Daphnetin ameliorated GM-induced renal injury through the suppression of oxidative stress and apoptosis in mice.

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.

Fan X ，Gu W ，Gao Y ，Ma N ，Fan C ，Ci X ... -  《-》

Daphnetin-mediated Nrf2 antioxidant signaling pathways ameliorate tert-butyl hydroperoxide (t-BHP)-induced mitochondrial dysfunction and cell death.

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.

Lv H ，Liu Q ，Zhou J ，Tan G ，Deng X ，Ci X ... -  《-》

Daphnetin activates the Nrf2-dependent antioxidant response to prevent arsenic-induced oxidative insult in human lung epithelial cells.

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 NH2-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.

Lv X ，Li Y ，Xiao Q ，Li D ... -  《-》

Enhanced Keap1-Nrf2/Trx-1 axis by daphnetin protects against oxidative stress-driven hepatotoxicity via inhibiting ASK1/JNK and Txnip/NLRP3 inflammasome activation.

Oxidative stress-triggered fatal hepatotoxicity is an essential pathogenic factor in acute liver failure (ALF). 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. 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. 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. 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.

Lv H ，Zhu C ，Wei W ，Lv X ，Yu Q ，Deng X ，Ci X ... -  《-》

Up-regulation of Nrf2/HO-1 and inhibition of TGF-β1/Smad2/3 signaling axis by daphnetin alleviates transverse aortic constriction-induced cardiac remodeling in mice.

Oxidative damage and accumulation of extracellular matrix (ECM) components play a crucial role in the adverse outcome of cardiac hypertrophy. Evidence suggests that nuclear factor erythroid-derived factor 2 related factor 2 (Nrf2) can modulate oxidative damage and adverse myocardial remodeling. Daphnetin (Daph) is a coumarin obtained from the plant genus Daphne species that exerts anti-oxidative and anti-inflammatory properties. Herein, we investigated the roles of Daph in transverse aortic constriction (TAC)-induced cardiac hypertrophy and fibrosis in mice. TAC-induced alterations in cardiac hypertrophy markers, histopathological changes, and cardiac function were markedly ameliorated by oral administration of Daph in mice. We found that Daph significantly reduced the reactive oxygen species (ROS) generation, increased the nuclear translocation of Nrf2, and consequently, reinstated the protein levels of NAD(P)H quinone dehydrogenase1 (NQO1), heme oxygenase-1 (HO-1), and other antioxidants in the heart. Besides, Daph significantly inhibited the TAC-induced accumulation of ECM components, including α-smooth muscle actin (α-SMA), collagen I, collagen III, and fibronectin, and interfered with the TGF-β1/Smad2/3 signaling axis. Further studies revealed that TAC-induced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive nuclei and the protein levels of Bax/Bcl2 ratio and cleaved caspase 3 were substantially decreased by Daph treatment. We further characterized the effect of Daph on angiotensin II (Ang-II)-stimulated H9c2 cardiomyoblast cells and observed that Daph markedly decreased the Ang-II induced increase in cell size, production of ROS, and proteins associated with apoptosis and fibrosis. Mechanistically, Daph alone treatment enhanced the protein levels of Nrf2, NQO1, and HO-1 in H9c2 cells. The inhibition of this axis by Si-Nrf2 transfection abolished the protective effect of Daph in H9c2 cells. Taken together, Daph effectively counteracted the TAC-induced cardiac hypertrophy and fibrosis by improving the Nrf2/HO-1 axis and inhibiting the TGF-β1/Smad2/3 signaling axis.

Syed AM ，Kundu S ，Ram C ，Kulhari U ，Kumar A ，Mugale MN ，Mohapatra P ，Murty US ，Sahu BD ... -  《-》