Search

The purpose of this research was to assess the impact of different doses of Daphnetin (DAP, a natural compound derived from coumarin) on hippocampus neuronal injury, neurobehavioral function, blood-brain barrier (BBB) integrity, expression of claudin-5, brain-derived neurotrophic factor (BDNF), superoxide dismutase (SOD), and inflammatory markers in a mouse model of cerebral ischemia. Cerebral ischemia was induced in mice through 30 minutes of bilateral common carotid occlusion (BCCAO), followed by 48 hours of reperfusion. The viability of hippocampal neurons was assessed using Cresyl violet staining and BBB function was determined by measuring Evans blue (E.B) dye leakage. Spatial memory was tested using the Radial Arm Water Maze (RAWM) task. Claudin-5 and BDNF were measured by immunofluorescence, while SOD, interleukin-1 beta (IL-1β), and nuclear factor-κB (NF-κB) expression were determined through western blotting. Administering DAP significantly increased neuron survival in the hippocampus CA1, CA3, and dentate gyrus (DG) regions and improved spatial memory dose-dependently (P<0.0001). Treatment with DAP (40 mg/kg IP) significantly reduced E.B leakage and brain water content (P<0.001). Furthermore, it increased the claudin-5, BDNF, and SOD levels and diminished NF-κB and IL-1β expression (P<0.0001). The research found that DAP protected neurons in the CA1, CA3, and DG areas of the hippocampus, enhanced behavioral functions, and preserved BBB integrity in a cerebral ischemia model. This positive impact is achieved by increasing the expression of claudin-5, BDNF, and SOD and diminishing neuroinflammation. Further research is required to clarify the mechanisms and possible clinical uses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

As a cancer with the highest incidence in recent years, lung cancer is mainly composed of three diseases: non-small cell lung cancer, small cell lung cancer and neuroendocrine tumor. The morbidity and mortality of this malignant tumor are the highest in both male and female populations worldwide. In my country, lung cancer has become the most common cancer disease and the leading cause of cancer death, so it is extremely important to find lung cancer therapeutic targets. Based on previous studies, we speculated that the TLR4-Myd88-NFκB pathway may be involved in hmgb1-induced EMT in A549 cells, and daphnetin may also inhibit hmgb1-induced EMT through the TLR4-Myd88-NFκB pathway in A549 cells, but related studies have not linked it to hmgb1-induced EMT. Therefore, the innovation of this study is to test these two conjectures and analyze how daphnetin affects the epithelial-mesenchymal transition (EMT) mechanism induced by HMGB1 in human lung adenocarcinoma cells (A549 cell line), aiming at lung adenocarcinoma cells, foundation for clinical treatment. The proliferation rate and the migrating cell number presented an obvious decrease in the HMGB1+TLR4-shRNA group and the HMGB1+daphnetin group relative to the HMGB1 group ( P  < 0.0001). The intracellular expression of TLR4, Myd88, NFκB, vimentin and snail1 proteins were significantly decreased ( P  < 0.001), while that of E-cadherin presented a remarkable increase ( P  < 0.001) in the HMGB1+TLR4-shRNA and HMGB1+daphnetin group compared with the HMGB1 group. TLR4-MyD88-NFκB pathway is associated with HMGB1-induced EMT in A549 cells. Daphnetin had an inhibitory effect on HMGB1-induced EMT via the TLR4-Myd88-NF-κB pathway in A549 cells.

Allergic asthma is a major health burden on society as a chronic respiratory disease characterized by inflammation and muscle tightening around the airways in response to inhaled allergens. Daphne kiusiana Miquel is a medicinal plant that can suppress allergic airway inflammation; however, its specific molecular mechanisms of action are unclear. In this study, we aimed to elucidate the mechanisms by which D. kiusiana inhibits allergic airway inflammation. We evaluated the anti-inflammatory effects of the ethyl acetate (EA) fraction of D. kiusiana and its major compound, daphnetin, on murine T lymphocyte EL4 cells stimulated with phorbol 12-myristate 13-acetate and ionomycin in vitro and on asthmatic mice stimulated with ovalbumin in vivo. The EA fraction and daphnetin inhibited T-helper type 2 (Th2) cytokine secretion, serum immunoglobulin E production, mucus secretion, and inflammatory cell recruitment in vivo. In vitro, daphnetin suppressed intracellular Ca 2+ mobilization (a critical regulator of nuclear factor of activated T cells) and functions of the activator protein 1 transcription factor to reduce interleukin (IL)-4 and IL-13 expression. Daphnetin effectively suppressed the IL-4/-13-induced activation of Janus kinase (JAK)/signal transducer and activator of transcription 6 (STAT6) signaling in vitro and in vivo, thereby inhibiting the expression of GATA3 and PDEF, two STAT6-target genes responsible for producing Th2 cytokines and mucins. These findings indicate that daphnetin suppresses allergic airway inflammation by stabilizing intracellular Ca 2+ levels and subsequently inactivating the JAK/STAT6/GATA3/PDEF pathway, suggesting that daphnetin is a promising alternative to existing asthma treatments., Competing Interests: Declaration of competing interest None: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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 × 10 4 M -1 was observed between OVA and daphnetin, with a static quenched mechanism identified during the fluorescence quenching processes. Metal ions' (Cu 2+ and Zn 2+ ) 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 Δ H ° [+(29.34 ± 1.526) kJ mol -1 ] and Δ S ° [+(181.726 ± 5.465) J mol -1 ] values, indicating the presence of the hydrophobic forces, while negative Δ G ° signifies spontaneous binding interactions. These experimental findings were further correlated with computational analysis, revealing daphnetin dynamics within the binding site of OVA.

Background: Ferroptosis, an emerging nonapoptotic, modulated cell death process characterized by iron accumulation and subsequent lipid peroxidation, has been intimately implicated in the development and progression of ovarian cancer (OC). Daphnetin (Daph), a natural product isolated from Daphne Korean Nakai, exhibits anticancer efficacy against various solid tumors. However, the specific role and potential mechanism underlying Daph-mediated modulation of ferroptosis in OC cells remain elusive., Purpose: This study aims to analyze the proferroptotic impacts of Daph on OC cells and to further explore the underlying mechanisms involved., Study Design and Methods: We used CCK-8, wound healing and Transwell assays to assess whether Daph can inhibit the proliferation and migration of OC cells. Additionally, transmission electron microscopy (TEM), iron measurement, reactive oxygen species (ROS) analysis, lipid peroxidation assays, qRT-PCR and western blotting were utilized to evaluate the impact of Daph on ferroptosis and elucidate the potential underlying mechanism. Furthermore, RNA sequencing analysis, molecular docking analysis, cellular thermal shift assays (CETSAs) and NQO1 activity assays were used to predict and validate the binding and mechanistic interactions between Daph and NQO1. Subcutaneous tumorigenesis models were utilized to examine the effectiveness of Daph (and/or cisplatin) in vivo., Results: Daph exerted antitumor effects by inducing the death and suppressing the migration of A2780 and SKOV3 cells. Further, Daph induced ferroptosis in OC cells, as evidenced by the accumulation of intracellular ferrous iron (Fe2+), ROS and lipid peroxides, as well as the decreases in the glutathione/oxidized glutathione disulfide (GSH/GSSG) ratio and the expression of ferroptosis indicators (SLC7A11 and GPX4). RNA sequencing and molecular docking analyses revealed that the direct interaction between NQO1 and Daph reduced both the activity and expression of NQO1. Importantly, NQO1 overexpression effectively alleviated the effects of Daph on proliferation, migration, and ferroptosis in vitro and in vivo. Interestingly, we also found that combination treatment with Daph, a negative regulator of NQO1, and cisplatin synergistically induced cytotoxicity in OC cells., Conclusion: Our findings are the firstly demonstrated that Daph acts as a novel ferroptosis inducer in OC cells by specifically targeting NQO1 and is thus a promising candidate agent for OC treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Daphnetin, a coumarin derivative isolated from Daphne odorifera, has anti-tumor effects. The MAPK, STAT3, and NF-κB signaling pathways are closely related to the pathogenesis of lung cancer. To investigate the effect of daphnetin on anti-lung adenocarcinoma A549 cells and its mechanism. The anti-tumor effects of daphnetin on the proliferation, clone formation, migration, and invasion of A549 lung adenocarcinoma cells were investigated. The results showed that daphnetin inhibited the proliferation, colony formation, migration, and invasion of A549 cells through the MAPK/STAT3/NF-KB pathway, and mainly inhibited the clonal formation and migration of A549 cells through the JNK pathway. These results provide a new research direction and theoretical basis for the use of daphnetin in the inhibition of lung adenocarcinoma.

Silicosis is a hazardous occupational disease caused by inhalation of silica, characterized by persistent lung inflammation that leads to fibrosis and subsequent lung dysfunction. Moreover, the complex pathophysiology of silicosis, the challenges associated with early detection, and the unfavorable prognosis contribute to the limited availability of treatment options. Daphnetin (DAP), a natural lactone, has demonstrated various pharmacological properties, including anti-inflammatory, anti-fibrotic, and pulmonary protective effects. However, the effects of DAP on silicosis and its molecular mechanisms remain uncover. This study aimed to evaluate the therapeutic effects of DAP against pulmonary inflammation and fibrosis using a silica-induced silicosis mouse model, and investigate the potential mechanisms and targets through network pharmacology, proteomics, molecular docking, and cellular thermal shift assay (CETSA). Here, we found that DAP significantly alleviated silica-induced lung injury in mice with silicosis. The results of H&E staining, Masson staining, and Sirius red staining indicated that DAP effectively reduced the inflammatory response and collagen deposition over a 28-day period following lung exposure to silica. Furthermore, DAP reduced the number of TUNEL-positive cells, increased the expression levels of Bcl-2, and decreased the expression of Bax and cleaved caspase-3 in the mice with silicosis. More importantly, DAP suppressed the expression levels of NLRP3 signaling pathway-related proteins, including NLRP3, ASC, and cleaved caspase-1, thereby inhibiting silica-induced lung inflammation. Further studies demonstrated that DAP possesses the ability to inhibit the epithelial mesenchymal transition (EMT) induced by silica through the inhibition of the TGF-β1/Smad2/3 signaling pathway. The experimental results of proteomic analysis found that the PI3K/AKT1 signaling pathway was the key targets of DAP to alleviate lung injury induced by silica. DAP significantly inhibited the activation of the PI3K/AKT1 signaling pathway induced by silica in lung tissues. The conclusion was also verified by the results of molecular and CETSA. To further verify this conclusion, the activity of PI3K/AKT1 signaling pathway was inhibited in A549 cells using LY294002. When the A549 cells were pretreated with LY294002, the protective effect of DAP on silica-induced injury was lost. In conclusion, the results of this study suggest that DAP alleviates pulmonary inflammation and fibrosis induced by silica by modulating the PI3K/AKT1 signaling pathway, and holds promise as a potentially effective treatment for silicosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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-1β, 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., (© 2023. The Author(s).)

Alkali burn is a significant contributor to corneal injury. Alkali burn-induced corneal inflammation often causes vision loss due to corneal neovascularization. Daphnetin (DAP) has been studied for its anti-inflammatory and antiangiogenic properties with encouraging results. Driven by those encouraging results, we sought to explore the effects of DAP in treating alkali burn-induced corneal inflammation and neovascularization and its mechanism of action. We found that the angiogenesis processes of human umbilical vein endothelial cells (HUVECs) induced by vascular endothelial growth factor A (VEGF-A) were primarily attenuated by treatment with DAP, including proliferation, migration, and tube formation. Treatment of DAP significantly suppressed the VEGF-A-induced protein expression of VEGF receptor2 (VEGFR2), as well as the activation of downstream signal transducer and activator of transcription 3 (STAT3), AKT, and extracellular signal-regulated kinase (ERK) signaling. In the mouse corneal alkali burn model, the inflammatory cell infiltrations and neovascularization in the cornea caused by alkali burn were inhibited by 10 µM DAP eye drops. Alkali burn-induced corneal protein expression of VEGF-A, VEGFR2, phosphorylated (p-)STAT3, p-AKT, and p-ERK in corneal tissue were reduced mainly by DAP. Moreover, the upregulation of inflammatory caused by alkali burn in the pathological process was significantly neutralized by DAP. Mechanistically, the inflammatory response could be alleviated by DAP in the way of inhibiting the expression levels of TLR4, p-NF-κB, NLRP3, ASC, Cleaved-caspase-1 (p20), mature-IL-1β (p17), and N-GSDM. In conclusion, our findings confirmed that the corneal inflammation and neovascularization caused by alkali burn could be inhibited by DAP in vitro and in vivo, elucidating the underlying mechanisms of its protective effects. DAP may have tremendous therapeutic potential for the treatment of corneal alkali burn., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Natural killer (NK) cells are a potent weapon against tumor and viral infection. Finding active compounds with the capacity of enhancing NK cell effector functions will be effective to develop new anti-cancer drugs. In this study, we initially screened 287 commercially available active compounds by co-culturing with peripheral blood mononuclear cells (PBMCs). We found that five compounds, namely, Daphnetin, MK-8617, LW6, JIB-04, and IOX1, increased the IFN-γ + NK cell ratio in the presence of IL-12. Further studies using purified human primary NK cells revealed that Daphnetin directly promoted NK cell IFN-γ production in the presence of IL-12 but not IL-15, while the other four compounds acted on NK cells indirectly. Daphnetin also improved the direct cytotoxicity of NK cells against tumor cells in the presence of IL-12. Through RNA-sequencing, we found that PI3K-Akt-mTOR signaling acted as a central pathway in Daphnetin-mediated NK cell activation in the presence of IL-12. This was further confirmed by the finding that both inhibitors of PI3K-Akt and its main downstream signaling mTOR, LY294002, and rapamycin, respectively, can reverse the increase of IFN-γ production and cytotoxicity in NK cells promoted by Daphnetin. Collectively, we identify a natural product, Daphnetin, with the capacity of promoting human NK cell activation via PI3K-Akt-mTOR signaling in the presence of IL-12. Our current study opens up a new potential application for Daphnetin as a complementary immunomodulator for cancer treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yao, Yang, Yang, Li, Peng, Wu, Wang, Zhang, Huang, Wang, Xiong, Luo, Li, Jia, Deng and Deng.)

Daphnetin is a 7, 8 dihydroxy coumarin isolated from different medicinal plants of the Thymelaeaceae family and exhibits copious pharmacological activities including neuroprotection, anti-cancer, anti-malarial, anti-inflammatory, anti-parasitic and anti-arthritic activity. It has been proved to be an effective neuroprotective agent in several preclinical animal studies and cell line examinations. It is found to interact with different cellular mediators and signaling pathways to confer protection against neurodegeneration. The reactive oxygen species and inflammatory mediators are the major culprits of different neurodegenerative diseases. Oxidative stress activates the pro-apoptotic proteins and inhibits anti-apoptotic proteins, leading to neuronal cell death. Daphnetin restores cellular redox balance by upregulating the antioxidants level (GSH and SOD), anti-apoptotic protein (Bcl-2), as well as by reducing the levels of proinflammatory cytokines, executioner caspase-3, pro-apoptotic-Bax, and oxidative stress markers. Furthermore, activation of Nrf-2/HO-1 signaling and upregulation of HSP-70 governs the protection elicited by daphnetin against oxidative stress-induced neuronal apoptosis. Daphnetin modulated inhibition of JNK-MAPK, JAK-STAT, and TLR-4/NF-κB signaling pathways also contributed to its neuroprotective effect. The positive effects of daphnetin have been also related to its AChE, BChE, and BACE-1 inhibitory potential. The present review has been designed to explore the mechanistic interplay of various mediators in mediating the neuroprotective effects of daphnetin., (© 2021. Maj Institute of Pharmacology Polish Academy of Sciences.)

Daphnetin (7, 8-dihydroxycoumarin, DAPH), a coumarin derivative isolated from Daphne odora var., recently draws much more attention as a promising drug candidate to treat neuroinflammatory diseases due to its protective effects against neuroinflammation. However, itscontribution to chronic inflammatory pain is largely unknown. In the current work, we investigated the effects of DAPH in a murine model of inflammatory pain induced by complete Freund's adjuvant (CFA) and its possible underlying mechanisms. Our results showed that DAPH treatment significantly attenuated mechanical allodynia provoked by CFA. A profound inhibition of spinal glial activation, followed by attenuated expression levels of spinal pro-inflammatory cytokines, was observed in DAPH-treated inflammatory pain mice. Further study demonstrated that DAPH mediated negative regulation of spinal NF-κB pathway, as well as its preferential activation of Nrf2/HO-1 signaling pathway in inflammatory pain mice. This study, for the first time, indicated that DAPH might preventthe development of mechanical allodynia in mice with inflammatory pain. And more importantly, these data provide evidence for the potential application of DAPH in the treatment of chronic inflammatory pain., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Daphnetin, a biologically active coumarin derivative found in plants of the genus Daphne, is a potent antioxidant phenolic compound. The present work describes the mechanisms and kinetics of the HO, NO, HOO, and NO 2 scavenging activities of daphnetin in physiological environments using quantum chemistry calculations. The main antiradical mechanisms have been studied: formal hydrogen transfer (FHT), sequential electron transfer proton transfer (SETPT), sequential proton loss electron transfer (SPLET), and radical adduct formation (RAF). Besides its good HO scavenging activity in physiological environments, daphnetin is expected to exhibit good HOO and NO 2 scavenging activities in water with k overall  = 1.51 × 10 7 and 4.79 × 10 8  M -1 s -1 , respectively. The FHT mechanism decides the HO scavenging activity in aqueous solution, as well as HO, HOO, and NO 2 scavenging activities in lipid media, while SPLET is the primary mechanism in water for HOO and NO 2 scavenging activities. The theoretical predictions were found to be in good agreement with the available experimental data, which supports the reliability of the calculations., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Gentamicin (GM), an aminoglycoside antibiotic, is one of the most effective drugs used in the treatment of various types of bacterial infections, but the major adverse effect and drug-induced nephrotoxicity of GM limit its clinical applications. Daphnetin (Daph) is a natural coumarin derivative that is clinically used to treat rheumatoid arthritis and coagulopathy and exhibits antioxidant effects. However, the effect of Daph on GM-induced nephrotoxicity has not yet been elucidated. This study investigated Daph-mediated protection against GM-induced nephrotoxicity in mice and explored the underlying mechanisms of GM-induced renal dysfunction in mice. We found that Daph treatment significantly reduced GM-induced nephrotoxicity mainly by ameliorating renal injury in mice and attenuating cell damage in vitro. Mechanistically, we found that Daph upregulated the expression level of Nrf2 and its regulated antioxidant enzymes HO-1, NQO1, GCLC and GCLM in vivo and in vitro. GM upregulated the expression levels of NOX4, cleaved Caspase-3 and p53 and the BAX/BCL2 ratio in vivo to stimulate oxidative stress and apoptosis. However, Daph treatment significantly improved the oxidative stress and apoptosis caused by GM, thereby exerting antioxidative and antiapoptotic effects. Our study was the first to suggest that the natural product Daph protects against GM-induced nephrotoxicity through the activation of Nrf2 which regulates oxidative stress and apoptosis. The pharmacological activation of Nrf2 may be useful as a novel therapy to prevent renal injury., (Copyright © 2021. Published by Elsevier B.V.)

Leukemia is a malignant tissue-forming disease, which induces the overproduction of large numbers of immature blood cells entering the peripheral blood. It is well documented that inflammation plays a crucial role in the expansion of leukemia. Daphnetin has confirmed anti-inflammatory effects against various diseases. In this experimental study, we evaluated the anti-leukemia and anti-inflammatory effect of daphnetin against benzene-induced leukemia in rats and explored the underlying mechanism. Benzene was used for inducing leukemia in experimental rats. The rats were divided into different groups and the body weight, hematological parameters, bone marrow cells, cytokines, and inflammatory mediators were estimated. Reverse transcription polymerase chain reaction (RT-PCR) was used for estimating the messenger RNA (mRNA) expression of sphingosine-1-phosphate receptor-1. Daphnetin-treated rats showed upregulation of body weight compared to other groups. Moreover, Daphnetin reduced blasts in leukemic rats. It also altered hematological parameters such as red blood cells, white blood cells, lymphocytes, neutrophils, monocytes, eosinophils, monocytes, and basophils, respectively. Daphnetin-treated rats showed a reduction of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β (IL-1β), IL-2, IL-6, and inflammatory mediators including nuclear factor-κB. RT-PCR showed upregulated mRNA expression of sphingosine-1-phosphate receptor-1 of daphnetin-treated group rats compared to other groups. The current study showed that the anti-inflammatory effect of daphnetin against the benzene-induced leukemia via alteration of cytokines., (© 2021 Wiley Periodicals LLC.)

Introduction: Most of the immunosuppressive drugs are used for the treatment of autoimmune disease, allergic diseases, and transplant rejection, but toxicity is the major obstacle for the potent drugs in the wide use of these immunosuppressive drugs. Daphnetin, a Chinese herbal product, has been reported that daphnetin possesses antimicrobial, anticoagulation, antimalarial, anticancer, and antioxidant activity. In a previous study, we found that daphnetin exhibited a potential immunosuppressive effect on LPS-induced B lymphocyte cells in vitro , therefore, in this research, we investigated the immunosuppressive effects of daphnetin in BALB/c mice use OVA as a prototype antigen., Methods: Sixty BALB/c mice were divided into six groups. The emulsion (100 μL containing 100 μg OVA) was injected subcutaneously with OVA + CFA into the shaved backs of the BALB/c mice on day 1, and a boosting injection was administered in OVA + IFA 2 weeks later. Beginning on the day of immunization, the immunized mice were administered intraperitoneally with daphnetin at a dose of 5, 10, and 20 mg/kg in saline solution for 28 consecutive days. We measured the effect of daphnetin on OVA-specific antibody, cytokine production, and Splenocyte proliferation in vivo ., Results: The results revealed that daphnetin significantly suppressed serum immunoglobulin G levels (IgG), and the OVA-specific IgG subclasses IgG1 and IgG2b, daphnetin was also significantly decreased the Th1 and Th2 cytokine productions, inhibited the splenocytes proliferation rate in vivo ., Conclusions: It proved that daphnetin could suppress humoral response activity on OVA-sensitized mice, suggesting a potential role on daphnetin as a new immunosuppressive drug.

In this study, the microbiological, physicochemical, and flavor changes of turbot (Scophthalmus maximus) coated with a composite active coating of locust bean gum (LBG) and sodium alginate (SA) supplemented with daphnetin emulsions (0.16, 0.32, 0.64 mg·mL -1 ) were determined during 18 days of refrigerated storage (4 ± 1 °C). Results showed that LBG-SA coatings containing 0.32 mg·mL -1 daphnetin emulsions could significantly lower the total viable count (TVC), psychrophiles, Pseudomonas spp. and H 2 S-producing bacteria counts, and inhibit the productions of off-flavor compounds including the total volatile basic nitrogen (TVB-N), trimethylamine (TMA) and ATP-related compounds. 32 volatile compounds were identified by solid phase microextraction combined with gas chromatography-mass spectrometer method (SPME-GC/MS) during refrigerated storage and the treated turbot samples significantly lowered the relative content of fishy flavor compounds. Further, the LBG-SA coatings containing daphnetin could also delay the myofibril degradation of the turbot samples. These results indicated that the LBG-SA coatings with 0.32 mg·mL -1 daphnetin were a potential alternative way to improve the quality of turbot during refrigerated storage., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Ovarian cancer is one of the most common gynecological malignancies in the world. Daphnetin (Daph) was previously reported to possess antitumor potential, but its potential and molecular mechanisms in ovarian cancer remain poorly understood., Purpose: In the current study, we aimed to explore the antitumor effect and detailed mechanisms of Daph in ovarian cancer cells., Methods: The cytotoxic effect of Daph on ovarian cells was determined in vitro and in vivo. Cell growth, proliferation, apoptosis and ROS generation were measured by CCK8 assays, colony formation assays and flow cytometry. Western blotting was used to evaluate the related signal proteins. Immunofluorescence and transmission electron microscopy were used to evaluate markers of autophagy and autophagic flux. The antitumor effects were observed in the A2780 xenograft model. Moreover, Daph-induced autophagy was observed by enhanced LC3-II accumulation and endogenous LC3 puncta, and an autophagy inhibitor further enhanced the antitumor efficacy of Daph, which indicated that the cytoprotective role of autophagy in ovarian cancer., Results: We found that Daph exhibited antitumor effects by inducing ROS-dependent apoptosis in ovarian cancer, which could be reversed by N-acetyl cysteine (NAC). The AMPK/Akt/mTOR pathway was involved in Daph-mediated cytoprotective autophagy, and when Daph-mediated the expression level of AMPK and autophagy were blocked, there was robust inhibition of cell proliferation and induction of apoptosis. In addition, in the A2780 xenograft model, combined treatment with Daph and an autophagy inhibitor showed obvious synergetic effects on the inhibition of cell viability and promotion of apoptosis, without any side effects., Conclusion: Our results suggest that Daph triggers ROS-induced cell apoptosis and induces cytoprotective autophagy by modulating the AMPK/Akt/mTOR pathway. Moreover, the combination of Daph and autophagy inhibitor may be a potential therapeutic strategy for ovarian cancer., (Copyright © 2021. Published by Elsevier GmbH.)

Background/aims: Daphnetin (7,8-dihydroxycoumarin, DAP) exhibits various bioactivities, such as anti-inflammatory and antioxidant activities. However, the role of DAP in myocardial ischaemia/reperfusion (I/R) injury and I/R-related arrhythmia is still uncertain. This study aimed to investigate the mechanisms underlying the effects of DAP on myocardial I/R injury and electrophysiological properties in vivo and in vitro., Methods: Myocardial infarct size was measured by triphenyltetrazolium chloride staining. Cardiac function was assessed by echocardiographic and haemodynamic analyses. The levels of creatine kinase-MB, lactate dehydrogenase, malondialdehyde, superoxide dismutase, interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-α) were detected using commercial kits. Apoptosis was measured by terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labelling staining and flow cytometry. The viability of H9c2 cells was determined by the Cell Counting Kit-8 assay. In vitro, the levels of IL-6 and TNF-α were measured by quantitative PCR. The expression levels of proteins associated with apoptosis, inflammation, and the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor kappa B (TLR4/MyD88/NF-κB) signalling pathway were detected by Western blot analysis. The RR, PR, QRS, and QTc intervals were assessed by surface ECG. The 90% action potential duration (APD90), threshold of APD alternans, and ventricular tachycardia inducibility were measured by the Langendorff perfusion technique., Results: DAP preconditioning decreased myocardial I/R injury and hypoxia/reoxygenation (H/R) injury in cells. DAP preconditioning improved cardiac function after myocardial I/R injury. DAP preconditioning also suppressed apoptosis, attenuated oxidative stress, and inhibited inflammatory responses in vivo and in vitro. Furthermore, DAP preconditioning decreased the susceptibility to ventricular arrhythmia after myocardial I/R. Finally, DAP preconditioning inhibited the expression of TLR4, MyD88, and phosphorylated NF-κB (p-NF-κB)/P65 in mice subjected to I/R and cells subjected to H/R., Conclusions: DAP preconditioning protected against myocardial I/R injury and decreased susceptibility to ventricular arrhythmia by inhibiting the TLR4/MyD88/NF-κB signalling pathway., (© 2021 The Author(s) Published by S. Karger AG, Basel.)

Glucocorticoids have been widely used in multiple inflammatory and autoimmune diseases. However, long-term glucocorticoid therapy may result in osteoporosis. The present study aimed to evaluate the potential therapeutic effects and investigate the underlying mechanisms of Daphnetin (Daph) on glucocorticoid-induced osteoporosis (GIOP). In vivo, male Sprague Dawley rats were intramuscularly injected with dexamethasone (DEX) to induce GIOP and Daph was given intraperitoneally. Bone histological changes, mineral content, microstructure parameters and bone turnover markers were detected. Gut microbiota composition and intestinal barrier function were further assessed. In vitro, MC3T3-E1 pre-osteoblasts were treated with DEX and the abilities of Daph on osteoblast proliferation, differentiation and mineralization were assessed. A Wnt signaling inhibitor, XAV939, was added additionally to evaluate the effect of Daph on Wnt signaling. The results showed that in vivo, Daph increased the DEX-induced reduction in body weight gain, bone mineral content and microstructure parameters and restored the levels of bone turnover markers in GIOP rats. In vitro, Daph promoted osteoblast proliferation, differentiation and mineralization in DEX-treated MC3T3-E1 pre-osteoblasts. Moreover, Daph activated the Wnt/GSK-3β/β-catenin signaling pathway. XAV939 successfully abolished the beneficial effects of Daph on GIOP in vitro. Besides, Daph showed improvement on gut microbiota disorder and intestinal barrier dysfunction post GIOP. Collectively, these data demonstrated that Daph effectively ameliorates GIOP and the possible mechanism may be that Daph activated Wnt/GSK-3β/β-catenin signaling., (Copyright © 2020. Published by Elsevier Inc.)

Background: Psoriasis is a common chronic inflammatory skin disease. Keratinocytes hyperproliferation and excessive inflammatory response contribute to psoriasis pathogenesis. The agents able to attenuate keratinocytes hyperproliferation and excessive inflammatory response are considered to be potentially useful for psoriasis treatment. Daphnetin exhibits broad bioactivities including anti-proliferation and anti-inflammatory. This study aims to evaluate the anti-psoriatic potential of daphnetin in vitro and in vivo, and explore underlying mechanisms., Methods: HaCaT keratinocytes was stimulated with the mixture of IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α (M5) to establish psoriatic keratinocyte model in vitro. Cell viability was measured using Cell Counting Kit-8 (CCK-8). Quantitative Real-Time PCR (qRT-PCR) was performed to measure the mRNA levels of hyperproliferative marker gene keratin 6 (KRT6), differentiation marker gene keratin 1 (KRT1) and inflammatory factors IL-1β, IL-6, IL-8, TNF-α, IL-23A and MCP-1. Western blotting was used to detect the protein levels of p65 and p-p65. Indirect immunofluorescence assay (IFA) was carried out to detect p65 nuclear translocation. Imiquimod (IMQ) was used to construct psoriasis-like mouse model. Psoriasis severity (erythema, scaling) was scored based on Psoriasis Area Severity Index (PASI). Hematoxylin and eosin (H&E) staining was performed to examine histological change in skin lesion. The expression of inflammatory factors including IL-6, TNF-α, IL-23A and IL-17A in skin lesion was measured by qRT-PCR., Results: Daphnetin attenuated M5-induced hyperproliferation in HaCaT keratinocytes. M5 stimulation significantly upregulated mRNA levels of IL-1β, IL-6, IL-8, TNF-α, IL-23A and MCP-1. However, daphnetin treatment partially attenuated the upregulation of those inflammatory cytokines. Daphnetin was found to be able to inhibit p65 phosphorylation and nuclear translocation in HaCaT keratinocytes. In addition, daphnetin significantly ameliorate the severity of skin lesion (erythema, scaling and epidermal thickness, inflammatory cell infiltration) in IMQ-induced psoriasis-like mouse model. Daphnetin treatment attenuated IMQ-induced upregulation of inflammatory cytokines including IL-6, IL-23A and IL-17A in skin lesion of mice., Conclusions: Daphnetin was able to attenuate proliferation and inflammatory response induced by M5 in HaCaT keratinocytes through suppression of NF-κB signaling pathway. Daphnetin could ameliorate the severity of skin lesion and improve inflammation status in IMQ-induced psoriasis-like mouse model. Daphnetin could be an attractive candidate for future development as an anti-psoriatic agent.

Rheumatoid arthritis (RA) is a chronic, symmetric, systemic autoimmune disease. Because insufficient apoptosis of fibroblast-like synoviocytes (FLS) is an important characteristic of RA, promoting apoptosis is considered a potential therapeutic tool for treating RA. We have previously found that daphnetin (7,8-dihydroxycoumarin, DAP) has a pro-apoptotic effect on fibroblast-like synoviocytes from collagen-induced arthritis (CIA) rats. In the present study, we further investigated the mechanisms of DAP-induced apoptosis in CIA-FLS. CIA-FLS were incubated with DAP for 48 h in the presence or absence of caspase inhibitors, including inhibitors of caspase-3, caspase-8, or caspase-9 or a pan-caspase inhibitor; then, a series of experiments were performed to evaluate the mechanisms of DAP-induced apoptosis. Our results showed that DAP markedly decreased cell viability and induced the apoptosis of CIA-FLS along with typical morphological and ultrastructural changes; moreover, DAP increased FasL, cytochrome c (Cyt-c), Bax, caspase-3, caspase-8, and caspase-9 mRNA expression and Bax, caspase-3, caspase-8, and caspase-9 protein expression. In contrast, DAP decreased Bcl-2 mRNA and protein expression and promoted the release of Cyt-c from the mitochondria into the cytosol; these effects were attenuated to varying degrees by pre-treatment with caspase inhibitors, especially with caspase-3 or caspase-9 inhibitors or a pan-caspase inhibitor. In conclusion, the current findings demonstrate that the DAP-induced apoptosis of CIA-FLS occurred mainly via a caspase-dependent pathway, in particular the mitochondrial pathway, and that the Bax/Bcl-2 ratio was involved in this process. Thus, DAP may be a potential therapeutic agent for RA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Background: Oxidative stress-triggered fatal hepatotoxicity is an essential pathogenic factor in acute liver failure (ALF)., Aims: To investigate the protective effect of daphnetin (Daph) on tert-butyl hydroperoxide (t-BHP) and acetaminophen (APAP)-induced hepatotoxicity through altering Nrf2/Trx-1 pathway activation., Materials and Methods: In vivo, male C57BL/6 mice with Wild-type (WT) and Nrf2 -/- were divided into five groups and acute liver injury model were established by APAP or LPS/GalN after injection with Daph (20, 40, or 80 mg/kg), seperately. Then, liver tissue and serum were collected for biochemical determination, TUNEL and H & E staining, and western blot analysis. In vitro, HepG2 cells were used to investigate the protective effect and mechanism of daphnetin against ROS and apoptosis induced by t-BHP via apoptosis detection, western blot, immunofluorescence analysis, and sgRNA transfection., Results: Our results indicated that Daph efficiently inhibited t-BHP-stimulated hepatotoxicity, and modulated Trx-1 expression and Nrf2 activation which decreased Keap1-overexpression in HepG2 cells. Moreover, Daph inhibited t-BHP-excited hepatotoxicity and enhanced Trx-1 expression, which was reversed in Nrf2 -/- HepG2 cells. In vivo, a survival rate analysis first suggested that Daph significantly reduced the lethality induced by APAP or GalN/LPS in a Nrf2-dependent or -independent manner by using Nrf2 -/- mice, respectively. Next, further results implicated that Daph not only effectively alleviated APAP-induced an increase of ALT and AST levels, histopathological changes, ROS overproduction, malondialdehyde (MDA) formation and GSH/GSSG reduction, but it also relieved hepatic apoptosis by strengthening the suppression of cleaved-caspase-3 and expression of P53 protein. Additionally, Daph attenuated mitochondrial dysfunction by suppressing ASK1/JNK activation and decreasing apoptosis-inducing factor (AIF) and Cytochrome c release and Bax mitochondrial translocation. Daph inhibited inflammatory responses by inactivating the thioredoxin-interacting protein (Txnip)/NLRP3 inflammasome. Furthermore, Daph efficiently enhanced Nrf2 nuclear translocation and Trx-1 expression. However, these effects in WT mice were eliminated in Nrf2 -/- mice., Conclusions: These investigations demonstrated that Daph treatment has protective potential against oxidative stress-driven hepatotoxicity by inhibition of ASK1/JNK and Txnip/NLRP3 activation, which may be strongly related to the Nrf2/Trx-1 upregulation., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

To assess the potential role of daphnetin, a clinically used anti-inflammatory agent, on the development of the inflammatory and neurodegenerative disease, we investigated its immune regulatory function in a murine model of experimental autoimmune encephalomyelitis (EAE). Significantly, lower levels of pro-inflammatory cytokines including interleukin (IL)-17, interferon-γ, Il6, Il12a, and Il23a were observed in brains of daphnetin-treated EAE mice, compared with those in control littermates. We also confirmed that daphnetin suppressed the production of IL-1β, IL-6, and tumor necrosis factor-α in lipopolysaccharide-stimulated mouse BV2 microglial cells. Mechanistically, heme oxygenase-1 (HO-1), a canonical anti-oxidant and anti-inflammatory factor, was found to be substantially induced by daphnetin treatment in BV2 cells. Also, a significantly higher level of HO-1, accompanied by a decreased level of malondialdehyde, was observed in daphnetin-treated EAE mice. More importantly, the deletion of HO-1 in BV2 microglia largely abrogated daphnetin-mediated inhibition of the inflammatory response. Together, our data demonstrate that daphnetin has an anti-inflammatory and neuroprotective role during the pathogenesis of EAE, which is partially at least, dependent on its regulation of HO-1.

Daphnetin (DAP), an active ingredient extracted from Daphne odora, has pharmacological effects such as anti-inflammatory, antioxidation and anti-tumor properties. The current study aims to investigate the relationship between the anti-rheumatoid effect of DAP and the inhibition of both the PI3K/AKT/mTOR and autophagy signaling pathways. DAP inhibited the proliferation of CIA-FLS in a dose-dependent manner and induce apoptosis, accelerated the G1/G0 phase and inhibited the S phase. DAP reduced the phosphorylation of AKT and mTOR and the expression of Atg5, Beclin-1 and LC3-II/LC3-I in CIA-FLS induced by TNF-α. DAP also reduced the inflammatory response in CIA-FLS induced by TNF-α by inhibiting the cytokine expression of TNF-α, IL-6, TGF-β, IL-17, and INF-γ and promoting IL-10 expression. Overall, DAP inhibited the proliferation of CIA-FLS by down-regulating the PI3K/AKT/mTOR signaling pathway and inhibited autophagy in order to induces apoptosis, which may be potential therapeutic approach in treatment of RA., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aims: Daphnetin (DAP) is a traditional Chinese drug usually used to treat cardiovascular diseases. Studies have confirmed the anti-inflammatory, antioxidant, anti-bacterial and insecticidal, anti-tumor and neuro-protective effects of DAP. However, its anti-arthritic potential remains unexplored. The aim of this study is to investigate the in vitro and in vivo chondroprotective effects of DAP., Main Methods: The effect of DAP on primary rabbit chondrocytes was examined using recombinant human IL-1β for 24 h. For the in vivo studies, rabbits were randomly divided into groups: a normal control group and osteoarthritis (OA) groups. The OA groups received three different doses of DAP for 4 or 8 weeks. The anti-arthritic effect of DAP was assessed using histopathological examinations, qRT-PCR, western blotting and immunohistochemical analysis., Key Findings: Both in vitro and in vivo results indicate that DAP exerts a protective effect against IL-1β in chondrocytes. In vitro, DAP inhibits the expression of IL-6, IL-12, MMP-3, MMP-9 and MMP-13, induced by IL-1β in rabbit chondrocytes, and stimulates the production of IL-10. The inhibitory effect of DAP on the MMPs is partially regulated by the inhibition of the PI3K/AKT, MAPK and NF-κB signaling pathways. The effect of DAP on OA may be attributed to the suppression of inflammatory factor secretion, chondrocyte apoptosis observed by the decrease in pro-apoptotic Caspase-3 and BAX, and the activation of anti-apoptotic BCL-2., Significance: DAP has a broad range of prospects in the treatment of OA, which provides a novel therapeutic strategy for OA., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Aseptic prosthetic loosening and periprosthetic infection resulting in inflammatory osteolysis is a leading complication of total joint arthroplasty (TJA). Excessive bone destruction around the bone and prosthesis interface plays a key role in the loosening prostheses leading to revision surgery. The bacterial endotoxins or implant-derived wear particles-induced inflammatory response is the major cause of the elevated osteoclast formation and activity. Thus, agents or compounds that can attenuate the inflammatory response and/or inhibit the elevated osteoclastogenesis and excessive bone resorption would provide a promising therapeutic avenue to prevent aseptic prosthetic loosening in TJA. Daphnetin (DAP), a natural coumarin derivative, is clinically used in Traditional Chinese Medicine for the treatment of rheumatoid arthritis due to its anti-inflammatory properties. In this study, we report for the first time that DAP could protect against lipopolysaccharide-induced inflammatory bone destruction in a murine calvarial osteolysis model in vivo. This protective effect of DAP can in part be attributed to its direct inhibitory effect on RANKL-induced osteoclast differentiation, fusion, and bone resorption in vitro. Biochemical analysis found that DAP inhibited the activation of the ERK and NFATc1 signaling cascades. Collectively, our findings suggest that DAP as a natural compound has potential for the treatment of inflammatory osteolysis., (© 2019 Wiley Periodicals, Inc.)

The bacterial pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA) is a potentially fatal disease, featured with extensive infection, inflammation, and airway dysfunction. With the increasing emerging of drug-resistant strains, new therapeutic strategies beyond canonical antibiotic treatment are pressingly needed. Daphnetin (DAPH) is a natural coumarin derivative with anti-inflammation, anti-microorganism and anti-oxidative properties. However, the protective effect of DAPH on S. aureus-caused pneumonia and the mechanism involved are never explored. Here we show that DAPH treatment conferred substantial protection against S. aureus-induced pneumonia, characterized by the reduced inflammatory responses, the augmented bacterial clearance and the alleviated tissue damage. Our study indicates that DAPH significantly enhanced mTOR-dependent autophagic pathway, leading to the boosted microphage bactericidal activity and the suppressed inflammatory responses. Inhibition of autophagic pathway therefore largely abolished DAPH-elicited repression of inflammatory response and macrophage anti-bacterial capability. Together, we herein not only identify a novel, natural agent to combat bacterial pneumonia, but also underscore the significance of autophagic pathway in orchestrating antimicrobial and anti-inflammatory responses, which may have important implication for the treatment of the infectious diseases, particularly that caused by obstinate, antibiotic-resistant pathogens such as MRSA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: To investigate the effect of daphnetin (DAP) combined with insulin-like growth factor 1 (IGF-1) gene transfection on chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) in rats., Methods: Rat ADSCs were isolated and amplified by enzymatic digestion. The third generation ADSCs were treated with IGF-1 gene transfection as experimental group and normal ADSCs as control group. The cells of the two groups were treated with different concentrations of DAP (0, 30, 60, 90 μg/mL), respectively. Cell proliferation was detected by cell counting kit 8 (CCK-8) after cultured for 72 hours. After 14 days, real-time fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expressions of chondrocyte markers (collagen type Ⅱ and Aggrecan) in each group; and toluidine blue staining and collagen type Ⅱ immunohistochemical staining were performed., Results: CCK-8 assay showed that with the increase of DAP concentration, the cell absorbance ( A ) value of the control group and the experimental group increased gradually ( P <0.05). At the same DAP concentration, the cell A value of the experimental group was significantly higher than that of the control group ( P <0.05). Real-time fluorescence quantitative PCR and Western blot showed that with the increase of DAP concentration, the relative mRNA and protein expressions of collagen type Ⅱ and Aggrecan in the control group did not change significantly, and there was no significant difference among the different concentration groups ( P >0.05). But the mRNA and protein expressions of collagen type Ⅱ and Aggrecan in the experimental group increased gradually, and the 60 and 90 μg/mL DAP concentration groups were significantly higher than 0 μg/mL DAP concentration group ( P <0.05). At the same DAP concentration, the relative mRNA and protein expressions of collagen type Ⅱ and Aggrecan were significantly higher in the experimental group than in the control group ( P <0.05). Toluidine blue staining showed that with the increase of DAP concentration, there was no significant difference in cell staining between the control group and the experimental group. At the same DAP concentration, the cells in the experimental group were slightly darker than those in the control group. Immunohistochemical staining of collagen type Ⅱ showed that with the increase of DAP concentration, there was no significant difference in the cytoplasmic brown-yellow coloring of the cells in the control group. The cytoplasmic brown-yellow coloring of the cells in the experimental group gradually deepened, with 60 and 90 μg/mL DAP concentration groups significantly deeper than 0 μg/mL DAP concentration group. At the same DAP concentration, the color of the cells in the experimental group was significantly deeper than that in the control group., Conclusion: DAP can promote the proliferation of ADSCs in rats. The differentiation of ADSCs into chondrocytes induced by DAP alone was slightly, but DAP combined with IGF-1 gene transfection has obvious synergistic effect to promote chondrogenic differentiation of ADSCs.

NF-E2 p45-related factor 2 (Nrf2), which regulates the cellular antioxidant response, is a target for limiting tissue damage due to exposure to environmental toxicants, including arsenic. Daphnetin (Daph), a natural coumarin derivative, has been shown to induce remarkable antioxidant activity. The present study aimed to examine the protective effects and molecular mechanisms of Daph on arsenic-induced cytotoxicity in human lung epithelial cells. Our results demonstrate that Daph dramatically upregulated the antioxidant enzyme in a dose dependent manner, in association with induction of Nrf2 nuclear translocation and decreased Keap1 protein expression. Importantly, Daph also markedly induced the activation of AMP-activated protein kinase (AMPK), c-Jun NH 2 -terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) phosphorylation. Furthermore, Daph antagonized the arsenic-induced decreases in cell viability and the generation of reactive oxygen species (ROS). Notably, Daph pretreatment reversed the arsenic-induced decrease in anti-apoptotic factor B-cell lymphoma-2 (Bcl-2) and the increase in pro-apoptotic factor Bcl-2-associated X protein (Bax). The effects of Daph on Nrf2 and HO-1 activation, and arsenic-induced cell viability were largely weakened when Nrf2 was depleted in vitro. Accordingly, Daph might ameliorate arsenic-induced cytotoxicity and apoptosis, which may be linked to the induction of Nrf2-dependent antioxidant responses as well as stabilization of the anti-apoptotic factor Bcl-2 in human lung epithelial cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Daphnetin, a coumarin derivative extracted from Daphne odora var., was reported to possess a neuroprotective effect. Recently, it has been demonstrated that daphnetin attenuates ischemia/reperfusion (I/R) injury. However, the role of daphnetin in cerebral I/R injury and the potential mechanism have not been fully understood. The present study aimed to explore the regulatory roles of daphnetin on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cell injury in a model of hippocampal neurons. Our results demonstrated that daphnetin improved cell viability and reduced the lactate dehydrogenase leakage in OGD/R-stimulated hippocampal neurons. In addition, daphnetin inhibited oxidative stress and cell apoptosis in hippocampal neurons after OGD/R stimulation. Furthermore, daphnetin significantly enhanced the nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in hippocampal neurons exposed to OGD/R. Knockdown of Nrf2 blocked the protective effect of daphnetin on OGD/R-induced hippocampal neurons. In conclusion, these findings demonstrated that daphnetin attenuated oxidative stress and neuronal apoptosis after OGD/R injury through the activation of the Nrf2/HO-1 signaling pathway in hippocampal neurons. Thus, daphnetin may be a novel therapeutic agent for cerebral I/R injury., (© 2018 Wiley Periodicals, Inc.)

Background: Antibiotic-resistant H. pylori was increasingly found in infected individuals, which resulted in treatment failure and required alternative therapeutic strategies. Daphnetin, a coumarin-derivative compound, has multiple pharmacological activities., Methods: The mechanism of daphnetin on H. pylori was investigated focusing on its effect on cell morphologies, transcription of genes related to virulence, adhesion, and cytotoxicity to human gastric epithelial (GES-1) cell line., Results: Daphnetin showed good activities against multidrug resistant (MDR) H. pylori clinical isolates, with minimal inhibitory concentration (MIC) values ranging from 25 to 100 μg/mL. In addition, daphnetin exposure resulted in H. pylori morphological changes. Moreover, daphnetin caused increased translocation of phosphatidylserine (PS), DNA damage, and recA expression, and RecA protein production vs. control group. Of great importance, daphnetin significantly decreased H. pylori adhesion to GES-1 cell line vs. control group, which may be related to the reduced expression of colonization related genes (e.g., babA and ureI )., Conclusions: These results suggested that daphnetin has good activity against MDR H. pylori. The mechanism(s) of daphnetin against H. pylori were related to change of membrane structure, increase of DNA damage and PS translocation, and decrease of H. pylori attachment to GES-1 cells.

Diabetic nephropathy (DN) is one of the most common causes of end-stage renal disease (ESRD). Oxidative stress and inflammation have been documented to play important roles in the pathogenesis of DN. Daphnetin, a natural coumarin compound, possesses antioxidant and anti-inflammatory activities. However, the role of daphnetin in DN has not yet been investigated. The aim of the present study was to explore the function of daphnetin in DN and the underlying mechanism in vitro. Our results demonstrated that daphnetin alleviated cell proliferation induced by high glucose (HG) in human mesangial cells (MCs). Daphnetin strikingly reduced reactive oxygen species (ROS) and malonaldehyde (MDA) levels, and induced the superoxide dismutase (SOD) activity in HG-stimulated MCs. Besides, the production of TNF-α, IL-1β, IL-6, fibronectin (FN) and collagen IV (Col IV) was also inhibited by daphnetin in HG-stimulated MCs. In addition, daphnetin enhanced the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) and inhibited the levels of p-Akt and p-p65 in HG-stimulated MCs. The results indicated that daphnetin inhibited HG-induced oxidative stress, inflammatory response, and ECM accumulation in human MCs. The effect is partially mediated by Nrf2/keap1 and Akt/NF-κB pathways. The findings suggested that daphnetin might be a therapeutic or preventive agent for DN., (Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Daphnetin, one of the major bioactive components isolated from Daphne odora, has been reported to have anti-inflammatory and anti-oxidative effects. Inflammation and oxidative stress have been known to play critical roles in cisplatin-induced nephrotoxicity. The purpose of this study was to investigate the protective effects of daphnetin on cisplatin-induced nephrotoxicity. The levels of blood urea nitrogen (BUN) and creatinine, as well as the kidney reactive oxygen species (ROS), and malondialdehyde (MDA) activity were measured in this study. The expression of inflammatory cytokines TNF-α and IL-1β were measured by ELISA. The results showed that daphnetin protected against cisplatin-induced nephrotoxicity by attenuating kidney histological changes, serum BUN and creatinine. Furthermore, the expression of TNF-α and IL-1β, as well as ROS and MDA in kidney tissues were decreased by daphnetin. In addition, daphnetin dose-dependently inhibited cisplatin-induced NF-κB activation and up-regulated the expression of Nrf2 and HO-1. In conclusion, the results of this study suggested that daphnetin inhibited cisplatin-induced nephrotoxicity by inhibiting NF-κB and activating Nrf2 signaling pathways. Daphnetin might be a promising agent in the treatment of kidney injury., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This paper describes the very simple and robust ratiometric photonic switching properties of daphnetin (DP) toward HPO 4 2- ions selectively in complex biological fluids, without any interference from other relevant anions under physiological conditions. The sensing ability of DP toward HPO 4 2- ions was first demonstrated using UV-Vis and fluorescence spectroscopy, dynamic light scattering (DLS), and one- and two-dimensional NMR spectroscopy. DP can detect HPO 4 2- ions at concentrations up to the sub-micromolar/nanomolar level very effectively, with a ratiometric response resulting from intramolecular charge transfer aided by aggregated-induced emission. The interactions between DP and HPO 4 2- ions resulted in new bands appearing in the UV-Vis (at 385 nm) and emission (at 535 nm) spectra. The noncovalently held HPO 4 2- ions induced pronounced specific aggregation of DP molecules, resulting in the new excimer band at 535 nm while retaining the monomer band centered at 445 nm. In contrast, reciprocal absorptivity changes were observed at 320 and 385 nm, with exponential decrements and increments, respectively. This probe could effectively monitor the consumption of dNTPs during various cycles of the polymerase chain reaction performed with relatively short oligonucleotides as well as genomic DNA from Agrobacterium tumefaciens (AcH5α strain)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Acute liver failure (ALF), an inflammation-mediated hepatocellular injury process, is a life-threatening and fatal clinical syndrome. Daphnetin (Daph) has been reported to exhibit various pharmacological activities, particularly its antiinflammatory property. The present study was designed to evaluate the protective effects and underlying mechanisms of Daph against lipopolysaccharide and d-galactosamine (LPS/GalN)-induced ALF in mice. Our findings suggested that Daph treatment efficiently protected against LPS/GalN-induced ALF by lessening the lethality, decreasing the alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 secretion, malondialdehyde (MDA) formation and myeloperoxidase (MPO) level, inhibiting nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) protein expression, and increasing the glutathione (GSH) and superoxide dismutase (SOD) contents. Moreover, Daph treatment effectively inhibited LPS/GalN-induced nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3), mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathway activation, and significantly induced autophagy activation by enhancing the level of pro-autophagy proteins expression. Taken together, these findings suggested that Daph plays a pivotal role in liver protection by suppressing inflammatory responses which may be closely associated with the inhibition of NLRP3 inflammasome, MAPK and NF-κB activation, as well as the induction of autophagy., (Copyright © 2018. Published by Elsevier B.V.)

Objective: The aim of this study was to develop a drug-in-adhesive patch for transdermal delivery of daphnetin (DA), which is a coumarin derivative in Girald Daphne, and to investigate the role of Transcutol P (TP) in the release and percutaneous permeation processes of DA., Methods: Backing films, permeation enhancers and enhancer content in the transdermal patch were investigated through in vitro experiments using rat skin. Anti-inflammatory and analgesic effects of the optimized formulation were evaluated using the adjuvant arthritis model and the pain model induced by acetic acid, respectively. In addition, the enhancement effect of TP was investigated using differential scanning calorimetry (DSC), FTIR, and molecular dynamic simulation., Results: The optimal formulation, composed of DURO-TAK ® 87-2852, CoTran TM 9680, 1% DA, and 10% TP showed anti-inflammatory and analgesic effects. It was found that TP only promoted the release process of DA from its transdermal patch. Furthermore, the decrease of interaction between drug and pressure sensitive adhesive (PSA) as well as the improvement of PSA mobility due to TP addition were the main factors that enhanced the release of DA from patch., Conclusions: This study successfully used TP to develop a DA patch with good anti-inflammatory and analgesic effects, proving that TP promotes the release of DA by reducing the interaction between DA and PSA and increasing the mobility of PSA.

In this study, daphnetin 1 was chosen as the lead compound, and C-3 or C-4-substituted daphnetins were designed and synthesized to explore the potential relationship between the antioxidant activities and the chemical structures of daphnetin derivatives. The antioxidant activities of the generated compounds were evaluated utilizing the free radical scavenging effect on 2,2'-diphenyl-1-picrylhydrazyl, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) cation, and the ferric reducing power assays, and were then compared with those of the standard antioxidant Trolox. The results showed that the catechol group was the key pharmacophore for the antioxidant activity of the daphnetins. The introduction of an electron-withdrawing hydrophilic group at the C-4 position of daphnetin enhanced the antioxidative capacity, but this trend was not observed for C-3 substitution. In addition, introduction of a a hydrophobic phenyl group exerted negative effects on the antioxidant activity in both the C-3 and C-4 substitutions. Among all of the derivatives tested, the most powerful antioxidant was 4-carboxymethyl daphnetin (compound 9 ), for which the strongest antioxidant activity was observed in all of the assays. In addition, compound 9 also displayed strong pharmaceutical properties in the form of metabolic stability. To summarize, compound 9 holds great potential to be developed as an antioxidant agent with excellent antioxidant activity and proper pharmacokinetic behavior.

Objective: Here, we used various approaches to investigate the suppressive role of daphnetin in LPS-induced inflammatory response, with the goal to understand the underlining molecular mechanism by which daphnetin regulated these processes., Methods: We examined the survival rate and the lung injury in the mice model of LPS-induced endotoxemia. The production of pro-inflammatory factors including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), IL-6, nitric oxide (NO), and prostaglandin E2 (PGE2) was measured by ELISA and nitrite analysis, respectively. The expression of inducible NO synthase (iNOS), cyclooxygenase 2 (COX-2), and the activation of signaling molecules was determined by immunoblotting. The production of reactive oxygen species (ROS) was measured by the ROS assay., Results: In vivo study showed that daphnetin enhanced the survival rate and reduced the lung injury in mice with LPS-induced endotoxemia. Both in vivo and in vitro study showed that daphnetin prevented the production of pro-inflammatory factors including TNF-α, IL-1β, IL-6, NO, and PGE2 after LPS challenge. In Raw264.7 cells, we found that daphnetin reduced LPS-induced expression of iNOS and COX-2, and suppressed LPS-induced ROS production. In addition, we found that daphnetin suppressed the activation of JAK/STATs pathway and inhibited the nucleus import of STAT1 and STAT3., Conclusions: Here, our results indicate that daphnetin shows anti-inflammatory properties, at least in part, through suppressing LPS-induced activation of JAK/STATs cascades and ROS production.

1. Finding and developing inhibitors of catechol-O-methyltransferase (COMT) from natural products is highly recommended. Daphnetin, a naturally occurring catechol from the family thymelaeaceae, has a chemical structure similar to several potent COMT inhibitors reported previously. Here the potential of daphnetin and its Phase II metabolites as inhibitors of COMT was investigated with human liver cytosol (HLC). 2. Daphnetin and its methylated metabolite (8-O-methyldaphnetin) were found to inhibit COMT-mediated dopamine O-methylation in a dose-dependent manner. The IC 50 values for daphnetin (0.51∼0.53 μM) and 8-O-methyldaphnetin (22.5∼24.3 μM) were little affected by changes in HLC concentrations. Further kinetic analysis showed the differences in inhibition type and parameters (K i ) between daphnetin (competitive, 0.37 μM) and 8-O-methyldaphnetin (noncompetitive, 25.7 μM). Other metabolites, including glucuronidated and sulfated species, showed negligible inhibition against COMT. By using in vitro-in vivo extrapolation (IV-IVE), a 24.3-fold increase in the exposure of the COMT substrates was predicted when they are co-administrated with daphnetin. 3. With high COMT-inhibiting activity, daphnetin could serve as a lead compound for the design and development of new COMT inhibitors. Also, much attention should be paid to the clinical impact of combination of daphnetin and herbal preparations containing daphnetin with the drugs primarily cleared by COMT.

To establish HPLC-MS/MS method for simultaneous determination of daphnetin, daphnoretin, and daphneticin in rat plasma after oral and intravenous administration of Daphne giraldii extract, and then use them in the calculation of pharmacokinetic parameters. Six sprague-dawley rats received intragastric administration of D. giraldii extract (daphnetin, daphnoretin and daphneticin were 88.40, 3.24 and 4.28 mg•kg⁻¹, respectively). Their drug plasma concentration was determined by LC-MS/MS with schisandrin as an internal standard to draw plasma concentration-time curve. The pharmacokinetic parameters were calculated by Kinetica 4.4. The results showed that the linear range was 5-1 000 μg•L⁻¹ for daphnetin, daphnoretin and daphneticin, and the method ological test showed conformance to the requirements.The intraday and inter-day variable coefficients (RSD) were both less than 15.0%, indicating that both of legitimate precise and accuracy were consistent with the analysis requirements of biological samples. For daphnetin, the pharmacokinetic parameters Tmax, Cmax, AUC0-t, T1/2 and MRT were 4 h, 858.96 μg•L⁻¹, 10 566.4 μg•L⁻¹•h, 5.19 h and 9.43 h, respectively. For daphnoretin, the pharmacokinetic parameters Tmax, Cmax, AUC0-t, T1/2 and MRT were 2.92 h, 178.00 μg•L⁻¹, 905.89 μg•L⁻¹•h, 3.50 h and 6.95 h, respectively. For daphneticin, the pharmacokinetic parameters Tmax, Cmax, AUC0-t, T1/2 and MRT were 2 h, 36.67 μg•L⁻¹, 355.11 μg•L⁻¹•h, 4.95 h and 8.27 h, respectively. The LC-MS/MS analysis method established in this study was proved to be so accurate and sensitive that it can be applied to the pharmacokinetic study of daphnetin, daphnoretin and daphneticin., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Daphnetin (Daph), a natural coumarin derivative isolated from plants of the Genus Daphne, possesses abundant biological activities, such as anti-inflammatory, antioxidant and anticancer properties. In the present study, we focused on investigating the protective effect of Daph against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage, mitochondrial dysfunction and the involvement of underlying molecular mechanisms. Our findings indicated that Daph effectively inhibited t-BHP-stimulated cytotoxicity, cell apoptosis, and mitochondrial dysfunction, which are associated with suppressed reactive oxygen species (ROS) generation, decreased malondialdehyde (MDA) formation, increased superoxide dismutase (SOD) levels and glutathione (GSH)/GSSG (oxidized GSH) ratio. Further investigation indicated that Daph significantly suppressed cytochrome c release and NLRP3 inflammasome activation and modulated apoptosis-related protein Bcl-2, Bax, and caspase-3 expression. Moreover, Daph dramatically induced the expression of the glutamate-cysteine ligase modifier (GCLM) subunit and the glutamate-cysteine ligase catalytic (GCLC) subunit, heme oxygenase-1 (HO-1), and NAD (P) H: quinone oxidoreductase (NQO1), which is largely dependent on upregulating the nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation, reducing the Keap1 protein expression, and strengthening the antioxidant response element (ARE) promoter activity. Additionally, Daph remarkably activated a c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) phosphorylation, but ERK and JNK inhibitor pretreatment exhibited an evident decrease of the level of Daph-enhanced Nrf2 nuclear translocation. Furthermore, Daph exposure suppressed t-BHP-induced cytotoxicity and ROS overproduction, which are mostly blocked in Nrf2 knockout RAW 264.7 cells and peritoneal macrophages. Accordingly, Daph exhibited protective roles against t-BHP-triggered oxidative damage and mitochondrial dysfunction by the upregulation of Nrf2 antioxidant signaling pathways, which may be involved in the activation of JNK and ERK., (Copyright © 2017. Published by Elsevier Inc.)

Background and Objective: Daphnetin (7, 8-dihydroxycoumarin), a natural coumarin compound, is known to exhibit antioxidant and anti-inflammatrory effects. However, the underlying mechanisms of its anti-apoptotic and antidiabetic effects yet not been examined. Therefore, the present work studied the anti-apoptotic and anti-diabetic effects of daphnetin by in vitro experiments., Methods: The rat insulinoma (INS-1) cells were pre-treated with daphnetin at different concentrations (1, 10, 20 and 40µM) for 24h followed by exposition to streptozotocin (STZ) (3mM) for 12h. Effects of daphnetin and STZ on INS-1 cells were determined by MTT assay, glucose stimulated insulin secretion (GSIS) assay, lipid peroxidation, antioxidant status (SOD, CAT, GPx, and GST) Apoptosis staining (DAPI, Hoechst 33342, AO/EB and ROS) was performed by fluorescence microscopy, and Bcl-2, Bax and NF-κB protein expression was detected by Western blotting., Results: MTT assay indicated that the viability of INS-1 cells was significantly reduced with exposure to STZ for 12h as compared to control cells, while pre-treated with daphnetin for 24h resulted in a significant improvement of cell viability. The effects daphnetin treatment in INS-1 cells on insulin secretion was tested and results showed that the pre-treatment of daphnetin could improve GSIS. Further, daphnetin pre-treatment significantly reduced the levels of lipid peroxidation markers and also improved antioxidant enzymes' activities in STZ-induced INS-1 cells. Western blotting assay revealed that daphnetin could suppress apoptosis through up-regulation of anti-apoptotic Bcl-2 protein expression and the down-regulation of pro-apoptotic Bax and nuclear factor NF-κB protein levels., Conclusion: The results showed that daphnetin might be used in treating diabetes due to its insulin stimulating property and subsequent regulation of apoptotic pathway., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Coumarins, identified as plant secondary metabolites possess diverse biological activities including anti-angiogenic properties. Daphnetin (DAP), a plant derived dihydroxylated derivative of coumarin has shown significant pharmacological properties such as anticancer, anti-arthritic and anti-inflammatory. The present study was performed to investigate the anti-angiogenic potential of DAP, focusing on the mechanism of action. The in vivo anti-angiogenic potential of DAP was evaluated by vascular endothelial growth factor (VEGF)-induced rat aortic ring (RAR) assay and chick chorioallantoic membrane (CAM) assay. For in vitro evaluation, wounding migration, transwell invasion, tube formation and apoptosis assays were performed on VEGF (8 ng/mL)-induced human umbilical vein endothelial cells (HUVECs). The cellular mechanism of DAP was examined on TNFα (10 ng/mL) and VEGF-induced HUVECs by extracting the mRNA and protein levels using RT-qPCR and western blotting. Our data demonstrated that DAP inhibited the in vivo angiogenesis in the RAR and CAM assay. DAP also inhibited the different steps of angiogenesis, such as migration, invasion, and tube formation in HUVECs. DAP inhibited nuclear factor-κB signalling together including TNF-α induced IκBα degradation; phosphorylation of IκB kinase (IKKα/β) and translocation of the NF-κB-p65 protein. Furthermore, western blotting revealed that DAP significantly down-regulated the VEGF-induced signalling such as c-Src, FAK, ERK1/2 and the related phosphorylation of protein kinase B (Akt) and VEGFR2 expressions. DAP reduced the elevated mRNA expression of iNOS, MMP2 and also, induced apoptosis in VEGF-stimulated HUVECs by the caspase-3 dependent pathway. Taken together, this study reveals that DAP may have novel prospective as a new multi-targeted medication for the anti-angiogenesis and cancer therapy., (© 2016 John Wiley & Sons Australia, Ltd.)

Ethnopharmacological Relevance: In Turkey, daphnetin-containing Daphne oleoides is used as a folk medicine for treating rheumatic pain and lumbago. A daphnetin-containing traditional Chinese medicine tablet, named Zushima-Pian, is available in China for treating rheumatoid arthritis. The present study aimed to investigate the metabolism of daphnetin through sulfation in cultured human cells and to identify the human cytosolic sulfotransferase(s) (SULT(s)) that is(are) capable of mediating the sulfation of daphnetin., Materials and Methods: Cultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells were labeled with [(35)S]sulfate in the presence of different concentrations of daphnetin. Thirteen known human SULTs, previously expressed and purified, as well as cytosols of human kidney, liver, lung, and small intestine, were examined for daphnetin-sulfating activity using an established sulfotransferase assay., Results: [(35)S]sulfated daphnetin was found to be generated and released by HepG2 cells and Caco-2 cells labeled with [(35)S] sulfate in the presence of daphnetin. Among the 13 known human SULTs, SULT1A1, SULT1A2, SULT1A3, SULT1B1, and SULT1C4 displayed significant sulfating activity toward daphnetin. Of the four human organ samples later tested, small intestine and liver cytosols displayed considerably higher daphnetin-sulfating activity than those of lung and kidney., Conclusion: The results derived from the present study showed unequivocally that daphnetin could be sulfated in cultured human cells and by purified human SULT enzymes as well as human organ cytosols. The information obtained provided a basis for further studies on the metabolism of daphnetin through sulfation in vivo., Competing Interests: The authors declare no conflict of interest., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Cancer is a faction of disorders that conjugated primarily with oxidative imbalance. In mammary carcinoma, oxidative stress secondarily changes various gene expressions and signalling pathways that bring genomic instability and mutagenic alterations that fascinating carcinogenesis. Several coumarin compounds are active against various malignancies. Among them, daphnetin (DAP) exhibits valuable safety and bioactivity profile that contributes towards its efficacy against cancer. In this study, the antioxidative and chemotherapeutic potential of DAP against 7,12-dimethylbenz(a)anthracene (DMBA)- induced mammary carcinogenesis was evaluated in female Sprague-Dawley rats. Besides this, we have determined the effect of DAP on Keap1-Nrf-2, associated HO-1 and NF-κB expressions behind the antioxidative and anti-proliferating activity. In our findings, a protective effect of DAP was established against lipid peroxidation, enzymic (Total SOD, MnSOD, CuZnSOD, CAT, GPx) and non-enzymic (GSH) antioxidative markers in serum, liver, kidney and breast tissue of both control and experimental groups. An up-regulation of protective Nrf-2 & HO-1 with a synchronized suppression in Keap1 & NF-κB mRNA and protein expressions were observed. DAP revealed the inhibition of p-AKT which accountable for decrease in NF-κB expressions but shown to be ineffective on p-ERK1/2. This study revealed that DAP inhibits mammary carcinogenesis through multiple mechanisms. Dual efficacy of DAP on Nrf-2-Keap1 pathway and NF-κB expressions propose it as a potential chemotherapeutic agent in mammary cancer management., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Aims: Daphnetin, a coumarin derivative extracted from Daphne odora var. marginata, has been reported to have antiinflammatory and immunosuppressive properties. Our previous study indicated that it was able to remarkably suppress the neuroinflammation and suggested its potential application in treating neuroinflammatory diseases. Multiple sclerosis (MS), a Th cell-mediated autoimmune disease, is the most common inflammatory demyelinating disease of the central nervous system (CNS). We examined whether daphnetin treatment can protect mice against experimental autoimmune encephalomyelitis (EAE), an animal model for MS., Methods: To assess the effect of daphnetin in neuroinflammatory diseases, the EAE mice were established and treated with daphnetin at 8 mg/kg for 28 days. The severity of neuroinflammation and demyelination in the spinal cords was examined histopathologically. Infiltration of CD4(+) T cells into the CNS was assessed by immunohistochemistry, and the cytokine production was determined by ELISA. Meanwhile, the effect of daphnetin on the activity of dendritic cells (DCs) was evaluated, as assessed by DCs' capability to express surface markers, secrete cytokines, and activate naïve CD4(+) T cells. Furthermore, we explored the molecular mechanisms whereby DAPH regulated DCs' activity and thereby CD4(+) T cell responses., Results: The administration of daphnetin markedly alleviated the clinical symptoms of EAE and reduced the CNS inflammation and demyelination in experimental mice. Th1 and Th17 cell responses were profoundly repressed in daphnetin-treated EAE mice. Mechanistically, daphnetin treatment significantly repressed the activation, maturation, and antigen-presenting capability of DCs. NF-κB signaling was significantly reduced in daphnetin-treated DCs, along with a concomitant induction of heme oxygenase-1, a negative regulator of inflammatory signaling., Conclusions: Our findings for the first time demonstrate the property of daphnetin in regulating DCs' function and subsequently Th development. Given the low or absent toxicity associated with daphnetin, our data may suggest a novel safe and effective approach to control autoimmune neuroinflammation., (© 2016 John Wiley & Sons Ltd.)

Coumarins are important plant-derived natural products with wide-ranging bioactivities and extensive applications. In this study, we evaluated for the first time the antibacterial activity and mechanisms of action of coumarins against the phytopathogen Ralstonia solanacearum, and investigated the effect of functional group substitution. We first tested the antibacterial activity of 18 plant-derived coumarins with different substitution patterns, and found that daphnetin, esculetin, xanthotol, and umbelliferone significantly inhibited the growth of R. solanacearum. Daphnetin showed the strongest antibacterial activity, followed by esculetin and umbelliferone, with MICs of 64, 192, and 256 mg/L, respectively, better than the archetypal coumarin with 384 mg/L. We further demonstrated that the hydroxylation of coumarins at the C-6, C-7 or C-8 position significantly enhanced the antibacterial activity against R. solanacearum. Transmission electron microscope (TEM) and fluorescence microscopy images showed that hydroxycoumarins may interact with the pathogen by mechanically destroying the cell membrane and inhibiting biofilm formation. The antibiofilm effect of hydroxycoumarins may relate to the repression of flagellar genes fliA and flhC. These physiological changes in R. solanacearum caused by hydroxycoumarins can provide information for integral pathogen control. The present findings demonstrated that hydroxycoumarins have superior antibacterial activity against the phytopathogen R. solanacearum, and thus have the potential to be applied for controlling plant bacterial wilt.

Daphnetin, 7,8-dihydroxycoumarin, present in main constituents of Daphne odora var. marginatai, has multiple pharmacological activities including anti-proliferative effects in cancer cells. In this study, using a Transwell system, we showed that daphnetin inhibited invasion and migration of highly metastatic murine osteosarcoma LM8 cells in a dose-dependent manner. Following treatment by daphnetin, cells that penetrated the Transwell membrane were rounder than non-treated cells. Immunofluorescence analysis revealed that daphnetin decreased the numbers of intracellular stress fibers and filopodia. Moreover, daphnetin treatment dramatically decreased the expression levels of RhoA and Cdc42. In summary, the dihydroxycoumarin derivative daphnetin inhibits the invasion and migration of LM8 cells, and therefore represents a promising agent for use against metastatic cancer., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Our previous study demonstrated that daphnetin is subject to glucuronidation in vitro. However, daphnetin metabolism is still poorly documented. This study aimed to investigate daphnetin metabolism and its consequent effect on the bioactivity. Metabolic profiles obtained by human liver S9 fractions and human hepatocytes showed that daphnetin was metabolized by glucuronidation, sulfonation, and methylation to form 6 conjugates which were synthesized and identified as 7-O-glucuronide, 8-O-glucuronide, 7-O-sulfate and 8-O-sulfate, 8-O-methylate, and 7-O-suflo-8-O-methylate. Regioselective 8-O-methylation of daphnetin was investigated using in silico docking calculations, and the results suggested that a close proximity (2.03 Å) of 8-OH to the critical residue Lysine 144 might be the responsible mechanism. Compared with glucuronidation and sulfonation pathways, the methylation of daphnetin had a high clearance rate (470 μL/min/mg) in human liver S9 fractions and contributed to a large amount (37.3%) of the methyl-derived metabolites in human hepatocyte. Reaction phenotyping studies showed the major role of SULT1A1, -1A2, and -1A3 in daphnetin sulfonation, and soluble COMT in daphnetin 8-O-methylation. Of the metabolites, only 8-O-methyldaphnetin exhibited an inhibitory activity on lymphocyte proliferation comparable to that of daphnetin. In conclusion, methylation is a crucial pathway for daphnetin clearance and might be involved in pharmacologic actions of daphnetin in humans., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)