A prospective cohort study regarding usability of serum RIPK3 as a biomarker in relation to early hematoma growth and neurological outcomes after acute intracerebral hemorrhage.

OBJECTIVE: The receptor-interacting protein kinase 3 (RIPK3) is a pivotal component for triggering necroptosis. We intended to investigate predictive effects of serum RIPK3 levels on early hematoma growth (EHG) and poor neurological outcome after acute intracerebral hemorrhage (ICH). METHODS: In this prospective cohort study, 183 ICH patients and 100 controls were enrolled for measuring serum RIPK3 levels. National Institutes of Health Stroke Scale (NIHSS) and hematoma volume were recorded as the severity indicators. EHG and poststroke 6-month unfavorable outcome (modified Rankin Scale scores of 3-6) were registered as the two prognostic parameters. Multivariate analyses were implemented to discern relevance of serum RIPK3 to ICH severity and prognosis. RESULTS: Serum RIPK3 levels of patients, which were dramatically higher than those of controls, were independently related to NIHSS scores, hematoma volume, EHG, 6-month mRS scores and unfavorable outcome. Risks of EHG and unfavorable outcome were linearly pertinent to and efficiently discriminated by RIPK3 levels under restricted cubic spline and receiver operating characteristic curve respectively. RIPK3 levels nonsignificantly interacted with age, gender, hypertension, etc. Predictive ability of RIPK3 levels resembled those of NIHSS scores and hematoma volume. The prediction models, in which serum RIPK3, NIHSS scores and hematoma volume were integrated, were visually displayed via nomograms. The models' predictive capabilities substantially surpassed that of serum RIPK3, NIHSS scores and hematoma volumes alone. The models kept stable under calibration curve. CONCLUSION: A profound increase of serum RIPK3 levels after ICH is tightly relevant to severity, EHG and poor neurological outcomes, assuming that serum RIPK3 may emerge as a valuable prognostic predictor of ICH.

Receptor-interacting protein kinase-3 (RIPK3): a new biomarker for necrotising enterocolitis in preterm infants.

OBJECTIVE: This study aimed to evaluate the role of receptor-interacting protein kinase-3 (RIPK3) in the diagnosis, estimation of disease severity, and prognosis of premature infants with necrotising enterocolitis (NEC). METHODS: RIPK3, lactic acid (LA), and C-reactive protein (CRP) levels were measured in the peripheral blood of 108 premature infants between 2019 and 2023, including 24 with stage II NEC, 18 with stage III NEC and 66 controls. Diagnostic values of the indicators for NEC were evaluated via receiver operating characteristic (ROC) curve analysis. RESULTS: Plasma RIPK3 and LA levels upon NEC suspicion in neonates with stage III NEC were 32.37 ± 16.20 ng/mL. The ROC curve for the combination of RIPK3, LA, CRP for NEC diagnosis were 0.925. The time to full enteral feeding (FEFt) after recovery from NEC was different between two expression groups of plasma RIPK3 (RIPK3 < 20.06 ng/mL and RIPK3 ≥ 20.06 ng/mL). CONCLUSION: Plasma RIPK3 can be used as a promising marker for the diagnosis and estimation of disease severity of premature infants with NEC and for the guidance on proper feeding strategies after recovery from NEC.

Type I Interferon: Monkeypox/Mpox Viruses Achilles Heel?

Poxviruses are notorious for having acquired/evolved numerous genes to counteract host innate immunity. Chordopoxviruses have acquired/evolved at least three different inhibitors of host necroptotic death: E3, which blocks ZBP1-dependent necroptotic cell death, and vIRD and vMLKL that inhibit necroptosis downstream of initial cell death signaling. While this suggests the importance of the necroptotic cell death pathway in inhibiting chordopoxvirus replication, several chordopoxviruses have lost one or more of these inhibitory functions. Monkeypox/mpox virus (MPXV) has lost a portion of the N-terminus of its E3 homologue. The N-terminus of the vaccinia virus E3 homologue serves to inhibit activation of the interferon-inducible antiviral protein, ZBP1. This likely makes MPXV unique among the orthopoxviruses in being sensitive to interferon (IFN) treatment in many mammals, including humans, which encode a complete necroptotic cell death pathway. Thus, IFN sensitivity may be the Achille's Heel for viruses like MPXV that cannot fully inhibit IFN-inducible, ZBP1-dependent antiviral pathways.

Analysis of the serum levels of RIP3 and Drp1 in patients with heart failure.

AIMS: As necroptosis involving receptor-interacting protein kinase 3 (RIP3) and dynamin-related protein 1 (Drp1)-mediated signalling is a crucial mechanism of cell loss in heart failure (HF), we aimed to determine the potential diagnostic use of these molecules. METHODS AND RESULTS: The serum samples of the healthy subjects (n = 8) and patients with HF with reduced ejection fraction (n = 31), being subdivided according to the aetiology and New York Heart Association (NYHA) class, were used to measure RIP3 and Drp1 levels by enzyme-linked immunosorbent assay. Although the serum levels of Drp1 in the patients with HF were comparable with those seen in healthy individuals, we found a trend of increase in the levels of RIP3 (P = 0.0697) in the diseased group. These changes were unlikely dependent on the HF aetiology or NYHA class. The circulating RIP3 correlated with neither the main parameters assessing cardiac function (left ventricular ejection fraction, left ventricular end-diastolic diameter, and N-terminal pro-brain natriuretic peptide) nor the marker of inflammation (C-reactive protein). CONCLUSIONS: In this pilot study, findings on serum RIP3 supported the importance of necroptosis in HF pathomechanisms. The potential diagnostic use of circulating RIP3, unlike Drp1, as an additional biomarker of HF has also been indicated; however, further large studies are needed to prove this concept.

Enhancing Fat Transplantation Efficiency in a Mouse Model through Pretreatment of Adipose-Derived Stem Cells with RIP3 Inhibitors.

BACKGROUND: Autologous fat transplantation, widely used in cosmetic and reparative surgery for volumetric enhancements, faces challenges with its inconsistent long-term survival rates. The technique's efficacy, crucial for its development, is hindered by unpredictable outcomes. Enriching fat grafts with adipose-derived stem cells (ADSCs) shows promise in improving survival efficiency. OBJECTIVES: This study aimed to explore the potential of receptor-interacting protein kinase 3 (RIP3) kinase inhibitors as a pretreatment for ADSCs in enhancing autologous fat graft retention over a long term. METHODS: ADSCs were isolated, cultured under normal or oxygen-glucose deprivation conditions, and mixed with particulate fat grafts to form distinct experimental groups in female nude mice. Fat graft mass and volume, along with underlying mechanisms, were evaluated using quantitative reverse transcription polymerase chain reaction (RT-qPCR), immunohistochemistry, and Western blot analysis. RESULTS: The experimental group, pretreated with RIP3 kinase inhibitors, had higher graft mass and volume, greater adipocyte integrity, and increased peroxisome proliferator-activated receptor gamma (PPARγ) mRNA levels than control groups. Furthermore, the experimental group demonstrated lower expression of necroptosis pathway proteins in the short term and an ameliorated inflammatory response as indicated by interleukin-1 beta (IL-1ß), interleukin-10 (IL-10) mRNA levels, and histological analyses. Notably, enhanced neovascularization was evident in the experimental group. CONCLUSIONS: These findings suggest that RIP3 kinase inhibitor pretreatment of ADSCs can improve fat graft survival, promote adipocyte integrity, potentially decrease inflammation, and enhance neovascularization. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Engineering M2 type macrophage-derived exosomes for autoimmune hepatitis immunotherapy via loading siRIPK3.

Autoimmune hepatitis (AIH) is a progressive liver disease mediated by the immune system that involves an imbalance in pro-inflammatory and regulatory mechanisms including regulatory T cells (Tregs), T helper 17 (Th17) cells, Th1, macrophages, and many other immune cells. Current steroid therapy for AIH has significant systemic side effects and is poorly tolerated by some individuals. Therefore, there is an urgent need for alternative treatments. Maintaining homeostasis in macrophage differentiation and activation is crucial for regulating immune responses in hepatitis. In this study, we loaded small interfering RNA (siRNA) targeting receptor-interacting protein kinase 3 (RIPK3) into M2-type macrophage-derived exosomes (M2 Exos) to create functionalized exosomes called M2 Exos/siRIPK3. These exosomes demonstrated a natural ability to target the liver in mice, as they were efficiently taken up by hepatic macrophages and showed significant and stable accumulation. M2 Exos/siRIPK3 effectively mitigated immune-mediated hepatitis by suppressing the expression of RIPK3, resulting in a reduced release of pro-inflammatory cytokines and chemokines in both liver tissues and serum. Additionally, M2 Exos/siRIPK3 exhibited immunomodulatory effects, as its administration resulted in a decreased proportion of hepatic and splenic Th17 cells, along with an increased ratio of Tregs. Overall, this study suggests that loading small molecule drugs onto M2 Exos could be a promising approach for developing immunomodulators that specifically target liver macrophages to treat AIH. This strategy has the potential to provide a safer and more effective alternative to current therapy for AIH patients.

RIPK3 activation promotes DAXX-dependent neuronal necroptosis after intracerebral hemorrhage in mice.

BACKGROUND: Necroptosis induced by receptor-interacting protein kinase 3 (RIPK3) is engaged in intracerebral hemorrhage (ICH) pathology. In this study, we explored the impact of RIPK3 activation on neuronal necroptosis and the mechanism of the death domain-associated protein (DAXX)-mediated nuclear necroptosis pathway after ICH. METHODS: Potential molecules linked to the progression of ICH were discovered using RNA sequencing. The level of DAXX was assessed by quantitative real-time PCR, ELISA, and western blotting. DAXX localization was determined by immunofluorescence and immunoprecipitation assays. The RIPK3 inhibitor GSK872 and DAXX knockdown with shRNA-DAXX were used to examine the nuclear necroptosis pathway associated with ICH. Neurobehavioral deficit assessments were performed. RESULTS: DAXX was increased in patients and mice after ICH. In an ICH mouse model, shRNA-DAXX reduced brain water content and alleviated neurologic impairments. GSK872 administration reduced the expression of DAXX. shRNA-DAXX inhibited the expression of p-MLKL. Immunofluorescence and immunoprecipitation assays showed that RIPK3 and AIF translocated into the nucleus and then bound with nuclear DAXX. CONCLUSIONS: RIPK3 revitalization promoted neuronal necroptosis in ICH mice, partially through the DAXX signaling pathway. RIPK3 and AIF interacted with nuclear DAXX to aggravate ICH injury.

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.

Regulatory mechanism of CaMKII δ mediated by RIPK3 on myocardial fibrosis and reversal effects of RIPK3 inhibitor GSK'872.

BACKGROUND: Myocardial fibrosis (MF) remains a prominent challenge in heart disease. The role of receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is evident in the pathogenesis of numerous heart diseases. Concurrently, the activation of Ca2+/calmodulin-dependent protein kinase (CaMKII) is pivotal in cardiovascular disease (CVD). This study aimed to evaluate the impact and underlying mechanisms of RIPK3 on myocardial injury in MF and to elucidate the potential involvement of CaMKII. METHODS: Building upon our previous research methods [1], wild-type (WT) mice and RIPK3 knockout (RIPK3 -/-) mice underwent random assignment for transverse aortic constriction (TAC) in vivo. Four weeks post-procedure, the MF model was effectively established. Parameters such as the extent of MF, myocardial injury, RIPK3 expression, necroptosis, CaMKII activity, phosphorylation of mixed lineage kinase domain-like protein (MLKL), mitochondrial ultrastructural details, and oxidative stress levels were examined. Cardiomyocyte fibrosis was simulated in vitro using angiotensin II on cardiac fibroblasts. RESULTS: TAC reliably produced MF, myocardial injury, CaMKII activation, and necroptosis in mice. RIPK3 depletion ameliorated these conditions. The RIPK3 inhibitor, GSK'872, suppressed the expression of RIPK3 in myocardial fibroblasts, leading to improved fibrosis and inflammation, diminished CaMKII oxidation and phosphorylation levels, and the rectification of CaMKIIδ alternative splicing anomalies. Furthermore, GSK'872 downregulated the expressions of RIPK1, RIPK3, and MLKL phosphorylation, attenuated necroptosis, and bolstered the oxidative stress response. CONCLUSIONS: Our data suggested that in MF mice, necroptosis was augmented in a RIPK3-dependent fashion. There seemed to be a positive correlation between CaMKII activation and RIPK3 expression. The adverse effects on myocardial fibrosis mediated by CaMKII δ through RIPK3 could potentially be mitigated by the RIPK3 inhibitor, GSK'872. This offered a fresh perspective on the amelioration and treatment of MF and myocardial injury.

Novel, non-conventional pathways of necroptosis in the heart and other organs: Molecular mechanisms, regulation and inter-organelle interplay.

Necroptosis, a cell death modality that is defined as a necrosis-like cell death depending on the receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL), has been found to underlie the injury of various organs. Nevertheless, the molecular background of this cell loss seems to also involve, at least under certain circumstances, some novel axes, such as RIPK3-PGAM5-Drp1 (mitochondrial protein phosphatase 5-dynamin-related protein 1), RIPK3-CaMKII (Ca2+/calmodulin-dependent protein kinase II) and RIPK3-JNK-BNIP3 (c-Jun N-terminal kinase-BCL2 Interacting Protein 3). In addition, endoplasmic reticulum stress and oxidative stress via the higher production of reactive oxygen species produced by the mitochondrial enzymes and the enzymes of the plasma membrane have been implicated in necroptosis, thereby depicting an inter-organelle interplay in the mechanisms of this cell death. However, the role and relationship between these novel non-conventional signalling and the well-accepted canonical pathway in terms of tissue- and/or disease-specific prioritisation is completely unknown. In this review, we provide current knowledge on some necroptotic pathways being not directly associated with RIPK3-MLKL execution and report studies showing the role of respective microRNAs in the regulation of necroptotic injury in the heart and in some other tissues having a high expression of the pro-necroptotic proteins.

The pathophysiological role of receptor-interacting protein kinase 3 in cardiovascular disease.

Recent studies have found that receptor interacting protein kinase 3 (RIPK3) can mediate CaMK Ⅱ phosphorylation and oxidation, open mitochondrial permeability transition pore (mPTP), and induce myocardial necroptosis. The increased expression or phosphorylation of RIPK3 is one of the important markers of necroptosis; Inhibition of CaMK Ⅱ phosphorylation or oxidation significantly reduces RIPK3 mediated myocardial necroptosis; Studies have shown that necroptosis plays an important role in the occurrence and development of cardiovascular diseases; Using the selective inhibitor GSK '872 of RIPK3 can effectively inhibit the occurrence and development of cardiovascular diseases, and can reverse cardiovascular and cardiac dysfunction caused by overexpression of RIPK3. In this review, we provide a brief overview of the current knowledge on RIPK3 in mediating necroptosis, inflammatory response, and oxidative stress, and discussed the role of RIPK3 in cardiovascular diseases such as atherosclerosis, myocardial ischaemia, myocardial infarction, and heart failure.

Reovirus Type 3 Dearing Variants Do Not Induce Necroptosis in RIPK3-Expressing Human Tumor Cell Lines.

Reoviruses are used as oncolytic viruses to destroy tumor cells. The concomitant induction of anti-tumor immune responses enhances the efficacy of therapy in tumors with low amounts of immune infiltrates before treatment. The reoviruses should provoke immunogenic cell death (ICD) to stimulate a tumor cell-directed immune response. Necroptosis is considered a major form of ICD, and involves receptor-interacting protein kinase 1 (RIPK1), RIPK3 and phosphorylation of mixed-lineage kinase domain-like protein (MLKL). This leads to cell membrane disintegration and the release of damage-associated molecular patterns that can activate immune responses. Reovirus Type 3 Dearing (T3D) can induce necroptosis in mouse L929 fibroblast cells and mouse embryonic fibroblasts. Most human tumor cell lines have a defect in RIPK3 expression and consequently fail to induce necroptosis as measured by MLKL phosphorylation. We used the human colorectal adenocarcinoma HT29 cell line as a model to study necroptosis in human cells since this cell line has frequently been described in necroptosis-related studies. To stimulate MLKL phosphorylation and induce necroptosis, HT29 cells were treated with a cocktail consisting of TNFα, the SMAC mimetic BV6, and the caspase inhibitor Z-VAD-FMK. While this treatment induced necroptosis, three different reovirus T3D variants, i.e., the plasmid-based reverse genetics generated virus (T3DK), the wild-type reovirus T3D isolate R124, and the junction adhesion molecule-A-independent reovirus mutant (jin-1) failed to induce necroptosis in HT29 cells. In contrast, these viruses induced MLKL phosphorylation in murine L929 cells, albeit with varying efficiencies. Our study shows that while reoviruses efficiently induce necroptosis in L929 cells, this is not a common phenotype in human cell lines. This study emphasizes the difficulties of translating the results of ICD studies from murine cells to human cells.

PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons.

PANoptosis is a newly identified type of regulated cell death that consists of pyroptosis, apoptosis, and necroptosis, which simultaneously occur during the pathophysiological process of infectious and inflammatory diseases. Although our previous literature mining study suggested that PANoptosis might occur in neuronal ischemia/reperfusion injury, little experimental research has been reported on the existence of PANoptosis. In this study, we used in vivo and in vitro retinal neuronal models of ischemia/reperfusion injury to investigate whether PANoptosis-like cell death (simultaneous occurrence of pyroptosis, apoptosis, and necroptosis) exists in retinal neuronal ischemia/reperfusion injury. Our results showed that ischemia/reperfusion injury induced changes in morphological features and protein levels that indicate PANoptosis-like cell death in retinal neurons both in vitro and in vivo. Ischemia/reperfusion injury also significantly upregulated caspase-1, caspase-8, and NLRP3 expression, which are important components of the PANoptosome. These results indicate the existence of PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons and provide preliminary experimental evidence for future study of this new type of regulated cell death.

Necroptosis in inflammatory bowel disease: A potential effective target. / ç‚Žç—‡æ€§è‚ ç— ä¸­çš„åæ­»æ€§å‡‹äº¡ï¼šä¸€ä¸ªæ½œåœ¨çš„æ²»ç–—é¶ç‚¹ï¼ˆè‹±æ–‡ï¼‰.

The morbidity of inflammatory bowel diseases (IBD) is rising rapidly but no curative therapies to prevent its recurrence. Cell death is crucial to maintaining homeostasis. Necroptosis is a newly identified programmed cell death and its roles played in IBD need to be explored. Necroptosis is mediated by receptor interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), which resulted in cell swelling, plasma membrane rupture, intracellular content leaking, and eventually cell death as well as the promotion of inflammation. Studies have found that inhibiting necroptosis alleviated IBD in animal models and IBD patients with an increased level of necroptosis in inflammatory tissues, indicating that necroptosis is related to the pathogenesis of IBD. However, due to the complexity in regulation of necroptosis and the involvement of multiple functions of relevant signaling molecules, the specific mechanism remains elusive. Necroptosis may play a vital regulatory role in the pathogenesis of IBD, which provides a new idea and method for further exploring the therapeutic target of IBD.

LCK-Mediated RIPK3 Activation Controls Double-Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A-ERK Axis.

Receptor-interacting protein kinase 3 (RIPK3) is the primary regulator of necroptotic cell death. RIPK3 expression is often silenced in various cancer cells, which suggests that it may have tumor suppressor properties. However, the exact mechanism by which RIPK3 negatively regulates cancer development and progression remains unclear. This report indicates that RIPK3 acts as a potent regulator of the homeostatic proliferation of CD4+ CD8+ double-positive (DP) thymocytes. Abnormal proliferation of RIPK3-deficient DP thymocytes occurs independently of the well-known role for RIPK3 in necroptosis (upstream of MLKL activation), and is associated with an incidental thymic mass, likely thymic hyperplasia. In addition, Ripk3-null mice develop increased thymic tumor formation accompanied by reduced host survival in the context of an N-ethyl-N-nitrosourea (ENU)-induced tumor model. Moreover, RIPK3 deficiency in p53-null mice promotes thymic lymphoma development via upregulated extracellular signal-regulated kinase (ERK) signaling, which correlates with markedly reduced survival rates. Mechanistically, lymphocyte-specific protein tyrosine kinase (LCK) activates RIPK3, which in turn leads to increases in the phosphatase activity of protein phosphatase 2 (PP2A), thereby suppressing hyper-activation of ERK in DP thymocytes. Overall, these findings suggest that a RIPK3-PP2A-ERK signaling axis regulates DP thymocyte homeostasis and may provide a potential therapeutic target to improve thymic lymphoma therapies.

Feedback loop between fatty acid transport protein 2 and receptor interacting protein 3 pathways promotes polymorphonuclear neutrophil myeloid-derived suppressor cells-potentiated suppressive immunity in bladder cancer.

BACKGROUND: Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) promote tumor immune tolerance and cause tumor immunotherapy failure. In this study, we found that high PMN-MDSCs infiltration, overexpressed fatty acid transporter protein 2 (FATP2) and underexpressed receptor-interacting protein kinase 3 (RIPK3) existed in the mouse and human bladder cancer tissues. However, the related mechanisms remain largely unknown. METHODS AND RESULTS: Both FATP2 and RIPK3 expressions were associated with clinical stage. FATP2 knockout or up-regulating RIPK3 reduced the synthesis of prostaglandin E2 (PGE2) in PMN-MDSCs, attenuated the suppressive activity of PMN-MDSCs on CD8+ T cells functions and inhibited the tumor growth. There was a PGE2-mediated feedback loop between FATP2 and RIPK3 pathways, which markedly promoted the immunosuppressive activity of PMN-MDSCs. Combination therapy with inhibition of FATP2 and activation of RIPK3 can effectively inhibit tumor growth. CONCLUSIONS: This study demonstrated that a feedback loop between FATP2 and RIPK3 pathways in PMN-MDSCs significantly promoted the synthesis of PGE2, which severely impaired the CD8+ T cell functions. This study may provide new ideas for immunotherapy of human bladder cancer.

Neuroprotection by Trans-Resveratrol in Rats With Intracerebral Hemorrhage: Insights into the Role of Adenosine A1 Receptors.

Given the neuroprotective effects of trans-resveratrol (RV), this study aimed to investigate the involvement of the adenosine A1 receptor (A1R) in RV-mediated neuroprotection in a rat intracerebral hemorrhage (ICH) model induced by intrastriatal injection of collagenase. Rats were divided into 5 groups: (1) control, (2) sham-operated, (3) ICH pretreated with vehicle, (4) ICH pretreated with RV, and (5) ICH pretreated with RV and the A1R antagonist DPCPX. At 48 hours after ICH, the rats were subjected to neurological testing. Brain tissues were assessed for neuronal density and morphological features using routine and immunohistochemical staining. Expression of tumor necrosis factor-α (TNF-α), caspase-3, and RIPK3 proteins was examined using ELISA. A1R, MAPK P38, Hsp90, TrkB, and BDNF genes were examined using RT-qPCR. RV protected against neurological deficits and neuronal depletion, restored the expression of TNF-α, CASP3, RIPK3, A1R, and Hsp90, and increased BDNF/TrkB. DPCPX abolished the effects of RV on neurological outcomes, neuronal density, CASP3, RIPK3, A1R, Hsp90, and BDNF. These data indicate that the neuroprotection by RV involves A1R and inhibits CASP3-dependent apoptosis and RIPK3-dependent necroptosis in the perihematoma region; this is likely to be mediated by crosstalk between A1R and the BDNF/TrkB pathway.

Ca2+/Calmodulin-Dependent Protein Kinase II Regulation by Inhibitor of Receptor Interacting Protein Kinase 3 Alleviates Necroptosis in Glycation End Products-Induced Cardiomyocytes Injury.

Necroptosisis a regulatory programmed form of necrosis. Receptor interacting protein kinase 3 (RIPK3) is a robust indicator of necroptosis. RIPK3 mediates myocardial necroptosis through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) in cardiac ischemia-reperfusion (I/R) injury and heart failure. However, the exact mechanism of RIPK3 in advanced glycation end products (AGEs)-induced cardiomyocytes necroptosis is not clear. In this study, cardiomyocytes were subjected to AGEs stimulation for 24 h. RIPK3 expression, CaMKII expression, and necroptosis were determined in cardiomyocytes after AGEs stimulation. Then, cardiomyocytes were transfected with RIPK3 siRNA to downregulate RIPK3 followed by AGEs stimulation for 24 h. CaMKIIδ alternative splicing, CaMKII activity, oxidative stress, necroptosis, and cell damage were detected again. Next, cardiomyocytes were pretreated with GSK'872, a specific RIPK3 inhibitor to assess whether it could protect cardiomyocytes against AGEs stimulation. We found that AGEs increased the expression of RIPK3, aggravated the disorder of CaMKII δ alternative splicing, promoted CaMKII activation, enhanced oxidative stress, induced necroptosis, and damaged cardiomyocytes. RIPK3 downregulation or RIPK3 inhibitor GSK'872 corrected CaMKIIδ alternative splicing disorder, inhibited CaMKII activation, reduced oxidative stress, attenuated necroptosis, and improved cell damage in cardiomyocytes.

RIP3 Translocation into Mitochondria Promotes Mitofilin Degradation to Increase Inflammation and Kidney Injury after Renal Ischemia-Reperfusion.

The receptor-interacting protein kinase 3 (RIP3) has been reported to regulate programmed necrosis-necroptosis forms of cell death with important functions in inflammation. We investigated whether RIP3 translocates into mitochondria in response to renal ischemia-reperfusion (I/R) to interact with inner mitochondrial protein (Mitofilin) and promote mtDNA release into the cytosol. We found that release of mtDNA activates the cGAS-STING pathway, leading to increased nuclear transcription of pro-inflammatory markers that exacerbate renal I/R injury. Monolateral C57/6N and RIP3-/- mice kidneys were subjected to 60 min of ischemia followed by either 12, 24, or 48 h of reperfusion. In WT mice, we found that renal I/R injury increased RIP3 levels, as well as its translocation into mitochondria. We observed that RIP3 interacts with Mitofilin, likely promoting its degradation, resulting in increased mitochondria damage and mtDNA release, activation of the cGAS-STING-p65 pathway, and increased transcription of pro-inflammatory markers. All of these effects observed in WT mice were decreased in RIP3-/- mice. In HK-2, RIP3 overexpression or Mitofilin knockdown increased cell death by activating the cGAS-STING-p65 pathway. Together, this study point to an important role of the RIP3-Mitofilin axis in the initiation and development of renal I/R injury.

Ca2+/Calmodulin-Dependent Protein Kinase II Regulation by RIPK3 Alleviates Necroptosis in Transverse Arch Constriction-Induced Heart Failure.

Some studies have reported that the activation of Ca2+/calmodulin dependent protein kinase (CaMKII) plays a vital role in the pathogenesis of cardiovascular disease. Moreover, receptor interacting protein kinase 3 (RIPK3)-mediated necroptosis is also involved in the pathological process of various heart diseases. In the present study, we aimed to investigate the effect of RIPK3-regulated CaMKII on necroptosis in heart failure (HF) and its underlying mechanism. Wild type (WT) and RIPK3-depleted (RIPK3-/-) mice were treated with transverse arch constriction (TAC). After 6 weeks, echocardiography, myocardial injury, CaMKII activity, necroptosis, RIPK3 expression, mixed lineage kinase domain-like protein (MLKL) phosphorylation, and mitochondrial ultrastructure were measured. The results showed that TAC aggravated cardiac dysfunction, CaMKII activation, and necroptosis in WT mice. However, depletion of RIPK3 alleviated cardiac insufficiency, CaMKII activation, and necroptosis in TAC-treated mice. To verify the experimental results, WT mice were transfected with AAV-vector and AAV-RIPK3 shRNA, followed by TAC operation. The findings were consistent with the expected results. Collectively, our current data indicated that the activation of CaMKII, MLKL and necroptosis in HF mice were increased in a RIPK3-dependent manner, providing valuable insights into the pathogenesis and treatment strategy of HF.