Binding Interaction of Coumarin Derivative Daphnetin with Ovalbumin: Molecular Insights into the Complexation Process and Effects of Metal Ions and pH in the Binding and Antifibrillation Studies.

This study investigates the interaction between daphnetin and ovalbumin (OVA) as well as its potential to inhibit OVA fibrillation using both spectroscopic and computational analysis. A moderate binding affinity of 1 × 104 M-1 was observed between OVA and daphnetin, with a static quenched mechanism identified during the fluorescence quenching processes. Metal ions' (Cu2+ and Zn2+) presence led to an increase in the binding affinities of daphnetin toward OVA, mirroring a similar trend observed with the pH variation. Synchronous and 3D fluorescence studies indicated an increase in the polarity of the microenvironment surrounding the Trp residues during binding. Interestingly, circular dichroism and Fourier transform infrared studies showed a significant change in the secondary structure of OVA upon binding with daphnetin. The efficacy of daphnetin in inhibiting protein fibrillation was confirmed through thioflavin T and Congo Red binding assays along with fluorescence microscopic imaging analysis. The thermodynamic assessment showed positive ΔH° [+(29.34 ± 1.526) kJ mol-1] and ΔS° [+(181.726 ± 5.465) J mol-1] values, indicating the presence of the hydrophobic forces, while negative ΔG° signifies spontaneous binding interactions. These experimental findings were further correlated with computational analysis, revealing daphnetin dynamics within the binding site of OVA.

Up-regulated CD38 by daphnetin alleviates lipopolysaccharide-induced lung injury via inhibiting MAPK/NF-κB/NLRP3 pathway.

BACKGROUND: Sepsis is a life-threatening organ dysfunction syndrome resulted from severe infection with high morbidity and mortality. Cluster of differentiation 38 (CD38) is a multifunctional type II transmembrane glycoprotein widely expressed on the surface of various immunocytes membranes that mediates host immune response to infection and plays an important role in many inflammatory diseases. Daphnetin (Daph), isolated from the daphne genus plant, is a natural coumarin derivative that possesses anti-inflammatory and anti-apoptotic effects. The current study aimed to investigate the role and mechanism of Daph in alleviating lipopolysaccharide (LPS)-induced septic lung injury, and to explore whether the protective effect of Daph in mice and cell models was related to CD38. METHODS: Firstly, network pharmacology analysis of Daph was performed. Secondly, LPS-induced septic lung injury in mice were treated with Daph or vehicle control respectively and then assessed for survival, pulmonary inflammation and pathological changes. Lastly, Mouse lung epithelial cells (MLE-12 cells) were transfected with CD38 shRNA plasmid or CD38 overexpressed plasmid, followed by LPS and Daph treatment. Cells were assessed for viability and transfection efficiency, inflammatory and signaling. RESULTS: Our results indicated that Daph treatment improved survival rate and alleviated pulmonary pathological damage of the sepsis mice, as well as reduced the excessive release of pro-inflammatory cytokines IL-1ß, IL-18, IL-6, iNOS and chemokines MCP-1 regulated by MAPK/NF-κB pathway in pulmonary injury. Daph treatment decreased Caspase-3 and Bax, increased Bcl-2, inhibited nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis in lung tissues of septic lung injury. Also, Daph treatment reduced the level of excessive inflammatory mediators, inhibited apoptosis and pyroptosis in MLE-12 cells. It is noteworthy that the protective effect of Daph on MLE-12 cells damage and death was assisted by the enhanced expression of CD38. CONCLUSIONS: Our results demonstrated that Daph offered a beneficial therapeutic effect for septic lung injury via the up-regulation of CD38 and inhibition of MAPK/NF-κB/NLRP3 pathway. Video Abstract.

Daphnetin attenuates intestinal inflammation, oxidative stress, and apoptosis in ulcerative colitis via inhibiting REG3A-dependent JAK2/STAT3 signaling pathway.

Daphnetin is a natural coumarin compound with anti-inflammatory, anti-oxidant, and anti-apoptotic effects, which has been previously demonstrated to ameliorate DSS-induced ulcerative colitis (UC). However, the molecular mechanism involved in the daphnetin-mediated pathological process of UC remains unclarified. The current study used DSS-induced mice and LPS-challenged Caco-2 cells as UC models. Bodyweight, disease activity index (DAI) score, and colon length were used to evaluate the severity of colitis. The histological changes in colon tissues were observed using H&E and PAS staining. Protein levels were detected by western blot. The malondialdehyde (MDA) and superoxide dismutase (SOD) activities were used to assess oxidative stress. Inflammatory responses were evaluated by detecting the levels of inflammatory cytokines (IFN-r, IL-1ß, IL-6, and TNF-α) using flow cytometry. CCK-8 and TUNEL assay were employed to determine cell growth and cell death, respectively. The results showed that daphnetin could ameliorate the severity of colitis and attenuate the damage to intestinal structure in DSS-induced mice. Compared with the DSS group, the expression of ZO-1, occludin, and anti-apoptotic protein (BCL-2) was increased while the level of pro-apoptotic proteins (Bax and cleaved caspase 3) was decreased in DSS + daphnetin group. The activity of MDA and SOD, as well as the levels of inflammatory cytokines were substantially suppressed by daphnetin. In consistency, in vitro assays indicated that daphnetin protected Caco-2 cells from LPS-stimulated viability impairment, apoptosis, oxidative stress, and inflammation. Furthermore, daphnetin suppressed the activity of JAK2/STAT signaling in LPS-induced Caco-2 cells in a REG3A-dependent manner. REG3A overexpression abated the ameliorative effects of daphnetin while JAK2/STAT signaling inhibition functioned synergically with daphnetin in LPS-stimulated Caco-2 cells. Collectively, this study deepened the understanding of the therapeutic effects of daphnetin on UC and uncovered for the first time that daphnetin functioned through REG3A-activated JAK2/STAT3 signaling in UC, which may provide novel insights for the treatment of UC.

Antiplatelet Effect of Daphnetin Is Regulated by cPLA2-Mediated Thromboxane A2 Generation in Mice.

Coumarin derivatives have been recognized for their antithrombotic, anti-inflammatory, and antioxidant properties, and daphnetin is one of the natural coumarin derivatives isolated from Daphne Koreana Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect has not been studied to date. Here, we characterized the role and underlying mechanism of daphnetin in the regulation of platelet activation using murine platelets. In order to check the effect of daphnetin on platelet function, we first measured the effect of daphnetin on platelet aggregation and secretion. Collagen-induced platelet aggregation and dense granule secretion were partially inhibited by daphnetin. Interestingly, 2-MeSADP-induced secondary waves of aggregation and secretion were completely inhibited by daphnetin. It is known that 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by the positive feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2-MeSADP-induced platelet aggregation in aspirinated platelets where the contribution of TxA2 generation was blocked. Additionally, platelet aggregation and secretion induced by a low concentration of thrombin, which is affected by the positive feedback effect of TxA2 generation, were partially inhibited in the presence of daphnetin. Importantly, 2-MeSADP- and thrombin-induced TxA2 generation was significantly inhibited in the presence of daphnetin, confirming the role of daphnetin on TxA2 generation. Finally, daphnetin significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets. Only cPLA2 phosphorylation, not ERK phosphorylation, was significantly inhibited by daphnetin in aspirinated platelets. In conclusion, daphnetin plays a critical role in platelet function by inhibiting TxA2 generation through the regulation of cPLA2 phosphorylation.

Daphnetin alleviates neuropathic pain in chronic constrictive injury rats via regulating the NF-κB dependent CXCL1/CXCR2 signaling pathway.

CONTEXT: Daphnetin is a natural product with anti-inflammatory, antioxidant, and neuroprotective properties. Reports have found that it has a strong analgesic effect; however, its analgesic mechanism is unknown. OBJECTIVE: We explored the effect and mechanism of daphnetin on neuropathic pain (NP). MATERIALS AND METHODS: The rat model of NP was established by ligation of the sciatic nerve. Male Sprague-Dawley rats were divided into six groups: Control, Model, Sham, morphine (0.375 mg/kg), and daphnetin (0.0625 and 0.025 mg/kg). Rats were intrathecally injected with drugs or normal saline once daily for three days. Hyperalgesia was evaluated by mechanical withdrawal threshold (MWT) and thermal withdrawal threshold (TWT). Protein levels were detected using ELISA, immunofluorescence, and western blotting. RESULTS: Compared to the Model group, daphnetin improved TWT (46.70 °C vs. 42.20 °C) and MWT (45.60 g vs. 23.60 g), reduced the expression of interleukin-1ß (0.99 ng/g vs. 1.42 ng/g), interleukin-6 (0.90 ng/g vs. 1.52 ng/g), and tumor necrosis factor-α (0.93 ng/g vs. 1.52 ng/g) in the sciatic nerve. Daphnetin decreased the expression of toll-like receptor 4 (TLR4) (0.47-fold), phosphorylated inhibitor of NF-κB (p-IKBα) (0.29-fold), nuclear factor kappaB (NF-κB) (0.48-fold), glial fibrillary acidic protein (GFAP) (0.42-fold), CXC chemokine ligand type 1 (CXCL1) (0.84-fold), CXC chemokine receptor type 2 (CXCR2) (0.78-fold) in the spinal cord. DISCUSSION AND CONCLUSIONS: Daphnetin alleviates NP by inhibiting inflammation and astrocyte activation in the spinal cord, providing theoretical support for the extensive clinical treatment of NP.

Daphnetin ameliorates Aß pathogenesis via STAT3/GFAP signaling in an APP/PS1 double-transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) has become a major public health problem that affects the elderly population. Therapeutic compounds with curative effects are not available due to the complex pathogenesis of AD. Daphnetin, a natural coumarin derivative and inhibitor of various kinases, has anti-inflammatory and antioxidant activities. In this study, we found that daphnetin improved spatial learning and memory in an amyloid precursor protein (APP)/presenilin 1 (PS1) double-transgenic mouse model of AD. Daphnetin markedly decreased the levels of amyloid-ß peptide 1-40 (Aß40) and 1-42 (Aß42) in the cerebral cortex, downregulated the expressions of enzymes involved in APP processing, e.g., beta-site APP-cleaving enzyme (BACE), nicastrin and presenilin enhancer protein 2 (PEN2). We further found the reduced serum levels of inflammatory factors, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and chemokine (C-C motif) ligand 3 (CCL3), while daphnetin increased total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) levels in the serum. Interestingly, daphnetin markedly decreased the expression of glial fibrillary acidic protein (GFAP) and the upstream regulatory molecule- phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in APP/PS1 mice, and mainly inhibited the phosphorylation of STAT3 at Ser727 to decrease GFAP expression evidenced in a LPS-activated glial cell model. These results suggest that daphnetin ameliorates cognitive deficits and that Aß deposition in APP/PS1 mice is mainly correlated with astrocyte activation and APP processing.

Daphnetin inhibits α-MSH-induced melanogenesis via PKA and ERK signaling pathways in B16F10 melanoma cells.

Daphnetin is a dehydroxylated derivative of coumarin isolated from Daphne species. However, the effect of daphnetin on melanogenesis has not been elucidated. This study aims to investigate the inhibitory effect of daphnetin on melanogenesis in α-melanocyte stimulating hormone (α-MSH)-treated B16F10 cells and its potential mechanism. Melanin content analysis and cellular tyrosinase activity assay showed that daphnetin inhibited melanin biosynthesis in α-MSH-treated B16F10 cells. Immunoblotting and qRT-PCR also indicated that daphnetin suppressed the expression of microphthalmia-associated transcription factor, a mastering transcription factor of melanogenesis and its downstream melanogenic enzymes including tyrosinase and tyrosinase-related proteins. Moreover, daphnetin downregulated the phosphorylation of PKA, ERK, MSK1, and CREB. Additionally, daphnetin inhibited melanin synthesis in UVB-irradiated HaCaT conditioned medium system suggesting that daphnetin has potential as an antipigmentation activity in a physiological skin condition. Our data propose that daphnetin inhibits melanogenesis via modulating both the PKA/CREB and the ERK/MSK1/CREB pathways.

Daphnetin, a Coumarin in Genus Stellera Chamaejasme Linn: Chemistry, Bioactivity and Therapeutic Potential.

Coumarins is a huge family of phenolic compounds containing a common structure of 2H-1-benzopyran-2-one. Nowadays, more than 1,300 natural-based coumarins have been identified in a variety of plants, bacteria and fungi, many of them exhibited promising biomedical performance. Daphnetin (7,8-dihydroxycoumarin), a typical coumarin, showed a couple of bioactivities such as anti-cancer, antibacterial, anti-inflammatory and anti-arthritis. In the treatment of diseases, it has been verified that daphnetin has outstanding therapeutic effects on diabetes, arthritis, transplant rejection, cancer and even on central nervous system diseases. In China, it is being used for clinical applications, about 93 patent publications were associated with daphnetin. Due to its wide therapeutic potentials in clinical applications, numerous research on the action mechanisms and synthetic methods of daphnetin have been performed to support the future developments. Herein, we summarized the chemical synthetic methodologies, bioactivities, therapeutic potentials and structure-activity relationships of daphnetin and its derivatives. Moreover, the state-of-the-arts in current daphnetin study and future perspective in this field were discussed. Hopefully, this review would be beneficial for the discovery of new coumarin-based biomedicine in the near future.

Daphnetin inhibits the survival of hepatocellular carcinoma cells through regulating Wnt/ß-catenin signaling pathway.

Evidence has demonstrated that Daphnetin has antiangiogenesis activity, indicating it might be a new multi-targeted medication for cancer therapy. Here, we aimed to reveal Daphnetin role in hepatocellular carcinoma (HCC) progression and the underlying mechanism. Huh7 and SK-HEP-1, two human HCC cell lines were used in this study. MTT （3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide）, colony formation, flow cytometry, and tumor-bearing experiments were applied to evaluate the effects of different concentrations of Daphnetin on cell viability, apoptosis, cell cycle, and in vivo tumor formation, respectively. Real-time PCR （Polymerase Chain Reaction）and western blotting were applied to measure the mRNA and protein levels of ß-catenin. We observed that Daphnetin inhibited cell viability and tumorigenesis, promoted cell apoptosis, and induced a G1 phase arrest in a dose-dependent manner in both Huh7 and SK-HEP-1 cells, which were rescued by SKL2001, an activator of the Wnt/ß-catenin signaling. Taken together, this study reveals that Daphnetin exerts an antitumor role in HCC through the inactivation of Wnt/ß-catenin signaling.

Daphnetin, a natural coumarin averts reserpine-induced fibromyalgia in mice: modulation of MAO-A.

Fibromyalgia is a common, chronic, and generalized pain syndrome that is often associated with comorbid depression. The etiology of fibromyalgia is complex; most researchers have documented that the hallmark symptoms are due to the central nervous system's abnormal functioning. Neurotransmitters such as serotonin, norepinephrine, and glutamate, have been reported to be key regulators of fibromyalgia syndrome. Daphnetin is a 7, 8 dihydroxy coumarin widely distributed in Thymelaeaceae family plants, possessing various activities such as anti-arthritic, anti-tumor, anti-malarial, and anti-parasitic. The present study was designed to explore the potential of daphnetin against reserpine-induced fibromyalgia in mice. In mice, a fibromyalgia-like state was achieved by injecting reserpine (0.5 mg/kg, s.c) continuously for 3 days. All behavioral tests were conducted on the 4th and 6th day of experimentation. Reserpine administration significantly increased the mechanical hypersensitivity in electronic von Frey (eVF) and pressure application measurement (PAM) tests. It also increased the immobility period and time to reach the platform in force swim test (FST) and Morris water maze (MWM) test, respectively. In the biochemical analysis, reserpine treatment upregulated the monoamine oxidase-A (MAO-A) activity and level of glutamate, tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), and thiobarbituric acid reactive substances (TBARS). Whereas, it decreased the level of glutathione (GSH), dopamine, serotonin, and norepinephrine. Daphnetin pretreatment attenuated the behavioral and biochemical changes induced by reserpine. Thus, the current investigation results delineate that daphnetin might exert its protective effect by inhibiting inflammatory stress and MAO-A-mediated neurotransmitter depletion and oxidative stress.