Receptor-interacting Protein Kinase Research Articles

Contribution to the study of inflammasome and programmed cell death in paracoccidioidomycosis oral lesions.

Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, with a high incidence in Brazil, Colombia and Venezuela, and constitutes a serious public health problem, a frequent cause of morbidity and disability for work. Some mechanisms of cell death are described as important tools in infectious processes. When apoptosis is blocked, RIPK (Receptor-interacting protein kinase) 3 dependent, a caspase-independent form of cell death, can limit the replication and spread of pathogens. Some molecules that mediate necroptosis include RIPK3 and have been extensively studied due to their signalling mechanism and pathological function. RIPK3 activates NLRP1 and NLRP3-mediated inflammasome formation. Caspase-1 has an important role in processing the cytokines ILβ and IL18 to their active form. Such molecules are part of the inflammasome characterization, whose caspase-1-dependent activation promotes the death of pyroptotic cells and the secretion of proinflammatory cytokines. Knowledge about the mechanisms of pathogen-mediated cell death can be useful for understanding of the pathogenesis of infections and inflammatory conditions. The objective of this work was to identify the mechanisms of programmed cell death and inflammasome components in human oral mucosal lesions of paracoccidioidomycosis through immunohistochemical methods and identification of RIPK-3, IL1β, IL18, NLRP-1 and caspase-1. Thirty specimens were included, and a histopathological analysis of the lesions was performed using haematoxylin-eosin staining. Our results on insitu expression of inflammasome elements and programmed cell death showed increased expression of IL-1β, NLRP-1, caspase-1 and RIPK-3. We suggest that inflammasome complex participate in the immunopathogenesis in paracoccidioidomycosis oral lesions in an interplay with RIPK3.

Receptor-interacting Protein Kinase 2 Is an Immunotherapy Target in Pancreatic Cancer.

PDAC is resistant to almost all available therapies, including immune checkpoint blockade. Through in vivo CRISPR screen, we identified that RIPK2 plays a crucial role in facilitating immune evasion by impeding antigen presentation and cytotoxic T-cell killing. Targeting tumor-intrinsic RIPK2 either genetically or pharmacologically improves PDAC to anti-PD-1 immunotherapy. See related commentary by Liu et al., p. 208 . This article is featured in Selected Articles from This Issue, p. 201.

The mechanism of cadmium exposure-induced lymph node necroptosis and inflammation in piglets: Activation of CYP450 through VDR / CREB1 pathway

Cadmium (Cd) is toxic non-essential heavy metal that precipitates adverse health effects in humans and animals, but the effect of Cd on lymph node toxicity of piglets is still unclear. In order to explore the possible molecular mechanism of Cd toxicity to lymph nodes of piglets, ten 6-week-old male weaned piglets were randomly divided into two groups, C group and Cd group. Group C was fed with basal diet, while group Cd was fed with basal diet supplemented with CdCl2 (20 mg/kg) for 40 days, the pigs were euthanized and the mesenteric, inguinal and submandibular lymph nodes (MLN, ILN, SLN) were collected. The results indicated that Cd could induce the inflammatory cell infiltration, microvascular hemorrhage, microthrombosis and cell necrosis in MLN, ILN and SLN of piglets, induced Cytochrome P450 proteins (CYP1A1、CYP2E1、CYP2A1 and CYP3A2) mRNA levels and the protein levels of Vitamin D receptor (VDR) and cAMP response element binding protein 1 (CREB1). In addition, Cd exposure upregulated the mRNA and protein levels of dynamin-related protein 1 (DRP1), receptor-interacting protein kinase 3 (RIP3), mixed lineage kinase domain-like protein (MLKL), and increased tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), interleukin-2 (IL-2), interleukin-4 (IL-4), cyclooxygenase 2 (COX-2) protein levels, and the damage degree of three kinds of lymph nodes was similar after Cd exposure. In general, these results manifest that Cd exposure regulates VDR/CREB1 pathway, activates CYP450s, induces necroptosis of lymph nodes, and leads to inflammation.

CIAPs control RIPK1 kinase activity-dependent and-independent cell death and tissue inflammation.

Cellular inhibitor of apoptosis proteins (cIAPs) are RING-containing E3 ubiquitin ligases that ubiquitylate receptor-interacting protein kinase 1 (RIPK1) to regulate TNF signalling. Here, we established mice simultaneously expressing enzymatically inactive cIAP1/2 variants, bearing mutations in the RING domains of cIAP1/2 (cIAP1/2 mutant RING, cIAP1/2MutR ). cIap1/2MutR/MutR mice died during embryonic development due to RIPK1-mediated apoptosis. While expression of kinase-inactive RIPK1D138N rescued embryonic development, Ripk1D138N/D138N /cIap1/2MutR/MutR mice developed systemic inflammation and died postweaning. Cells expressing cIAP1/2MutR and RIPK1D138N were still susceptible to TNF-induced apoptosis and necroptosis, implying additional kinase-independent RIPK1 activities in regulating TNF signalling. Although further ablation of Ripk3 did not lead to any phenotypic improvement, Tnfr1 gene knock-out prevented early onset of systemic inflammation and premature mortality, indicating that cIAPs control TNFR1-mediated toxicity independent of RIPK1 and RIPK3. Beyond providing novel molecular insights into TNF-signalling, the mouse model established in this study can serve as a useful tool to further evaluate ongoing therapeutic protocols using inhibitors of TNF, cIAPs and RIPK1.

Receptor-interacting protein kinase 2 (RIPK2) profoundly contributes to post-stroke neuroinflammation and behavioral deficits with microglia as unique perpetrators

BackgroundReceptor-interacting protein kinase 2 (RIPK2) is a serine/threonine kinase whose activity propagates inflammatory signaling through its association with pattern recognition receptors (PRRs) and subsequent TAK1, NF-κB, and MAPK pathway activation. After stroke, dead and dying cells release a host of damage-associated molecular patterns (DAMPs) that activate PRRs and initiate a robust inflammatory response. We hypothesize that RIPK2 plays a damaging role in the progression of stroke injury by enhancing the neuroinflammatory response to stroke and that global genetic deletion or microglia-specific conditional deletion of Ripk2 will be protective following ischemic stroke.MethodsAdult (3–6 months) male mice were subjected to 45 min of transient middle cerebral artery occlusion (tMCAO) followed by 24 h, 48 h, or 28 days of reperfusion. Aged male and female mice (18–24 months) were subjected to permanent ischemic stroke and sacrificed 48 h later. Infarct volumes were calculated using TTC staining (24–48 h) or Cresyl violet staining (28d). Sensorimotor tests (weight grip, vertical grid, and open field) were performed at indicated timepoints. Blood–brain barrier (BBB) damage, tight junction proteins, matrix metalloproteinase-9 (MMP-9), and neuroinflammatory markers were assessed via immunoblotting, ELISA, immunohistochemistry, and RT-qPCR. Differential gene expression profiles were generated through bulk RNA sequencing and nanoString®.ResultsGlobal genetic deletion of Ripk2 resulted in decreased infarct sizes and reduced neuroinflammatory markers 24 h after stroke compared to wild-type controls. Ripk2 global deletion also improved both acute and long-term behavioral outcomes with powerful effects on reducing infarct volume and mortality at 28d post-stroke. Conditional deletion of microglial Ripk2 (mKO) partially recapitulated our results in global Ripk2 deficient mice, showing reductive effects on infarct volume and improved behavioral outcomes within 48 h of injury. Finally, bulk transcriptomic profiling and nanoString data demonstrated that Ripk2 deficiency in microglia decreases genes associated with MAPK and NF-κB signaling, dampening the neuroinflammatory response after stroke injury by reducing immune cell activation and peripheral immune cell invasion.ConclusionsThese results reveal a hitherto unknown role for RIPK2 in the pathogenesis of ischemic stroke injury, with microglia playing a distinct role. This study identifies RIPK2 as a potent propagator of neuroinflammatory signaling, highlighting its potential as a therapeutic target for post-stroke intervention.

Molecular mechanism by which RRM2-inhibitor (cholagogue osalmid) plus bafilomycin A1 cause autophagic cell death in multiple myeloma

Despite significant improvement in the prognosis of multiple myeloma (MM), the disease remains incurable; thus, more effective therapies are required. Ribonucleoside-diphosphate reductase subunit M2 (RRM2) is significantly associated with drug resistance, rapid relapse, and poor prognosis. Previously, we found that 4-hydroxysalicylanilide (osalmid), a specific inhibitor of RRM2, exhibits anti-MM activity in vitro, in vivo, and in human patients; however, the mechanism remains unclear. Osalmid inhibits the translocation of RRM2 to the nucleus and stimulates autophagosome synthesis but inhibits subsequent autophagosome–lysosome fusion. We confirm that RRM2 binds to receptor-interacting protein kinase 3 (RIPK3) and reduces RIPK3, inhibiting autophagosome–lysosome fusion. Interestingly, the combination of osalmid and bafilomycin A1 (an autophagy inhibitor) depletes RIPK3 and aggravates p62 and autophagosome accumulation, leading to autophagic cell death. Combination therapy demonstrates synergistic cytotoxicity both in vitro and in vivo. Therefore, we propose that combining osalmid and bafilomycin A1(BafA1) may have clinical benefits against MM.

Anti-Necroptotic Effects of Itaconate and its Derivatives.

Itaconate is an unsaturated dicarboxylic acid that is derived from the decarboxylation of the Krebs cycle intermediate cis-aconitate and has been shown to exhibit anti-inflammatory and anti-bacterial/viral properties. But the mechanisms underlying itaconate's anti-inflammatory activities are not fully understood. Necroptosis, a lytic form of regulated cell death (RCD), is mediated by receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) signaling. It has been involved in the pathogenesis of organ injury in many inflammatory diseases. In this study, we aimed to explore whether itaconate and its derivatives can inhibit necroptosis in murine macrophages, a mouse MPC-5 cell line and a human HT-29 cell line in response to different necroptotic activators. Our results showed that itaconate and its derivatives dose-dependently inhibited necroptosis, among which dimethyl itaconate (DMI) was the most effective one. Mechanistically, itaconate and its derivatives inhibited necroptosis by suppressing the RIPK1/RIPK3/MLKL signaling and the oligomerization of MLKL. Furthermore, DMI promoted the nuclear translocation of Nrf2 that is a critical regulator of intracellular redox homeostasis, and reduced the levels of intracellular reactive oxygen species (ROS) and mitochondrial superoxide (mtROS) that were induced by necroptotic activators. Consistently, DMI prevented the loss of mitochondrial membrane potential induced by the necroptotic activators. In addition, DMI mitigated caerulein-induced acute pancreatitis in mice accompanied by reduced activation of the necroptotic signaling in vivo. Collectively, our study demonstrates that itaconate and its derivatives can inhibit necroptosis by suppressing the RIPK1/RIPK3/MLKL signaling, highlighting their potential applications for treating necroptosis-associated diseases.

The Regulatory Effect of Receptor-Interacting Protein Kinase 3 on CaMKIIδ in TAC-Induced Myocardial Hypertrophy.

Necroptosis is a newly discovered mechanism of cell death, and its key regulatory role is attributed to the interaction of receptor-interacting protein kinases (RIPKs) RIPK1 and RIPK3. Ca2+/calmodulin-dependent protein kinase (CaMKII) is a newly discovered RIPK3 substrate, and its alternative splicing plays a fundamental role in cardiovascular diseases. In the present study, we aimed to explore the role and mechanism of necroptosis and alternative splicing of CaMKIIδ in myocardial hypertrophy. Transverse aortic constriction (TAC) was performed on wild-type and knockout mice to establish the model of myocardial hypertrophy. After 3 weeks, echocardiography, cardiac index, cross-sectional area of myocardial cells, hypertrophic gene expression, myocardial damage, and fibers were assessed. Moreover, we detected the levels of inflammatory factors (IL-6 and TNF-α) and examined the expressions of necroptosis-related proteins RIPK3, RIPK1, and phosphorylated MLKL. Meanwhile, we tested the expression levels of splicing factors ASF/SF2 and SC-35 in an attempt to explore CaMKII δ. The relationship between variable splicing disorder and the expression levels of splicing factors ASF/SF2 and SC-35. Further, we also investigated CaMKII activation, oxidative stress, and mitochondrial ultrastructure. In addition, wild-type mice were administered with a recombinant adeno-associated virus (AAV) carrying RIPK3, followed by TAC surgery to construct a model of myocardial hypertrophy, and the above-mentioned indicators were tested after 3 weeks. The results showed that RIPK3 deficiency could alleviate cardiac dysfunction, myocardial injury, aggravation of necrosis, and CaMKII activation induced by TAC surgery in mice with myocardial hypertrophy. Tail vein injection of AAV could reverse cardiac dysfunction, myocardial damage, aggravation of necrosis, and CaMKII activation in mice with myocardial hypertrophy. These results proved that RIPK3 could be used as a molecular intervention target for the prevention and treatment of myocardial hypertrophy.

RIP3 in Necroptosis: Underlying Contributions to Traumatic Brain Injury.

Traumatic brain injury (TBI) is a global public safety issue that poses a threat to death, characterized by high fatality rates, severe injuries and low recovery rates. There is growing evidence that necroptosis regulates the pathophysiological processes of a variety of diseases, particularly those affecting the central nervous system. Thus, moderate necroptosis inhibition may be helpful in the management of TBI. Receptor-interacting protein kinase (RIP) 3 is a key mediator in the necroptosis, and its absence helps restore the microenvironment at the injured site and improve cognitive impairment after TBI. In this report, we review different domains of RIP3, multiple analyses of necroptosis, and associations between necroptosis and TBI, RIP3, RIP1, and mixed lineage kinase domain-like. Next, we elucidate the potential involvement of RIP3 in TBI and highlight how RIP3 deficiency enhances neuronal function.

Dexamethasone-induced mitochondrial ROS-mediated inhibition of AMPK activity facilitates osteoblast necroptosis.

Long-term or high-dose glucocorticoid use can lead to serious orthopedic complications, including femoral head necrosis. Both basic and clinical studies have shown that high doses dexamethasone (Dex) can directly induce osteoblasts death. This study investigated the mechanism underlying Dex induced osteoblast death. In this study, we showed that Dex induces osteoblast necroptosis, rather than apoptosis, through the inhibition of AMP-activated protein kinase (AMPK) activity. We also demonstrated that inactivation of AMPK-mediated necroptosis is through receptor-interacting protein kinase 3 (RIP3), but not RIP1. Furthermore, we found that Dex-induced necroptosis is dependent on mitochondrial reactive oxygen species (ROS) following with directly activation of RIP1 and inactivation of AMPK. These findings provide new insights into the mechanism of Dex-induced osteoblast death and may have implications for the development of new therapies for osteoporosis and other bone-related diseases.

High RIPK3 expression is associated with a higher risk of early kidney transplant failure

Renal ischemia-reperfusion injury (IRI) is associated with reduced allograft survival, and each additional hour of cold ischemia time increases the risk of graft failure and mortality following renal transplantation. Receptor-interacting protein kinase 3 (RIPK3) is a key effector of necroptosis, a regulated form of cell death. Here, we evaluate the first-in-human RIPK3 expression dataset following IRI in kidney transplantation. The primary analysis included 374 baseline biopsy samples obtained from renal allografts 10minutes after onset of reperfusion. RIPK3 was primarily detected in proximal tubular cells and distal tubular cells, both of which are affected by IRI. Time-to-event analysis revealed that high RIPK3 expression is associated with a significantly higher risk of one-year transplant failure and prognostic for one-year (death-censored) transplant failure independent of donor and recipient associated risk factors in multivariable analyses. The RIPK3 score also correlated with deceased donation, cold ischemia time and the extent of tubular injury.

MEG3 activates necroptosis in human neuron xenografts modeling Alzheimer's disease.

Neuronal cell loss is a defining feature of Alzheimer's disease (AD), but the underlying mechanisms remain unclear. We xenografted human or mouse neurons into the brain of a mouse model of AD. Only human neurons displayed tangles, Gallyas silver staining, granulovacuolar neurodegeneration (GVD), phosphorylated tau blood biomarkers, and considerable neuronal cell loss. The long noncoding RNA MEG3 was strongly up-regulated in human neurons. This neuron-specific long noncoding RNA is also up-regulated in AD patients. MEG3 expression alone was sufficient to induce necroptosis in human neurons in vitro. Down-regulation of MEG3 and inhibition of necroptosis using pharmacological or genetic manipulation of receptor-interacting protein kinase 1 (RIPK1), RIPK3, or mixed lineage kinase domain-like protein (MLKL) rescued neuronal cell loss in xenografted human neurons. This model suggests potential therapeutic approaches for AD and reveals a human-specific vulnerability to AD.

The involvement of NETs in ANCA-associated vasculitis.

Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is a serious autoimmune disease that is characterized by vascular necrosis. The pathogenesis of AAV includes ANCA-mediated neutrophil activation, subsequent release of inflammatory cytokines and reactive oxygen species (ROS), and formation of neutrophil extracellular traps (NETs). Excessive NETs could participate not only in ANCA-mediated vascular injury but also in the production of ANCAs per se as autoantigens. Thus, a vicious cycle of NET formation and ANCA production is critical for AAV pathogenesis. Elucidating the molecular signaling pathways in aberrant neutrophil activation and NETs clearance systems will allow specific therapeutics to regulate these pathways. Currently, standard therapy with high doses of glucocorticoids and immunosuppressants has improved outcomes in patients with AAV. However, AAV frequently develops in elderly people, and adverse effects such as severe infections in the standard regimens might contribute to the mortality. Mechanistically, cytokines or complement factors activate and prime neutrophils for ANCA-binding; thus, C5a receptor blocker has garnered attention as potential replacement for glucocorticoids in clinical settings. Recent studies have demonstrated that receptor-interacting protein kinases (RIPK3) and cyclophilin D (CypD), which regulate cell necrosis, may be involved in ANCA-induced NETs formation. Meanwhile, targeting NETs clearance, including the addition of deoxyribonuclease I (DNase I) and macrophage engulfment, may improve vasculitis. In this review, we focus on the pathogenesis of NETs and discuss potential targeted therapies for AAV based on recent experimental evidence.

Gut-derived 4-hydroxyphenylacetic acid attenuates sepsis-induced acute kidney injury by upregulating ARC to inhibit necroptosis

BackgroundStudies have found that the plasma content of gut-derived 4-hydroxyphenylacetic acid (4-HPA) was significantly increased in septic patients. However, the mechanism of 4-HPA elevation during sepsis and its relationship with sepsis-induced acute kidney injury (SAKI) remain unclear. MethodsCecal ligation and puncture (CLP) was performed in C57BL/6 mice to establish the SAKI animal model. Human renal tubular epithelial (HK-2) cells stimulated with lipopolysaccharide were used to establish the SAKI cell model. The widely targeted metabolomics was applied to analyze the renal metabolite changes after CLP. Proteomics was used to explore potential target proteins regulated by 4-HPA. The blood sample of clinical sepsis patients was collected to examine the 4-HPA content. ResultsWe found that renal gut-derived 4-HPA levels were significantly increased after CLP. The high permeability of intestinal barrier after sepsis contributed to the dramatic increase of renal 4-HPA. Intriguingly, we demonstrated that exogenous 4-HPA administration could further significantly reduce CLP-induced increases in serum creatinine, urea nitrogen, and cystatin C, inhibit renal pathological damage and apoptosis, and improve the survival of mice. Mechanistically, 4-HPA inhibited necroptosis in renal tubular epithelial cells by upregulating the protein expression of apoptosis repressor with caspase recruitment domain (ARC) and enhancing the interaction between ARC and receptor-interacting protein kinase 1 (RIPK1). ConclusionsThe increase of gut-derived 4-HPA in the kidney after sepsis could play a protective effect in SAKI by upregulating ARC to inhibit necroptosis.

Neutrophil extracellular traps aggravate intestinal epithelial necroptosis in ischaemia-reperfusion by regulating TLR4/RIPK3/FUNDC1-required mitophagy.

Neutrophil extracellular trap (NET) has been confirmed to be related to gut barrier injury during intestinal ischaemia-reperfusion (II/R). However, the specific molecular regulatory mechanism of NETs in II/R-induced intestinal barrier damage has yet to be fully elucidated. Here, we reported increased NETs infiltration accompanied by elevated inflammatory cytokines, cellular necroptosis and tight junction disruption in the intestine of human II/R patients. Meanwhile, NETs aggravated Caco-2 intestinal epithelial cell necroptosis, impairing the monolayer barrier in vitro. Moreover, Pad4-deficient mice were used further to validate the role of NETs in II/R-induced intestinal injury. In contrast, NET inhibition via Pad4 deficiency alleviated intestinal inflammation, attenuated cellular necroptosis, improved intestinal permeability, and enhanced tight junction protein expression. Notably, NETs prevented FUN14 domain-containing 1 (FUNDC1)-required mitophagy activation in intestinal epithelial cells, and stimulating mitophagy attenuated NET-associated mitochondrial dysfunction, cellular necroptosis, and intestinal damage. Mechanistically, silencing Toll-like receptor 4 (TLR4) or receptor-interacting protein kinase 3 (RIPK3) via shRNA relieved mitophagy limitation, restored mitochondrial function and reduced NET-induced necroptosis in Caco-2 cells, whereas this protective effect was reversed by TLR4 or RIPK3 overexpression. The regulation of TLR4/RIPK3/FUNDC1-required mitophagy by NETs can potentially induce intestinal epithelium necroptosis.

A scalable human iPSC-based neuromuscular disease model on suspended biobased elastomer nanofiber scaffolds.

Many devastating neuromuscular diseases currently lack effective treatments. This is in part due to a lack of drug discovery platforms capable of assessing complex human neuromuscular disease phenotypes in a scalable manner. A major obstacle has been generating scaffolds to stabilise mature contractile myofibers in a multi-well assay format amenable to high content image (HCI) analysis. This study describes the development of a scalable human induced pluripotent stem cell (iPSC)-neuromuscular disease model, whereby suspended elastomer nanofibers support long-term stability, alignment, maturation, and repeated contractions of iPSC-myofibers, innervated by iPSC-motor neurons in 96-well assay plates. In this platform, optogenetic stimulation of the motor neurons elicits robust myofiber-contractions, providing a functional readout of neuromuscular transmission. Additionally, HCI analysis provides rapid and automated quantification of axonal outgrowth, myofiber morphology, and neuromuscular synapse number and morphology. By incorporating amyotrophic lateral sclerosis (ALS)-related TDP-43G298Smutant motor neurons and CRISPR-corrected controls, key neuromuscular disease phenotypes are recapitulated, including weaker myofiber contractions, reduced axonal outgrowth, and reduced number of neuromuscular synapses. Treatment with a candidate ALS drug, the receptor-interacting protein kinase-1 (RIPK1)-inhibitor necrostatin-1, rescues these phenotypes in a dose-dependent manner, highlighting the potential of this platform to screen novel treatments for neuromuscular diseases.

SARS-CoV-2 envelope protein induces necroptosis and mediates inflammatory response in lung and colon cells through receptor interacting protein kinase 1.

SARS-CoV-2 Envelope protein (E) is one of the crucial components in virus assembly and pathogenesis. The current study investigated its role in the SARS-CoV-2-mediated cell death and inflammation in lung and gastrointestinal epithelium and its effect on the gastrointestinal-lung axis. We observed that transfection of E protein increases the lysosomal pH and induces inflammation in the cell. The study utilizing Ethidium bromide/Acridine orange and Hoechst/Propidium iodide staining demonstrated necrotic cell death in E protein transfected cells. Our study revealed the role of the necroptotic marker RIPK1 in cell death. Additionally, inhibition of RIPK1 by its specific inhibitor Nec-1s exhibits recovery from cell death and inflammation manifested by reduced phosphorylation of NFκB. The E-transfected cells' conditioned media induced inflammation with differential expression of inflammatory markers compared to direct transfection in the gastrointestinal-lung axis. In conclusion, SARS-CoV-2 E mediates inflammation and necroptosis through RIPK1, and the E-expressing cells' secretion can modulate the gastrointestinal-lung axis. Based on the data of the present study, we believe that during severe COVID-19, necroptosis is an alternate mechanism of cell death besides ferroptosis, especially when the disease is not associated with drastic increase in serum ferritin.

Role of Necroptosis, a Regulated Cell Death, in Seizure and Epilepsy.

Epilepsy is a chronic neurological disease that is characterized by spontaneous and recurrent seizures. Regulated cell death is a controlled process and has been shown to be involved in neurodegenerative diseases. Necroptosis is a type of regulated cell death, and its association with epilepsy has been documented. Necroptosis signaling can be divided into two pathways: canonical and non-canonical pathways. Inhibition of caspase-8, dimerization of receptor-interacting protein kinase 1 (RIP1) and RIP3, activation of mixed-lineage kinase domain-like protein (MLKL), movement of MLKL to the plasma membrane, and cell rupture occurred in these pathways. Through literature review, it has been revealed that there is a relationship between seizure, neuroinflammation, and oxidative stress. The seizure activity triggers the activation of various pathways within the central nervous system, including TNF-α/matrix metalloproteases, Neogenin and Calpain/ Jun N-terminal Kinase 1, which result in distinct responses in the brain. These responses involve the activation of neurons and astrocytes, consequently leading to an increase in the expression levels of proteins and genes such as RIP1, RIP3, and MLKL in a time-dependent manner in regions such as the hippocampus (CA1, CA3, dentate gyrus, and hilus), piriform cortex, and amygdala. Furthermore, the imbalance in calcium ions, depletion of adenosine triphosphate, and elevation of extracellular glutamate and potassium within these pathways lead to the progression of necroptosis, a reduction in seizure threshold, and increased susceptibility to epilepsy. Therefore, it is plausible that therapeutic targeting of these pathways could potentially provide a promising approach for managing seizures and epilepsy.

Necrostatin-1s Suppresses RIPK1-driven Necroptosis and Inflammation in Periventricular Leukomalacia Neonatal Mice.

Periventricular leukomalacia (PVL), a predominant form of brain injury in preterm survivors, is characterized by hypomyelination and microgliosis and is also the major cause of long-term neurobehavioral abnormalities in premature infants. Receptor-interacting protein kinase 1 (RIPK1) plays a pivotal role in mediating cell death and inflammatory signaling cascade. However, very little is known about the potential effect of RIPK1 in PVL and the underlying mechanism. Herein, we found that the expression level of RIPK1 was drastically increased in the brain of PVL neonatal mice models, and treatment of PVL neonatal mice with Necrostatin-1s (Nec-1s), an inhibitor of RIPK1, greatly ameliorated cerebral ischemic injury, exhibiting an increase of body weights, reduction of cerebral infarct size, neuronal loss, and occurrence of necrosis-like cells, and significantly improved the long-term abnormal neurobehaviors of PVL. In addition, Nec-1s significantly suppressed hypomyelination and promoted the differentiation of oligodendrocyte precursor cells (OPCs), as demonstrated by the increased expression levels of MBP and Olig2, the decreased expression level of GPR17, a significant increase in the number of CC-1-positive cells, and suppression of myelin ultrastructure impairment. Moreover, the mechanism of neuroprotective effects of Nec-1s against PVL is closely associated with its suppression of the RIPK1-mediated necrosis signaling molecules, RIPK1, PIPK3, and MLKL. More importantly, inhibition of RIPK1 could reduce microglial inflammatory injury by triggering the M1 to M2 microglial phenotype, appreciably decreasing the levels of M1 marker CD86 and increasing the levels of M2 markers Arg1 or CD206 in PVL mice. Taken together, inhibition of RIPK1 markedly ameliorates the brain injury and long-term neurobehavioral abnormalities of PVL mice through the reduction of neural cell necroptosis and reversing neuroinflammation.

Potential of circulating receptor-interacting protein kinase 3 levels as a marker of acute liver injury

The pathogenesis of acute liver failure (ALF) involves cell death. Necroptosis is a newly suggested programmed cell death, and receptor-interacting protein kinase 3 (RIPK3) has been reported as a marker for necroptosis. However, there are few reports on necroptosis in ALF. Therefore, we evaluated the role of cell death markers such as cytokeratin (CK) 18, cleaved CK (cCK) 18, and RIPK3 in ALF, as well as cytokines and hepatocyte growth factor (HGF). Seventy-one hospitalized patients with acute liver injury (38 nonsevere hepatitis [non-SH]/22 severe hepatitis [SH]/11 ALF) were studied. No significant difference was found for cytokines, but a substantial increase in HGF levels was found following the severity of hepatitis. The non-SH group had lower levels of CK18 and cCK18 than the SH/ALF group. RIPK3 was significantly lower in the non-SH/SH group than in the ALF group. HGF, RIPK3, and albumin levels were found to be important predictive variables. The present study suggests that cCK18, CK18, and RIPK3 are associated with the severity of hepatitis. RIPK3 and other markers related cell death may be useful for understanding the pathogenesis of ALF and as a prognostic marker of acute liver injury.