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The extracts from Psoralea corylifolia Linn. (P. corylifolia) seeds have been shown to display antitumor activity. To date, the prospects of this plant and its active compounds in the treatment of non-small-cell lung cancer (NSCLC) have not been thoroughly studied. In this study, we identified a novel psorachromene compound that displays selective cytotoxic effects on all NSCLC cells tested, including NSCLC cells harboring epidermal growth factor receptor (EGFR) activation mutants (H1975L858R/T790M and H1975-MS35L858R/T790M/C797S ). Psorachromene induces G1 arrest in NSCLC cells harboring wild-type EGFR but induces apoptosis in NSCLC cells harboring activating EGFR mutations. Psorachromene inhibits activated EGFR signaling and kinase activity and suppresses tumor growth of implanted H1975-MS35L858R/T790M/C797S cells in nude mice. Molecular docking analysis revealed that psorachromene could form stronger bonds with mutant EGFR than wild-type EGFR, which might account for the greater cytotoxic effects observed in NSCLC cells harboring activating EGFR mutations (H1975 and H1975-MS35) than wild-type EGFR (A549). In conclusion, it is suggested that psorachromene is an attractive agent to be further explored for its use in the treatment of NSCLC patients harboring EGFR L858R/T790M/C797S. [ABSTRACT FROM AUTHOR]

Background: Immune system dysfunction, including higher levels of peripheral monocytes and inflammatory cytokines, is an important feature of Parkinson's disease (PD) pathogenesis, although the mechanism underlying the process remains to be investigated. In the central nervous system, it is well-known that α-synuclein (α-syn), a key protein involved in PD, activates microglia potently, and it is also reported that α-syn exists in the peripheral system, especially in erythrocytes or red blood cells (RBC) at exceedingly high concentration. The current study focused on the possibility that RBC-derived α-syn mediates the sensitization of peripheral monocytes in PD patients.Methods: The hyperactivation of monocytes was assessed quantitatively by measuring mRNA levels of typical inflammatory cytokines (including IL-1β, IL-6 and TNF-α) and protein levels of secreted inflammatory cytokines (including pro-inflammatory cytokines: IL-1β, IL-6, TNF-α, IL-8, IFN-γ, IL-2, and IL-12p70 and anti-inflammatory cytokines: IL-4, IL-10, and IL-13). Western blot, nanoparticle tracking analysis and electron microscopy were used to characterize RBC-derived extracellular vesicles (RBC-EVs). Inhibitors of endocytosis and leucine-rich repeat kinase 2 (LRRK2), another key protein involved in PD, were used to investigate how these two factors mediated the process of monocyte sensitization by RBC-EVs.Results: Increased inflammatory sensitization of monocytes was observed in PD patients and PD model mice. We found that α-syn-containing RBC-EVs isolated from PD model mice or free form oligomeric α-syn induced the inflammatory sensitization of THP-1 cells, and demonstrated that endocytosis was a requirement for this pathophysiological pathway. Furthermore, the hyperactivation of THP-1 cells induced by RBC-EVs was associated with increased LRRK2 production and kinase activity. The phenomenon of inflammatory sensitization of human monocytes and increased LRRK2 were also observed by the treatment of RBC-EVs isolated from PD patients.Conclusions: Our data provided new insight into how hyperactivation of monocytes occurs in PD patients, and identified the central role played by α-syn-containing RBC-EVs in this process. [ABSTRACT FROM AUTHOR]

Phosphatidylinositide 3-kinases (PI3Ks) are intracellular signal transducer enzymes that recruit protein kinase B (aka Akt) to the cell membrane, the subsequent activation of which regulates many cellular functions. PI3K/Akt activity is up-regulated within mesocorticolimbic structures in animal models of alcoholism, but less is known regarding PI3K/Akt activity in animal models of cocaine addiction. Given that prefrontal cortex (PFC) is grossly dysregulated in addiction, we studied how cocaine affects protein indices of PFC PI3K/Akt activity in rat and mouse models and examined the relevance of PI3K activity for cocaine-related learning. Immunoblotting of mouse medial PFC at 3 weeks withdrawal from a cocaine-sensitization regimen (seven injections of 30 mg/kg, intraperitoneal [IP]) revealed increased kinase activity, as did immunoblotting of tissue from the ventral PFC of rats with a history of long-access intravenous cocaine self-administration (0.25 mg/0.1 mL infusion; 10 days of 6 h/d cocaine access). Interestingly, increased Akt phosphorylation was observed in rat ventromedial PFC at both 3- and 30-day withdrawal only in animals re-exposed to cocaine-associated cues. A conditioned place-preference paradigm in mice and a cue-elicited drug-seeking test in rats were conducted to determine the functional relevance for elevated PI3K activity for addiction-related behavior. In both cases, an intra-PFC infusion of the PI3K inhibitor wortmannin (50μM) reduced drug-seeking behavior. Taken together, this cross-species, interdisciplinary, study provides convincing evidence that cocaine history produces an enduring increase in PI3K/Akt-dependent signaling within the more ventral aspect of the PFC that is relevant to behavioral reactivity to drug-associated cues/contexts. As such, PI3K inhibitors may well serve as an effective strategy for reducing drug cue reactivity and craving in cocaine addiction. [ABSTRACT FROM AUTHOR]

Background: Transient receptor potential vanilloid-1 (TRPV1), activated by heat, acidic pH, endogenous vanilloids and capsaicin, is essential for thermal hyperalgesia. Under inflammatory conditions, phosphorylation of TRPV1 by protein kinase C (PKC) can sensitize the channel and decrease the activation threshold. Src kinase also phosphorylates TRPV1, promoting channel trafficking to the plasma membrane. These post-translational modifications are important for several chronic pain conditions. This study presents a previously undescribed relationship between Src and PKC phosphorylation of TRPV1, influencing the thermal hypersensitivity associated with TRPV1 activation.Methods: We assessed TRPV1 channel activity using intracellular calcium imaging and patch-clamp electrophysiology in mouse dorsal root ganglion cultures. Additionally, we used behavioural experiments to evaluate plantar thermal sensitivity following intraplantar injections of activators of known modulators of TRPV1 with and without an Src antagonist.Results: Using calcium imaging and patch-clamp techniques, we demonstrated that pharmacological inhibition of Src kinase or mutation of the Src phosphorylation site on TRPV1 prevented PKC but not PKA-mediated sensitization of TRPV1 in vitro. We found that intraplantar injection of the PKC activator phorbol 12-myristate 13-acetate (PMA) or bradykinin produces thermal hypersensitivity that can be attenuated by pharmacological inhibition of Src. Additionally, complete Freund's Adjuvant (CFA)-induced inflammatory hypersensitivity could also be attenuated by local Src kinase inhibition.Conclusions: Our data demonstrate that Src phosphorylation is critical for PKC-mediated sensitization of TRPV1. Further, in a model of inflammatory pain, CFA, Src kinase inhibition could reduce thermal hypersensitivity. Targeting of Src kinase may have analgesic benefits in inflammatory pain conditions.Significance: Src kinase-mediated phosphorylation of TRPV1 is a critical regulator of the PKC-induced sensitization induced by multiple inflammatory mediators. This suggest a new regulatory mechanism governing TRPV1 function and a potential therapeutic target for inflammatory type pain, including cancer pain where Src antagonists are currently utilized. [ABSTRACT FROM AUTHOR]

Simple Summary: Patients with treatable cancers all too often return to the clinic with untreatable tumors, requiring highly toxic secondary treatments or palliative care. In this study, we aim to determine whether activation of the Anaphase Promoting Complex (APC) in multiple-drug-resistant (MDR) breast cancer cells will re-sensitize them to chemotherapeutic agents. The APC is required for the targeted degradation of proteins that inhibit passage through mitosis. We found that APC activity is indeed impaired in MDR cells and that chemical activation of the APC increased the sensitivity of these cells to doxorubicin. Mitotic progression was slowed in MDR cells, compared to matched parental drug sensitive cells, with an accumulation of APC substrate proteins. APC activation in nocodazole-arrested cells resulted in increased passage through mitosis with lower APC substrate levels. Mice growing a patient-derived xenografted (PDX) tumor were treated with increasing doses of a chemical APC activator, resulting in a dose-dependent reduction in tumor size. Taken together, our data show that APC activity is reduced in MDR cells, with APC activation resulting in a species- and cancer-type-independent reversal of the MDR phenotype. The development of multiple-drug-resistant (MDR) cancer all too often signals the need for toxic alternative therapy or palliative care. Our recent in vivo and in vitro studies using canine MDR lymphoma cancer cells demonstrate that the Anaphase Promoting Complex (APC) is impaired in MDR cells compared to normal canine control and drug-sensitive cancer cells. Here, we sought to establish whether this phenomena is a generalizable mechanism independent of species, malignancy type, or chemotherapy regime. To test the association of blunted APC activity with MDR cancer behavior, we used matched parental and MDR MCF7 human breast cancer cells, and a patient-derived xenograft (PDX) model of human triple-negative breast cancer. We show that APC activating mechanisms, such as APC subunit 1 (APC1) phosphorylation and CDC27/CDC20 protein associations, are reduced in MCF7 MDR cells when compared to chemo-sensitive matched cell lines. Consistent with impaired APC function in MDR cells, APC substrate proteins failed to be effectively degraded. Similar to our previous observations in canine MDR lymphoma cells, chemical activation of the APC using Mad2 Inhibitor-1 (M2I-1) in MCF7 MDR cells enhanced APC substrate degradation and resensitized MDR cells in vitro to the cytotoxic effects of the alkylating chemotherapeutic agent, doxorubicin (DOX). Using cell cycle arrest/release experiments, we show that mitosis is delayed in MDR cells with elevated substrate levels. When pretreated with M2I-1, MDR cells progress through mitosis at a faster rate that coincides with reduced levels of APC substrates. In our PDX model, mice growing a clinically MDR human triple-negative breast cancer tumor show significantly reduced tumor growth when treated with M2I-1, with evidence of increased DNA damage and apoptosis. Thus, our results strongly support the hypothesis that APC impairment is a driver of aggressive tumor development and that targeting the APC for activation has the potential for meaningful clinical benefits in treating recurrent cases of MDR malignancy. [ABSTRACT FROM AUTHOR]

Background: Alzheimer’s disease (AD) is a multifactorial neurodegenerative condition. The search for multi-target traditional Chinese medicines or ingredients for treating AD has attracted much attention. Corydalis rhizome (CR) is a traditional Chinese medicine. Its main components are alkaloids, which have therapeutic effects that can potentially be used for treating AD. However, no systematic study has been conducted to explore the anti-AD efficacy of CR, as well as its active compounds and mechanisms of action. Objective: The present study aimed to clarify CR’s active constituents and its pharmacological mechanisms in treating AD. Methods: A D-galactose & scopolamine hydrobromide-induced AD mouse model was used and CR was administered orally. The prototypical alkaloid components were identified in the serum. The core components, key targets, and possible mechanisms of action of these alkaloids were revealed through network pharmacology. Molecular docking of the key target was performed. Finally, the mechanism was validated by lipopolysaccharide (LPS)-induced activation of BV2 microglia. Results: The results showed that CR improved anxiety-like behavior, spatial and non-spatial recognition, and memory capacity in AD mice. It also achieved synergistic AD treatment by modulating neurotransmitter levels, antineuroinflammation, and anti-oxidative stress. The core components that enhance CR’s efficacy in treating AD are protoberberine-type alkaloids. The CR may induce the polarization of LPS-activated BV2 microglia from phenotype M1 to M2. This is partially achieved by modulating the IL-6/JAK2/STAT3 signaling pathway, which could be the mechanism by which CR treats AD through anti-inflammation. Conclusion: The present study provided a theoretical and experimental basis for the clinical application of CR in treating AD. It also provides information that aids the secondary development, and precise clinical use of CR. [ABSTRACT FROM AUTHOR]

Recent study indicated that glutamine prevents alcoholic tissue injury in mouse gut and liver. Here we investigated the potential role of Epidermal Growth Factor Receptor (EGFR) in glutamine-mediated prevention of ethanol-induced colonic barrier dysfunction, endotoxemia and liver damage. Wild-type and EGFR*Tg transgenic (expressing dominant negative EGFR) mice were fed 1-6% ethanol in Lieber-DeCarli diet. Gut permeability was measured by vascular-to-luminal flux of FITC-inulin, and junctional integrity assessed by confocal microscopy. Liver injury was evaluated by plasma transaminases, histopathology and triglyceride analyses. Glutamine effect on acetaldehyde-induced tight junction disruption was investigated in Caco-2 cell monolayers. Doxycycline-induced expression of EGFR* blocked glutamine-mediated prevention of ethanol-induced disruption of colonic epithelial tight junction, mucosal permeability and endotoxemia. Ethanol activated cofilin and disrupted actin cytoskeleton, which was blocked by glutamine in an EGFR-dependent mechanism. Ethanol down-regulated antioxidant gene expression and up-regulated cytokine and chemokine gene expression, which were blocked by glutamine in wild-type mice in the presence or absence of doxycycline, but not in EGFR*Tg mice in the presence of doxycycline. Histopathology, plasma transaminases, triglyceride and expression of chemokine and antioxidant genes indicated ethanol-induced liver damage, which were blocked by glutamine in an EGFR-dependent mechanism. Src kinase activity and extracellular ligand binding domain of EGFR are required for glutamine-mediated protection of barrier function in Caco-2 cell monolayers. Glutamine released metalloproteinases into the medium, and metalloproteinase inhibitors blocked glutamine-mediated protection of barrier function. Results demonstrate that EGFR plays an important role in glutamine-mediated prevention of alcoholic gut permeability, endotoxemia and liver damage. [ABSTRACT FROM AUTHOR]

Background and Purpose: The pathogenesis of osteoarthritis implicates a low-grade inflammation associated to the innate immune system activation. Toll like receptor (TLR) stimulation triggers the release of inflammatory mediators, which aggravate osteoarthritis. We studied the preventive effect of 6-shogaol, a potential TLR4 inhibitor, on the treatment of experimental knee osteoarthritis.Experimental Approach: Osteoarthritis was induced in C57BL6 mice by surgical section of the medial meniscotibial ligament, which received 6-shogaol for eight weeks. Cartilage damage, inflammatory mediator presence and disease markers were assessed in joint tissues by immunohistochemistry. Computational modelling was used to predict binding modes of 6-shogaol into the TLR4/MD2 receptor and its permeability across cellular membranes. Employing LPS-stimulated chondrocytes and MAPK assay, we elucidated 6-shogaol action mechanisms.Key Results: 6-Shogaol treatment prevented articular cartilage lesions, synovitis and the presence of pro-inflammatory mediators, and disease markers in osteoarthritis animals. Molecular modelling studies predicted 6-shogaol interaction with the TLR4/MD-2 heterodimer in an antagonist conformation through its binding into the MD-2 pocket. In cell culture, we confirmed that 6-shogaol reduced LPS-induced TLR4 inflammatory signalling pathways. Besides, MAPK assay demonstrated that 6-shogaol directly inhibits the ERK1/2 phosphorylation activity.Conclusion and Implications: 6-Shogaol evoked a preventive action on cartilage and synovial inflammation in osteoarthritis mice. 6-shogaol effect may take place not only by hindering the interaction between TLR4 ligands and the TLR4/MD-2 complex in chondrocytes, but also through inhibition of ERK phosphorylation, implying a pleiotropic effect on different mediators activated during osteoarthritis, which proposes it as an attractive drug for osteoarthritis treatments. [ABSTRACT FROM AUTHOR]

Objective: We previously reported that alterations of the tumor microenvironment (TME) by programmed death receptor-1 (PD1) blockade affected tumor glucose metabolism and tumor 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) uptake. In cancer cells, high glycolysis allows cells to sustain rapid proliferation since glycolysis is closely related to the proliferation of cancer cells. Therefore, imaging of cellular proliferation may provide more detail of TME alterations. In this study, we investigated how TME alterations by PD1 blockade affects the uptake of 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT), which is a 18F-radiolabeled thymidine derivative and is taken up by proliferating cells.Methods: Mice inoculated with murine colon carcinoma CT26 cells were intraperitoneally administered an anti-PD1 antibody on Day 0, when the tumor volume exceeded 50 mm3, and Day 5. [18F]FLT-PET imaging was performed pre-treatment (Day 0) and post treatment (Day 7). Tumor infiltrating lymphocytes (TILs) were identified by flow cytometry. [18F]FLT accumulation and localization in tumor tissue was evaluated by autoradiography and immunohistochemistry. The cell-cycle distribution of tumors and CT26 cells exposed to cytokines (interleukin-2, interferon [INF]-γ, and tumor necrosis factor [TNF]-α) was analyzed by flow cytometry.Results: PD1 blockade increased CD8+ and CD4+ T cells in tumor tissue and significantly suppressed tumor proliferation; however, tumor [18F]FLT uptake remained unchanged. Autoradiography and immunohistochemistry showed that [18F]FLT was mainly taken up by cancer cells, but not TILs. Flow cytometric analysis demonstrated that the population of cells in G2/M phase increased after PD1 blockade. Moreover, INF-γ and TNF-α significantly increased cells in G2/M phase in vitro.Conclusion: PD1 blockade-induced alteration of the TME increased CT26 tumor cells in the G2/M phase, which have high thymidine kinase 1 activity. Therefore, [18F]FLT is taken up by tumor cells even if tumor proliferation is suppressed. This observation may be useful for evaluating the response to immunotherapy. [ABSTRACT FROM AUTHOR]

Long-term glucagon receptor (GCGR) agonism is associated with hyperglycemia and glucose intolerance, while acute GCGR agonism enhances whole-body insulin sensitivity and hepatic AKTSer473 phosphorylation. These divergent effects establish a critical gap in knowledge surrounding GCGR action. mTOR complex 2 (mTORC2) is composed of seven proteins, including RICTOR, which dictates substrate binding and allows for targeting of AKTSer473. We used a liver-specific Rictor knockout mouse (RictorΔLiver) to investigate whether mTORC2 is necessary for insulin receptor (INSR) and GCGR cross talk. RictorΔLiver mice were characterized by impaired AKT signaling and glucose intolerance. Intriguingly, RictorΔLiver mice were also resistant to GCGR-stimulated hyperglycemia. Consistent with our prior report, GCGR agonism increased glucose infusion rate and suppressed hepatic glucose production during hyperinsulinemic-euglycemic clamp of control animals. However, these benefits to insulin sensitivity were ablated in RictorΔLiver mice. We observed diminished AKTSer473 and GSK3α/βSer21/9 phosphorylation in RictorΔLiver mice, whereas phosphorylation of AKTThr308 was unaltered in livers from clamped mice. These signaling effects were replicated in primary hepatocytes isolated from RictorΔLiver and littermate control mice, confirming cell-autonomous cross talk between GCGR and INSR pathways. In summary, our study reveals the necessity of RICTOR, and thus mTORC2, in GCGR-mediated enhancement of liver and whole-body insulin action. [ABSTRACT FROM AUTHOR]

Purpose: Intervertebral disc (IVD) degeneration is accompanied by mechanical and gene expression changes to IVDs. SPARC-null mice display accelerated IVD degeneration, and treatment with (toll-like receptor 4 (TLR4) inhibitor) TAK-242 decreases proinflammatory cytokines and pain. This study examined if chronic TAK-242 treatment impacts mechanical properties and gene expression associated with IVD degeneration in SPARC-null mice.Methods: Male and female SPARC-null and WT mice aged 7-9 months were given intraperitoneal injections with TAK-242 or an equivalent saline vehicle for 8 weeks (3x/per week, M-W-F). L2-L5 spinal segments were tested in cyclic axial tension and compression. Gene expression analysis (RT-qPCR) was performed on male IVD tissues using Qiagen RT2 PCR arrays.Results: SPARC-null mice had decreased NZ length (p = 0.001) and increased NZ stiffness (p < 0.001) compared to WT mice. NZ length was not impacted by TAK-242 treatment (p = 0.967) despite increased hysteresis energy (p = 0.024). Tensile stiffness was greater in SPARC-null mice (p = 0.018), and compressive (p < 0.001) stiffness was reduced from TAK-242 treatment in WT but not SPARC-null mice (p = 0.391). Gene expression analysis found upregulation of 13 ECM and 5 inflammatory genes in SPARC-null mice, and downregulation of 2 inflammatory genes after TAK-242 treatment.Conclusions: TAK-242 had limited impacts on SPARC-null mechanical properties and did not attenuate NZ mechanical changes associated with IVD degeneration. Expression analysis revealed an increase in ECM and inflammatory gene expression in SPARCnull mice with a reduction in inflammatory expression due to TAK-242 treatment. This study provides insight into the role of TLR4 in SPARC-null mediated IVD degeneration. [ABSTRACT FROM AUTHOR]

Background and Purpose: Recent evidence links brain insulin resistance with neurodegenerative diseases, where hyperphosphorylated tau protein contributes to neuronal cell death. In the present study, we aimed to evaluate if d-pinitol inositol, which acts as an insulin sensitizer, affects the phosphorylation status of tau protein.Experimental Approach: We studied the pharmacological effect of d-pinitol on insulin signalling and tau phosphorylation in the hippocampus of Wistar and Zucker rats. To this end, we evaluated by western blotting the Akt pathway and its downstream proteins as being one of the main insulin-mediator pathways. Also, we explored the functional status of additional kinases phosphorylating tau, including PKA, ERK1/2, AMPK and CDK5. We utilized the 3xTg mouse model as a control for tauopathy, since it carries tau mutations that promote phosphorylation and aggregation.Key Results: Surprisingly, we discovered that oral d-pinitol treatment lowered tau phosphorylation significantly, but not through the expected kinase GSK-3 regulation. An extensive search for additional kinases phosphorylating tau revealed that this effect was mediated through a mechanism dependent on the reduction of the activity of the CDK5, affecting both its p35 and p25 subunits. This effect disappeared in leptin-deficient Zucker rats, uncovering that the association of leptin deficiency, obesity, dyslipidaemia and hyperinsulinaemia abrogates d-pinitol actions on tau phosphorylation. The 3xTg mice confirmed d-pinitol effectiveness in a genetic AD-tauopathy.Conclusion and Implications: The present findings suggest that d-pinitol, by regulating CDK5 activity through a decrease of CDK5R1, is a potential drug for developing treatments for neurological disorders such as tauopathies. [ABSTRACT FROM AUTHOR]

Rationale: Although persistent fibroblast activation is a hallmark of idiopathic pulmonary fibrosis (IPF), mechanisms regulating persistent fibroblast activation in the lungs have not been fully elucidated. Objectives: On the basis of our observation that lung fibroblasts express TBXA2R (thromboxane-prostanoid receptor) during fibrosis, we investigated the role of TBXA2R signaling in fibrotic remodeling. Methods: We identified TBXA2R expression in lungs of patients with IPF and mice and studied primary mouse and human lung fibroblasts to determine the impact of TBXA2R signaling on fibroblast activation. We used TBXA2R-deficient mice and small-molecule inhibitors to investigate TBXA2R signaling in preclinical lung fibrosis models. Measurements and Main Results: TBXA2R expression was upregulated in fibroblasts in the lungs of patients with IPF and in mouse lungs during experimental lung fibrosis. Genetic deletion of TBXA2R, but not inhibition of thromboxane synthase, protected mice from bleomycin-induced lung fibrosis, thereby suggesting that an alternative ligand activates profibrotic TBXA2R signaling. In contrast to thromboxane, F2-isoprostanes, which are nonenzymatic products of arachidonic acid induced by reactive oxygen species, were persistently elevated during fibrosis. F2-isoprostanes induced TBXA2R signaling in fibroblasts and mediated a myofibroblast activation profile due, at least in part, to potentiation of TGF-β (transforming growth factor-β) signaling. In vivo treatment with the TBXA2R antagonist ifetroban reduced profibrotic signaling in the lungs, protected mice from lung fibrosis in three preclinical models (bleomycin, Hermansky-Pudlak mice, and radiation-induced fibrosis), and markedly enhanced fibrotic resolution after bleomycin treatment. Conclusions: TBXA2R links oxidative stress to fibroblast activation during lung fibrosis. TBXA2R antagonists could have utility in treating pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Background: Coronary atherosclerosis (CA) is the most common type of atherosclerosis. However, the inherent pathogenesis and mechanisms of CA are unclear, and the relationship with ferroptosis-related genes (FRGs) has not been reported. The purpose of this study was to use bioinformatics techniques to evaluate potential therapeutic targets for CA.Please provide the given name for author "Dingshun".Please provide the given name for author "Dingshun".Methods: First, the GSE132651 dataset was acquired from the Gene Expression Omnibus database. Gene Ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and Protein-Protein interaction network were successively conducted. Next, overlapping genes between hub genes and CA genes were found. FRGs were found when comparing the CA group with the normal group. The correlation between overlapping genes and FRGs was further analyzed. At last, we performed Elisa to validate the expression of these genes in human blood specimens. Mice aortic tissues were used for western blot to detect the expression of proteins.Results: Based on the GSE132651 dataset, 102 differentially expressed genes were identified. Five overlapping genes between hub genes and CA genes were found (CCNA2, RRM2, PBK, PCNA, CDK1). TFRC and GPX4 were found to be FRGs. TFRC was positively correlated with CCNA2, PBK, PCNA, CDK1, RRM2, with CDK1 being the strongest correlation. GPX4 was negatively correlated with these genes, among which CCNA2 was the strongest correlation. The ELISA results showed that CCNA2, CDK1, and TFRC expression were markedly increased in serum of the CA samples compared with controls, while GPX4 expression was markedly decreased in the CA samples. The western blot results show that GPX4 expression was lower in the model group, TFRC, CDK1, and CCNA2 protein expression were high in the model group.Conclusions: Ferroptosis-related genes GPX4 and TFRC were closely correlated with the identified overlapping genes CCNA2 and CDK1, which may serve as targeted therapies for the treatment of CA. [ABSTRACT FROM AUTHOR]

Background: Midostaurin combined with chemotherapy is currently used to treat newly diagnosed acute myeloid leukemia (AML) patients with FMS-like tyrosine kinase 3 (FLT3)-mutations. However, midostaurin acts as an antagonist to some chemotherapeutic agents in leukemia cell lines without FLT3 mutations. All-trans retinoic acid (ATRA) induces apoptosis when used in combination with midostaurin in FLT3-mutated AML cells. This combination has been shown to be safe in AML patients. However, the effect of this combination has not been investigated in AML without FLT3 mutations.Methods: Cell proliferation was assessed by a cell counting assay. Cell death was evaluated by cell viability and Annexin-V assays. Cell differentiation was assessed by CD11b expression profiling and morphological analysis. To explore the underlying mechanisms, we studied the role of caspase3/7, Lyn, Fgr, Hck, RAF, MEK, ERK, AKT, PU.1, CCAAT/enhancer binding protein β (C/EBPβ) and C/EBPε by Western blot analysis and immunoprecipitation assays. Antitumor activity was also confirmed in mouse xenograft models established with AML cells.Results: In this study, 0.1 - 0.25 μM midostaurin (mido(L)) combined with ATRA induced differentiation while 0.25 - 0.5 μM midostaurin (mido(H)) combined with ATRA triggered apoptosis in some AML cell lines without FLT3-mutations. Midostaurin combined with ATRA (mido-ATRA) also exhibited antitumor activity in mouse xenograft models established with AML cells. Mechanistically, mido(H)-ATRA-induced apoptosis was dependent on caspase-3/7. Mido(L)-ATRA inhibited Akt activation which was associated with decreased activity of Lyn/Fgr/Hck, resulted in dephosphorylation of RAF S259, activated RAF/MEK/ERK, along with upregulating the protein levels of C/EBPβ, C/EBPε and PU.1. A MEK specific inhibitor was observed to suppress mido(L)-ATRA-induced increases in the protein levels of C/EBPs and PU.1 and mido(L)-ATRA-induced differentiation. Furthermore, inhibition of Akt activity promoted mido(L)-ATRA-induced downregulation of RAF S259 phosphorylation and mido(L)-ATRA-induced differentiation. Therefore, Lyn/Fgr/Hck-associated Akt inhibition activated RAF/MEK/ERK and controlled mido(L)-ATRA-induced differentiation by upregulation of C/EBPs and PU.1. Mido(L)-ATRA also promoted assembly of the signalosome, which may facilitate RAF activation.Conclusions: Midostaurin combined with ATRA exerts antitumor activity against AML with wild-type FLT3 mutations in vitro and in vivo. These findings may provide novel therapeutic strategies for some AML patients without FLT3 mutations and imply a new target of midostaurin. [ABSTRACT FROM AUTHOR]

Background: Excessively deposited fibrotic scar after spinal cord injury (SCI) inhibits axon regeneration. It has been reported that platelet-derived growth factor receptor beta (PDGFRβ), as a marker of fibrotic scar-forming fibroblasts, can only be activated by platelet-derived growth factor (PDGF) B or PDGFD. However, whether the activation of the PDGFRβ pathway can mediate fibrotic scar formation after SCI remains unclear.Methods: A spinal cord compression injury mouse model was used. In situ injection of exogenous PDGFB or PDGFD in the spinal cord was used to specifically activate the PDGFRβ pathway in the uninjured spinal cord, while intrathecal injection of SU16f was used to specifically block the PDGFRβ pathway in the uninjured or injured spinal cord. Immunofluorescence staining was performed to explore the distributions and cell sources of PDGFB and PDGFD, and to evaluate astrocytic scar, fibrotic scar, inflammatory cells and axon regeneration after SCI. Basso Mouse Scale (BMS) and footprint analysis were performed to evaluate locomotor function recovery after SCI.Results: We found that the expression of PDGFD and PDGFB increased successively after SCI, and PDGFB was mainly secreted by astrocytes, while PDGFD was mainly secreted by macrophages/microglia and fibroblasts. In addition, in situ injection of exogenous PDGFB or PDGFD can lead to fibrosis in the uninjured spinal cord, while this profibrotic effect could be specifically blocked by the PDGFRβ inhibitor SU16f. We then treated the mice after SCI with SU16f and found the reduction of fibrotic scar, the interruption of scar boundary and the inhibition of lesion and inflammation, which promoted axon regeneration and locomotor function recovery after SCI.Conclusions: Our study demonstrates that activation of PDGFRβ pathway can directly induce fibrotic scar formation, and specific blocking of this pathway would contribute to the treatment of SCI. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) is responsible for significant mortality among hospitalized patients that is especially troubling aged people. An effective self-made Chinese medicine formula, Xiaoyu Xiezhuo Drink (XXD), displayed therapeutic effects on AKI. However, the compositions and underlying mechanisms of XXD remain to be elucidated. In this study, we used the ultra-high-performance liquid chromatography method coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) to investigate the chemical components in XXD. Then, the absorbable components of XXD were identified based on the five principles and inputted into the SwissTargetPrediction and STITCH databases to identify the drug targets. AKI-related targets were collected from the GenCLiP 3, GeneCards, and DisGeNET databases. The crossover genes of XXD and AKI were identified for functional enrichment analysis. The protein-protein interaction (PPI) network of crossover genes was constructed, followed by the identification of hub genes. Subsequently, the effects and potential mechanisms of XXD on AKI predicted by the network pharmacology and bioinformatics analyses were experimentally validated in ischemia-reperfusion (I/R) injury-induced AKI aged mouse models. A total of 122 components in XXD were obtained; among them, 58 components were found that could be absorbed in the blood. There were 800 potential drug targets predicted from the 58 absorbable components in AKI which shared 36 crossover genes with AKI-related targets. The results of functional enrichment analysis indicated that crossover genes mostly associated with the response to oxidative stress and the HIF1 signaling pathway. In the PPI network analysis, 12 hub genes were identified, including ALB, IL-6, TNF, TP53, VEGFA, PTGS2, TLR4, NOS3, EGFR, PPARG, HIF1A, and HMOX1. In AKI aged mice, XXD prominently alleviated I/R injury-induced renal dysfunction, abnormal renal pathological changes, and cellular senescence, inflammation, and oxidative damage with a reduction in the expression level of the inflammatory mediator, α-SMA, collagen-1, F4/80, TP53, VEGFA, PTGS2, TLR4, NOS3, EGFR, PPARG, HIF1A, ICAM-1, TGF-β1, Smad3, and p-Smad3 and an increase of nephridial tissue p-H3, Ki67, HMOX1, MMP-9, and Smad7 levels. In summary, our findings suggest that XXD has renoprotective effects against AKI in aged mice via inhibiting the TGF-β1/Smad3 and HIF1 signaling pathways. [ABSTRACT FROM AUTHOR]

FLT3 mutations, which are found in a third of patients with acute myeloid leukemia (AML), are associated with poor prognosis. Responses to currently available FLT3 inhibitors in AML patients are typically transient and followed by disease recurrence. Thus, FLT3 inhibitors with new inhibitory mechanisms are needed to improve therapeutic outcomes. AMG925 is a novel, potent, small-molecule dual inhibitor of FLT3 and CDK4/6. In this study. we determined the antileukemic effects and mechanisms of action of AMG925 in AML cell lines and primary samples, in particular AML stem/progenitor cells. AMG925 inhibited cell growth and promoted apoptosis in AML cells with or without FLT3 mutations. Reverse-phase protein array profiling confirmed its on-target effects on FLT3-CDK4/6-regulated pathways and identified unrevealed signaling network alterations in AML blasts and stem/progenitor cells in response to AMG925. Mass cytometry identified pathways that may confer resistance to AMG925 in phenotypically defined AML stem/progenitor cells and demonstrated that combined blockade of FLT3-CDK4/6 and AKT/mTOR signaling facilitated stem cell death. Our findings provide a rationale for the mechanism-based inhibition of FLT3-CDK4/6 and for combinatorial approaches to improve the efficacy of FLT3 inhibition in both FLT3 wild-type and FLT3-mutated AML. [ABSTRACT FROM AUTHOR]

Background: The EGFR ligand betacellulin (BTC) has been previously shown to protect mice against experimentally induced acute pancreatitis (AP). BTC binds both autonomous ERBB receptors EGFR and ERBB4. In this study, we evaluated the mechanism underlying the protection from AP-associated inflammation in detail.Methods: AP was induced with cerulein or L-arginine and investigated in a pancreas-specific ERBB4 knockout and in an EGFR knockdown mouse model (EgfrWa5/+). Pancreatitis was evaluated by scoring inflammation, necrosis, and edema, while microarrays were performed to analyze alterations in the transcriptome between mice with AP and animals which were protected against AP. The intracellular domain (ICD) of ERBB4 was analyzed in different cell compartments.Results: While the pancreas of BTC transgenic mice in the background of EgfrWa5/+ is still protected against AP, the BTC-mediated protection is no longer present in the absence of ERBB4. We further demonstrate that BTC activates the ICD of ERBB4, and increases the expression of the extracellular matrix (ECM) proteins periostin and matrix gla protein as well as the ECM modulators matrix metalloproteinases 2 and 3, but only in the presence of ERBB4. Notably, the increased expression of these proteins is not accompanied by an increased ECM amount.Conclusions: These findings suggest that BTC derivates, as a drug, or the ERBB4 receptor, as a druggable target protein, could play an important role in modulating the course of AP and even prevent AP in humans. [ABSTRACT FROM AUTHOR]

Berberine, a natural isoquinoline alkaloid isolated from the berberis species, has a wide array of biological properties such as anti-inflammatory, antibacterial, antifungal, and antihelminthic effects. We evaluated the antiviral effect of berberine against influenza A/FM1/1/47 (H1N1) in vivo and in vitro. The results showed that berberine strongly suppressed viral replication in A549 cells and in mouse lungs. Meanwhile, berberine relieved pulmonary inflammation and reduced necrosis, inflammatory cell infiltration, and pulmonary edema induced by viral infection in mice when compared with vehicle-treated mice. Berberine suppressed the viral infection-induced up-regulation of TLR7 signaling pathway, such as TLR7, MyD88, and NF-κB (p65), at both the mRNA and protein levels. Furthermore, berberine significantly inhibited the viral infection-induced increase in Th1/Th2 and Th17/Treg ratios as well as the production of inflammatory cytokines. Our data provide new insight into the potential of berberine as a therapeutic agent for viral infection via its antiviral activity. [ABSTRACT FROM AUTHOR]

Background: Interactions between the protein synthesis inhibitor homoharringtonine (HHT) and the proteasome inhibitor bortezomib were investigated in DLBCL and mantle cell lymphoma cells (MCL).Methods: Various DLBCL and MCL cells were exposed to HHT and bortezomib alone or together after which apoptosis and signaling pathway perturbations were monitored by flow cytometry and Western blot analysis. Xenograft mouse models were used to assess tumor growth and animal survival.Results: HHT and bortezomib co-administration synergistically induced apoptosis in GC-, ABC- and double-hit DLBCL cells. Similar interactions were observed in MCL cells and in primary lymphoma cells. HHT/bortezomib co-administration diminished binding of MCL-1 to both BAK and NOXA. Knock-down of NOXA significantly diminished lethality whereas MCL-1 knock-down or ectopic NOXA expression increased cell death. Notably, HHT/bortezomib lethality was dramatically reduced in BAK knockout or knockdown cells. Finally, HHT/bortezomib co-administration significantly improved survival compared to single agents in GC- and ABC- xenograft models while exhibiting little toxicity.Conclusions: These findings indicate that HHT and bortezomib cooperate to kill DLBCL and MCL cells through a process involving MCL-1 down-regulation, NOXA up-regulation, and BAK activation. They also suggest that a strategy combining HHT with bortezomib warrants attention in DLBCL and MCL. [ABSTRACT FROM AUTHOR]

Background: Aberrant hyperactivation of epithelial proliferation, AKT signaling, and association with unopposed estrogen (E2) exposure is the most common endometrial cancer dysfunction. In the normal uterus, progesterone (P4) inhibits proliferation by coordinating stromal-epithelial cross-talk, which we previously showed is mediated by the function of Mitogen-inducible gene 6 (Mig-6). Despite their attractive characteristics, non-surgical conservative therapies based on progesterone alone have not been universally successful. One barrier to this success has been the lack of understanding of the P4 effect on endometrial cells.Method: To further understand the role of Mig-6 and P4 in controlling uterine proliferation, we developed a Sprr2f-cre driven mouse model where Mig-6 is specifically ablated only in the epithelial cells of the uterus (Sprr2f cre+ Mig-6 f/f ). We examined P4 effect and regulation of AKT signaling in the endometrium of mutant mice.Results: Sprr2f cre+ Mig-6 f/f mice developed endometrial hyperplasia. P4 treatment abated the development of endometrial hyperplasia and restored morphological and histological characteristics of the uterus. P4 treatment reduced cell proliferation which was accompanied by decreased AKT signaling and the restoration of stromal PGR and ESR1 expression. Furthermore, our in vitro studies revealed an inhibitory effect of MIG-6 on AKT phosphorylation as well as MIG-6 and AKT protein interactions.Conclusions: These data suggest that endometrial epithelial cell proliferation is regulated by P4 mediated Mig-6 inhibition of AKT phosphorylation, uncovering new mechanisms of P4 action. This information may help guide more effective non-surgical interventions in the future. [ABSTRACT FROM AUTHOR]

Background: Multicellular Tumor Spheroids are frequently used to mimic the regionalization of proliferation and the hypoxic environment within avascular tumors. Here we exploit these features to study the activation of DNA damage repair pathways and their correlation to developing hypoxia.Methods: Activation of DNA damage repair markers, proliferation, cell death, glycogen accumulation and developing hypoxia were investigated using immunofluorescence, immuno-histochemistry, EdU incorporation, Western blots, COMET assays, and pharmacological agents in A673 Ewing sarcoma spheroids and monolayer cultures.Results: DNA damage marker γ-H2AX is observed in the hypoxic, peri-necrotic region of growing spheroids. While most proliferating cells are seen on the spheroid surface, there are also a few Ki-67 positive cells in the hypoxic zone. The hypoxia-induced phosphorylation of H2AX to form γ-H2AX in spheroids is attenuated by the ATM inhibitor KU55933, but not the ATR inhibitor VE-821.Conclusion: Tumor spheroids mimic tumor microenvironments such as the anoxic, hypoxic and oxic niches within solid tumors, as well as populations of cells that are viable, proliferating, and undergoing DNA damage repair processes under these different micro-environmental conditions. ATM, but not ATR, is the primary kinase responsible for γ-H2AX formation in the hypoxic core of A673 spheroids. Spheroids could offer unique advantages in testing therapeutics designed to target malignant cells that evade conventional treatment strategies by adapting to the hypoxic tumor microenvironment. [ABSTRACT FROM AUTHOR]

Background: Neural stem cells (NSCs) display tissue trophic and immune modulatory therapeutic activities after transplantation in central nervous system disorders. The intercellular interplay between stem cells and target immune cells is increased in NSCs exposed to inflammatory cues. Here, we hypothesize that inflammatory cytokine signalling leads to metabolic reprogramming of NSCs regulating some of their immune modulatory effects.Methods: NSC lines were prepared from the subventricular zone (SVZ) of 7-12-week-old mice. Whole secretome-based screening and analysis of intracellular small metabolites was performed in NSCs exposed to cocktails of either Th1-like (IFN-γ, 500 U/ml; TNF-α, 200 U/ml; IL-1β, 100 U/ml) or Th2-like (IL-4, IL-5 and IL-13; 10 ng/ml) inflammatory cytokines for 16 h in vitro. Isotopologues distribution of arginine and downstream metabolites was assessed by liquid chromatography/mass spectrometry in NSCs incubated with U-(13)C6 L-arginine in the presence or absence of Th1 or Th2 cocktails (Th1 NSCs or Th2 NSCs). The expression of arginase I and II was investigated in vitro in Th1 NSCs and Th2 NSCs and in vivo in the SVZ of mice with experimental autoimmune encephalomyelitis, as prototypical model of Th1 cell-driven brain inflammatory disease. The effects of the inflammatory cytokine signalling were studied in NSC-lymph node cells (LNC) co-cultures by flow cytometry-based analysis of cell proliferation following pan-arginase inhibition with N(ω)-hydroxy-nor-arginine (nor-NOHA).Results: Cytokine-primed NSCs showed significantly higher anti-proliferative effect in co-cultures vs. control NSCs. Metabolomic analysis of intracellular metabolites revealed alteration of arginine metabolism and increased extracellular arginase I activity in cytokine-primed NSCs. Arginase inhibition by nor-NOHA partly rescued the anti-proliferative effects of cytokine-primed NSCs.Conclusions: Our work underlines the use of metabolic profiling as hypothesis-generating tools that helps unravelling how stem cell-mediated mechanisms of tissue restoration become affected by local inflammatory responses. Among different therapeutic candidates, we identify arginase signalling as novel metabolic determinant of the NSC-to-immune system communication. [ABSTRACT FROM AUTHOR]

Diet-induced muscle insulin resistance is associated with expansion of extracellular matrix (ECM) components, such as collagens, and the expression of collagen-binding integrin, α2β1. Integrins transduce signals from ECM via their cytoplasmic domains, which bind to intracellular integrin-binding proteins. The integrin-linked kinase (ILK)-PINCH-parvin (IPP) complex interacts with the cytoplasmic domain of β-integrin subunits and is critical for integrin signaling. In this study we defined the role of ILK, a key component of the IPP complex, in diet-induced muscle insulin resistance. Wild-type (ILK(lox/lox)) and muscle-specific ILK-deficient (ILK(lox/lox)HSAcre) mice were fed chow or a high-fat (HF) diet for 16 weeks. Body weight was not different between ILK(lox/lox) and ILK(lox/lox)HSAcre mice. However, HF-fed ILK(lox/lox)HSAcre mice had improved muscle insulin sensitivity relative to HF-fed ILK(lox/lox) mice, as shown by increased rates of glucose infusion, glucose disappearance, and muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. Improved muscle insulin action in the HF-fed ILK(lox/lox)HSAcre mice was associated with increased insulin-stimulated phosphorylation of Akt and increased muscle capillarization. These results suggest that ILK expression in muscle is a critical component of diet-induced insulin resistance, which possibly acts by impairing insulin signaling and insulin perfusion through capillaries. [ABSTRACT FROM AUTHOR]

Background: Klebsiella pneumoniae causes serious infections and healthcare burdens in humans. We have previously reported that the deficiency of autophagy-related gene (Atg) 7 in macrophages (murine alveolar macrophage cell line [MH-S]) induced irregular host immunity against K. pneumoniae and worsened pathologic effects in the lung. In the current study, we investigated the molecular mechanism by which Atg7 influenced K. pneumoniae-induced inflammatory responses. Methods: Expression levels of Atg7, ubiquitin (Ub), and tumor necrosis factor (TNF) α and phosphorylation of IκBα (p-IκBα) were determined with immunoblotting. Ubiquitylation of p-IκBα was determined with immunoprecipitation. Results: We noted an interaction between Atg7 and p-IκBα, which was decreased in MH-S after K. pneumoniae infection, whereas the interaction between Ub and p-IκBα was increased. Knock-down of Atg7 with small interfering RNA increased p-IκBα ubiquitylation, promoted nuclear factor κB translocation into the nucleus, and increased the production of TNF-α. Moreover, knock-down of Ub with lentivirus-short hairpin RNA Ub particles decreased binding of p-IκBα to Ub and inhibited TNF-α expression in the primary alveolar macrophages and lung tissue of atg7-knockout mice on K. pneumoniae infection. Conclusions: Loss of Atg7 switched binding of p-IκBα from Atg7 to Ub, resulting in increased ubiquitylation of p-IκBα and intensified inflammatory responses against K. pneumoniae. Our findings not only reveal a regulatory role of Atg7 in ubiquitylation of p-IκBα but also indicate potential therapeutic targets for K. pneumoniae control. [ABSTRACT FROM AUTHOR]

Background: Secondary brain damage caused by the innate immune response and subsequent proinflammatory factor production is a major factor contributing to the high mortality of intracerebral haemorrhage (ICH). Nucleotide-binding oligomerization domain 1 (NOD1)/receptor-interacting protein 2 (RIP2) signalling has been reported to participate in the innate immune response and inflammatory response. Therefore, we investigated the role of NOD1/RIP2 signalling in mice with collagenase-induced ICH and in cultured primary microglia challenged with hemin.Methods: Adult male C57BL/6 mice were subjected to collagenase for induction of ICH model in vivo. Cultured primary microglia and BV2 microglial cells (microglial cell line) challenged with hemin aimed to simulate the ICH model in vitro. We first defined the expression of NOD1 and RIP2 in vivo and in vitro using an ICH model by western blotting. The effect of NOD1/RIP2 signalling on ICH-induced brain injury volume, neurological deficits, brain oedema, and microglial activation were assessed following intraventricular injection of either ML130 (a NOD1 inhibitor) or GSK583 (a RIP2 inhibitor). In addition, levels of JNK/P38 MAPK, IκBα, and inflammatory factors, including tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, and inducible nitric oxide synthase (iNOS) expression, were analysed in ICH-challenged brain and hemin-exposed cultured primary microglia by western blotting. Finally, we investigated whether the inflammatory factors could undergo crosstalk with NOD1 and RIP2.Results: The levels of NOD1 and its adaptor RIP2 were significantly elevated in the brains of mice in response to ICH and in cultured primary microglia, BV2 cells challenged with hemin. Administration of either a NOD1 or RIP2 inhibitor in mice with ICH prevented microglial activation and neuroinflammation, followed by alleviation of ICH-induced brain damage. Interestingly, the inflammatory factors interleukin (IL)-1β and tumour necrosis factor-α (TNF-α), which were enhanced by NOD1/RIP2 signalling, were found to contribute to the NOD1 and RIP2 upregulation in our study.Conclusion: NOD1/RIP2 signalling played an important role in the regulation of the inflammatory response during ICH. In addition, a vicious feedback cycle was observed between NOD1/RIP2 and IL-1β/TNF-α, which could to some extent result in sustained brain damage during ICH. Hence, our study highlights NOD1/RIP2 signalling as a potential therapeutic target to protect the brain against secondary brain damage during ICH. [ABSTRACT FROM AUTHOR]