DDX3X alleviates doxorubicin-induced cardiotoxicity by regulating Wnt/ß-catenin signaling pathway in an in vitro model.

The life-threatening adverse effects of doxorubicin (Dox) caused by its cardiotoxic properties limit its clinical application. DDX3X has been shown to participate in a variety of physiological processes, and it acts as a regulator of Wnt/ß-catenin signaling. However, the role of DDX3X in Dox-induced cardiotoxicity (DIC) remains unclear. In this study, we found that DDX3X expression was significantly decreased in H9c2 cardiomyocytes treated with Dox. Ddx3x knockdown and RK-33 (DDX3X ATPase activity inhibitor) pretreatment exacerbated cardiomyocyte apoptosis and mitochondrial dysfunction induced by Dox treatment. In contrast, Ddx3x overexpression ameliorated the DIC response. Moreover, Wnt/ß-catenin signaling in cardiomyocytes treated with Dox was suppressed, but this suppression was reversed by Ddx3x overexpression. Overall, this study demonstrated that DDX3X plays a protective role in DIC by activating Wnt/ß-catenin signaling.

DDX3X deficiency alleviates LPS-induced H9c2 cardiomyocytes pyroptosis by suppressing activation of NLRP3 inflammasome.

Increasing evidence suggest that NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis may be the underlying pathological mechanism of sepsis-induced cardiomyopathy. DDX3X, an ATP-dependent RNA helicase, plays a vital role in the formation of the NLRP3 inflammasome by directly interacting with cytoplasmic NLRP3. However, whether DDX3X has a direct impact on lipopolysaccharide (LPS)-induced cardiomyocyte injury by regulating NLRP3 inflammasome assembly remains unclear. The present study aimed to investigate the role of DDX3X in the activation of the NLRP3 inflammasome and determine the molecular mechanism of DDX3X action in LPS-induced pyroptosis in H9c2 cardiomyocytes. H9c2 cardiomyocytes were treated with LPS to simulate sepsis in vitro. The results demonstrated that LPS stimulation upregulated DDX3X expression in H9c2 cardiomyocytes. Furthermore, Ddx3x knockdown significantly attenuated pyroptosis and cell injury in LPS-treated H9c2 cells by suppressing NLRP3 inflammasome activation. Taken together, these results suggest that DDX3X is involved in LPS-induced cardiomyocyte pyroptosis, and DDX3X deficiency mitigates cardiomyocyte damage induced by LPS treatment.

Apoptotic cell death induced by Z-Ligustilidein human ovarian cancer cells and role of NRF2.

Z-Ligustilide is the most potent bioactive component of Angelica sinensis, which is widely used in Chinese traditional medicine. Z-Ligustilide selectively affected ovarian cancer cell survival in a dose dependent manner. Z-Ligustilide induced apoptotic cell death was determined by flow cytometry. We also demonstrated that apoptotic cell death was triggered by Z-Ligustilideinduced oxidative stress and mitochondria played an active role. Mitochondrial polarization was reduced by Z-Ligustilidewhereas mitochondrial superoxide formation was increased. NRF2 was induced by Z-Ligustilide in OVCAR-3 cells at epigenetic level and its downstream antioxidant defense genesHeme oxygenase-1,NAD(P)H Quinone Dehydrogenase 1, UDP Glucuronosyltransferase Family 1 Member A1and Glutamate-Cysteine Ligase. NRF2 knockdown by siRNA resulted increased cell death by Z-Ligustilide in ovarian cancer cells. Our result demonstrated the pro-survival role of NRF2 in Z-Ligustilide induced ovarian cancer cell death.

Cyanidin ameliorates endotoxin-induced myocardial toxicity by modulating inflammation and oxidative stress through mitochondria and other factors.

Cyanidin, an anthocyanin pigment, demonstrates anti-oxidant and anti-inflammatory properties. Here, we examined the mechanistic role of cyanidin in endotoxin induced myocardial injury in inflammation and oxidative stress. In lipopolysaccharide (LPS) induced myocardial injury model, cyanidin ameliorated cardiac injury (Lactate dehydrogenase or LDH, Creatine Kinase or CK, cardiac troponin I or cTnI and cardiac myosin light chains 1 or cMLC1), cell death (caspase 3 activity and PARP activity), and improved cardiac function (ejection fraction or EF and end diastolic left ventricular inner dimension or LVID). Cyanidin also attenuated endotoxin induced myocardial injury by modulating inflammatory cytokines (Tumor necrosis factor alpha or TNFα, Interleukin-1 beta or IL-1ß, macrophage inflammatory protein 2 or MIP-2 and chemokine (C-C motif) ligand 2 also known as monocyte chemoattractant protein 1 or MCP1) and oxidative stress (protein nitration). Cyanidin modulated redox homeostasis through intracellular oxidized/reduced glutathione. The most striking properties of cyanidin in endotoxin induced mediated myocardial injury was the modulation of mitochondria, its oxidative damage and associated factor Opa1 and Trx1. Thus, our study demonstrated that cyanidin as a constituent of our food chain may be beneficial and has therapeutic potential in sepsis treatment or other myocardial oxidative and/or inflammation induced injuries.

Apigenin Alleviates Endotoxin-Induced Myocardial Toxicity by Modulating Inflammation, Oxidative Stress, and Autophagy.

Apigenin, a component in daily diets, demonstrates antioxidant and anti-inflammatory properties. Here, we intended to explore the mechanism of apigenin-mediated endotoxin-induced myocardial injury and its role in the interplay among inflammation, oxidative stress, and autophagy. In our lipopolysaccharide- (LPS-) induced myocardial injury model, apigenin ameliorated cardiac injury (lactate dehydrogenase (LDH) and creatine kinase (CK)), cell death (TUNEL staining, DNA fragmentation, and PARP activity), and tissue damage (cardiac troponin I (cTnI) and cardiac myosin light chain-1 (cMLC1)) and improved cardiac function (ejection fraction (EF) and end diastolic left ventricular inner dimension (LVID)). Apigenin also alleviated endotoxin-induced myocardial injury by modulating oxidative stress (nitrotyrosine and protein carbonyl) and inflammatory cytokines (TNF-α, IL-1ß, MIP-1α, and MIP-2) along with their master regulator NFκB. Apigenin modulated redox homeostasis, and its anti-inflammatory role might be associated with its ability to control autophagy. Autophagy (determined by LAMP1, ATG5, and p62), its transcriptional regulator transcription factor EB (TFEB), and downstream target genes including vacuolar protein sorting-associated protein 11 (Vps11) and microtubule-associated proteins 1A/1B light chain 3B (Map1lc3) were modulated by apigenin. Thus, our study demonstrated that apigenin may lead to potential development of new target in sepsis treatment or other myocardial oxidative and/or inflammation-induced injuries.

Role of TFEB Mediated Autophagy, Oxidative Stress, Inflammation, and Cell Death in Endotoxin Induced Myocardial Toxicity of Young and Aged Mice.

Elderly patients are susceptible to sepsis. LPS induced myocardial injury is a widely used animal model to assess sepsis induced cardiac dysfunction. The age dependent mechanisms behind sepsis susceptibility were not studied. We analyzed age associated changes to cardiac function, cell death, inflammation, oxidative stress, and autophagy in LPS induced myocardial injury. Both young and aged C57BL/6 mice were used for LPS administration. The results demonstrated that LPS induced more cardiac injury (creatine kinase, lactate dehydrogenase, troponin I, and cardiac myosin-light chains 1), cardiac dysfunction (left ventricular inner dimension, LVID, and ejection fraction (EF)), cell death, inflammation, and oxidative stress in aged mice compared to young mice. However, a significant age dependent decline in autophagy was observed. Translocation of Transcription Factor EB (TFEB) to nucleus and formation of LC3-II were significantly reduced in LPS administered aged mice compared to young ones. In addition to that, downstream effector of TFEB, LAMP-1, was induced in response to LPS challenge in young mice. The present study newly demonstrates that TFEB mediated autophagy is crucial for protection against LPS induced myocardial injury particularly in aging senescent heart. Targeting this autophagy-oxidative stress-inflammation-cell death axis may provide a novel therapeutic strategy for cardioprotection in the elderly.

Apoptotic Cell Death Induced by Resveratrol Is Partially Mediated by the Autophagy Pathway in Human Ovarian Cancer Cells.

Resveratrol (trans-3,4,5'-trihydroxystilbene) is an active compound in food, such as red grapes, peanuts, and berries. Resveratrol exhibits an anticancer effect on various human cancer cells. However, the mechanism of resveratrol-induced anti-cancer effect at the molecular level remains to be elucidated. In this study, the mechanism underlying the anti-cancer effect of resveratrol in human ovarian cancer cells (OVCAR-3 and Caov-3) was investigated using various molecular biology techniques, such as flow cytometry, western blotting, and RNA interference, with a major focus on the potential role of autophagy in resveratrol-induced apoptotic cell death. We demonstrated that resveratrol induced reactive oxygen species (ROS) generation, which triggers autophagy and subsequent apoptotic cell death. Resveratrol induced ATG5 expression and promoted LC3 cleavage. The apoptotic cell death induced by resveratrol was attenuated by both pharmacological and genetic inhibition of autophagy. The autophagy inhibitor chloroquine, which functions at the late stage of autophagy, significantly reduced resveratrol-induced cell death and caspase 3 activity in human ovarian cancer cells. We also demonstrated that targeting ATG5 by siRNA also suppressed resveratrol-induced apoptotic cell death. Thus, we concluded that a common pathway between autophagy and apoptosis exists in resveratrol-induced cell death in OVCAR-3 human ovarian cancer cells.

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis.

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX's cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury.

Cadmium induces vascular permeability via activation of the p38 MAPK pathway.

The vasculature of various organs is a targeted by the environmental toxin, cadmium (Cd). However, mechanisms leading to pathological conditions are poorly understood. In the present study, we examined the effect of cadmium chloride (CdCl2) on human umbilical vein endothelial cells (HUVECs). At 4 µM, CdCl2 induced a hyper-permeability defect in HUVECs, but not the inhibition of cell growth up to 24h. This effect of CdCl2 was dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. The p38 MAPK inhibitor SB203850 suppressed the CdCl2-induced alteration in trans-endothelial electrical resistance in HUVEC monolayers, a model measurement of vascular endothelial barrier integrity. SB203850 also inhibited the Cd-induced membrane dissociation of vascular endothelial (VE) cadherin and ß-catenin, the important components of the adherens junctional complex. In addition, SB203850 reduces the Cd-induced expression and secretion of tumor necrosis factor α (TNF-α). Taken together, our findings suggest that Cd induces vascular hyper-permeability and disruption of endothelial barrier integrity through stimulation of p38 MAPK signaling.

Trastuzumab alters the expression of genes essential for cardiac function and induces ultrastructural changes of cardiomyocytes in mice.

Treatment with trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of Human Epidermal Growth Factor Receptor 2 (HER2), very successfully improves outcomes for women with HER2-positive breast cancer. However, trastuzumab treatment was recently linked to potentially irreversible serious cardiotoxicity, the mechanisms of which are largely elusive. This study reports that trastuzumab significantly alters the expression of myocardial genes essential for DNA repair, cardiac and mitochondrial functions, which is associated with impaired left ventricular performance in mice coupled with significant ultrastructural alterations in cardiomyocytes revealed by electron microscopy. Furthermore, trastuzumab treatment also promotes oxidative stress and apoptosis in myocardium of mice, and elevates serum levels of cardiac troponin-I (cTnI) and cardiac myosin light chain-1 (cMLC1). The elevated serum levels of cMLC1 in mice treated with trastuzumab highlights the potential that cMLC1 could be a useful biomarker for trastuzumab-induced cardiotoxicity.

Resveratrol alleviates endotoxin-induced myocardial toxicity via the Nrf2 transcription factor.

BACKGROUND/AIMS: Septic cardiomyopathy is a severe condition that remains a challenge for clinical management. This study investigated whether the natural polyphenolic compound resveratrol could be used as a prophylactic treatment to alleviate sepsis-related myocardial injury; the underlying molecular mechanisms were deciphered by both in vitro and in vivo experiments. METHODS: A mouse model of endotoxin-induced cardiomyopathy was developed by intraperitoneal injection of LPS, and resveratrol was administered prophylatically to the animals. Serum LDH and CK activities were measured to detect myocardial injury, and echocardiography was performed to monitor cardiac structure and function. Various cytokines/chemokines and the Nrf2 antioxidant defense system were examined in the heart tissue. The effects of resveratrol on LPS-induced Nrf2 activation, ROS generation, and apoptotic cell death were further investigated in cultured primary human cardiomyocytes. An Nrf2 specific siRNA was used to define its role in resveratrol-mediated cardiomyocyte protective effect. RESULTS: Resveratrol pretreatment significantly attenuated LPS-induced myocardial injury in mice, which was associated with suppressed proinflammatory cytokine production and enhanced Nrf2 activation in the heart. In cultured primary human cardiomyocytes, resveratrol activated Nrf2, inhibited LPS-induced ROS generation, and effectively protected the cells from LPS-induced apoptotic cell death. Knockdown of Nrf2 abrogated resveratrol-mediated protection of the cells from LPS-induced cell death. CONCLUSION: Resveratrol effectively alleviates endotoxin-induced cardiac toxicity through mechanisms that involve the Nrf2 antioxidant defense pathway. Our data suggest that resveratrol might be developed as a useful prophylactic management for septic cardiomyopathy.

Monoacylglycerol lipase controls endocannabinoid and eicosanoid signaling and hepatic injury in mice.

BACKGROUND & AIMS: The endocannabinoid and eicosanoid lipid signaling pathways have important roles in inflammatory syndromes. Monoacylglycerol lipase (MAGL) links these pathways, hydrolyzing the endocannabinoid 2-arachidonoylglycerol to generate the arachidonic acid precursor pool for prostaglandin production. We investigated whether blocking MAGL protects against inflammation and damage from hepatic ischemia/reperfusion (I/R) and other insults. METHODS: We analyzed the effects of hepatic I/R in mice given the selective MAGL inhibitor JZL184, in Mgll(-/-) mice, fatty acid amide hydrolase(-/-) mice, and in cannabinoid receptor type 1(-/-) (CB1-/-) and cannabinoid receptor type 2(-/-) (CB2-/-). Liver tissues were collected and analyzed, along with cultured hepatocytes and Kupffer cells. We measured endocannabinoids, eicosanoids, and markers of inflammation, oxidative stress, and cell death using molecular biology, biochemistry, and mass spectrometry analyses. RESULTS: Wild-type mice given JZL184 and Mgll(-/-) mice were protected from hepatic I/R injury by a mechanism that involved increased endocannabinoid signaling via CB2 and reduced production of eicosanoids in the liver. JZL184 suppressed the inflammation and oxidative stress that mediate hepatic I/R injury. Hepatocytes were the major source of hepatic MAGL activity and endocannabinoid and eicosanoid production. JZL184 also protected from induction of liver injury by D-(+)-galactosamine and lipopolysaccharides or CCl4. CONCLUSIONS: MAGL modulates hepatic injury via endocannabinoid and eicosanoid signaling; blockade of this pathway protects mice from liver injury. MAGL inhibitors might be developed to treat conditions that expose the liver to oxidative stress and inflammatory damage.

Chinese medicinal herb Radix Astragali suppresses cardiac contractile dysfunction and inflammation in a rat model of autoimmune myocarditis.

Radix Astragali, a Chinese medicinal herb, consists of polysaccharides and flavonoids as its main active ingredients. It has been widely used for treatment of cardiovascular diseases such as heart failure, angina pectoris, myocardial infarction and stroke in Asian countries. This study was designed to evaluate the effect of Radix Astragali on myocardial dysfunction, cardiac remodeling and morphological alteration in an experimental model of autoimmune myocarditis, a clinical condition often resulting in dilated cardiomyopathy. Experimental autoimmune myocarditis was established with a subcutaneous injection of porcine cardiac myosin into rear footpad in Lewis rats. Radix Astragali treatment was delivered via an intravenous injection (0.2 ml/100g body weight, daily) for 3 weeks. Results from transthoracic echocardiography indicated that experimental autoimmune myocarditis led to impaired myocardial contractile function which was reconciled by Radix Astragali. The experimental autoimmune myocarditis triggered profound inflammation and fibrosis in myocardium as assessed by hematoxylin and eosin (H and E) and Masson's trichrome staining. Interestingly, Radix Astragali significantly attenuated autoimmune myocarditis-induced myocardial inflammation and fibrosis. Similarly, Radix Astragali treatment alleviated autoimmune myocarditis-triggered overt lymphocyte proliferation. Furthermore, Radix Astragali significantly attenuated elevated levels of the Th1 cytokines (IFN-gamma and IL-2), and increased the Th2 cytokines (IL-4 and IL-10) in autoimmune myocarditis. Collectively, our data revealed that Radix Astragali effectively protected against cardiac functional and morphological aberrations in experimental autoimmune myocarditis.