Programmed myofibre necrosis in critical illness acquired muscle wasting

PurposeTo reveal the role of receptor-interacting protein kinase 3 (RIPK3) in regulating macrophage inflammation and corneal neovascularization (CoNV) induced by alkali burn.MethodsA corneal alkali burn (AB) model was established in C57BL/6J (wild-type) and RIPK3fl/flCx3cr1+/cre (RIPK3−/−, RIPK3 knockout [KO]) mice using sodium hydroxide. Anterior segment optical coherence tomography and hematoxylin and eosin staining were used to evaluate the impact of RIPK3 on corneal edema and morphology. CoNV was detected by slit-lamp microscopy and whole-mount immunofluorescence staining of cornea. Corneal macrophage and necroptotic cell death was analyzed through immunofluorescence staining and propidium iodide (PI) staining. Activation of the necroptosis pathway was examined after corneal AB by western blot.ResultsNecroptosis-related proteins RIPK1, RIPK3, and mixed lineage kinase domain-like (MLKL) were upregulated and activated following corneal AB. Among these, RIPK3 demonstrated the most pronounced increase. Notably, the elevated level of RIPK3 was prominently colocalized with the infiltrating F4/80+ macrophages. RIPK3 KO significantly alleviated corneal edema and morphology defects. Additionally, as the corneal morphological defects progressed, macrophages became activated, and CoNV and lymphangiogenesis (LyG) were enhanced. RIPK3 KO markedly reduced AB-induced macrophage accumulation, as well as CoNV and LyG. RIPK3 KO mice also showed a meaningful decrease in PI+ necroptotic cells. Mechanistically, AB-induced necroptosis stimulated the expression of MLKL and fibroblast growth factor 2 (FGF2), whereas RIPK3 deficiency decreased their expression.ConclusionsThis study revealed that RIPK3-mediated necroptosis drives macrophage inflammation and CoNV. Targeting RIPK3 could effectively suppress these responses by inhibiting the MLKL/FGF2 pathway, making it a promising therapeutic strategy for corneal AB.

Necroptosis is one of several programmed lytic cell death processes for which key effector proteins have only been defined and experimentally dissected in the last decade. Unlike apoptotic cell death, where cellular contents undergo membrane-contained caspase-mediated disassembly, necroptosis leads to the release of highly inflammatory intracellular components e.g. histones into the surrounding milieu.1 Necroptotic cell death is thought to have originally evolved as a pathogen defence mechanism. As such, necroptosis can be induced by a series of cytokine- and pathogen detecting- receptors binding their appropriate ligands. Many of these signaling routes to necroptosis employ the apex kinase receptor-interacting serine/threonine protein kinase 1 (RIPK1). All upstream necroptotic signaling culminates in the activation of the effector protein Mixed Lineage Kinase domain-Like (MLKL) by its obligate activating kinase, receptor interacting protein kinase 3 (RIPK3). Following activation, MLKL associates with phospholipids to promote potassium efflux and eventual membrane bilayer destabilization, oncosis and cell death.1 This lytic form of programmed cell death has been implicated in the etiology of a range of human pathologies. Specifically, its role in lung disease has recently come to the fore following prominent publications implicating necroptosis in lung epithelial cell damage and inflammatory neutrophil influx. Following a lethal dose of influenza A virus (IAV), the absence of necroptosis reduces mortality, protecting mice from lung epithelial cell damage and influx of Neutrophil Extracellular TRAP (NET)-forming neutrophils.2 Mice lacking necroptosis were also seen to be protected from airway remodelling and inflammation in a model of cigarette smoke-induced chronic obstructive pulmonary disease (COPD).3 These and other studies illustrate the contribution of necroptosis to the progression of lung pathologies and present an intriguing basis for recent work by Oikonomou et al.4 The relative contribution of necroptosis in specific tissues or cell types can be investigated using whole body or tissue-specific genetic modification of upstream and core necroptotic machinery. Genetic deletion of obligate necroptotic machinery (MLKL or RIPK3), or ablation of RIPK1 kinase activity, enables examination of necroptotic contribution through elimination. Conversely, genetic manipulation of certain upstream pathway components can be used to induce spontaneous activation of necroptosis. The most commonly used models of spontaneous necroptosis in mice are Fas-associated protein with death domain (FADD) and Caspase-8 knockouts. In addition to their adapter and enzymatic roles in apoptosis pathways, FADD and Caspase-8 also function as gate keepers to the necroptosis-inducing signaling platform comprising RIPK1 and RIPK3. The absence of FADD or Caspase-8 makes way for the unimpeded assembly of this necroptosis activation platform, the activation of MLKL and cell death. Whole body FADD or Caspase-8 gene knockout mice die before birth. Oikonomou et al. employ both airway epithelial cell (AEC)-specific ablation of FADD (necroptosis activation) and complementary whole body RIPK3 knockout, MLKL knockout or RIPK1 kinase activity knock-in (necroptosis elimination) to dissect the role of this pathway in a model of asthma induced by house dust mite extract sensitization and challenge. Regulation of necroptosis in barrier tissues, such as skin and intestines, is known to be an important mechanism in maintaining immune homeostasis. Targeted gene deletion of FADD, Caspase-8, or RIPK1 sensitises epithelial cells to necroptosis. This strategy has been used by the Pasparakis lab and others to demonstrate that dysregulated necroptosis in intestinal epithelial cells or keratinocytes alone is sufficient to induce the spontaneous development of inflammatory gut or skin damage.5-8 In barrier tissues like the gut or skin, where abundant microbially derived stimuli favour the expression and unmitigated assembly of RIPK1-RIPK3, the specific genetic ablation of FADD alone is sufficient to trigger the cascade of necroptosis and inflammation. Oikonomou et al. conclude that mice with a FADD-deficient airway epithelium (FADDAEC-KO) do not exhibit this same capacity for spontaneous necroptosis in the lung at steady state. They propose this may be due to a lower microbial load in the lung relative to the gut or skin. However, priming the airway epithelium with a physiologically-relevant stimulus in the form of house dust mite extract unmasks a capacity for an inflammatory response in the lungs of FADDAEC-KO mice that is over and above that of FADD-sufficient mice (Figure 1). This augmented inflammatory response in FADDAEC-KO mice manifests in both enhanced inflammatory cytokines and immune cell infiltrates. Oikonomou et al. used genetic deletion of Ripk3 to assess the contribution of FADD-deficient AEC necroptosis in house dust mite-induced airway inflammation. FADDAEC-KO, Ripk3-/- mice exhibited limited immune cell infiltration and similar inflammatory cytokines levels when compared to control mice. Consistent with this finding, FADDAEC-KO, Ripk1D138N kinase dead, and FADDAEC-KO, Mlkl-/- mice also showed an attenuation of this house dust mite-induced pathology. FADDAEC-KO mice were protected from induced airway hyperresponsiveness, resembling mice that had not been treated with house dust mite extract. Despite exaggerated inflammation induced by house dust mite extract, FADDAEC-KO mice exhibited reduced mucus production stemming from reduced numbers of mucus-producing goblet cells in the lung relative to fadd fl/fl controls. This loss of goblet cells was not attributed to enhanced necroptosis in these mice, but to the expression of Cre-recombinase itself. While this confounding experimental artifact precluded examination of the full physiological impact of enhanced airway epithelial cell necroptosis in this model, it still stands that inflammation, mucus production and airway hyperresponsiveness certainly go hand-in-hand in real life. It is thus reasonable to predict that necroptosis-enhanced airway inflammation would also enhance mucus production and airway hyperresponsiveness. These carefully controlled experimental data reveal an important caveat to the future use of the Scgb1a1 (AEC-specific promoter)-Cre transgene for the study of airway epithelial cell death itself, with airway epithelial cell death unrelated to FADD activity being observed on the microscopic level. This study by Oikonomou et al. also lends further credence to previous research that examined the effectiveness of targeting downstream mucus hypersecretion for the amelioration of airway hyperresponsiveness,9 but certainly does not detract from the potential benefits of stopping the train further up the etiological line at necroptosis. An enhanced propensity for airway epithelial cell necroptosis (through conditional FADD ablation) quantitatively intensifies inflammation in the lungs. While unable to directly demonstrate or quantify necroptotic epithelial cell death in situ in FADDAEC-KO mice, the protein level of the MLKL-activating kinase, RIPK3, is clearly upregulated in the lungs. Detection of dead or near-dead necroptotic cells remains highly challenging in mouse tissues however, improvements in necroptosis-detecting tools and techniques will soon permit a more reliable detection in such scenarios.10 The work of Oikonomou et al. prompts further questions regarding the potential role of necroptotic cell death in human asthma: Is the propensity for necroptosis what distinguishes patients with severe life-long chronic asthma from those with milder forms? Is it the activation and perpetuation of necroptosis that underpins the role of common respiratory viruses in inducing asthma? Is the enhanced propensity for airway epithelial cell necroptosis mediated purely by infection history and the adaptive immune response, and/or is there a direct genetic influence on propensity for necroptosis that contributes to asthma risk in humans? Etiology aside, this work provides an important precedent for further preclinical exploration of whether asthmatics could benefit from the emerging class of necroptosis-inhibitor drugs currently in development. JMM and JMH contribute to a project developing necroptosis inhibitors in collaboration with Anaxis Pty Ltd. Sarah E Garnish: Writing-original draft. Emma C Tovey Crutchfield: Writing-original draft. James M Murphy: Writing-original draft. Joanne M Hildebrand: Conceptualization; Writing-original draft; Writing-review & editing.

RIPK3 deficiency ameliorates diabetic sarcopenia through proteostasis regulation by suppressing inflammatory signaling and cellular stress pathways.

Objective: Aortic smooth muscle cell (SMC) depletion is a major pathological feature of abdominal aortic aneurysm (AAA). Our lab has previously reported elevated levels of Receptor Interacting Protein Kinase-3 (RIPK3), a major mediator of necroptosis, in human AAA tissues. Pharmacologic inhibition of RIPK3 or deletion of the Ripk3 gene prevents aortic SMC death and attenuates aneurysm growth in murine models. In the current study, we tested the hypothesis that manipulation of Ripk3 gene expression would alter AAA development. Methods: We manipulated Ripk3 gene expression by mutating a conserved putative transcription enhancer site using CRISPR-Cas9-mediated gene editing. AAA was induced in 8-week old male CRISPR mutated (CM) and wild-type (WT) mice by perivascular application of CaCl 2 or NaCl (sham). Aortic tissues were harvested on days 4 or 28 post-surgery and analyzed by immunohistochemistry (IHC). Primary aortic SMCs isolated from both groups were stimulated with inflammation-related cytokines and Ripk3 mRNA was examined by RT-PCR. Results: Both CM and WT mice developed AAA in response to CaCl 2 . Change in aortic diameter was greater in CM mice than WT at 28 days (66.81±4.512 vs 44.87±6.685%, P =0.0108). IHC showed CM aneurysm tissue to exhibit fewer SMCs and a greater number of non-SMC cells in the tunica media, presumably infiltrated immune cells. Furthermore, mutant tissues contained higher levels of RIPK3 positivity than WT aortas. Consistently, CM aortic SMCs expressed more Ripk3 mRNA than WT SMCs, both with vehicle and following stimulation with IL-1β, IL-25, and IL-33. Conclusions: Our data show a positive correlation between Ripk3 expression and aneurysm size. We are currently analyzing data from experiments aimed to prove the causal relationship between Ripk3 gene editing, SMC necroptosis, inflammation, and aneurysm growth. This study further illustrates the potential of RIPK3 as a therapeutic target of AAA.

The aim of this study was to explore the function of receptor-interacting protein kinase 3 (RIPK3) on retinal neuron damage induced by retinal ischemia/reperfusion (IR). Microglia-specific RIPK3 knockout (KO) mice were employed to establish retinal IR models. Retinal structural and functional status was assessed using hematoxylin and eosin staining along with electroretinogram. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was employed to detect the situations of apoptotic cell death. Immunofluorescence and western blot were applied to detect the proteins associated with necroptosis and retinal inner neurons. Following retinal IR, necroptosis-related protein RIPK3 became activated within microglia, inducing the activation of mixed lineage kinase domain-like protein (MLKL). RIPK3 KO significantly alleviated IR-induced retinal morphological defects and protected against IR-induced visual dysfunction by preserving neurons within the retina. Additionally, the counts of TUNEL+ apoptotic cells were markedly reduced within RIPK3 KO mice after IR, along with a decrease in retinal inflammatory responses. Mechanistically, IR injury promoted retinal ganglion cells (RGCs) death by activating RIPK3 to induce MLKL and fibroblast growth factor 2 (FGF2) activation; however, RIPK3 KO suppressed this process. After IR, RIPK3-mediated necroptosis in microglia induced its activation, promoting inflammatory responses and thereby facilitating RGCs death. Targeting RIPK3 could protect retinal neurons from injury after IR through suppressing the MLKL/FGF2 pathway, rendering this a potential curative measure for RGCs degeneration in ischemic retinopathy.

Necroptosis is a form of programmed necrotic cell death that is rapidly emerging as an important pathophysiological pathway in numerous disease states. Necroptosis is dependent on receptor-interacting protein kinase 3 (RIPK3), a protein shown to play an important role in experimental models of critical illness. However, there is limited clinical evidence regarding the role of extracellular RIPK3 in human critical illness. Plasma RIPK3 levels were measured in 953 patients prospectively enrolled in 5 ongoing intensive care unit (ICU) cohorts in both the USA and Korea. RIPK3 concentrations among groups were compared using prospectively collected phenotypic and outcomes data. In all 5 cohorts, extracellular RIPK3 levels in the plasma were higher in patients who died in the hospital compared with those who survived to discharge. In a combined analysis, increasing RIPK3 levels were associated with elevated odds of in-hospital mortality (odds ratio [OR] 1.7 for each log10-unit increase in RIPK3 level, P < 0.0001). When adjusted for baseline severity of illness, the OR for in-hospital mortality remained statistically significant (OR 1.33, P = 0.007). Higher RIPK3 levels were also associated with more severe organ failure. Our findings suggest that elevated levels of RIPK3 in the plasma of patients admitted to the ICU are associated with in-hospital mortality and organ failure. Supported by NIH grants P01 HL108801, R01 HL079904, R01 HL055330, R01 HL060234, K99 HL125899, and KL2TR000458-10. Supported by Samsung Medical Center grant SMX1161431.

IntroductionSepsis is defined as a systemic hyper-inflammatory immune response, with a subsequent immune-suppressive phase, which leads to multiple organ dysfunction and late lethality. Receptor-interacting protein kinase 3 (RIPK3)-dependent necrosis is implicated in driving tumor necrosis factor alpha (TNF-α)- and sepsis-induced mortality in mice. However, it is unknown if RIPK3 deficiency has any impact on immune cell trafficking, which contributes to organ damage in sepsis.MethodsTo study this, male wild-type (WT) and RIPK3-deficient (Ripk3-/-) mice on C57BL/6 background were subjected to sham operation or cecal ligation and puncture (CLP)-induced sepsis. Blood and tissue samples were collected 20 hours post-CLP for various measurements.ResultsIn our severe sepsis model, the mean survival time of Ripk3-/- mice was significantly extended to 68 hours compared to 41 hours for WT mice. Ripk3-/- mice had significantly decreased plasma levels of TNF-α and IL-6 and organ injury markers compared to WT mice post-CLP. In the lungs, Ripk3-/- mice preserved better integrity of microscopic structure with reduced apoptosis, and decreased levels of IL-6, macrophage inflammatory protein (MIP)-2 and keratinocyte-derived chemokine (KC), compared to WT. In the liver, the levels of MIP-1, MIP-2 and KC were also decreased in septic Ripk3-/- mice. Particularly, the total number of neutrophils in the lungs and liver of Ripk3-/- mice decreased by 59.9% and 66.7%, respectively, compared to WT mice post-CLP. In addition, the number of natural killer (NK) and CD8T cells in the liver decreased by 64.8% and 53.4%, respectively, in Ripk3-/- mice compared to WT mice post-sepsis.ConclusionsOur data suggest that RIPK3 deficiency modestly protected from CLP-induced severe sepsis and altered the immune cell trafficking in an organ-specific manner attenuating organ injury. Thus, RIPK3 acts as a detrimental factor in contributing to the organ deterioration in sepsis.

The dwindling list of antimicrobial agents exhibiting broad efficacy against clinical strains of Mycobacterium tuberculosis (Mtb) has forced the medical community to redefine current approaches to the treatment of tuberculosis (TB). Host receptor-interacting protein kinase 3 (RIPK3) has been flagged recently as a potential target, given that it is believed to regulate necroptosis-independent signaling pathways, which have been implicated in exacerbating several inflammatory conditions and which reportedly play a role in the necrosis of Mtb-infected macrophages. To examine the therapeutic potential of inhibiting RIPK3, we infected RIPK3-deficient mice with aerosolized Mtb. We found that the loss of RIPK3 did not alter overall disease outcomes, with deficient animals harboring similar bacterial numbers in the lungs and spleens compared to their wild-type counterparts. Mtb-infected macrophages were not rescued from dying by Ripk3 deletion, nor did this affect production of the pro-inflammatory cytokine IL-1β, both in vitro and in vivo. Infiltration of immune cells into the lungs, as well as the activation of adaptive immunity, similarly was not overtly affected by the loss of RIPK3 signaling. Collectively, our data argue against a role of RIPK3 in mediating pathological inflammation or macrophage necrosis during Mtb disease pathogenesis and thus suggest that this host protein is unlikely to be an attractive therapeutic target for TB.

BackgroundTanshinone I (TI) is a primary component of Salvia miltiorrhiza Bunge (Danshen), which confers a favorable role in a variety of pharmacological activities including cardiovascular protection. However, the exact mechanism of the cardiovascular protection activity of TI remains to be illustrated. In this study, the cardiovascular protective effect and its mechanism of TI were investigated.MethodsIn this study, tert-butyl hydroperoxide (t-BHP)-stimulated H9c2 cells model was employed to investigate the protective effect in vitro. The cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) kit. The reactive-oxygen-species (ROS) level and mitochondrial membrane potential (MMP) were investigated by the flow cytometry and JC-1 assay, respectively. While in vivo experiment, the cardiovascular protective effect of TI was determined by using myocardial ischemia–reperfusion (MI/R) model including hematoxylin–eosin (H&E) staining assay and determination of superoxide dismutase (SOD) and malondialdehyde (MDA). Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release were detected by Enzyme-linked immunosorbent assay (ELISA). Receptor interacting protein kinase 1 (RIP1), receptor interacting protein kinase 3 (RIP3), receptor interacting protein kinase 3 (MLKL), protein kinase B (Akt), Nuclear factor erythroid 2 related factor 2 (Nrf2), Heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase-1 (NQO-1) were determined by western blotting.ResultsOur data demonstrated that TI pretreatment attenuated t-BHP and MI/R injury-induced necroptosis by inhibiting the expression of p-RIP1, p-RIP3, and p-MLKL. TI activated the Akt/Nrf2 pathway to promote the expression of antioxidant-related proteins such as phosphorylation of Akt, nuclear factor erythroid 2 related factor 2 (Nrf2), quinone oxidoreductase-1 (NQO-1) and heme oxygenase-1 (HO-1) expression in t-BHP-stimulated H9c2 cells. TI relieved oxidative stress by mitigating ROS generation and reversing MMP loss. In vivo experiment, TI made electrocardiograph (ECG) recovery better and lessened the degree of myocardial tissue damage. The counts of white blood cell (WBC), neutrophil (Neu), lymphocyte (Lym), and the release of TNF-α and IL-6 were reversed by TI treatment. SOD level was increased, while MDA level was decreased by TI treatment.ConclusionCollectively, our findings indicated that TI exerted cardiovascular protective activities in vitro and in vivo through suppressing RIP1/RIP3/MLKL and activating Akt/Nrf2 signaling pathways, which could be developed into a cardiovascular protective agent.

Acute ischemic stroke (AIS) is a common cerebrovascular disease with high mortality. AIS patients in the intensive care unit (ICU) often have severe conditions that require close monitoring and timely treatment. Receptor-interacting protein kinase 1 (RIPK1) and RIPK3 play important roles in cell apoptosis and inflammation. However, the relevance of serum RIPK1/3 to AIS patients in the ICU has not been clarified. To explore the correlation of serum RIPK1 and RIPK3 with the prognosis of AIS patients in the ICU. One hundred and twenty AIS patients were selected as the research subjects for the retrospective analysis. The subjects were grouped based on the volume of cerebral infarction and the score of the National Institute of Health Stroke Scale (NIHSS) and mRS. The correlation was explored using Pearson analysis. The predictive value was valued using the ROC curve. The content of serum RIPK1 and RIPK3 was gradually elevated with increased cerebral infarction volume and the severity of the disease (p < 0.05). Patients with poor prognosis had a higher content of serum RIPK1 and RIPK3 than those with good prognosis (p < 0.05). Serum RIPK1 and RIPK3 levels were positively correlated with infarct volume, NHISS, and mRS scores (p < 0.001). The area under the curve (AUC) of RIPK1 and RIPK3 for predicting the severity of AIS was 0.703, 0.883, and 0.912, respectively. The AUC for predicting poor prognosis of AIS was 0.797, 0.721, and 0.893, respectively. The cooperative detection of RIPK1 and RIPK3 had higher clinical value. AIS patients in the ICU had abnormally elevated content of serum RIPK1 and RIPK3, which was closely related to the volume of cerebral infarction, severity, and prognosis. Combined detection of RIPK1 and RIPK3 might help to early identify the severity and evaluate the prognosis, providing a reference basis for clinical doctors to develop treatment strategies.

Chronic kidney disease (CKD)-associated cardiac injury is a common complication in patients with CKD. Indole-3 acetic acid (IAA) is a uremic toxin that injures the cardiovascular system. Saikosaponin A (SSA) protects against pressure overload-induced cardiac fibrosis. However, the role and molecular mechanisms of IAA and SSA in CKD-associated cardiac injury remain unclear. The present study investigated the effects of IAA and SSA on CKD-associated cardiac injury in neonatal mouse cardiomyocytes and a mouse model of CKD. The expression of tripartite motif-containing protein 16 (Trim16), receptor interacting protein kinase 2 (RIP2) and phosphorylated-p38 were assessed using western blotting. The ubiquitination of RIP2 was measured by coimmunoprecipitation, and mouse cardiac structure and function were evaluated using hematoxylin and eosin staining and echocardiography. The results demonstrated that, SSA inhibited IAA-induced cardiomyocyte hypertrophy, upregulated Trim16 expression, downregulated RIP2 expression and decreased p38 phosphorylation. Furthermore, Trim16 mediated SSA-induced degradation of RIP2 by ubiquitination. In a mouse model of IAA-induced CKD-associated cardiac injury, SSA upregulated the protein expression levels of Trim16 and downregulated those of RIP2. Moreover, SSA alleviated heart hypertrophy and diastolic dysfunction in IAA-treated mice. Taken together, these results suggest that SSA is a protective agent against IAA-induced CKD-associated cardiac injury and that Trim16-mediated ubiquitination-related degradation of RIP2 and p38 phosphorylation may contribute to the development of CKD-associated cardiac injury.

Mouse Skeletal Muscle Fiber-Type-Specific Macroautophagy and Muscle Wasting Are Regulated by a Fyn/STAT3/Vps34 Signaling Pathway

Necrostatin-1 protects corneal epithelial cells by inhibiting the RIPK1/RIPK3/MLKL cascade in a benzalkonium chloride-induced model of necroptosis

Recent studies have shown that exposure to sevoflurane in developing brains causes neuronal apoptosis and cognitive dysfunction. "Necroptosis" is a novel pathway of necrosis. We introduced the caspase-specific inhibitor Z-VAD in addition to the receptor-interacting protein kinase 1 (RIPK1) inhibitor Nec-1, to ascertain the existence and importance of necroptosis. Sprague-Dawley rat pups postnatal day 7 were randomly assigned into one of five groups: control, sevoflurane + Z-VAD, sevoflurane + Nec-1, sevoflurane + Z-VAD + Nec-1 and 3% sevoflurane group. Neuronal apoptosis was evaluated by hematoxylin and eosin staining. The MTT assay was performed to evaluate cell viability. Immunofluorescence was employed to measure expression of RIPK1 and RIPK3. Western blots showing expression of RIPK1, RIPK3 and phosphorylation of mixed lineage kinase domain-like (p-MLKL) were used to explore the role of necroptosis. Binding of RIPK1/RIPK3 was detected via co-immunoprecipitation. Finally, the Morris water maze test was used to determine cognitive function. Exposure to 3% sevoflurane for 6h induced neurotoxicity and inhibited cell viability. Neuron viability was low in the SEV, SEV + Z-VAD and SEV + Nec-1 groups. The study revealed that RIPK1 and RIPK3 protein expression increased significantly, but there was no significant differences between the SEV and SEV + Z-VAD groups. The expression of p-MLKL significantly increased in the SEV and SEV + Z-VAD groups, but not in the SEV + Nec-1 group or SEV + Z-VAD + Nec-1 group compared to the control group. Co-immunoprecipitation results showed that sevoflurane exposure enhanced binding of RIPK1/RIPK3 protein significantly. Blockade of apoptosis and necroptosis alleviated sevoflurane-induced cognitive impairment. Sevoflurane exposure elicited neurotoxicity within neonatal hippocampal neurons and tissues. Blockade of apoptosis or necroptosis alone did not attenuate sevoflurane-induced neurotoxicity (SIN). RIPK1/RIPK3-mediated necroptosis was involved in SIN in hippocampal neurons. SIN could be attenuated only by inhibiting both apoptosis and necroptosis.

Receptor-interacting protein kinase 3: A macromolecule with multiple cellular actions and its perspective in the diagnosis and treatment of heart disease.