Search Results (36)

Dorsomorphin, a pyrazolo[1,5-a]pyrimidine derivative, inhibits the bone morphogenetic protein (BMP) pathway by targeting the type I BMP receptors active in receptor-like kinases. However, the investigation of its—and its derivatives’—antiproliferative activity towards endothelial and cancer cell lines still requires reinforcement with additional studies. In the presented work, several dorsomorphin derivatives have been efficiently synthesized, based on a previously reported synthetic protocol with minor modifications. The endeavor was reinforced by a molecular docking study on the interactions of the designed derivatives with various protein targets, while the inhibitory effects of the synthesized novel molecules on the proliferation of murine leukemia cells (L1210), human T-lymphocyte cells (CEM), human cervix carcinoma cells (HeLa), and endothelial cells (human dermal microvascular, HMEC-1, and bovine aortic endothelial cells, BAECs) were investigated. Among the compounds tested, diphenol 22, emerged as the most promising bioactive lead since it demonstrated half-maximal inhibitory concentration (IC₅₀) values below 9 μM in all tested lines except HeLa cells. In the same context, the carbamate derivative 6 was determined as a potent inhibitor of endothelial cell proliferation in BAECs at a low micromolar range. In conclusion, the presented work not only reveals promising antiproliferative dorsomorphin derivatives but also sets the basis for further exploitation of dorsomorphin’s bioactive portfolio, based on bioactivity results and molecular modeling calculations. Full article

Alcoholic liver disease (ALD) caused by excessive alcohol consumption poses a serious threat to human health. Callistephus chinensis (L.) Nees is an herb of the Asteraceae family that has good results in the prevention and treatment of a variety of liver diseases, including multifactorial liver injury, non-alcoholic fatty liver disease/non-alcoholic steatohepatitis, liver fibrosis. Sesquiterpenes are thought to be biologically active components that typically have anti-inflammatory, immunomodulatory, and cardioprotective effects. Callistephus A (CA) is a sesquiterpene compound with a rare 6/7 ring skeleton, which has been isolated only from the Callistephus chinensis Nees. However, the mechanism of action of CA on alcoholic liver disease remains unclear. In this study, alcoholic liver mice were fed with 20 and 40 mg/kg CA, respectively, for 11 days. This study showed that CA improved hepatic steatosis and oxidative stress associated with alcohol consumption. CA alleviated liver inflammation by inhibiting the TLR4/MYD88/NF-κB pathway, ameliorating gut imbalance by restoring the abundance of Akkermansia, and restoring short-chain fatty acids in the gut. Transcriptome analysis revealed that CA primarily affects genes involved in lipid metabolism and inflammation. In vitro, by adding inhibitors of TLR4 (TAK-242) and AMPK (Dorsomorphin), it was confirmed that CA alleviates ALD by inhibiting TLR4 and activating AMPK. This study is the first to demonstrate that CA protects against alcoholic liver disease through the regulation of the gut flora and modulation of the AMPK/NF-κB pathway. In conclusion, CA can effectively improve alcoholic liver disease and can be used as an effective candidate drug with liver-protective effects. Full article

Background/Objectives: Pain is a growing public health concern worldwide, and the use of combinations of drugs can improve their analgesic effects while minimizing their adverse effects. Drugs such as metformin (antidiabetic) and melatonin (sleep regulator) have analgesic potential in combination. In this study, we evaluated the pharmacological interaction between metformin and melatonin when orally administered in a rat model, using the formalin test. Methods: Female Wistar rats (220–350 g) were injected with 50 µL of 1% formalin in the dorsal surface of the right hind paw. Formalin produces pain-related flinching behavior, and antinociception was evaluated as the reduction in this response. The percentage of the antinociceptive effect was determined after the oral administration of metformin (30–1000 mg/kg), melatonin (10–150 mg/kg), and their combination (MMC). To establish the nature of the interaction, isobolographic analysis was performed in a fixed-dose ratio (0.5:0.5), based on the effective dose 50 (ED₅₀) values for each drug: metformin (947.46 ± 242.60 mg/kg) and melatonin (126.86 ± 37.98 mg/kg). To evaluate the mechanism of action, the receptor antagonist for metformin compound C (dorsomorphin) for AMPK inhibition, MT1 and MT2 melatonin receptor antagonists (4-P-PDOT, luzindole), and an opioid antagonist (naloxone) were employed. The rotarod test was used to evaluate the safety profile of the combination. Results: The metformin–melatonin combination significantly reduced the number of flinches in the second phase of the formalin test. The theoretical ED₅₀ for the combination (ED₅₀ T) was 537.15 ± 122.76 mg/kg. Experimentally, the ED₅₀ (ED₅₀ E) was significantly lower (360.83 ± 23.36 mg/kg), indicating a synergistic interaction for the combination involving opioidergic pathways, MT2 receptors, and AMPK activation. Conclusions: Oral metformin–melatonin coadministration could provide a therapeutic alternative for inflammatory pain. Full article

Mesenchymal stromal cells (MSCs), such as bone marrow-derived cells (BMSCs) and adipose-derived cells (ASCs), are key candidates for bone regeneration therapies but have not yet been integrated into standard clinical practice due to heterogeneity in their osteogenic capacities. This study investigated the osteogenic differentiation of porcine BMSCs and ASCs by analyzing BMP-2-induced receptor expression and the effects of inhibiting BMP, TGF-β, and FGF signaling pathways. While pBMSCs underwent osteogenesis in standard differentiation medium, pASCs required BMP-2 stimulation to initiate this process. BMP signaling inhibition via dorsomorphin suppressed osteogenic differentiation, but this effect was reversed by co-inhibition of TGF-β or FGF signaling. Notably, simultaneous inhibition of TGF-β and FGF in the presence of BMP-2 optimized osteogenic differentiation in both pMSC types. In pASCs, successful differentiation correlated with early activation of p38 MAPK and Wnt signaling pathways, with BMP-2 serving as a primary driver, while TGF-β and FGF pathways acted as modulators. These findings highlight the importance of signaling context and MSC tissue origin in bone formation and suggest that tailored modulation of BMP, TGF-β, and FGF signaling will be necessary in future in vivo applications to maximize the regenerative potential of MSC-based therapies. Full article

Pyrazolo[1,5-a]pyrimidine is a nitrogen-containing fused heterocycle that imitates the nitrogenous base adenine with varying degrees of reliability. This fact determines its frequent use in drug design, including the development of ATP-competitive kinase inhibitors. These include dorsomorphin which shows compromised kinase selectivity but is still widely used as an AMPK inhibitor. ATP-binding pockets of many proteins have a fairly conservative spatial structure and there is a high probability of obtaining a compound with low target selectivity during drug development. In the case of a common scaffold, the careful selection of side substituents that determine the activity and selectivity of the final compound plays an important role. In this work, a convergent strategy for the synthesis of dorsomorphin and its close analogs was developed and implemented. The resulting small series of compounds is distinguished by the maximum possible diversification and allows for an assessment of the biological activity towards AMPK. An original route to obtain variants of the phenoxy-alkylamine moiety of dorsomorphin via the Mitsunobu reaction will be useful for generating targeted-focused libraries of ATP-competitive kinase inhibitors and highly active receptor ligands. Full article

Plant secondary metabolites are known to be valuable agents to hamper inflammation owing to their multiple mechanisms of action. This study investigates the molecular mechanisms underlying the anti-inflammatory effects of vicenin-2 in lipopolysaccharide (LPS)-stressed THP-1 cells. After ascertaining the safety of vicenin-2 in our in vitro model, we assessed the anti-inflammatory potential of this flavonoid. Indeed, it counteracted the increase of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 levels, as well as the overexpression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 caused by the exposure of THP-1 cells to LPS. Acknowledged the role of SIRT1 in the inflammatory process, we focused our attention on this enzyme. Our results showed that LPS dramatically decreased the expression of SIRT1, whereas vicenin-2 restored the levels of this enzyme to those of unexposed cells. These effects were also observed in terms of acetylated p53, a SIRT1 substrate. Notably, we observed that vicenin-2 did not act as a direct activator of SIRT1. Therefore, we investigated the potential involvement of AMP-activated protein kinase (AMPK), an upstream activator of SIRT1. Of note, by blocking AMPK by dorsomorphin, the protective effects of vicenin-2 on SIRT1 expression and activity were lost, suggesting the engagement of this kinase. Consequently, the blockage of AMPK caused a downstream loss of the anti-inflammatory effect of vicenin-2, which was no longer able to decrease both the activation of nuclear factor (NF)-κB and the production of cytokines induced by LPS. Finally, docking simulation suggested that vicenin-2 might act as an activator of Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ), one of the regulators of AMPK. Overall, our results suggest that the anti-inflammatory effects of vicenin-2 may be due to the interaction with the CaMKKβ-AMPK-SIRT1 axis. Full article

Background/Objectives: Allergic contact dermatitis (ACD), an inflammatory skin condition, is commonly treated with topical corticosteroids; however, long-term use of these drugs is associated with various risks, such as skin atrophy and steroid resistance. Triacetin (TA), a triglyceride metabolized to acetate, exerts anti-inflammatory affects by activating AMP-activated protein kinase (AMPK) and suppressing mast cell degranulation. Here, we aimed to assess the immediate and long-term effects of TA on ACD suppression, focusing on AMPK activation, using a 2,4-dinitrofluorobenzene-induced rodent model. Methods: Various concentrations of TA were topically applied to rats with 2,4-dinitrofluorobenzene-induced dermatitis. Ear thickness was measured, and histological analysis was performed to assess the inflammation, mast cell infiltration, and degranulation in the established models. AMPK activation was analyzed via Western blotting, and TA degradation was assessed via gas chromatography-mass spectrometry. Dorsomorphin (an AMPK inhibitor) was used to evaluate the effects of AMPK on ACD. Results: TA significantly inhibited inflammation and mast cell degranulation in a dose-dependent manner, with 0.25 mmol/L showing the most potent effects. It also activated AMPK activation. Notably, AMPK inhibition reversed the effects of TA. Conclusions: Overall, TA exerted immediate and long-term anti-inflammatory effects via AMPK activation and inhibition of mast cell degranulation, showing potential as a non-steroidal therapeutic for ACD. Full article

Background/Objective: This study examined the anti-obesity effect of royal jelly (RJ) in rats fed with a high-fat diet by targeting the major pathways involved in adipogenesis and lipolysis. In addition, it examined whether this effect is AMPK-dependent. Methods: Five groups of adult male albino rats were used (n = 6 each as 1); the control rats were fed with a normal diet (2.9 kcal), and the other groups were as follows: control + RJ (300 mg/kg), HFD (4.75 kcal), HFD + RJ (300 mg/kg), and HFD + RJ (300 mg/kg) + dorsomorphin (an AMPK inhibitor) (0.2 mg/kg). Results: RJ was administered orally to all rats. With no changes in food and energy intake, RJ significantly reduced gains in body weight, fat weight, body mass index (BMI), the Lee index, abdominal circumference (AC), and the adiposity index (AI). It also reduced fasting glucose and insulin levels, HOMA-IR, and the circulatory levels of free fatty acids (FFAs), triglycerides, cholesterol, and LDL-c in the HFD-fed rats. RJ also increased serum glycerol levels and adiponectin levels, but reduced the serum levels of leptin, IL-6, and TNF-α. Moreover, RJ reduced the secretion of IL-6 and TNF-α from isolated WAT. At the tissue level, the HFD + RJ rats exhibited a smaller adipocyte size compared to the HFD rats. At the molecular level, RJ increased the phosphorylation of AMPK, SREBP1, and ACC-1 and increased the mRNA and protein levels of HSL and ATG in the WAT of the HFD rats. In concomitance, RJ increased the mRNA levels of PGC-α1, reduced the protein levels of PPARγ, and repressed the transcriptional activities of PPARγ, SREBP1, and C/EBPαβ in the WAT of these rats. All the aforementioned effects of RJ were prevented by co-treatment with dorsomorphin. Conclusions: RJ exerts a potent anti-obesity effect in rats that is mediated by the AMPk-dependent suppression of WAT adipogenesis and the stimulation of lipolysis. Full article

Hundreds of millions of people worldwide are expected to suffer from diabetes mellitus. Diabetes is characterized as a dynamic and heterogeneous disease that requires deeper understanding of the pathophysiology, genetics, and metabolic shaping of this disease and its macro/microvascular complications. Macrophages play an essential role in regulating local immune responses, tissue homeostasis, and disease pathogenesis. Here, we have analyzed transforming growth factor beta 1 (TGFβ1)/Smad signaling in primary human macrophages grown in normal (NG) and high-glucose (HG; +25 mM glucose) conditions. Cell culture lactate concentration and cellular phosphofructokinase (PFK) activity were increased in HG concentrations. High glucose levels in the growth media led to increased macrophage mRNA expression of TGFβ1, and TGFβ-regulated HAMP and PLAUR mRNA levels, while the expression of TGFβ receptor II remained unchanged. Stimulation of cells with TGFβ1 protein lead to Smad2 phosphorylation in both NG and HG conditions, while the phosphorylation of Smad1/5 was detected only in response to TGFβ1 stimulation in HG conditions. The use of the specific Alk1/2 inhibitor dorsomorphin and the Alk5 inhibitor SB431542, respectively, revealed that HG conditions led TGFβ1 to activation of Smad1/5 signaling and its downstream target genes. Thus, high-glucose activates TGFβ1 signaling to the Smad1/5 pathway in primary human macrophages, which may contribute to cellular homeostasis in a harmful manner, priming the tissues for diabetic complications. Full article

Vascular complications in Type 2 diabetes mellitus (T2DM) patients increase morbidity and mortality. In T2DM, angiogenesis is impaired and can be enhanced or reduced in different tissues (“angiogenic paradox”). The present study aimed to delineate differences between macrovascular and microvascular endothelial cells that might explain this paradox. In a monoculture system of human macrovascular (EaHy926) or microvascular (HMEC-1) endothelial cell lines and a monocytic cell line (U937), high glucose concentrations (25 mmole/L) increased the secretion of the pro-angiogenic factors CD147/EMMPRIN, VEGF, and MMP-9 from both endothelial cells, but not from monocytes. Co-cultures of EaHy926/HMEC-1 with U937 enhanced EMMPRIN and MMP-9 secretion, even in low glucose concentrations (5.5 mmole/L), while in high glucose HMEC-1 co-cultures enhanced all three factors. EMMPRIN mediated these effects, as the addition of anti-EMMPRIN antibody decreased VEGF and MMP-9 secretion, and inhibited the angiogenic potential assessed through the wound assay. Thus, the minor differences between the macrovascular and microvascular endothelial cells cannot explain the angiogenic paradox. Metformin, a widely used drug for the treatment of T2DM, inhibited EMMPRIN, VEGF, and MMP-9 secretion in high glucose concentration, and the AMPK inhibitor dorsomorphin enhanced it. Thus, AMPK regulates EMMPRIN, a key factor in diabetic angiogenesis, suggesting that targeting EMMPRIN may help in the treatment of diabetic vascular complications. Full article

With the increase in the age of laying chickens, the aging of follicles is accelerated, and the reproductive ability is decreased. Increased oxidative stress and mitochondrial malfunction are indispensable causes of ovarian aging. In this study, the physiological condition of prehierarchical small white follicles (SWFs) was compared between D280 high-producing chickens and D580 aging chickens, and the effect of a plant-derived flavonoid nobiletin (Nob), a natural antioxidant, on senescence of SWFs granulosa cells (SWF-GCs) was investigated. The results showed that Nob treatment activated cell autophagy by activating the AMP-activated protein kinase (AMPK) and Sirtuin-1 (SIRT1) pathways in D-galactose (D-gal)-generated senescent SWF-GCs, restoring the expression of proliferation-related mRNAs and proteins. In addition, the expression of inflammation-related protein NF-κB was significantly enhanced in aging GCs that were induced by D-gal. Nob supplementation significantly increased the antioxidant capacity and decreased the expression of several genes associated with cell apoptosis. Furthermore, Nob promoted activation of PINK1 and Parkin pathways for mitophagy and alleviated mitochondrial edema. Either the AMPK inhibitor dorsomorphin (Compound C) or SIRT1 inhibitor selisistat (EX-527) attenuated the effect of Nob on mitophagy. The protective effect of Nob on natural aging, GC proliferation, and elimination of the beneficial impact on energy regulation of naturally aging ovaries was diminished by inhibition of Nob-mediated autophagy. These data suggest that Nob treatment increases the expression of mitophagy-related proteins (PINK1 and Parkin) via the AMPK/SIRT1 pathways to prevent ovarian aging in the laying chickens. Full article

Glucosamine hydrochloride (GAH) is a natural component of glycoproteins present in almost all human tissues and participates in the construction of human tissues and cell membranes. GAH has a wide range of biological activities, particularly in anti-inflammatory and osteogenic damage repair. At present, little is known about how GAH functions in angiogenesis. To determine the role of GAH on vascular development and impairment repair, we used the inhibitors VRI, DMH1, and dorsomorphin (DM) to construct vascular-impaired models in Tg(kdrl: mCherry) transgenic zebrafish. We then treated with GAH and measured its repair effects on vascular impairment through fluorescence intensity, mRNA, and protein expression levels of vascular-specific markers. Our results indicate that GAH promotes vascular development and repairs impairment by regulating the vascular endothelial growth factor (VEGF) signaling pathway through modulation of bone morphogenetic protein (BMP) signaling. This study provides an experimental basis for the development of GAH as a drug to repair vascular diseases. Full article

The browning of white adipocytes may be an innovative approach to address obesity. This study investigated the effects of rabbit meat extract on 3T3−L1 adipocytes, with a specific emphasis on inducing browning. The browning effects of rabbit meat extract were evaluated by analyzing genes specifically expressed in 3T3−L1 adipocytes using quantitative PCR and immunoblotting. Rabbit meat extract increased the expression of brown adipocyte−specific markers, UCP1 and PGC1α, and mitochondrial biogenesis factors, TFAM and NRF1, without affecting cell viability in fully differentiated 3T3−L1 adipocytes. Moreover, adipocyte differentiation and the triglyceride content were decreased; hormone−sensitive lipase activity was promoted. Rabbit meat extract activated the AMPK pathway in the differentiated 3T3−L1 cells. However, in adipocytes treated with rabbit meat extract, the expression of genes related to browning was reduced by the AMP−activated protein kinase (AMPK) inhibitor, dorsomorphin dihydrochloride. To the best of our knowledge, this is the first study to demonstrate that rabbit meat extract induces the browning of white adipocytes via the activation of the AMPK pathway, thereby demonstrating its therapeutic potential in preventing obesity. Full article

Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrosis and, thus, build the “soil” for hepatocarcinogenesis. Furthermore, HSCs are known to promote the progression of hepatocellular carcinoma (HCC), but the molecular mechanisms are only incompletely understood. Recently, we newly described the expression of bone morphogenetic protein 13 (BMP13) by HSCs in fibrotic liver tissue. In addition, BMP13 has mostly been studied in the context of cartilage and bone repair, but not in liver disease or cancer. Thus, we aimed to analyze the expression and function of BMP13 in HCC. Expression analyses revealed high BMP13-expression in activated human HSCs, but not in human HCC-cell-lines. Furthermore, analysis of human HCC tissues showed a significant correlation between BMP13 and α-smooth muscle actin (α-SMA), and immunofluorescence staining confirmed the co-localization of BMP13 and α-SMA, indicating activated HSCs as the cellular source of BMP13 in HCC. Stimulation of HCC cells with recombinant BMP13 increased the expression of the inhibitors of differentiation 1 (ID1) and 2 (ID2), which are known targets of BMP-signaling and cell-cycle promotors. In line with this, BMP13-stimulation caused an induced SMAD 1/5/9 and extracellular signal-regulated kinase (ERK) phosphorylation, as well as reduced expression of cyclin-dependent kinase inhibitors 1A (CDKN1A) and 2A (CDKN2A). Furthermore, stimulation with recombinant BMP13 led to increased proliferation and colony size formation of HCC cells in clonogenicity assays. The protumorigenic effects of BMP13 on HCC cells were almost completely abrogated by the small molecule dorsomorphin 1 (DMH1), which selectively blocks the intracellular kinase domain of ALK2 and ALK3, indicating that BMP13 acts via these BMP type I receptors on HCC cells. In summary, this study newly identifies stroma-derived BMP13 as a potential new tumor promotor in HCC and indicates this secreted growth-factor as a possible novel therapeutic target in HCC. Full article

Background: In vertebrates, the development of the inner ear is a delicate process, whereas its relating molecular pathways are still poorly understood. LMO4, an LIM domain-only transcriptional regulator, is drawing an increasing amount of interest for its multiple roles regarding human embryonic development and the modulation of ototoxic side effects of cisplatin including cochlear apoptosis and hearing loss. The aim of the present study is to further explore the role of lmo4a in zebrafish inner ear development and thus explore its functional role. Methods: The Spatial Transcript Omics DataBase was referred to in order to evaluate the expression of lmo4a during the first 24 h of zebrafish development. In situ hybridization was applied to validate and extend the expression profile of lmo4a to 3 days post-fertilization. The morpholino (MO) knockdown and CRISPR/Cas9 knockout (KO) of lmo4a was applied. Morphological analyses of otic vesical, hair cells, statoacoustic ganglion and semicircular canals were conducted. The swimming pattern of lmo4a KO and MO zebrafish was tracked. In situ hybridization was further applied to verify the expression of genes of the related pathways. Rescue of the phenotype was attempted by blockage of the bmp pathway via heat shock and injection of Dorsomorphin. Results: lmo4a is constitutively expressed in the otic placode and otic vesicle during the early stages of zebrafish development. Knockdown and knockout of lmo4a both induced smaller otocysts, less hair cells, immature statoacoustic ganglion and malformed semicircular canals. Abnormal swimming patterns could be observed in both lmo4a MO and KO zebrafish. eya1 in preplacodal ectoderm patterning was downregulated. bmp2 and bmp4 expressions were found to be upregulated and extended in lmo4a morphants, and blockage of the Bmp pathway partially rescued the vestibular defects. Conclusions: We concluded that lmo4a holds a regulative effect on the Bmp pathway and is required for the normal development of zebrafish inner ear. Our study pointed out the conservatism of LMO4 in inner ear development between mammals and zebrafish as well as shed more light on the molecular mechanisms behind it. Further research is needed to distinguish the relationships between lmo4 and the Bmp pathway, which may lead to diagnostic and therapeutic approaches towards human inner ear malformation. Full article