Necrostatin-1 as a Potential Anticonvulsant: Insights from Zebrafish Larvae Model of PTZ-Induced Seizures.

Epilepsy is a common neurological disorder affecting around 70 million people worldwide. Despite significant research and advancements in pharmaceutical therapies, the exact mechanisms underlying epileptogenesis remain unclear. As a result, current antiepileptic drug treatments are ineffective for approximately 30% of patients, providing only symptomatic relief. The epileptic process is influenced by the signaling of tumor necrosis factor alpha (TNFα), which affects neuronal excitability. The TNFα/TNFR1 signaling pathway and the role of RIPK1 in initiating inflammatory cell death pathways are well studied in human disorders. Dysregulation of RIPK1 is linked to inflammation and neurodegenerative diseases. Necrostatin-1 (Nec-1) selectively inhibits RIPK1 in various pathological conditions. The current study aimed to investigate the anticonvulsant properties of Nec-1 (a selective RIPK1 inhibitor) in PTZ-induced seizures in zebrafish larvae. Before the onset of seizures, zebrafish were treated with Nec-1 (15 µM) for 24 h at 6 days post-fertilization (dpf), followed by exposure to 15 mM of PTZ for 30 min. Behavioral assessments were conducted to observe changes in locomotor activity. Additionally, c-Fos expression was measured as an indicator of neuronal activation and analyzed mRNA levels of the astrocyte activation marker (GFAP) along with various inflammatory cytokines. Western blot analysis was conducted to evaluate GABAA receptor expression. Pretreatment with Nec-1 restored normal behavior and reversed the expression of c-Fos in zebrafish larvae induced by PTZ. Additionally, Nec-1 reduced the elevated mRNA expression of inflammatory cytokines and significantly suppressed TNF/TNFR1 signaling, thereby inhibiting the enhanced internalization of GABAA receptors. Our findings indicate that Nec-1 reduced the severity of PTZ-induced seizures in zebrafish by regulating behavior changes, suppressing inflammatory mediators, and enhancing the expression of GABAA receptors, suggesting potential anticonvulsant properties.

Developing patient-derived organoids to demonstrate JX24120 inhibits SAMe synthesis in endometrial cancer by targeting MAT2B.

Endometrial cancer (EC) is one of the most common gynecologic malignancies, which lacking effective drugs for intractable conditions or patients unsuitable for surgeries. Recently, the patient-derived organoids (PDOs) are found feasible for cancer research and drug discoveries. Here, we have successfully established a panel of PDOs from EC and conducted drug repurposing screening and mechanism analysis for cancer treatment. We confirmed that the regulatory ß subunit of methionine adenosyltransferase (MAT2B) is highly correlated with malignant progression in endometrial cancer. Through drug screening on PDOs, we identify JX24120, chlorpromazine derivative, as a specific inhibitor for MAT2B, which directly binds to MAT2B (Kd = 4.724 µM) and inhibits the viability of EC PDOs and canonical cell lines. Correspondingly, gene editing assessment demonstrates that JX24120 suppresses tumor growth depending on the presence of MAT2B in vivo and in vitro. Mechanistically, JX24120 induces inhibition of S-adenosylmethionine (SAMe) synthesis, leading to suppressed mTORC1 signaling, abnormal energy metabolism and protein synthesis, and eventually apoptosis. Taken together, our study offers a novel approach for drug discovery and efficacy assessment by using the PDOs models. These findings suggest that JX24120 may be a potent MAT2B inhibitor and will hopefully serve as a prospective compound for endometrial cancer therapy.

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

PURPOSE: Benzalkonium chloride (BAC) is commonly used as a preservative in ophthalmic medications, despite its potential to induce chemical injury. Extensive research has demonstrated that BAC can lead to adverse effects, including injuries to the ocular surface. Our study aimed to elucidate the underlying mechanism of necroptosis induced by BAC. METHODS: Human corneal epithelial (HCE) cells and mouse corneas were subjected to chemical injury, and the necrostatin-1 (Nec1) group was compared to the dimethylsulfoxide (DMSO) group. The extent of damage to HCE cells was assessed using CCK-8 and flow cytometry. Hematoxylin and eosin staining, as well as fluorescein sodium staining, were used to detect and characterize corneal injury. The activation of inflammatory cytokines and necroptosis-related proteins and genes was evaluated using Western blotting, immunofluorescence staining, and quantitative RT‒PCR. RESULTS: In our study, the induction of necroptosis by a hypertonic solution was not observed. However, necroptosis was observed in HCE cells exposed to NaOH and BAC, which activated the receptor-interacting protein kinase 1 (RIPK1) - receptor-interacting protein kinase 3 (RIPK3) - mixed lineage kinase domain-like protein (MLKL) signaling pathway. In mouse corneal tissues, BAC could induce necroptosis and inflammation. The administration of Nec1 mitigated the inflammatory response and ocular surface damage caused by BAC-induced necroptosis in our experimental models. Furthermore, our in vivo experiments revealed that the severity of necroptosis was greater in the 3-day group than in the 7-day group. CONCLUSIONS: Necroptosis plays a role in the pathological development of ocular surface injury caused by exposure to BAC. Furthermore, our study demonstrated that the administration of Nec1 could mitigate the pathological effects of necroptosis induced by BAC in clinical settings.

Necrostatin-1: a promising compound for neurological disorders.

Necrostatin-1, a small molecular alkaloid, was identified as an inhibitor of necroptosis in 2005. Investigating the fundamental mechanism of Necrostatin-1 and its role in various diseases is of great significance for scientific and clinical research. Accumulating evidence suggests that Necrostatin-1 plays a crucial role in numerous neurological disorders. This review aims to provide a comprehensive overview of the potential functions of Necrostatin-1 in various neurological disorders, offering valuable insights for future research.

Unlocking cardioprotection: iPSC exosomes deliver Nec-1 to target PARP1/AIFM1 axis, alleviating HF oxidative stress and mitochondrial dysfunction.

BACKGROUND: Heart failure (HF) is characterized by oxidative stress and mitochondrial dysfunction. This study investigates the therapeutic potential of Necrostatin-1 (Nec-1) delivered through exosomes derived from induced pluripotent stem cells (iPSCs) to address these pathologies in HF. METHODS: An HF rat model was established, and comprehensive assessments were performed using echocardiography, hemodynamics, and ventricular mass index measurements. iPSCs were used to isolate exosomes, loaded with Nec-1, and characterized for efficient delivery into cardiomyocytes. The interaction between Nec-1-loaded exosomes (Nec-1-Exos), poly (ADP-ribose) polymerase 1 (PARP1), and apoptosis-inducing factor mitochondria-associated 1 (AIFM1) was explored. Gain-of-function experiments assessed changes in cardiomyocyte parameters, and histological analyses were conducted on myocardial tissues. RESULTS: Cardiomyocytes successfully internalized Nec-1-loaded exosomes, leading to downregulation of PARP1, inhibition of AIFM1 nuclear translocation, increased ATP and superoxide dismutase levels, reduced reactive oxygen species and malonaldehyde levels, and restored mitochondrial membrane potential. Histological examinations confirmed the modulation of the PARP1/AIFM1 axis by Nec-1, mitigating HF. CONCLUSIONS: iPSC-derived exosomes carrying Nec-1 attenuate oxidative stress and mitochondrial dysfunction in HF by targeting the PARP1/AIFM1 axis. This study proposes a promising therapeutic strategy for HF management and highlights the potential of exosome-mediated drug delivery.

Silencing of tropomodulin 1 inhibits acute myeloid leukemia cell proliferation and tumor growth by elevating karyopherin alpha 2-mediated autophagy.

Evidence shows that tropomodulin 1 (TMOD1) is a powerful diagnostic marker in the progression of several cancer types. However, the regulatory mechanism of TMOD1 in tumor progression is still unclear. Here, we showed that TMOD1 was highly expressed in acute myeloid leukemia (AML) specimens, and TMOD1-silencing inhibited cell proliferation by inducing autophagy in AML THP-1 and MOLM-13 cells. Mechanistically, the C-terminal region of TMOD1 directly bound to KPNA2, and TMOD1-overexpression promoted KPNA2 ubiquitylation and reduced KPNA2 levels. In contrast, TMOD1-silencing increased KPNA2 levels and facilitated the nuclear transfer of KPNA2, then subsequently induced autophagy and inhibited cell proliferation by increasing the nucleocytoplasmic transport of p53 and AMPK activation. KPNA2/p53 inhibitors attenuated autophagy induced by silencing TMOD1 in AML cells. Silencing TMOD1 also inhibited tumor growth by elevating KPNA2-mediated autophagy in nude mice bearing MOLM-13 xenografts. Collectively, our data demonstrated that TMOD1 could be a novel therapeutic target for AML treatment.

The autophagy protein RUBCNL/PACER represses RIPK1 kinase-dependent apoptosis and necroptosis.

Mesenchymal stem cells (MSCs) are used in cell therapy; nonetheless, their application is limited by their poor survival after transplantation in a proinflammatory microenvironment. Macroautophagy/autophagy activation in MSCs constitutes a stress adaptation pathway, promoting cellular homeostasis. Our proteomics data indicate that RUBCNL/PACER (RUN and cysteine rich domain containing beclin 1 interacting protein like), a positive regulator of autophagy, is also involved in cell death. Hence, we screened MSC survival upon various cell death stimuli under loss or gain of function of RUBCNL. MSCs were protected from TNF (tumor necrosis factor)-induced regulated cell death when RUBCNL was expressed. TNF promotes inflammation by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We determine that MSCs succumb to RIPK1 kinase-dependent apoptosis upon TNF sensing and necroptosis when caspases are inactivated. We show that RUBCNL is a negative regulator of both RIPK1-dependent apoptosis and necroptosis. Furthermore, RUBCNL mutants that lose the ability to regulate autophagy, retain their function in negatively regulating cell death. We also found that RUBCNL forms a complex with RIPK1, which disassembles in response to TNF. In line with this finding, RUBCNL expression limits assembly of RIPK1-TNFRSF1A/TNFR1 complex I, suggesting that complex formation between RUBCNL and RIPK1 represses TNF signaling. These results provide new insights into the crosstalk between the RIPK1-mediated cell death and autophagy machineries and suggest that RUBCNL, due to its functional duality in autophagy and apoptosis/necroptosis, could be targeted to improve the therapeutic efficacy of MSCs. Abbreviations: BAF: bafilomycin A1; CASP3: caspase 3; Caspases: cysteine-aspartic proteases; cCASP3: cleaved CASP3; CQ: chloroquine; CHX: cycloheximide; cPARP: cleaved poly (ADP-ribose) polymerase; DEPs: differential expressed proteins; ETO: etoposide; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain-like; MSC: mesenchymal stem cell; MTORC1: mechanistic target of rapamycin kinase complex 1; Nec1s: necrostatin 1s; NFKB/NF-kB: nuclear factor of kappa light polypeptide gene enhancer in B cells; PLA: proximity ligation assay; RCD: regulated cell death; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RUBCNL/PACER: RUN and cysteine rich domain containing beclin 1 interacting protein like; siCtrl: small interfering RNA nonsense; siRNA: small interfering RNA; TdT: terminal deoxynucleotidyl transferase; Tm: tunicamycin; TNF: tumor necrosis factor; TNFRSF1A/TNFR1: tumor necrosis factor receptor superfamily, member 1a.

Genista cephalantha Spach. protects against acetaminophen-induced liver failure via preserving the glutathione redox system, reducing inflammatory response, and inhibiting hepatocyte death in rats.

Objectives: The current study was conducted to assess the protective mechanisms of n-BuOH fraction from the aerial part of Genista cephontala (BEGC) on APAP-induced liver injury compared to necrostatine-1 (Nec-1). Materials and Methods: A model of APAP-induced hepatotoxicity was created in male rats by injecting a single dose; 1000 mg/kg APAP, the protective effect was performed with (200 mg/kg; 10 days) BEGC compared to Nec-1, (1.8 mg/kg). Results: BEGC or NeC-1 pretreatment significantly abolished impaired effects in APAP-rats, by decreasing the generation of TBARS and ROS in mitochondrial and cytosolic fractions and maintaining liver function activities. A marked response was observed in the levels of both GSH and GSH-system enzymes in liver homogenates and mitochondrial fractions to BEGC. BEGC/ Nec-1 successfully regulated the inflammatory mediators (IL-ß, TNF-α, HMGB1, and acHMGB1) and MPO levels. During APAP treatment, no caspase-3 or -8 activity was detected, and the level of fk18; M30 was higher than the levels of cck18; M65. Moreover, RIPK3 and MLKL levels were increased in the APAP group. These results suggested that necroptosis predominates during the APAP liver injury model. Interestingly, these necroptotic factors were significantly down-regulated by BEGC treatment. Both biochemical and histopathological findings were consistent with each other. Conclusion: From all these findings, the hepatoprotective effect of BEGC could be due to the abundance of polyphenols identified by LC-MS/MS analysis, as well as the synergistic interactions of all contents.

Necrostatin-1 releasing nanoparticles: In vitro and in vivo efficacy for supporting immunoisolated islet transplantation outcomes.

Immunoisolation of pancreatic islets in alginate microcapsules allows for transplantation in the absence of immunosuppression but graft survival time is still limited. This limited graft survival is caused by a combination of tissue responses to the encapsulating biomaterial and islets. A significant loss of islet cells occurs in the immediate period after transplantation and is caused by a high susceptibility of islet cells to inflammatory stress during this period. Here we investigated whether necrostatin-1 (Nec-1), a necroptosis inhibitor, can reduce the loss of islet cells under stress in vitro and in vivo. To this end, we developed a Nec-1 controlled-release system using poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) as the application of Nec-1 in vivo is limited by low stability and possible side effects. The PLGA NPs stably released Nec-1 for 6 days in vitro and protected beta cells against hypoxia-induced cell death in vitro. Treatment with these Nec-1 NPs at days 0, 6, and 12 post-islet transplantation in streptozotocin-diabetic mice confirmed the absence of side effects as graft survival was similar in encapsulated islet grafts in the absence and presence of Nec-1. However, we found no further prolongation of graft survival of encapsulated grafts which might be explained by the high biocompatibility of the alginate encapsulation system that provoked a very mild tissue response. We expect that the Nec-1-releasing NPs could find application to immunoisolation systems that elicit stronger inflammatory responses, such as macrodevices and vasculogenic biomaterials.

ASSESSMENT OF CARDIOPROTECTIVE EFFECT OF NECROSTATIN-1 STABLE IN A MICE MODEL OF ACUTE DOXORUBICIN-INDUCED CARDIOTOXICITY.

OBJECTIVE: The aim: To evaluate the effect of Necrostatin-1s (Nec-1s), an inhibitor of necroptosis, on acute Dox-induced cardiotoxicity in a mice model. PATIENTS AND METHODS: Materials and methods: Fifteen male mice were used. The animals were allocated into three groups. On the third day of the experiment, a single intraper¬itoneal dose of 20 mg/kg Dox was used to induce cardiotoxicity. Mice in the control group were given vehicle (DMSO) intraperitoneally, whereas mice in the third group were given 5 mg/kg Nec-1s two days before Dox treatment and continued for a total of five days. Animals were euthanized at the conclusion of the research. ELISA was used to assess the following parameters: cTnI, TNF-α, IL-1ß, GPX-4, and Hmox-1. The expression of TNF-R1 and phosphorylated NF-κß p65 was measured using immunohistochemistry. In addition, a histopathologic evaluation of the cardiac lesions was conducted. RESULTS: Results: Our results showed that Dox treatment substantially elevated serum cTnI levels, increased tissue inflammatory biomarkers (TNF-α, IL-1ß, phospho NF-κß p65 and TNF-R1), and reduced tissue antioxidant enzymes (GPX-4, Hmox-1). A histopathological analysis showed pronounced necrosis and vacuolization. These results were drastically changed by pretreatment with Nec-1s, with serum cTnI levels in this group being much lower than in the Dox group. In addition to a significant decrease in inflammatory markers, antioxidant enzymes were partially recovered. Moreover, there was preservation of the cardiac morphology to a level that was roughly normal. CONCLUSION: Conclusions: Our findings demonstrate that pretreatment with Nec-1s protected against acute Dox-induced cardiotoxicity. This cardioprotective effect was mainly due to amelioration of inflammation that reflected by inhibition of NF-κß/TNF-α/TNF-R1 pathway, with partial restoration of antioxidant enzymes, GPX-4 and Hmox1.

Atezolizumab Induces Necroptosis and Contributes to Hepatotoxicity of Human Hepatocytes.

Atezolizumab is an immune checkpoint inhibitor (ICI) targeting PD-L1 for treatment of solid malignancies. Immune checkpoints control the immune tolerance, and the adverse events such as hepatotoxicity induced by ICIs are often considered as an immune-related adverse event (irAE). However, PD-L1 is also highly expressed in normal tissues, e.g., hepatocytes. It is still not clear whether, targeting PD-L1 on hepatocytes, the atezolizumab may cause damage to liver cells contributing to hepatotoxicity. Here, we reveal a novel mechanism by which the atezolizumab induces hepatotoxicity in human hepatocytes. We find that the atezolizumab treatment increases a release of LDH in the cell culture medium of human hepatocytes (human primary hepatocytes and THLE-2 cells), decreases cell viability, and inhibits the THLE-2 and THLE-3 cell growth. We demonstrate that both the atezolizumab and the conditioned medium (T-CM) derived from activated T cells can induce necroptosis of the THLE-2 cells, which is underscored by the fact that the atezolizumab and T-CM enhance the phosphorylation of RIP3 and MLKL proteins. Furthermore, we also show that necrostatin-1, a necrosome inhibitor, decreases the amount of phosphorylated RIP3 induced by the atezolizumab, resulting in a reduced LDH release in the culture media of the THLE-2 cells. This finding is further supported by the data that GSK872 (a RIP3 inhibitor) significantly reduced the atezolizumab-induced LDH release. Taken together, our data indicate that the atezolizumab induces PD-L1-mediated necrosome formation, contributing to hepatotoxicity in PD-L1+-human hepatocytes. This study provides the molecular basis of the atezolizumab-induced hepatotoxicity and opens a new avenue for developing a novel therapeutic approach to reducing hepatotoxicity induced by ICIs.

Regulation of RIP1-Mediated necroptosis via necrostatin-1 in periodontitis.

OBJECTIVE: To explore the mechanism of receptor-interacting protein 1 (RIP1)-mediated necroptosis during periodontitis progression. BACKGROUND: RIP3 and mixed lineage kinase domain-like protein (MLKL) have been detected to be upregulated in periodontitis models. Because RIP1 is involved in necroptosis, it might also play a role in the progression of periodontitis. METHODS: An experimental periodontitis model in BALB/c mice was established by inducing oral bacterial infection. Western blotting and immunofluorescence analyses were used to detect RIP1 expression in the periodontal ligament. Porphyromonas gingivalis was used to stimulate L929 and MC3T3-E1. RIP1 was inhibited using small-interfering RNA. Western blotting, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) analyses were used to detect the effect of necroptosis inhibition on the expression of damage-associated molecular patterns and inflammatory cytokines. Necrostatin-1 (Nec-1) was intraperitoneally injected to inhibit RIP1 expression in mice. Necroptosis activation and inflammatory cytokine expression in periodontal tissue were verified. Tartrate-resistant acid phosphatase staining was applied to observe osteoclasts in the bone tissues of different groups. RESULTS: RIP1-mediated necroptosis was activated in mice with periodontitis. P. gingivalis induced RIP1-mediated necroptosis in L929 and MC3T3-E1 cells. After RIP1 inhibition, the expression levels of high mobility group protein B1 (HMGB1) and inflammatory cytokines were downregulated. After inhibiting RIP1 with Nec-1 in vivo, necroptosis was also inhibited, the expression levels of HMGB1 and inflammatory cytokines were downregulated, and osteoclast counts in the periodontal tissue decreased. CONCLUSION: RIP1-mediated necroptosis plays a role in the pathological process of periodontitis in mice. Nec-1 inhibited necroptosis, alleviated inflammation in periodontal tissue, and reduced bone resorption in periodontitis.

Administration of necrostatin-1 ameliorates glucocorticoid-induced osteonecrosis of the femoral head in rats.

Glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH) is a serious complication of glucocorticoid treatment and is characterized by dysfunctional bone reconstruction at necrotic sites. Our previous study confirmed the protective potential of necrostatin-1, a selective blocker of necroptosis, in glucocorticoid-induced osteoporosis. In this study, rat models of GC-induced ONFH were established to evaluate the effects of necrostatin-1 on osteonecrotic changes and repair processes. Osteonecrosis was verified by histopathological staining. An analysis of trabecular bone architecture was performed to evaluate osteogenesis in the osteonecrotic zone. Then, necroptotic signaling molecules such as RIP1 and RIP3 were examined by immunohistochemistry. Histopathological observations indicated that necrostatin-1 administration reduced the incidence of osteonecrosis and the osteogenic response in subchondral areas. Additionally, bone histomorphometry demonstrated that necrostatin-1 intervention could restore bone reconstruction in the necrotic zone. The protective mechanism of necrostatin-1 was related to the inhibition of RIP1 and RIP3. Necrostatin-1 administration alleviated GC-induced ONFH in rats by attenuating the formation of necrotic lesions, recovering the function of osteogenesis, and suppressing glucocorticoid-induced osteocytic necroptosis by inhibiting the expression of RIP1 and RIP3.

RIPK1 inhibition contributes to lysosomal membrane stabilization in ischemic astrocytes via a lysosomal Hsp70.1B-dependent mechanism.

Receptor-interacting protein kinase 1 (RIPK1) contributes to necroptosis. Our previous study showed that pharmacological or genetic inhibition of RIPK1 protects against ischemic stroke-induced astrocyte injury. In this study, we investigated the molecular mechanisms underlying RIPK1-mediated astrocyte injury in vitro and in vivo. Primary cultured astrocytes were transfected with lentiviruses and then subjected to oxygen and glucose deprivation (OGD). In a rat model of permanent middle cerebral artery occlusion (pMCAO), lentiviruses carrying shRNA targeting RIPK1 or shRNA targeting heat shock protein 70.1B (Hsp70.1B) were injected into the lateral ventricles 5 days before pMCAO was established. We showed that RIPK1 knockdown protected against OGD-induced astrocyte damage, blocked the OGD-mediated increase in lysosomal membrane permeability in astrocytes, and inhibited the pMCAO-induced increase in astrocyte lysosome numbers in the ischemic cerebral cortex; these results suggested that RIPK1 contributed to the lysosomal injury in ischemic astrocytes. We revealed that RIPK1 knockdown upregulated the protein levels of Hsp70.1B and increased the colocalization of Lamp1 and Hsp70.1B in ischemic astrocytes. Hsp70.1B knockdown exacerbated pMCAO-induced brain injury, decreased lysosomal membrane integrity and blocked the protective effects of the RIPK1-specific inhibitor necrostatin-1 on lysosomal membranes. On the other hand, RIPK1 knockdown further exacerbated the pMCAO- or OGD-induced decreases in the levels of Hsp90 and the binding of Hsp90 to heat shock transcription factor-1 (Hsf1) in the cytoplasm, and RIPK1 knockdown promoted the nuclear translocation of Hsf1 in ischemic astrocytes, resulting in increased Hsp70.1B mRNA expression. These results suggest that inhibition of RIPK1 protects ischemic astrocytes by stabilizing lysosomal membranes via the upregulation of lysosomal Hsp70.1B; the mechanism underlying these effects involves decreased Hsp90 protein levels, increased Hsf1 nuclear translocation and increased Hsp70.1B mRNA expression.

ALOX5 inhibition protects against dopaminergic neurons undergoing ferroptosis.

Oxidative disruption of dopaminergic neurons is regarded as a crucial pathogenesis in Parkinson's disease (PD), eventually causing neurodegenerative progression. (-)-Clausenamide (Clau) is an alkaloid isolated from plant Clausena lansium (Lour.), which is well-known as a scavenger of lipid peroxide products and exhibiting neuroprotective activities both in vivo and in vitro, yet with the in-depth molecular mechanism unrevealed. In this study, we evaluated the protective effects and mechanisms of Clau on dopaminergic neuron. Our results showed that Clau directly interacted with the Ser663 of ALOX5, the PKCα-phosphorylation site, and thus prevented the nuclear translocation of ALOX5, which was essential for catalyzing the production of toxic lipids 5-HETE. LC-MS/MS-based phospholipidomics analysis demonstrated that the oxidized membrane lipids were involved in triggering ferroptotic death in dopaminergic neurons. Furthermore, the inhibition of ALOX5 was found to significantly improving behavioral defects in PD mouse model, which was confirmed associated with the effects of attenuating the accumulation of lipid peroxides and neuronal damages. Collectively, our findings provide an attractive strategy for PD therapy by targeting ALOX5 and preventing ferroptosis in dopaminergic neurons.

Donepezil Reduces H2O2-Inflicted Oxidative Stress and Necroptosis in Cardiomyocytes.

OBJECTIVE: Necroptosis, as a form of regulated cell necrosis, could participate in myocardial oxidative damage. We investigated whether donepezil attenuates H2O2-induced oxidative stress injury and necroptosis in rat cardiomyocytes. METHODS: H9c2 cells were incubated with H2O2 (final concentration of 1 mM) and then intervened with donepezil at doses of 2.5 and 10 µM. Subsequently, the necroptosis inhibitor necrostatin-1 (Nec-1) was introduced to treat H9c2 cells. For cell function experiments, cell proliferation; the contents of creatine kinase (CK), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA); the protein and mRNA levels of the necroptosis-related proteins receptor-interacting serine-threonine kinase 3 (RIP3) and mixed lineage kinase-like (MLKL); and calcium ion fluorescence intensity were detected using Cell Counting Kit-8, enzyme-linked immunosorbent assay (ELISA), Western blotting, quantitative reverse transcription polymerase chain reaction, and flow cytometry, respectively. RESULTS: Cell viability was conspicuously decreased; CK and LDH contents, RIP3 and MLKL expression levels, and MDA production were preeminently elevated; and the production of SOD, CAT, and GSH was prominently reduced under H2O2 stimulation, which were dose-dependently countered by donepezil intervention. Nec-1 decreased the cell necroptosis, oxidative stress, and calcium overload caused by H2O2. However, on the premise of donepezil intervention, the addition of Nec-1 failed to further improve the situation, suggesting that donepezil exerts cardioprotective effects partly by inhibiting RIP3 and MLKL levels. CONCLUSION: Donepezil reduced H2O2-inflicted oxidative stress and necroptosis in cardiomyocytes by suppressing RIP3 and MLKL levels and calcium ion overload.

Dimethyl fumarate inhibits necroptosis and alleviates systemic inflammatory response syndrome by blocking the RIPK1-RIPK3-MLKL axis.

Necroptosis has been implicated in various inflammatory diseases including tumor-necrosis factor-α (TNF-α)-induced systemic inflammatory response syndrome (SIRS). Dimethyl fumarate (DMF), a first-line drug for treating relapsing-remitting multiple sclerosis (RRMS), has been shown to be effective against various inflammatory diseases. However, it is still unclear whether DMF can inhibit necroptosis and confer protection against SIRS. In this study, we found that DMF significantly inhibited necroptotic cell death in macrophages induced by different necroptotic stimulations. Both the autophosphorylation of receptor-interacting serine/threonine kinase 1 (RIPK1) and RIPK3 and the downstream phosphorylation and oligomerization of MLKL were robustly suppressed by DMF. Accompanying the suppression of necroptotic signaling, DMF blocked the mitochondrial reverse electron transport (RET) induced by necroptotic stimulation, which was associated with its electrophilic property. Several well-known anti-RET reagents also markedly inhibited the activation of the RIPK1-RIPK3-MLKL axis accompanied by decreased necrotic cell death, indicating a critical role of RET in necroptotic signaling. DMF and other anti-RET reagents suppressed the ubiquitination of RIPK1 and RIPK3, and they attenuated the formation of necrosome. Moreover, oral administration of DMF significantly alleviated the severity of TNF-α-induced SIRS in mice. Consistent with this, DMF mitigated TNF-α-induced cecal, uterine, and lung damage accompanied by diminished RIPK3-MLKL signaling. Collectively, DMF represents a new necroptosis inhibitor that suppresses the RIPK1-RIPK3-MLKL axis through blocking mitochondrial RET. Our study highlights DMF's potential therapeutic applications for treating SIRS-associated diseases.

RIPK1 Regulates Microglial Activation in Lipopolysaccharide-Induced Neuroinflammation and MPTP-Induced Parkinson's Disease Mouse Models.

Increasing evidence suggests a pivotal role of receptor-interacting protein kinase 1 (RIPK1), an initiator of necroptosis, in neuroinflammation. However, the precise role of RIPK1 in microglial activation remains unclear. In the present study, we explored the role of RIPK1 in lipopolysaccharide (LPS)-induced neuroinflammation and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model mice by using RIPK1-specific inhibitors necrostatin-1 (Nec-1) and necrostatin-1 stable (Nec-1s). Nec-1/Nec-1s or RIPK1 siRNA inhibited the production of proinflammatory molecules and the phosphorylation of RIPK1-RIPK3-MLKL and cell death in LPS-induced inflammatory or LPS/QVD/BV6-induced necroptotic conditions of BV2 microglial cells. Detailed mechanistic studies showed that Nec-1/Nec-1s exerted anti-inflammatory effects by modulating AMPK, PI3K/Akt, MAPKs, and NF-κB signaling pathways in LPS-stimulated BV2 cells. Subsequent in vivo studies showed that Nec-1/Nec-1s inhibited microglial activation and proinflammatory gene expression by inhibiting the RIPK1 phosphorylation in the brains of LPS-injected mice. Furthermore, Nec-1/Nec-1s exert neuroprotective and anti-inflammatory effects in MPTP-induced PD mice. We found that p-RIPK1 is mainly expressed in microglia, and thus RIPK1 may contribute to neuroinflammation and subsequent cell death of dopaminergic neurons in MPTP-induced PD model mice. These data suggest that RIPK1 is a key regulator of microglial activation in LPS-induced neuroinflammation and MPTP-induced PD mice.

Anti-oxidative and anti-neuroinflammatory role of Necrostatin-1s and docosahexaenoic acid in RIP-1-mediated neurotoxicity in MPTP-induced Parkinson's disease model.

Parkinson's disease (PD) is a neuromuscular ailment that affects people in their later years and causes both motor and non-motor deficits. Receptor-interacting protein-1 (RIP-1) is a critical participant in necroptotic cell death, possibly through an oxidant-antioxidant imbalance and cytokine cascade activation in PD pathogenesis. The present study examined the role of RIP-1-mediated necroptosis and neuroinflammation in the MPTP-induced PD mouse model, as well as their protection by Necrostatin-1s (an RIP signalling inhibitor), antioxidant DHA and their functional interaction. BALB/c mice were given acute MPTP therapy (4 injections of 15 mg/kg i.p. at 2-h intervals) on day 1. After MPTP intoxication, Necrostatin-1s (Nec-1s; 8 mg/kg/day, i.p.) and DHA (300 mg/kg/day, p.o.) treatments were given once daily for 7 days. The Nec-1s treatment prevented MPTP-induced behavioural, biochemical and neurochemical alterations, and the addition of DHA increases Nec-1s' neuroprotective impact. In addition, Nec-1s and DHA significantly improve the survival of TH-positive dopaminergic neurons and lower expression levels of the inflammatory cytokines, IL-1ß and TNF-α. Furthermore, Nec-1s dramatically reduced RIP-1 expression, whereas DHA had little effect. Our research raises the possibility that neuroinflammatory signalling and acute MPTP-induced necroptosis are both mediated by TNFR1-driven RIP-1 activity. In this study, RIP-1 ablation through Nec-1s and the addition of DHA showed a reduction in the levels of pro-inflammatory and oxidative markers, as well as protection from MPTP-driven dopaminergic degeneration and neurobehavioural changes, suggesting potential therapeutic applications. For a better understanding, additional research about the mechanism(s) behind Nec-1s and DHA is required.

An in vitro evaluation of the antioxidant activities of necroptosis and apoptosis inhibitors: the potential of necrostatin-1 and necrostatin-1i to have radical scavenging activities.

BACKGROUND: Necroptosis inhibitors, including necrostatin-1 (Nec-1), are attracting attention as potential therapeutic agents against various diseases, such as acute lung injury, chronic obstructive pulmonary disease, acute kidney injury, nonalcoholic fatty liver, and neurodegenerative disease, where necroptosis is thought to act as a contributing factor. Nec-1 suppresses necroptosis by inhibiting receptor-interacting protein (RIP) 1 kinase and can also reduce reactive oxygen species (ROS) production; however, the underlying molecular mechanisms mediating ROS reduction remain unclear. METHODS: The antioxidant effects of necroptosis inhibitors, including Nec-1 and apoptosis inhibitors, were quantified by performing a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. Nec-1-related compounds were subsequently assayed for cupric ion-reducing capacity and superoxide dismutase (SOD)-like activity. RESULTS: Considering all examined apoptosis and necroptosis inhibitors, Nec-1and Nec-1i exhibited antioxidant activity in DPPH radical scavenging assay. In the cupric ion-reducing capacity assay, Nec-1i showed stronger antioxidant capacity than Nec-1. In the SOD-like activity assay, both Nec-1 and Nec-1i were found to have stronger antioxidant capacity than ascorbic acid (IC50 = 4.6 ± 0.040 and 61 ± 0.54 µM, respectively). CONCLUSION: These results suggest that Nec-1 and Nec-1i may exhibit direct radical scavenging ability against superoxide anions, independent of RIP1 inhibition.