mice (p < 0.05), but none reached that of CON mice in these measures. T cell frequencies of the spleen, blood, and mesenteric lymph nodes were reduced in ADF, PF, and HF compared to the CON group. Importantly, there were no significant differences between the ADF and PF groups in any of the measurements made in the current study. These data suggest that ADF, PF, and LF diets each lead to improved markers of health relative to high-fat diet-induced obese mice, and that the caloric restriction associated with ADF is the major factor for the noted improvements.

Dietary fatty acid quality affects systemic parameters and promotes prostatitis and pre-neoplastic lesions

Environmental and nutritional factors, including fatty acids (FA), are associated with prostatitis, benign prostate hyperplasia and prostate cancer. We hypothesized that different FA in normolipidic diets (7%) affect prostate physiology, increasing the susceptibility to prostate disorders. Thus, we fed male C57/BL6 mice with normolipidic diets based on linseed oil, soybean oil or lard (varying saturated and unsaturated FA contents and ω-3/ω-6 ratios) for 12 or 32 weeks after weaning and examined structural and functional parameters of the ventral prostate (VP) in the systemic metabolic context. Mongolian gerbils were included because they present a metabolic detour for low water consumption (i.e., oxidize FA to produce metabolic water). A linseed oil-based diet (LO, 67.4% PUFAs, ω-3/ω-6 = 3.70) resulted in a thermogenic profile, while a soybean oil-based diet (SO, 52.7% PUFAs, ω-3/ω-6 = 0.11) increased body growth and adiposity. Mice fed lard (PF, 13.1% PUFA, ω-3/ω-6 = 0.07) depicted a biphasic growth, resulting in decreased adiposity in adulthood. SO and PF resulted in hepatic steatosis and steatohepatitis, respectively. PF and SO increased prostate epithelial volume, and lard resulted in epithelial hyperplasia. Animals in the LO group had smaller prostates with predominant atrophic epithelia and inflammatory loci. Inflammatory cells were frequent in the VP of PF mice (predominantly stromal) and LO mice (predominantly luminal). RNAseq after 12 weeks revealed good predictors of a later-onset inflammation. The transcriptome unveiled ontologies related to ER stress after 32 weeks on PF diets. In conclusion, different FA qualities result in different metabolic phenotypes and differentially impact prostate size, epithelial volume, inflammation and gene expression.

Factor XIII Contributes to Clot Formation and Thrombin Generation

BackgroundFactor XIII (FXIII) stabilizes fibrin clots, minimizing the amount of thrombin generation required to stop bleeding. It also has a long half-life (>7 days) and supratherapeutic levels are not associated with thrombosis making it an attractive therapeutic agent to augment factor replacement in hemophilia. FXIII is a transglutaminase that cross-links fibrin and localizes alpha 2-antiplasmin to fibrin inhibiting fibrinolysis. It also interacts with numerous other proteins, most of which have not been well studied and their physiologic significance is unknown. FXIII is classically considered to be activated by thrombin, which may limit its utility in low thrombin states such as hemophilia. However, activation also occurs physiologically through other mechanisms including calpain, neutrophil elastase and calcium influx. In addition, low levels of thrombin are generated by Factor Xa in the absence of the Factor VIII (FVIII)/Factor IX (FIX) complex that may contribute to both FXIII activation and clot formation. Thus, it is not clear if thrombin generated through the participation of FVIII and FIX is necessary for FXIII activation.HypothesisWe hypothesize that FXIII (recombinant FXIII-A2) may contribute to both clot formation and thrombin generation in hemophilia.MethodsAnimal models include Exon 16-deleted FVIII-deficient mice (FVIII-KO), wild-type mice (WT), and exon 16-deleted Factor VIII deficient/GP1bα-FVIII knock-in mice (PF). The PF mice are FVIII-deficient mice expressing human FVIII driven from the glycoprotein 1bα promoter resulting in approximately 3% circulating FVIII.Various combinations of factors were given via tail-vein injection. Citrated blood was collected by cardiac puncture 1.5 - 4 hours post-injection, depending on the factor type. Clotting was characterized using thromboelastography (TEG). Thrombin generation was measured on a fluorescence reader using a reagent comprised of a low concentration of phospholipid micelles containing tissue factor in HEPES buffer.ResultsTEG characterization shows differences in clotting times (R), speed of clot formation (K), and the kinetics of the formation of the clot (α angle) between the various groups without changes in overall clot stability (MA) or degree of fibrinolysis (LY30). R, K, and α angle are all measurements of clot formation, with prolongation of R and K and reduced α angle characteristic of hemophilia. PF mice have similar R, K, and α angle compared to FVIII-KO (p = 0.5, 0.68, and 0.89, respectively), and elongated R and K, and reduced α angle compared to WT (p = 4.0E-3, 2.0E-3, and 1.37E-6, respectively). Giving supratherapeutic FXIII to PF mice results in normalization of these values compared to WT, with a trend towards elongated K and α angle (p = 0.21, 0.08, and 0.13, respectively), and differences compared to FVIII-KO (p = 8.1E-3, 7.4E-3, and 2.1E-3, respectively). Administering FXIII to FVIII-KO mice did not alter R, K, and α angle compared to untreated FVIII-KO mice (p = 0.25, 0.37, and 0.67, respectively).PF mice have similar peak thrombin generation compared to FVIII-KO (p = 0.56) and reduced peak thrombin generation compared to WT (p = 6.69E-5). Giving supratherapeutic FXIII to the PF mice results in peak thrombin generation similar to that of WT (p = 0.97). In contrast, giving supratherapeutic FXIII to FVIII-KO mice did not alter peak thrombin generation levels compared to untreated FVIII-KO mice (p = 0.72). The administration of a cocktail containing both FVIII (2.5 U/kg) and FXIII resulted in a trend for improved peak thrombin generation when compared to an injection of FVIII alone (p = 0.12).ConclusionsThe function of FXIII has classically been considered to be secondary to its transglutaminase activity. With a direct impact on early clot formation and thrombin generation, these data suggest that FXIII has other roles beyond its known activities. The implication of these findings is that FXIII may be an effective hemostatic agent in mild and moderate hemophilia. DisclosuresTaylor:Novo Nordisk: Research Funding; Kedrion: Research Funding; Baxalta/Shire: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding.

Krüppel-like factor 4 is a transcriptional regulator of M1/M2 macrophage polarization in alcoholic liver disease.

Macrophages play an important role in inflammation and liver injury. In ALD, activated macrophages, including M1 (proinflammatory) and M2 (anti-inflammatory) macrophages, are present in the liver. As KLF4 has been described as a regulator of macrophage polarization, we investigated its role in ALD. Chronic alcohol feeding in C57Bl/6 mice led to increased expression of M1 (TNF-α, MCP1, and IL-1β) and M2 (Arg1, Mrc1, and IL-10) genes and the frequency of CD206+CD163+ M2 macrophages in the liver. KLF4 mRNA and protein levels were increased in the livers of EtFed compared with PF mice. In macrophages, in vivo and in vitro, EtOH increased KLF4 levels, transcriptional activity, and expression of M1 and M2 genes. KLF4 knockdown and overexpression experiments demonstrated alcohol-dependent and -independent functions of KLF4 in regulating M1 and M2 markers. KLF4 siRNA treatment, alone and in synergy with alcohol, increased the levels of M1 markers. In contrast, KLF4 overexpression increased the levels of M2 and decreased M1 markers, and this was enhanced further by alcohol. KLF4 was regulated by alcohol and its metabolites. KLF4 mRNA and activity were increased in the presence of 4-MP, an inhibitor of ADH, and CYP2E1. However, inhibition of acetaldehyde breakdown attenuated KLF4 induction and promoted M1 polarization. We conclude that KLF4 regulates M1 and M2 markers in ALD. EtOH promotes KLF4 and M2 phenotype, whereas acetaldehyde attenuates KLF4 and promotes M1 macrophage, which may explain the increased presence of M1 and M2 macrophage populations in ALD.

Acyl Ghrelin Acts in the Brain to Control Liver Function and Peripheral Glucose Homeostasis in Male Mice

Recent evidence suggests that peripheral ghrelin regulates glucose metabolism. Here, we designed experiments to examine how central acyl ghrelin infusion affects peripheral glucose metabolism under pair-fed or ad libitum feeding conditions. Mice received intracerebroventricular (icv) infusion of artificial cerebrospinal fluid (aCSF), ghrelin, and allowed to eat ad libitum (icv ghrelin ad lib) or ghrelin and pair-fed to the aCSF group (icv ghrelin pf). Minipumps delivered acyl ghrelin at a dose of 0.25 μg/h at 0.5 μL/h for 7 days. There was no difference in daily blood glucose, insulin, glucagon, triglycerides, or nonesterified fatty acids. Body weight gain and food intake was significantly higher in icv ghrelin ad lib mice. However, both icv ghrelin ad lib and icv ghrelin pf groups exhibited heavier white adipose mass. Icv ghrelin pf mice exhibited better glucose tolerance than aCSF or icv ghrelin ad lib mice during a glucose tolerance test, although both icv ghrelin ad lib and icv ghrelin pf increased insulin release during the glucose tolerance test. Central acyl ghrelin infusion and pair feeding also increased breakdown of liver glycogen and triglyceride, and regulated genes involved in hepatic lipid and glucose metabolism. Icv ghrelin pf mice had an increase in plasma blood glucose during a pyruvate tolerance test relative to icv ghrelin ad lib or aCSF mice. Our results suggest that under conditions of negative energy (icv ghrelin pf), central acyl ghrelin engages a neural circuit that influences hepatic glucose function. Metabolic status affects the ability of central acyl ghrelin to regulate peripheral glucose homeostasis.

Abstract LB-86: Dependent and independent tumor suppression function of ARF and p53 in murine BCC carcinogenesis in Ptch1+/- mice

Abstract Basal cell carcinoma (BCC), the most commonly diagnosed human cancer, is driven by aberrant activation of hedgehog (HH) signaling. In addition, we have found that p53 is a potent inhibitor of HH-driven BCC carcinogenesis - ablation of p53 in Ptch1+/- K14CreER2 p53fl/fl (PF mice) dramatically accelerates BCC formation with a shorter tumor latency and a 10 fold increase of tumor burden. To begin to determine how p53 is activated in this Ptch1+/- murine BCC model, we investigated the role of p19ARF in translating oncogene-induced stress (OIS) into p53 activation. We bred Ptch1+/- mice with ARFGFP/GFP to generate Ptch1+/- ARFGFP/GFP (ARF mice), lacking functional ARF. In addition, we crossed ARF mice and PF mice to generate ARF/p53 double deletion mice (DD mice), treated ARF, PF and DD mice as well as Ptch1+/- (ARF+/+, p53+/+) with IR at mouse age 8w, and quantified BCC formation. .As assessed in age 5m skin biopsies, PF and DD mice had a significantly higher microscopic BCC tumor burden than did ARF and Ptch1+/- mice. There was no significant difference between PF and DD or between ARF and Ptch1+/-. ARF and PF mice developed visible BCC starting at 5-6 m. By contrast, DD mice had visible BCCs as early as age 4m, a statistically different age of onset. Consistent with our previous findings, Ptch1+/- mice did not form any visible BCCs until age 9 m or later. These data indicate that loss of functional ARF or of p53 promotes malignant conversion of microscopic BCCs to visible BCCs. However, only loss of p53, but not loss of ARF, enhances tumor initiation. To gain further insights in the molecular mechanisms differentiating these tumors, we examined the whole genome expression analysis by RNA-seq. Taken together, our findings suggest that both ARF-dependent and -independent activation of p53 is involved in BCC carcinogenesis. ARF-mediated p53 activation, presumably via the OIS pathway, appears to be crucial in regulating malignant transformation of BCC, while ARF-independent p53 activation inhibits tumor initiation. Citation Format: Grace Y. Wang, Carla Wood, Hiroe Hu, John Dolorito, Eileen Libove, Ervin H. Eptein. Dependent and independent tumor suppression function of ARF and p53 in murine BCC carcinogenesis in Ptch1+/- mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-86. doi:10.1158/1538-7445.AM2014-LB-86

The development of diet-induced obesity and glucose intolerance in C57BL/6 mice on a high-fat diet consists of distinct phases.

High–fat (HF) diet-induced obesity and insulin insensitivity are associated with inflammation, particularly in white adipose tissue (WAT). However, insulin insensitivity is apparent within days of HF feeding when gains in adiposity and changes in markers of inflammation are relatively minor. To investigate further the effects of HF diet, C57Bl/6J mice were fed either a low (LF) or HF diet for 3 days to 16 weeks, or fed the HF-diet matched to the caloric intake of the LF diet (PF) for 3 days or 1 week, with the time course of glucose tolerance and inflammatory gene expression measured in liver, muscle and WAT. HF fed mice gained adiposity and liver lipid steadily over 16 weeks, but developed glucose intolerance, assessed by intraperitoneal glucose tolerance tests (IPGTT), in two phases. The first phase, after 3 days, resulted in a 50% increase in area under the curve (AUC) for HF and PF mice, which improved to 30% after 1 week and remained stable until 12 weeks. Between 12 and 16 weeks the difference in AUC increased to 60%, when gene markers of inflammation appeared in WAT and muscle but not in liver. Plasma proteomics were used to reveal an acute phase response at day 3. Data from PF mice reveals that glucose intolerance and the acute phase response are the result of the HF composition of the diet and increased caloric intake respectively. Thus, the initial increase in glucose intolerance due to a HF diet occurs concurrently with an acute phase response but these effects are caused by different properties of the diet. The second increase in glucose intolerance occurs between 12 - 16 weeks of HF diet and is correlated with WAT and muscle inflammation. Between these times glucose tolerance remains stable and markers of inflammation are undetectable.

Lactobacillus rhamnosus GG Treatment Potentiates Intestinal Hypoxia-Inducible Factor, Promotes Intestinal Integrity and Ameliorates Alcohol-Induced Liver Injury

Gut-derived endotoxin is a critical factor in the development and progression of alcoholic liver disease (ALD). Probiotics can treat alcohol-induced liver injury associated with gut leakiness and endotoxemia in animal models, as well as in human ALD; however, the mechanism or mechanisms of their beneficial action are not well defined. We hypothesized that alcohol impairs the adaptive response-induced hypoxia-inducible factor (HIF) and that probiotic supplementation could attenuate this impairment, restoring barrier function in a mouse model of ALD by increasing HIF-responsive proteins (eg, intestinal trefoil factor) and reversing established ALD. C57BJ/6N mice were fed the Lieber DeCarli diet containing 5% alcohol for 8 weeks. Animals received Lactobacillus rhamnosus GG (LGG) supplementation in the last 2 weeks. LGG supplementation significantly reduced alcohol-induced endotoxemia and hepatic steatosis and improved liver function. LGG restored alcohol-induced reduction of HIF-2α and intestinal trefoil factor levels. In vitro studies using the Caco-2 cell culture model showed that the addition of LGG supernatant prevented alcohol-induced epithelial monolayer barrier dysfunction. Furthermore, gene silencing of HIF-1α/2α abolished the LGG effects, indicating that the protective effect of LGG is HIF-dependent. The present study provides a mechanistic insight for utilization of probiotics for the treatment of ALD, and suggests a critical role for intestinal hypoxia and decreased trefoil factor in the development of ALD.

Weight Loss via Exercise with Controlled Dietary Intake May Affect Phospholipid Profile for Cancer Prevention in Murine Skin Tissues

Exercise has been linked to a reduced cancer risk in animal models. However, the underlying mechanisms are unclear. This study assessed the effect of exercise with dietary consideration on the phospholipid profile in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin tissues. CD-1 mice were randomly assigned to one of the three groups: ad libitum-fed sedentary control; ad libitum-fed treadmill exercise at 13.4 m/min for 60 min/d, 5 d/wk (Ex+AL); and treadmill-exercised but pair-fed with the same amount as the control (Ex+PF). After 14 weeks, Ex+PF but not Ex+AL mice showed approximately 25% decrease in both body weight and body fat when compared with the controls. Of the total 338 phospholipids determined by electrospray ionization-tandem mass spectrometry, 57 were significantly changed, and 25 species could distinguish effects of exercise and diet treatments in a stepwise discriminant analysis. A 36% to 75% decrease of phosphatidylinositol (PI) levels in Ex+PF mice occurred along with a significant reduction of PI 3-kinase in TPA-induced skin epidermis, as measured by both Western blotting and immunohistochemistry. In addition, approximately 2-fold increase of the long-chain polyunsaturated fatty acids, docosahexaenoic and docosapentaenoic acids, in phosphatidylcholines, phosphatidylethanolamines, and lysophosphatidylethanolamines was observed in the Ex+PF group. Microarray analysis indicated that the expression of fatty acid elongase-1 increased. Taken together, these data indicate that exercise with controlled dietary intake, but not exercise alone, significantly reduced body weight and body fat as well as modified the phospholipid profile, which may contribute to cancer prevention by reducing TPA-induced PI 3-kinase and by enhancing omega-3 fatty acid elongation.