The receptor-interacting kinase-3 (RIP3) and its downstream substrate mixed lineage kinase domain-like protein (MLKL) have emerged as the key cellular components in programmed necrotic cell death. Receptors for the cytokines of tumor necrosis factor (TNF) family and Toll-like receptors (TLR) 3 and 4 are able to activate RIP3 through receptor-interacting kinase-1 and Toll/IL-1 receptor domaincontaining adapter inducing IFN-β, respectively. This form of cell death has been implicated in the host-defense system. However, the molecular mechanisms that drive the activation of RIP3 by a variety of pathogens, other than the above-mentioned receptors, are largely unknown. Here, we report that human herpes simplex virus 1 (HSV-1) infection triggers RIP3-dependent necrosis. This process requires MLKL but is independent of TNF receptor, TLR3, cylindromatosis, and host RIP homotypic interaction motif-containing protein DNA-dependent activator of IFN regulatory factor. After HSV-1 infection, the viral ribonucleotide reductase large subunit (ICP6) interacts with RIP3. The formation of the ICP6-RIP3 complex requires the RHIM domains of both proteins. An HSV-1 ICP6 deletion mutant failed to cause effective necrosis of HSV-1-infected cells. Furthermore, ectopic expression of ICP6, but not RHIM mutant ICP6, directly activated RIP3/MLKL-mediated necrosis. Mice lacking RIP3 exhibited severely impaired control of HSV-1 replication and pathogenesis. Therefore, this study reveals a previously uncharacterized host antipathogen mechanism.programmed necrosis | HSV-1 | ICP6 | RIP3 | MLKL C ell death triggered by pathogens is a crucial component of mammalian host-defense system. Apoptosis, a predominant programmed cell death in mammals, functions as an effective host-defense mechanism for preventing pathogen replication. Apoptosis is initiated by either mitochondria or cell-death receptors, and it is executed by a group of cysteine proteases called caspases (1). The apoptotic pathway can be subverted by pathogen-encoded apoptotic suppressors such as caspase inhibitors (2). Recent studies have revealed that caspase inhibition can lead to alternative activation of necrosis, releasing the damage-associated molecular patterns (DAMPs) signal to trigger the activation of the host immune system (3, 4).Cytokines of the TNF family are classical inducers of programmed necrosis that are morphologically characterized by the swelling of intracellular organelles and disrupted plasma membranes. Programmed necrosis triggered by death cytokines such as TNF, also known as necroptosis (5-7), is tightly regulated by receptor-interacting kinase-1 (RIP1) (8), its deubiquitin enzyme cylindromatosis (CYLD) (9), and receptor-interacting kinase-3 (RIP3) (10-12). The RIP homotypic interaction motif (RHIM) domains of RIP1 and RIP3 are required for the formation of the RIP1-RIP3 complex that is called a necrosome (13). Recently, mixed lineage kinase domain-like (MLKL) protein has been identified as a functional substrate of RIP3 kinase (14, 15). Upon phosphorylation, ...
Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) (PL) is a naturally occurring yellow pigment found in the plants of the Plumbaginaceae, Droseraceae, Ancistrocladaceae, and Dioncophyllaceae families. It has been reported that PL exhibits anticarcinogenic, anti-inflammatory, and analgesic activities. However, the mechanism underlying its anti-inflammatory action remains unknown. In the current study, we investigated and characterized the anti-inflammatory and analgesic effects of PL orally administrated in a range of dosages from 5 to 20 mg/kg. We also examined the role of nuclear factor B (NF-B) and proinflammatory cytokines and mediators in this effect. The results showed that PL significantly and dose-dependently suppressed the paw edema of rats induced by carrageenan and various proinflammatory mediators, including histamine, serotonin, bradykinin, and prostaglandin E 2 . PL reduced the number of writhing episodes of mice induced by the intraperitoneal injection of acetic acid, but it did not reduce the writhing episode numbers induced by MgSO 4 in mice or prolong the tail-flick reaction time of rats to noxious thermal pain. Mechanistic studies showed that PL effectively decreased the production of the proinflammatory cytokines interleukin 1, interleukin 6, and tumor necrosis factor ␣. It also inhibited the expression of the proinflammatory mediators inducible nitricoxide synthase and cyclooxygenase 2, whereas it did not inhibit the expression of cyclooxygenase 1. Further studies demonstrated that PL suppressed inhibitor of B␣ phosphorylation and degradation, thus inhibiting the phosphorylation of the p65 subunit of NF-B. This study suggests that PL has a potential to be developed into an anti-inflammatory agent for treating inflammatory diseases.
Tumor necrosis factor-α (TNF-α) is a critical proinflammatory cytokine regulating neuroinflammation. Elevated levels of TNF-α have been associated with various neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. However, the signaling events that lead to TNF-α-initiated neurotoxicity are still unclear. Here, we report that RIP3-mediated necroptosis, a form of regulated necrosis, is activated in the mouse hippocampus after intracerebroventricular injection of TNF-α. RIP3 deficiency attenuates TNF-α-initiated loss of hippocampal neurons. Furthermore, we characterized the molecular mechanism of TNF-α-induced neurotoxicity in HT-22 hippocampal neuronal cells. HT-22 cells are sensitive to TNF-α only upon caspase blockage and subsequently undergo necrosis. The cell death is suppressed by knockdown of CYLD or RIP1 or RIP3 or MLKL, suggesting that this necrosis is necroptosis and mediated by CYLD-RIP1-RIP3-MLKL signaling pathway. TNF-α-induced necroptosis of HT-22 cells is largely independent of both ROS accumulation and calcium influx although these events have been shown to be critical for necroptosis in certain cell lines. Taken together, these data not only provide the first in vivo evidence for a role of RIP3 in TNF-α-induced toxicity of hippocampal neurons, but also demonstrate that TNF-α promotes CYLD-RIP1-RIP3-MLKL-mediated necroptosis of hippocampal neurons largely bypassing ROS accumulation and calcium influx.
Introduction Since December 2019, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has caused the coronavirus disease 2019 (COVID-19) pandemic in China and worldwide. New drugs for the treatment of COVID-19 are in urgent need. Considering the long development time for new drugs, the identification of promising inhibitors from FDA-approved drugs is an imperative and valuable strategy. Recent studies have shown that the S1 and S2 subunits of the spike protein of SARS-CoV-2 utilize human angiotensin-converting enzyme 2 (hACE2) as the receptor to infect human cells. Methods We combined molecular docking and surface plasmon resonance (SPR) to identify potential inhibitors for ACE2 from available commercial medicines. We also designed coronavirus pseudoparticles that contain the spike protein assembled onto green fluorescent protein or luciferase reporter gene-carrying vesicular stomatitis virus core particles. Results We found that thymoquinone, a phytochemical compound obtained from the plant Nigella sativa , is a potential drug candidate. SPR analysis confirmed the binding of thymoquinone to ACE2. We found that thymoquinone can inhibit SARS-CoV-2, SARS-CoV, and NL63 pseudoparticles infecting HEK293-ACE2 cells, with half-maximal inhibitory concentrations of 4.999, 7.598, and 6.019 μM, respectively. The SARS-CoV-2 pseudoparticle inhibition had half-maximal cytotoxic concentration of 35.100 μM and selection index = 7.020. Conclusion Thymoquinone is a potential broad-spectrum inhibitor for the treatment of coronavirus infections. Supplementary Information The online version contains supplementary material available at 10.1007/s40121-021-00400-2.
Objective To investigate the mechanism through which tacrolimus, often used to treat refractory nephropathy, protects against puromycin-induced podocyte injury. Methods An in vitro model of puromycin-induced podocyte injury was established by dividing podocytes into three groups: controls, puromycin only (PAN group), and puromycin plus tacrolimus (FK506 group). Podocyte morphology, number, apoptosis rate and microtubule associated protein 1 light chain 3 alpha ( LC3) expression were compared. Results Puromycin caused podocyte cell body shrinkage and loose intercellular connections, but podocyte morphology in the FK506 group was similar to controls. The apoptosis rate was lower in the FK506 group versus PAN group. The low level of LC3 mRNA observed in untreated podocytes was decreased by puromycin treatment; however, levels of LC3 mRNA were higher in the FK506 group versus PAN group. Although LC3-I and LC3-II protein levels were decreased by puromycin, levels in the FK506 group were higher than the PAN group. Fewer podocyte autophagosomes were observed in the control and FK506 groups versus the PAN group. Cytoplasmic LC3-related fluorescence intensity was stronger in control and FK506 podocytes versus the PAN group. Conclusions Tacrolimus inhibited puromycin-induced mouse podocyte damage by regulating LC3 expression and enhancing autophagy.
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