Custodiol® Supplemented with Synthetic Human Relaxin Decreases Ischemia-Reperfusion Injury after Porcine Kidney Transplantation.

Objective. Ischemia-reperfusion injury (IRI) is inevitable after kidney transplantation (KT), impairing outcomes. Relaxin-2 (RLX) is a promising insulin-related peptide hormone that protects against renal IRI in rodents, although large animal models are needed before RLX can be tested in a human setting. Methods. In this blinded, randomized, and placebo-controlled experimental study kidneys from 19 donor pigs were retrieved after perfusion with Custodiol® ± RLX (5 or 20 nmol/L) and underwent static cold storage (SCS) for 24 and 48 h, respectively. Subsequently, KT was performed after unilateral right nephrectomy. Study outcomes included markers for kidney function, oxidative stress, lipid peroxidation, and endothelial cell damage. PCR analysis for oxidative stress and apoptosis-related gene panels as well as immunohistochemistry were performed. Results. RLX upregulated SOD2 and NFKB expression to 135% (p = 0.042) and 125% (p = 0.019), respectively, while RIPK1 expression was downregulated to 82% (p = 0.016) of corresponding controls. Further RLX significantly downregulated RIPK1 and MLKL expression and decreased the number of Caspase 3- and MPO-positive cells in grafts after SCS. Conclusions. RLX supplemented Custodiol® significantly decreased IRI via both antioxidant and anti-apoptotic mechanisms. Clinical trials are warranted to implement synthetic human RLX as a novel additive to preservation solutions against IRI.

Chlorpromazine and promethazine reduces Brain injury through RIP1-RIP3 regulated activation of NLRP3 inflammasome following ischemic stroke.

Objectives: Stroke is an important cause of death and disability. Recent evidence suggests that post-stroke inflammation is an important factor in stroke pathology and a root cause of its lasting consequences. Phenothiazine drugs, like chlorpromazine and promethazine (C + P), induce hypothermia and have been shown to play a major role in neuroprotection. In the present study, we investigated this neuroprotective mechanism by assessing the anti-inflammatory effect of these drugs.Methods: Adult Sprague-Dawley rats underwent 2 h of middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion, with or without C + P (8 mg/kg). Infarct volumes, neurological deficits, along with mRNA and protein quantities of receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), NLRPyrin domain containing 3 (NLRP3), and interleukin-1ß (IL-1ß) were assessed, as well as the infiltration of neutrophils and macrophages.Results: C + P induced hypothermia that significantly reduced RIP1, RIP3, NLRP3 and IL-1ß expression, infarction, and immune cell infiltration, while C + P treatment with temperature control at 37°C induced lesser effect.Conclusion: These findings suggest that the anti-inflammatory effect of C + P may be dependent on drug-induced hypothermia and regulation of the NLRP3 inflammasome via the RIP1/RIP3 complex. Future investigations are needed regarding C + P as potential treatment of ischemic stroke.

RIP3-mediated necroptosis was essential for spiral ganglion neuron damage.

To identify the role of RIP3 in ouabain-induced necroptosis and offer clinical implications to prevent spiral ganglion neurons (SGNs) from death, ouabain was applied in SGNs derived from fetal rats and injected into Sprague-Dawley rats to construct injury model in vitro and in vivo, respectively. The necroptosis rate of SGNs was determined by flow cytometry and MTT assays. The protein levels and phosphorylation of RIP3 were evaluated using western blotting and immunofluorescence. SGNs injury was observed using H&E staining and immunofluorescence. The hearing function of rats was evaluated by the auditory brainstem response (ABR) and Distortion Product Otoacoustic Emissions (DPOAE) methods. Ouabain caused dose-dependent necroptosis in SGNs and significant loss of SGNs of the cochlear axis in vivo. RIP3 and pRIP3 were upregulated with SGNs injury promoted, and RIP3 overexpression promoted ouabain-induced necroptosis in SGNs in vitro, which could be suppressed by necrostatin-1. RIP3 knockdown inhibited ouabain-induced necroptosis and reduced the phosphorylation of MLKL but no RIP3-dependent effect on the level of MLKL. RIP3 inhibition in vivo protected rats from ouabain-induced hearing damage with reducing ABR threshold shifts and promoting DPOAE amplitudes, while overexpression of RIP3 enhanced ouabain-induced injury that could be partially reversed by necrostatin-1. A decrease of SGNs density and an upregulation of pRIP3 were observed with RIP3 overexpression, which was in contrast when RIP3 was silenced. Therefore, RIP3 was essential for mediating necroptosis in ouabain-induced SGNs damage. Targeting RIP3 may prevent SGNs from death in clinical practice, and finally help the treatment of sensorineural hearing loss.

RIPK1 Expression Associates With Inflammation in Early Atherosclerosis in Humans and Can Be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice.

BACKGROUND: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. We previously showed that macrophages in the atherogenic plaque undergo RIPK3 (receptor-interacting serine/threonine-protein kinase 3)-MLKL (mixed lineage kinase domain-like protein)-dependent programmed necroptosis in response to sterile ligands such as oxidized low-density lipoprotein and damage-associated molecular patterns and that necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1 (receptor-interacting serine/threonine-protein kinase 1), which acts as a master switch that controls whether the cell undergoes NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells)-dependent inflammation, caspase-dependent apoptosis, or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is driven largely by NF-κB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NF-κB-dependent inflammation in early atherogenic lesions, and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. METHODS: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and used loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 antisense oligonucleotides to Apoe-/- mice fed a cholesterol-rich (Western) diet for 8 weeks. RESULTS: We find that RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 antisense oligonucleotides led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, P<0.01) and plasma inflammatory cytokines (IL-1α [interleukin 1α], IL-17A [interleukin 17A], P<0.05) in comparison with controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NF-κB, TNFα [tumor necrosis factor α], IL-1α) and in vivo lipopolysaccharide- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B, E-selectin, and monocyte attachment. CONCLUSIONS: We identify RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

Hypothermia Inhibits the Expression of Receptor Interacting Protein Kinases 1 and 3 After Transient Spinal Cord Ischaemia in Rabbits.

OBJECTIVES: Necroptosis, a form of regulated necrosis, might be a potential mechanism of delayed paraplegia; therefore, its role in transient spinal cord ischaemia was investigated by immunohistochemical analysis of necroptosis related protein receptor interacting protein kinase (RIP) 1, RIP3, and cellular inhibitor of apoptosis protein (cIAP) 1/2. METHODS: This study used rabbit normothermic (n = 24) and hypothermic (n = 24) transient spinal cord ischaemia models and sham controls (n = 6). Neurological function was assessed according to a modified Tarlov score at 8 h, 1, 2, and 7 days after reperfusion (n = 6 each). Morphological changes in the spinal cord were examined using haematoxylin and eosin staining in the sham, 2, and 7 day groups. Western blot and histochemical analyses of RIP1, RIP3, and cIAP1/2, and double label fluorescent immunocytochemical studies of RIP3 and cIAP1/2 were performed at 8 h, 1, and 2 days after reperfusion (n = 6 each). RESULTS: There were significant differences in neurological function between the normothermic and hypothermic groups (median scores 0 and 5 at 7 days, p = .023). In the normothermic group, most motor neurons were lost seven days after reperfusion (p = .046 compared with sham), but they were preserved in the hypothermic group. Western blot analysis revealed the upregulation of RIP1, RIP3, and cIAP1/2 at 8 h in the normothermic group (RIP1, p = .032; RIP3, p < .001; cIAP1/2, p = .041 compared with sham), and the overexpression of RIP3 was prolonged for two days. In the hypothermic group, the expression of these proteins was not observed. The double label fluorescent immunocytochemical study revealed the induction of RIP3 and cIAP1/2 in the same motor neurons. CONCLUSIONS: These data suggest that transient normothermic ischaemia induces necroptosis, a potential factor in delayed motor neuron death, and that hypothermia may inhibit necroptosis.

Inhibition of PLK1 by capped-dose volasertib exerts substantial efficacy in MDS and sAML while sparing healthy haematopoiesis.

INTRODUCTION: Targeting the cell cycle machinery represents a rational therapeutic approach in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). Despite substantial response rates, clinical use of the PLK inhibitor volasertib has been hampered by elevated side effects such as neutropenia and infections. OBJECTIVES: The primary objective was to analyse whether a reduced dose of volasertib was able to limit toxic effects on the healthy haematopoiesis while retaining its therapeutic effect. METHODS: Bone marrow mononuclear cells (BMMNCs) of patients with MDS/sAML (n = 73) and healthy controls (n = 28) were treated with volasertib (1 µM to 1 nM) or vehicle control. Short-term viability analysis was performed by flow cytometry after 72 hours. For long-term viability analysis, colony-forming capacity was assessed after 14 days. Protein expression of RIPK3 and MCL-1 was quantified via flow cytometry. RESULTS: Reduced dose levels of volasertib retained high cell death-inducing efficacy in primary human stem and progenitor cells of MDS/sAML patients without affecting healthy haematopoiesis in vitro. Interestingly, volasertib reduced colony-forming capacity and cell survival independent of clinical stage or mutational status. CONCLUSIONS: Volasertib offers a promising therapeutic approach in patients with adverse prognostic profile. RIPK3 and MCL-1 might be potential biomarkers for sensitivity to volasertib treatment.

Immunohistochemical expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the normal human kidney development.

AIM: Present study analyses the co-localisation of RIP5 with FGFR1, FGFR2 and HIP2 in the developing kidney, as RIP5 is a major determinant of urinary tract development, downstream of FGF-signaling. METHODS: Paraffin embedded human kidney tissues of 16 conceptuses between the 6th-22th developmental week were analysed using double-immunofluorescence method with RIP5/FGFR1/FGFR2 and HIP2 markers. Quantification of positive cells were performed using Kruskal-Wallis test. RESULTS: In the 6th week of kidney development RIP5 (89.6%) and HIP2 (39.6%) are strongly expressed in the metanephric mesenchyme. FGFR1 shows moderate/strong expression in the developing nephrons (87.3%) and collecting ducts (70.5%) (p < 0.05). RIP5/FGFR1 co-localized at the marginal zone and the ureteric bud with predominant FGFR1 expression. FGFR2 (26.1%) shows similar expression pattern as FGFR1 (70.5%) in the same kidney structures. RIP5/FGFR2 co-localized at the marginal zone and the collecting ducts (predominant expression of FGFR2). HIP2 is strongly expressed in collecting ducts (96.7%), and co-localized with RIP5. In 10th week, RIP5 expression decrease (74.2%), while the pattern of expression of RIP5 and FGFR1 in collecting ducts (33.4% and 91.9%) and developing nephrons (21.9% and 32.4%) (p < 0.05) is similar to that in the 6th developmental week. Ureter is moderately expressing RIP5 while FGFR1 is strongly expressed in the ureteric wall. FGFR2 is strongly expressed in the collecting ducts (84.3%) and ureter. HIP2 have 81.1% positive cells in the collecting duct. RIP5/FGFR1 co-localize in collecting ducts and Henley's loop. CONCLUSIONS: The expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the human kidney development might indicate their important roles in metanephric development and ureteric muscle layer differentiation through FGF signaling pathways.

TAK1 suppresses RIPK1-dependent cell death and is associated with disease progression in melanoma.

Melanoma cells are highly resistant to conventional genotoxic agents, and BRAFV600/MEK-targeted therapies as well as immunotherapies frequently remain inefficient. Alternative means to treat melanoma, in particular through the induction of programmed cell death modalities such as apoptosis or necroptosis, therefore still need to be explored. Here, we report that melanoma cell lines expressing notable amounts of RIPK1, RIPK3 and MLKL, the key players of necroptosis signal transduction, fail to execute necroptotic cell death. Interestingly, the activity of transforming growth factor ß-activated kinase 1 (TAK1) appears to prevent RIPK1 from contributing to cell death induction, since TAK1 inhibition by (5Z)-7-Oxozeaenol, deletion of MAP3K7 or the expression of inactive TAK1 were sufficient to sensitize melanoma cells to RIPK1-dependent cell death in response to TNFα or TRAIL based combination treatments. However, cell death was executed exclusively by apoptosis, even when RIPK3 expression was high. In addition, TAK1 inhibitor (5Z)-7-Oxozeaenol suppressed intrinsic or treatment-induced pro-survival signaling as well as the secretion of cytokines and soluble factors associated with melanoma disease progression. Correspondingly, elevated expression of TAK1 correlates with reduced disease free survival in patients diagnosed with primary melanoma. Overall, our results therefore demonstrate that TAK1 suppresses the susceptibility to RIPK1-dependent cell death and that high expression of TAK1 indicates an increased risk for disease progression in melanoma.

High-mobility group box 1 protein-mediated necroptosis contributes to dasatinib-induced cardiotoxicity.

Dasatinib shows remarkable activity against imatinib-refractory chronic myelogenous leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL). However, severe cardiovascular toxicity limits the clinical applications of dasatinib. Since the underlying mechanism of dasatinib-induced cardiotoxicity is still elusive, we aim to clarify this. Recent studies have shown that necroptosis and apoptosis participate in multiple toxicity development. Here, we first report that dasatinib could directly induce cardiomyocytes death, as analyzed by the Sulforhodamine B (SRB) assay. This type of cardiomyocytes death was mediated by the necrosis pathway rather than apoptosis, as determined by using flow cytometry to characterize the mode of dasatinib-induced cell death. Inhibition of receptor-interacting protein kinase 1 (RIP1)activity and knockdown of receptor-interacting protein kinase 3 (RIP3)expression can block dasatinib-evoked cardiotoxicity, which further confirmed the involvement of necroptosis. We next found that the classic substrates of RIP3, mixed lineage kinase domain-like protein (MLKL) and Ca2+-calmodulin-dependent protein kinase II (CaMKII) were not involved in dasatinib-induced cardiomyocytes necroptosis. What's more, unlike the inflammation-associated necroptosis, dasatinib-triggered necroptosis was dependent on intracellular instead of secreted High-mobility group box 1 (HMGB1) protein. Collectively, our study revealed that dasatinib-induced cardiotoxicity acted via leading cardiomyocytes to HMGB1-mediated necroptosis, indicating a viable strategy for prevention of dasatinib-induced cardiotoxicity.

Necroptosis in 3-chloro-1, 2-propanediol (3-MCPD)-dipalmitate-induced acute kidney injury in vivo and its repression by miR-223-3p.

Fatty acid esters of 3-chloro-1, 2-propanediol (3-MCPD) are a group of processing-induced food contaminants with nephrotoxicity. This study investigated whether and how necroptosis played a role in the nephrotoxic effect of 3-MCPD-dipalmitate (2.5 g/kg BW) in C57 BL/6 mice. The results showed that the principal components in necroptosis pathway including receptor-interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) were up-regulated in 3-MCPD-dipalmitate-induced acute kidney injury (AKI). Deletion of RIPK3 or MLKL, and inhibition of RIPK1 suppressed AKI. The up-regulation of inflammatory cytokines in the kidney of 3-MCPD-dipalmitate treated mice were attenuated in RIPK3- or MLKL- deficient mice, suggesting a positive feedback loop involving necroptosis and inflammation. The microRNA analysis revealed that 38 known miRNAs and 40 novel miRNAs were differentially expressed (DE) in the kidney treated with 3-MCPD-dipalmitate. Of these miRNAs, miR-223-3p was significantly up-regulated during 3-MCPD-dipalmitate-induced AKI. In cultured mouse proximal tubular cells, a miR-223-3p mimic suppressed RIPK3 expression, which was blocked by miR-223-3p inhibitor. The luciferase reporter assay confirmed that miR-223-3p was able to inhibit RIPK3 expression by targeting the 3' un-translated region of RIPK3. These results suggest that necroptosis contributes to 3-MCPD-dipalmitate-induced acute kidney injury, and that may be attenuated by miR-223-3p.

TWEAK and RIPK1 mediate a second wave of cell death during AKI.

Acute kidney injury (AKI) is characterized by necrotic tubular cell death and inflammation. The TWEAK/Fn14 axis is a mediator of renal injury. Diverse pathways of regulated necrosis have recently been reported to contribute to AKI, but there are ongoing discussions on the timing or molecular regulators involved. We have now explored the cell death pathways induced by TWEAK/Fn14 activation and their relevance during AKI. In cultured tubular cells, the inflammatory cytokine TWEAK induces apoptosis in a proinflammatory environment. The default inhibitor of necroptosis [necrostatin-1 (Nec-1)] was protective, while caspase inhibition switched cell death to necroptosis. Additionally, folic acid-induced AKI in mice resulted in increased expression of Fn14 and necroptosis mediators, such as receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage domain-like protein (MLKL). Targeting necroptosis with Nec-1 or by genetic RIPK3 deficiency and genetic Fn14 ablation failed to be protective at early time points (48 h). However, a persistently high cell death rate and kidney dysfunction (72-96 h) were dependent on an intact TWEAK/Fn14 axis driving necroptosis. This was prevented by Nec-1, or MLKL, or RIPK3 deficiency and by Nec-1 stable (Nec-1s) administered before or after induction of AKI. These data suggest that initial kidney damage and cell death are amplified through recruitment of inflammation-dependent necroptosis, opening a therapeutic window to treat AKI once it is established. This may be relevant for clinical AKI, since using current diagnostic criteria, severe injury had already led to loss of renal function at diagnosis.

A novel damage mechanism: Contribution of the interaction between necroptosis and ROS to high glucose-induced injury and inflammation in H9c2 cardiac cells.

Recently, a novel mechanism known as 'programmed necrosis' or necroptosis has been shown to be another important mechanism of cell death in the heart. In this study, we investigated the role of necroptosis in high glucose (HG)-induced injury and inflammation, as well as the underlying mechanisms. In particular, we focused on the interaction between necroptosis and reactive oxygen species (ROS) in H9c2 cardiac cells. Our results demonstrated that the exposure of H9c2 cardiac cells to 35 mM glucose (HG) markedly enhanced the expression level of receptor-interacting protein 3 (RIP3), a kinase which promotes necroptosis. Importantly, co-treatment of the cells with 100 µM necrostatin-1 (a specific inhibitor of necroptosis) and HG for 24 h attenuated not only the increased expression level of RIP3, but also the HG-induced injury and inflammation, as evidenced by an increase in cell viability, a decrease in ROS generation, the attenuation of the dissipation of mitochondrial membrane potential and a decrese in the secretion levels of inflammatory cytokines, i.e., interleukin (IL)-1ß and tumor necrosis factor (TNF)-α. Furthermore, treatment of the cells with 1 mM N-acetyl­L­cysteine (a scavenger of ROS) for 60 min prior to exposure to HG significantly reduced the HG-induced increase in the RIP3 expression level, as well as the injury and inflammatory response described above. Taken together, the findings of this study clearly demonstrate a novel damage mechanism involving the positive interaction between necroptosis and ROS attributing to HG-induced injury and inflammation in H9c2 cardiac cells.

A Three-Dimensional Lymphatic Endothelial Cell Tube Formation Assay to Identify Novel Kinases Involved in Lymphatic Vessel Remodeling.

The lymphatic system is a series of vessels that transport cells and excess fluid from tissues to the blood vascular system. Normally quiescent, the lymphatics can grow or remodel in response to developmental, immunological, or cells pathological stimuli. Lymphatic vessels comprise lymphatic endothelial cells (LECs) that can respond to external growth factors by undergoing proliferation, migration, adhesion, and tube and lumen formation into new vessel structures, a process known as lymphangiogenesis. To understand the key gene and signaling pathways necessary for lymphangiogenesis and lymphatic vessel remodeling, we have developed a three-dimensional LEC tube formation assay to explore the role of kinase signaling in these processes. The collagen-overlay-based assay was used with primary human adult dermal LECs to investigate a library of 60 tyrosine kinase (TK) and TK-like genes by siRNA knockdown. Nine candidate genes were identified and characterized for their ability to modify key parameters of lymphatic tube formation, including tube length, area, thickness, branching, and number of blind-ended sacs. Four genes-ZAP70, IRAK4, RIPK1, and RIPK2-were identified as high-confidence hits after tertiary deconvolution screens and demonstrate the utility of the assay to define LEC genes critical for the formation of tube structures. This assay facilitates the identification of potential molecular targets for novel drugs designed to modulate the remodeling of lymphatics that is important for the metastatic spread of cancer and other pathologies.

Protective effect of necrostatin-1 on myocardial tissue in rats with acute myocardial infarction.

The aim of this study was to investigate the protective effect of necrostatin-1 on myocardial tissue of acute myocardial infarction (AMI) rats and to provide a basis for necrostatin-1 for the treatment of acute myocardial infarction. AMI rats (45) were established by ligating the anterior descending branch of the left coronary artery. The rats were randomly divided into the model group and necrostatin-1 low-dose and high-dose groups. The control group rats (15) underwent the sham operation. The rats in the necrostatin-1 low-dose and high-dose groups were injected with 1 and 4 mg/kg necrostatin-1, respectively, via the tail vein. The rats in the control and model groups were injected with isometric dimethyl sulfoxide, once daily, for 3 consecutive days. The levels of RIP1 and RIP3 mRNA and phosphorylated protein in the myocardial tissue of rats were detected by real time polymerase chain reaction and western blot. The myocardial infarct size was detected by tetrazolium chloride. Compared with that in the control group, the levels of RIP1 and RIP3 mRNA and phosphorylated protein significantly increased in the myocardial tissue of model group rats, necrostatin-1 low-dose group, and high-dose group. The levels of RIP1 and RIP3 mRNA and phosphorylated protein in the myocardial tissue of rats in the necrostatin-1 low-dose and high-dose groups decreased significantly compared with that in the model group (P < 0.05). The levels of RIP1 and RIP3 mRNA in the myocardium of the high-dose group rats were significantly lower than those of the low-dose group rats (P < 0.05). The myocardial infarct sizes significantly increased in model, low-dose, and high-dose group rats. The apoptotic level of myocardial cells significantly decreased in the low-dose group and high-dose group after treatment with necrostatin-1 but was still higher than that of the control group (P < 0.05). In conclusion, necrostatin-1 can inhibit myocardial tissue apoptosis and necrosis in acute myocardial infarct rats and has a protective effect on myocardial tissue.

MLKL inhibition attenuates hypoxia-ischemia induced neuronal damage in developing brain.

Mixed lineage kinase domain-like protein (MLKL) is a critical molecule mediating cell necroptosis. However, its role in brain injury remains obscure. We first investigated the functions and mechanisms of MLKL in mediating neuronal damage in developing brain after hypoxia-ischemia. Neuronal necroptosis was induced by oxygen-glucose deprivation (OGD) plus caspase inhibitor zVAD treatment (OGD/zVAD). We found that two important necroptosis related proteins, receptor-interacting protein 1 and 3 (RIP1, RIP3) were upregulated. Furthermore, the interaction of RIP1-RIP3 with MLKL increased. Inhibition of MLKL through siRNA diminished RIP1-RIP3-MLKL interaction and attenuated neuronal death induced by OGD/zVAD. The translocation of oligomerized MLKL to the neuronal membrane leading to the injury of cellular membrane is the possible new mechanism of neuronal necroptosis. Animal experiment with neonatal rats further proved that MLKL inhibition attenuated brain damage induced by hypoxia-ischemia. These findings suggest that MLKL is a target to attenuate brain damage in developing brain.

Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury.

Chronic alcohol exposure increased hepatic receptor-interacting protein kinase (RIP) 3 expression and necroptosis in the liver but its mechanisms are unclear. In the present study, we demonstrated that chronic alcohol feeding plus binge (Gao-binge) increased RIP3 but not RIP1 protein levels in mouse livers. RIP3 knockout mice had decreased serum alanine amino transferase activity and hepatic steatosis but had no effect on hepatic neutrophil infiltration compared with wild type mice after Gao-binge alcohol treatment. The hepatic mRNA levels of RIP3 did not change between Gao-binge and control mice, suggesting that alcohol-induced hepatic RIP3 proteins are regulated at the posttranslational level. We found that Gao-binge treatment decreased the levels of proteasome subunit alpha type-2 (PSMA2) and proteasome 26S subunit, ATPase 1 (PSMC1) and impaired hepatic proteasome function. Pharmacological or genetic inhibition of proteasome resulted in the accumulation of RIP3 in mouse livers. More importantly, human alcoholics had decreased expression of PSMA2 and PSMC1 but increased protein levels of RIP3 compared with healthy human livers. Moreover, pharmacological inhibition of RIP1 decreased Gao-binge-induced hepatic inflammation, neutrophil infiltration and NF-κB subunit (p65) nuclear translocation but failed to protect against steatosis and liver injury induced by Gao-binge alcohol. In conclusion, results from this study suggest that impaired hepatic proteasome function by alcohol exposure may contribute to hepatic accumulation of RIP3 resulting in necroptosis and steatosis while RIP1 kinase activity is important for alcohol-induced inflammation.

Naringenin protects cardiac hypercholesterolemia-induced oxidative stress and subsequent necroptosis in rats.

BACKGROUND: In earlier studies, the supplementation of the natural compound Naringenin (NGEN), improved the liver oxidative and inflammatory status, which indicates its direct effect via inhibition of the nuclear factor κB pathway on high cholesterol-induced hepatic damages. In this regard, the present study highlights the mechanisms associated with the protective efficacy of NGEN in the heart tissue of hypercholesterolemic diet rats. RESULTS: The animals exposed to a high cholesterol diet (HCD) for 90 days exhibited a significant increase in the levels of serum lactate dehydrogenase (LDH) and creatine kinase (CK) activities, nitric oxide (NO) levels, protein and lipid oxidative markers and cardiac lipids profile. Moreover, hypercholesterolemia decreased the levels of enzymatic and non enzymatic antioxidants associated with mitochondrial dysfunctions as proved by the decrease in the mitochondrial complexes in comparison to controls. Importantly, cholesterol-feeding significantly increased myocardial reactive oxygen species (ROS) and nuclear DNA damage and led to the activation of gene expression of the tumor necrosis factor-α (TNF-α) and receptor-interacting protein kinase 3 (RIP3) mRNA that contributed to the elucidation of cholesterol-induced necroptosis, a recently described type of programmed necrosis, in the cardiac tissue. CONCLUSIONS: Our results show that the co-administration of NGEN (50 mg/kg/bw) in HCD rats improved all the altered parameters and provided insight into a possible molecular mechanism underlying NGEN suppression of necroptosis pathway in the heart.

The natural compound celastrol inhibits necroptosis and alleviates ulcerative colitis in mice.

Ulcerative colitis (UC) is a chronic intestinal inflammatory disease. Necroptosis plays an important role in the pathogenesis of UC. Celastrol, a triterpene from the root bark of the Chinese medicinal plant Tripterygium wilfordii, has been reported to have anti-oxidant and anti-inflammatory activities in colitis. It is not known, however, how celastrol exerts its beneficial effects. The aim of this study is to investigate the effects and possible mechanism of celastrol in UC. Colitis was induced in mice by administration of 5% dextran sulfate sodium (DSS) in drinking water for 4days. Celastrol was administered intraperitoneally (1mg/kg) for 7days after colitis was induced. Our results showed that celastrol treatment ameliorated the severity of colitis, decreased the level of interleukin (IL)-1ß, IL-6 and myeloperoxidase (MPO) and upregulated the level of E-cadherin in colitis mice. Moreover, the TUNEL staining and cleaved caspase-3 immunohistochemistry staining proved decreased necrotic cell death after celastrol treatment. On the mechanism, decreased level of necroptosis factors RIP3 and MLKL, and increased level of active caspase-8 were detected after celastrol treatment. Taken together, our results demonstrated that celastrol exerted beneficial effects in colitis treatment via suppressing the RIP3/MLKL necroptosis pathway.

RIPK3 expression in cervical cancer cells is required for PolyIC-induced necroptosis, IL-1α release, and efficient paracrine dendritic cell activation.

Previous studies have shown that cervical cancer cells only release low levels of pro-inflammatory cytokines owing to infection with human papillomaviruses. This results in low immunogenicity of the cancer cells. The viral dsRNA analog PolyIC has been suggested as a promising adjuvant for cervical cancer immunotherapy. However, little is known about the molecular requirements resulting in successful immune activation. Here, we demonstrate that stimulation of cervical cancer cells with PolyIC induced necroptotic cell death, which was strictly dependent on the expression of the receptor-interacting protein kinase RIPK3. Necroptotic cancer cells released interleukin-1α (IL-1α), which was required for powerful activation of dendritic cells (DC) to produce IL-12, a cytokine critical for anti-tumor responses. Again both, IL-1α release and DC activation, were strictly dependent on RIPK3 expression in the tumor cells. Of note, our in situ analyses revealed heterogeneous RIPK3 expression patterns in cervical squamous cell carcinomas and adenocarcinomas. In summary, our study identified a novel RIPK3-dependent mechanism that explains how PolyIC-treatment of cervical cancer cells leads to potent DC activation. Our findings suggest that the RIPK3 expression status in cervical cancer cells might critically influence the outcome of PolyIC-based immunotherapeutic approaches and should therefore be assessed prior to immunotherapy.

Nucleotide-binding oligomerization domain 2 receptor is expressed in platelets and enhances platelet activation and thrombosis.

BACKGROUND: Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. METHOD AND RESULTS: Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1ß maturation and accumulation in human and mouse platelets NOD2 dependently. CONCLUSIONS: NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.