Dibenzazepine Research Articles

Dibenzazepine, a γ-Secretase Enzyme Inhibitor, Protects Against Doxorubicin-Induced Cardiotoxicity by Suppressing NF-κB, iNOS, and Hes1/Hey1 Expression.

Doxorubicin (DOX) is an effective chemotherapeutic drug; however, its cardiotoxicity and resistance compromise its therapeutic index. The Notch pathway was reported to contribute to DOX cancer resistance. The role of Notch pathway in DOX cardiotoxicity has not been identified yet. Notch receptors are characterized by their extracellular (NECD) and intracellular(NICD) domains (NICD). The γ-secretase enzyme helps in the release of NICD. Dibenzazepine (DBZ) is a γ-secretase inhibitor. The present study investigated the effect of Notch pathway inhibition on DOX cardiotoxicity. Twenty-four male Wistar rats were divided into four groups: control group, DOX group, acute cardiotoxicity was induced by a single dose of DOX (20 mg/kg) i.p., DOX (20 mg/kg) plus DBZ group, and DBZ group. The third and fourth groups received i.p. injection of DBZ daily for 14 days at 2 mg/kg dose. DOX cardiotoxicity increased the level of serum creatine kinase-MB and cardiac troponin I, and it was confirmed by the histopathological examination. Moreover, the antioxidants glutathione peroxidase and superoxide dismutase levels were markedly decreased, and the inflammatory markers, inducible nitric oxide synthase, nuclear factor-ķB, and tumor necrosis factor-α were markedly increased. Furthermore, DOX increased BAX protein and downregulated BCL-2. In addition, DOX upregulated Notch pathway-related parameters: Hes1 and Hey1 mRNA levels, and increased Hes1 protein levels. DBZ ameliorated DOX-induced cardiotoxicity, evidenced by reducing the cardiac injury biomarkers, improving cardiac histopathological changes, correcting antioxidant levels, and reducing inflammatory and apoptotic proteins. Our study indicates the protective effect of Notch inhibitor against DOX-induced cardiotoxicity.

Reactive Oxygen Species-Responsive Delivery of a Notch Inhibitor to Alleviate Nonalcoholic Steatohepatitis by Inhibiting Hepatic de Novo Lipogenesis and Inflammation.

With the increased prevalence of nonalcoholic steatohepatitis (NASH) in the world, effective pharmacotherapy in clinical practice is still lacking. Previous studies have shown that dibenzazepine (DBZ), a Notch inhibitor, could alleviate NASH development in a mouse model. However, low bioavailability, poor water solubility, and extrahepatic side effects restrict its clinical application. To overcome these barriers, we developed a reactive oxygen species (ROS)-sensitive nanoparticle based on the conjugation of bilirubin to poly(ethylene glycol) (PEG) chains, taking into account the overaccumulation of hepatic ROS in the pathologic state of nonalcoholic steatohepatitis (NASH). The PEGylated bilirubin can self-assemble into nanoparticles in an aqueous solution and encapsulate insoluble DBZ into its hydrophobic cavity. DBZ nanoparticles (DBZ Nps) had good stability, rapidly released DBZ in response to H2O2, and effectively scavenged intracellular ROS of hepatocytes. After systemic administration, DBZ Nps could accumulate in the liver of the NASH mice, extend persistence in circulation, and improve the bioavailability of DBZ. Furthermore, DBZ Nps significantly improved glucose intolerance, relieved hepatic lipid accumulation and inflammation, and ameliorated NASH-induced liver fibrosis. Additionally, DBZ Nps had no significant extrahepatic side effects. Taken together, our results highlight the potential of the ROS-sensitive DBZ nanoparticle as a promising therapeutic strategy for NASH.

Intestinal Stem Cells Remodel in Response to Acute Notch Inhibition and Paneth Cell Loss

INTRODUCTION: Intestinal epithelial cells are rapidly renewed throughout life by intestinal stem cells (ISCs). ISC function is regulated by niche factors that orchestrate cellular proliferation and differentiation. Of these, Notch signaling is essential for ISC self-renewal and regulates the cell fate choice between absorptive cells (enterocyte) vs secretory (Paneth, goblet, enteroendocrine, and tuft) cells. Yet, the Notch niche is not fully defined. In the mature small intestine, Paneth cells neighbor each ISC and act as Notch niche cells, providing the Notch signal by expressing the Notch ligands DLL1 and DLL4 on their surface. However, studies in mice have shown that ISCs can function in the immature intestine before Paneth cells are formed, and when Paneth cells are depleted in the adult intestine. We previously observed that acute Notch inhibition via the inhibitor dibenzazepine (DBZ) resulted in Paneth cell apoptosis with intact ISCs displaying transient dysfunction. Here, we aimed to define the Notch niche after Paneth cell apoptosis in adult mice and in the developing intestine. We hypothesized that during acute Notch inhibition, ISCs remodel to compensate for Paneth cell loss by activating Notch ligand expression, and that this remodeling mimics the state of immature ISCs prior to Paneth cell emergence. METHODS: We isolated Lgr5+ ISCs by FACS from Lgr5-GFP-CreERT2 mice 24 hours after DBZ or vehicle treatment and performed bulk RNAseq analysis. To define the Notch niche during development, we analyzed postnatal intestines from Notch ligand reporter mice (Dll1-mCherry and Dll4-mCherry) crossed with the ISC reporter mice (Lgr5-GFP-CreERT2). RESULTS: ISCs isolated from Paneth cell-depleted intestines upregulated expression of Notch ligands Dll4 and Dll4 and various secretory cell markers, with expected downregulation of Notch target genes, but no alteration of other stem cell genes. This suggests that after acute Notch inhibition, Lgr5+ ISCs maintain partial stem cell identity while acquiring a signature resembling that of secretory progenitors. In the immature intestine, we showed by immunohistology that sporadic cells expressing secretory cell markers (LYZ, MUC2, and CHGA) localized to the intervillus region and co-stained with Dll1- or Dll4-mCherry+. This suggests that multiple differentiated cell types could provide the Notch signal to immature ISCs. We also observed occasional Lgr5-GFP+ ISCs that were co-positive for Dll1-mCherry, suggesting that immature ISCs can express Notch ligands at homeostasis. Further experimentation will determine whether these Dll1+ ISCs are similar to the remodeled adult ISCs after Paneth cell depletion. CONCLUSIONS: Acute Notch inhibition directly remodels ISCs to express the Notch ligands, Dll1 and Dll4 in the adult intestine. In the immature intestine, multiple secretory cell types appear to comprise the immature Notch niche. This project was funded by NIH R01 DK118023 awarded to Dr. Linda Samuelson. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids

Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide an unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using colon organoids generated from healthy tissue of colorectal cancer patients. Culture of these organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1, FABP2) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the γ-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 showing only weak signals of cell differentiation, and were unresponsive to DBZ. We also investigated the effects of 1α,25-dihydroxyvitamin D3 (calcitriol) on differentiation. Calcitriol attenuated the effects of BMP4 and DBZ on colon normal organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features.

Process induced degradation of carbamazepine during hot melt extrusion

The aim of this studywas to evaluate hot melt extrudates of carbamazepine(CBZ) for presence of process induced degradation product iminostilbene (IMB) and quantifying using highperformance liquid chromatography (HPLC). CBZ was used as model drug. Hot-melt extrusion (HME) was performed to preparesolid dispersions. Different grades of compatible polymers were used. Drug loading,processing temperature and screw rotations per minute (RPM) showed significanteffect on degradation of CBZ The extrudates were characterized by differentialscanning calorimetry (DSC), X-ray diffraction (XRD), Fourier transform infrared(FTIR) studies. DSC and XRD data confirmed formation of amorphous soliddispersions of CBZ. HPLC was performed to quantify percentage amount of CBZ andIMB. Process induced degradation was significantly varied depending on polymergrade and (w/w) percentage combinations. In addition, processing temperatureand RPM had direct effect on percentage of IMB formation. 

Research on solubility behavior of iminostilbene in twelve mono-solvents: Measurement, modeling, molecular simulation and thermodynamic properties

Iminostilbene (IMB) is an important pharmaceutical intermediate that can be used to prepare the antiepileptic drug omazepine. The measurement of solid–liquid equilibrium solubility parameters in various solvents can be employed for crystallization and separation processes of IMB. In this research, mole-fraction solubility of IMB in twelve individual organic solvents namely: “methyl acetate (MeAC), ethyl acetate (EtAC), n-propyl acetate (NPAC), isopropyl acetate (IPAC), n-butyl acetate (NBAC), isobutyl acetate (IBAC), N-methylpyrrolidone (NMP), acetone (DMK), N,N-dimethylformamide (DMF), acetonitrile (MeCN), N,N- dimethylacetamide (DMAC) and 1,4-dioxane (Diox)” was critically evaluated ranged from 278.15/288.15 K to 323.15 K at 0.1 MPa using laser monitoring method. Experimental solubility data had a general uptrend with the incremental temperature. Simultaneously, IMB solubility in selected mono-solvents showed an obvious order from high to low of: NMP > DMAC > DMF > Diox > DMK > NBAC > NPAC > EtAC > IBAC > IPAC > MeAC > MeCN. In order to explore dissolution behavior, Hirshfeld surface as well as molecular electrostatic potential surface (MEPs) were employed to analyze intermolecular interaction, and result indicated hydrogen bonding was easily formed on >NH group and its opposite position CHCH group. Meanwhile, Hansen solubility parameters (HSPs) were used to investigate solubility order, the result of which suggested that dispersion interaction could be the main factor influenced on solubility order in homologous solvents. Moreover, four activity coefficient models (Margules, UNIQUAC, NRTL, NRTL-SAC model) as well as a combined equation (Jouyban model) were applied for correlation of solubility data with twelve solvents. UNIQUAC model provided the highest regression accuracy among five models in terms of average relative deviation (ARD) lower than 1.178 % and root mean square deviation (RMSD) not higher than 1.176 ‰. Furthermore, based on UNIQUAC equation, thermodynamic properties including dissolution and mixing process were strictly evaluated for further research on solubility behavior. The results demonstrated that both of mixing and dissolution behaviors were spontaneous and entropically driven.

Synthesis of indene-fused spiro-dibenz(ox)azepines via Rh(III)-catalyzed cascade regioselective C-H activation/annulation.

We report an unprecedented atom-economic one-pot Cp*Rh(III)-catalyzed regioselective [3+2]-spiroannulation reaction between dibenz(ox)azepines and ynones, allowing the synthesis of biologically relevant novel spirocyclic dibenz(ox)azepines under operationally simple and mild reaction conditions. The reaction proceeds without any silver additive or external oxidant implementing a redox-neutral pathway. A broad substrate scope with diverse functional group tolerance permitted the regioselective synthesis of a wide spectrum of indene-containing spirocyclic dibenz(ox)azepines in good to excellent yields. Also, we showcased detailed mechanistic studies to justify the formation of spirocycles. In addition, the synthetic utility of this process was also demonstrated by the modular synthesis of various steroid conjugates.

Effects of Notch signaling pathway inhibition by dibenzazepine in acute experimental toxoplasmosis

Notch signaling pathway plays a crucial role in cellular fate across species, being important for the differentiation and development of several cell types. The aim of this study was to evaluate the effect of Notch inhibition pathway by dibenzazepine (DBZ) in histological and inflammatory alterations and, tissue parasitism in acute Toxoplasma gondii infection. For this, C57BL/6 mice were treated with DBZ before infection with T. gondii, and the small intestine, lungs and liver were analyzed. The genes related to Notch signaling pathway were assayed through qPCR in the organs, and cytokine measurement was performed in serum samples. In the small intestine, T. gondii infection impaired the Hes1 and Math1 mRNA expressions, increased the inflammation and decreased goblet and Paneth cell numbers. The DBZ-treatment was able to partially preserve these cells, however, the parasitism and inflammation were not altered. In parallel, the high IL-2, IL-6, TNF and, IFN-γ levels induced by infection were not changed with the DBZ treatment, with the IFN-γ levels even higher. In contrast, in the liver and lungs, the DBZ-treatment diminished parasitism and inflammation. Our results highlight that Notch pathway inhibition in T.gondii infection results in different parasitological and inflammatory outcomes depending on the organ analyzed.

2-deoxy-D-glucose mitigates Citrobacter rodentium and dibenzazepine-induced gastrointestinal damage and colitis: novel implications of 2-DG polypharmacopea

Purpose Citrobacter rodentium (CR) infection coupled with blocking Notch/Wnt signaling via γ-secretase inhibitor dibenzazepine (DBZ) disrupts the gastro-intestinal (GI) barrier and induces colitis, akin to ionizing radiation (IR)-induced GI-injury. We investigated the effects of 2-deoxy-D-glucose (2-DG) to ameliorate the CR-DBZ-induced GI damage. Materials and methods NIH:Swiss outbred mice were inoculated with 109CFUs of CR orally. DBZ was administered intraperitoneally (10 μM/kg b.wt; for 10 days 2 days post-CR infection). Mice were fed with 0.4% 2-DG (w/v) daily in drinking water. For microbiota depletion, antibiotics (Abx), 1 g/l metronidazole, and 0.2 g/l ciprofloxacin were administered for 10 days in drinking water. Oxidative stress, survival assay, colonic crypt hyperplasia, Notch/Wnt downstream signaling, immunomodulation, and bacterial dysbiosis were measured. Results We show that real-time visualization of reactive oxygen species (ROS) is similar during CR-induced colonic infection and IR-induced GI-damage. The histology revealed that dietary 2-DG mitigates CR + DBZ-induced colitis and improves survival compared with CR + DBZ alone. These changes were phenocopied in Abx-treated mice. Both 2-DG and Abx reduced dysbiosis, increased proliferation, inhibited pro-inflammatory response, and restored Hes-1 and β-catenin protein levels, in the crypts. Conclusion The energy disruptor 2-DG mitigates bacterial infection and its responsive hyperplasia/colitis, indicating its utility as a mitigator of infection/IR-induced GI-damage.

Carbamazepine polymorphism: A re-visitation using Raman imaging

Raman imaging methods have appeared in the last years as a powerful approach to monitoring the quality of pharmaceutical compounds. Because polymorphism occurs in many crystalline pharmaceutical compounds, it is essential to monitor polymorphic transformations induced by different external stimulus, such as temperature changes, to which those compounds may be submitted. Raman imaging with k-means cluster analysis (CA) is used here as an essential technique to investigate structural and chemical transformations occurring in carbamazepine p-monoclinic (CBZ III) into carbamazepine triclinic (CBZ I) when submitted to temperatures near the melting point of CBZ III (178 °C) and CBZ I (193 °C).CBZ III commercial powder and laboratorial prepared CBZ I were analyzed by differential scanning calorimetry, powder X-ray diffraction and Raman spectroscopy with variable temperature. After thermal treatment, the resultant CBZ powder was evaluated by Raman imaging, in which all imaging data was analyzed using CA. Raman imaging allowed the identification of different polymorphs of CBZ (CBZ III and CBZ I) and iminostilbene (IMS), a degradation product of CBZ, in the treated samples, depending on the heating treatment method.

The radical scavenging activity of monosubstituted iminostilbenes: Theoretical insights

Iminostilbene (IS) (5H‐dibenz[b,f]azepine) is a common polymerization inhibitor used in industrial processes to control radical polymerization reactions. Here a library of 90 monosubstituted IS derivatives was screened in silico to establish an activity scale for radical scavenging. Substituents on the aromatic rings had only weak effect on bond dissociation energies of the N-H moiety; however, ionization energies were much more affected. Thus the IS derivatives 3-NMe2-IS, 1-OH-IS, and 10-OH-IS are predicted to be potent radical scavengers in organic solvents suitable for capturing radicals in polymerization reactions and improving polymer stability.

Cell differentiation is disrupted by MYO5B loss through Wnt/Notch imbalance.

Functional loss of myosin Vb (MYO5B) induces a variety of deficits in intestinal epithelial cell function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). The impact of MYO5B loss on differentiated cell lineage choice has not been investigated. We quantified the populations of differentiated epithelial cells in tamoxifen-induced, epithelial cell–specific MYO5B-knockout (VilCreERT2 Myo5bfl/fl) mice utilizing digital image analysis. Consistent with our RNA-sequencing data, MYO5B loss induced a reduction in tuft cells in vivo and in organoid cultures. Paneth cells were significantly increased by MYO5B deficiency along with expansion of the progenitor cell zone. We further investigated the effect of lysophosphatidic acid (LPA) signaling on epithelial cell differentiation. Intraperitoneal LPA significantly increased tuft cell populations in both control and MYO5B-knockout mice. Transcripts for Wnt ligands were significantly downregulated by MYO5B loss in intestinal epithelial cells, whereas Notch signaling molecules were unchanged. Additionally, treatment with the Notch inhibitor dibenzazepine (DBZ) restored the populations of secretory cells, suggesting that the Notch pathway is maintained in MYO5B-deficient intestine. MYO5B loss likely impairs progenitor cell differentiation in the small intestine in vivo and in vitro, partially mediated by Wnt/Notch imbalance. Notch inhibition and/or LPA treatment may represent an effective therapeutic approach for treatment of MVID.

Cigarette Smoke Activates NOTCH3 to Promote Goblet Cell Differentiation in Human Airway Epithelial Cells.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the United States and is primarily caused by cigarette smoking. Increased numbers of mucus-producing secretory ("goblet") cells, defined as goblet cell metaplasia or hyperplasia (GCMH), contributes significantly to COPD pathophysiology. The objective of this study was to determine whether NOTCH signaling regulates goblet cell differentiation in response to cigarette smoke. Primary human bronchial epithelial cells (HBECs) from nonsmokers and smokers with COPD were differentiated in vitro on air-liquid interface and exposed to cigarette smoke extract (CSE) for 7 days. NOTCH signaling activity was modulated using 1) the NOTCH/γ-secretase inhibitor dibenzazepine (DBZ), 2) lentiviral overexpression of the NICD3 (NOTCH3-intracellular domain), or 3) NOTCH3-specific siRNA. Cell differentiation and response to CSE were evaluated by quantitative PCR, Western blotting, immunostaining, and RNA sequencing. We found that CSE exposure of nonsmoker airway epithelium induced goblet cell differentiation characteristic of GCMH. Treatment with DBZ suppressed CSE-dependent induction of goblet cell differentiation. Furthermore, CSE induced NOTCH3 activation, as revealed by increased NOTCH3 nuclear localization and elevated NICD3 protein levels. Overexpression of NICD3 increased the expression of goblet cell-associated genes SPDEF and MUC5AC, whereas NOTCH3 knockdown suppressed CSE-mediated induction of SPDEF and MUC5AC. Finally, CSE exposure of COPD airway epithelium induced goblet cell differentiation in a NOTCH3-dependent manner. These results identify NOTCH3 activation as one of the important mechanisms by which cigarette smoke induces goblet cell differentiation, thus providing a novel potential strategy to control GCMH-related pathologies in smokers and patients with COPD.

Modelling the mechanism and kinetics of the radical scavenging activity of iminostilbene

Iminostilbene (IS) (5H‐dibenz[b,f]azepine) is frequently used as a polymerization inhibitor to stop the reactions of radical polymerization. The compound itself is considered an antioxidant; however, little is known about the mechanism and kinetics of its activity. In this study, the antiradical activity of IS against typical radicals i.e. HO•, HOO• and TBO• (tert-butoxy radical) has been evaluated by computational methods. It was found that IS exhibits excellent HO• and TBO• scavenging activity in gas phase and in lipid-mimetic environment (benzene), whereas its activity against HOO• is moderate with koverall = ~ 104 M−1 s−1. The overall rate constants of the HO• antiradical activity are koverall = 7.06 × 1011 and 7.20 × 109 M−1 s−1 in gas phase and benzene, respectively, whereas for the TBO• antiradical activity in benzene it is 2.10 × 108 M−1 s−1. Comparison with the experimentally determined rate constant in benzene (k(BTO•) = 7.8 × 108 M−1s−1) yields the kcalculated/kexperimetal ratio of 0.3; thus, the computed kinetic data are reasonably accurate. It was shown that the TBO• and HOO• radicals scavenging of IS mostly follows the FHT mechanism at the NH bond; however, in case of the HO• radical both FHT (at NH bond) and RAF (at C10 position) mechanisms contribute to the observable activity.

Generation of intestinal chemosensory cells from nonhuman primate organoids

Several gastrointestinal epithelial cells are involved in taste signal transduction. Although rodent tissues are extensively used as a human gut model, recent studies show that the chemical sensing system in rodents differs from that in humans. Nonhuman primates in biomedical research are valuable animal models to advance our understanding of biological responses in humans. The 3D organoid culture produces functional gastrointestinal epithelial cells in vitro and can be generated from animal and human tissues. Here, we report the generation of intestinal chemosensory cells from nonhuman primates, macaques, using an organoid culture system. We were able to maintain macaque intestinal organoids in the proliferation medium for more than six months. Upon switching to differentiation medium, we observed a drastic change in organoid morphology and chemosensory cell marker protein expression. This switch from proliferation to differentiation was confirmed by transcriptome analysis of the duodenum, jejunum, and ileum organoids. We further observed that the supplementation of culture media with interleukin (IL)-4 or the Notch inhibitor dibenzazepine (DBZ) accelerated terminal cell differentiation into chemosensory cells.Overall, we generated monkey intestinal organoids for the first time. These organoids are suitable for studying the function of primate chemosensory cells.

Preparation and Photoluminescent Properties of Three 5-Amino Benzothiadiazoles (5-amBTDs).

Three D-A compounds were designed and synthesized based on a benzothiadiazole acceptor. Azepane (AP), iminodubenzyl (IDB) and iminostilbene (ISB) were used, respectively, as donors and installed on the 5-position of BTD to afford 1, 2 and 3, respectively. Their photophysical properties in different states (solution, film, crystal, and powder) are systematically investigated. Among them, AIE-active compounds 2 and 3 were found to have good sensitivity toward viscosity and display quite good linear relationship with an increase in viscosity. Compound 2 displayed dual emission in solutions which largely depended on the polarity of the solvent. Meanwhile, compound 2 exhibits a mechanochromic character with disappearance and reappearance of a dual-emissive peak induced by mechanical grinding and solvent fuming. Furthermore, these three compounds can be used in the fabrication of blue OLED devices.

Iminostilbene, a novel small-molecule modulator of PKM2, suppresses macrophage inflammation in myocardial ischemia–reperfusion injury

IntroductionInflammation is a key factor in myocardial ischemia/reperfusion (MI/R) injury. Targeting leucocyte-mediated inflammation is an important strategy for MI/R therapy. Iminostilbene (ISB), a simple dibenzoazepine small molecule compound, has a strong anti-neurodegenerative effect. However, no study has shown the cardioprotective effect of ISB. ObjectivesThis study aimed to investigate the role of ISB against MI/R injury and identify its molecular target. MethodsTo verify the cardiac protection of ISB in vivo and in vitro, we performed rat MI/R surgery and subjected inflammatory modeling of macrophages. In terms of molecular mechanisms, we designed and synthesized a small molecular probe of ISB and employed it on the click chemistry-activity-based protein profiling technique to fish for ISB targets in macrophages. To identify the target, we applied the competitive inhibition assay, surface-plasmon resonance (SPR), cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay. ResultsIn vivo, ISB showed robust anti-myocardial injury activity by improving cardiac function, reducing myocardial infarction, and inhibiting macrophage-mediated inflammation. In vitro, ISB strongly inhibited the transcription and the expression levels of inflammatory cytokines in macrophages. The pyruvate kinase isozyme type M2 (PKM2) was identified as the potential target of ISB through proteomic analysis and the competitive assay was performed for specific binding verification. Further thermodynamic and kinetic experiments showed that ISB was bound to PKM2 in a dose-dependent manner. Moreover, in terms of the biological function of ISB on PKM2, ISB reduced the expression of PKM2, thereby reducing the expression of HIF1α and the phosphorylation of STAT3. ConclusionThis study for the first time demonstrated that ISB targeted PKM2 to reduce macrophage inflammation thereby significantly alleviating MI/R injury.

Dibenzazepine promotes cochlear supporting cell proliferation and hair cell regeneration in neonatal mice.

ObjectivesTo investigate the role of dibenzazepine (DBZ) in promoting supporting cell (SC) proliferation and hair cell (HC) regeneration in the inner ear.Materials and MethodsPostnatal day 1 wild‐type or neomycin‐damaged mouse cochleae were cultured with DBZ. Immunohistochemistry and scanning electron microscopy were used to examine the morphology of cochlear cells, and high‐throughput RNA‐sequencing was used to measure gene expression levels.ResultsWe found that DBZ promoted SC proliferation and HC regeneration in a dose‐dependent manner in both normal and damaged cochleae. In addition, most of the newly regenerated HCs induced by DBZ had visible and relatively mature stereocilia bundle structures. Finally, RNA sequencing detected the differentially expressed genes between DBZ treatment and controls, and interaction networks were constructed for the most highly differentially expressed genes.ConclusionsOur study demonstrates that DBZ can significantly promote SC proliferation and increase the number of mitotically regenerated HCs with relatively mature stereocilia bundles in the neonatal mouse cochlea by inhibiting Notch signalling and activating Wnt signalling, suggesting the DBZ might be a new therapeutic target for stimulating HC regeneration.

Nanoparticle-Mediated Inhibition of Notch Signaling Promotes Mitochondrial Biogenesis and Reduces Subcutaneous Adipose Tissue Expansion in Pigs.

SummaryInhibition of Notch signaling has been shown to induce white to beige transformation of adipocytes and reduce the risk of obesity in mice. However, it remains unknown whether the metabolic benefits of Notch inhibition are dependent on uncoupling protein 1 (UCP1)-mediated thermogenesis and evolutionarily relevant in other mammalian species. Here we report the effect of Notch inhibition in adipocytes of pigs, which lost the UCP1 gene during evolution. Notch inhibition using a γ-secretase inhibitor dibenzazepine (DBZ) promoted beige adipogenesis and mitochondrial biogenic gene expression in porcine adipocytes. Moreover, encapsulation of DBZ into poly(lactide-co-glycolide) nanoparticles enabled rapid cellular internalization and enhanced bioactivity to achieve sustained Notch inhibition, thereby inducing beige-specific gene expression and reducing subcutaneous adipose tissue expansion in pigs. These results demonstrate for the first time a role of Notch signaling in regulating adipose plasticity in large animals, highlighting the therapeutic potential of targeting Notch signaling in obesity treatment.

Hydrogen-Bonded Tetramers of Carbamazepine

In this study, the pharmaceutical carbamazepine (CBZ) and the related molecule dibenzazepine (DBZ) are prepared on the Au(111) surface and visualized using scanning tunneling microscopy. Monolayers...