Mixed Lineage Kinase Domain-like Protein Research Articles

Linolenic acid stimulates eryptosis and hemolysis through oxidative stress and CK1α/MLKL: protective role of melatonin, urea, and polyethylene glycol

Anticancer medications cause anemia in patients through ill-defined mechanisms, including hemolysis and eryptosis. Although α-linolenic acid (ALA) possesses anticancer properties against a variety of cancer cells, there is a dearth of evidence regarding how it modulates red blood cell (RBC) physiology. RBCs from healthy donors were subjected to anticancer concentrations of ALA (2.5, 5, 10, 20, 40, 80, and 100 μM) at 37 °C for 24 h, and colorimetric tests were used to determine hemolysis and acetylcholinesterase (AChE) activity. Meanwhile, flow cytometry was employed to identify eryptotic cells using annexin-V-FITC and forward scatter (FSC), Fluo4/AM to detect Ca2+, and H2DCFDA to assess oxidative stress. ALA significantly increased hemolysis and eryptosis in a concentration-dependent manner, along with elevated Fluo4 and DCF fluorescence, and erythrocyte sedimentation rate, and reduced FSC and AChE activity. Moreover, the addition of D4476, necrosulfonamide, melatonin, isosmotic urea, and polyethylene glycol 8000 – but not sucrose – significantly inhibited ALA toxicity. In conclusion, ALA stimulates hemolysis and eryptosis through Ca2+ buildup, oxidative stress, anticholinesterase activity, casein kinase 1α (CK1α), and mixed lineage kinase domain-like protein (MLKL). The anticancer activity of ALA may be potentiated by the use of Ca2+ channel blockers and chelators, antioxidants, and CK1α and MLKL inhibitors to ameliorate its toxicity to RBCs.

Role of Z-DNA Binding Protein 1 Sensing Mitochondrial Z-DNA and Triggering Necroptosis in Oxalate-Induced Acute Kidney Injury.

Calcium oxalate-induced acute kidney injury is a severe condition in which the kidneys suffer rapid damage due to the deposition of oxalate crystals. Known factors contributing to cell death induced by calcium oxalate include receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) protein dependent necroptosis, as well as necrosis involving peptidylprolyl isomerase F (PPIF) mediated mitochondrial permeability transition. However, the detailed molecular mechanisms linking mitochondrial dysfunction to RIPK3 activation are not fully understood. Mice with gene knock-out of Zbp1, Ripk3, or Mlkl and mice with mutations in the Z-nucleic acid sensing domain of ZBP1 or deletion of Zα1 were used in an oxalate-induced AKI model. Proximal renal tubule cells were isolated and cultured for further investigation. Human oxalate nephropathy biopsy samples were analyzed. Specific gene deletions of Zbp1, Ripk3, or Mlkl in proximal renal tubules significantly reduced the severity of oxalate-induced AKI by preventing necroptosis and subsequent inflammation. Notably, mice with mutations in the Z-nucleic acid sensing domain of ZBP1 or deletion of Zα1 were protected from AKI. In cultured proximal tubular cells, calcium oxalate damaged mitochondria, accompanied by an increase in Bax and a decrease in BCL2 and TAFM, leading to the release of mitochondrial Z-DNA. ZBP1 sensed this mitochondrial Z-DNA and then recruited RIPK3 via the RIP homotypic interaction motifs (RHIM), which in turn activated MLKL through RIPK3 phosphorylation, leading to necroptosis and contributing to AKI. ZBP1 plays a critical role in sensing mitochondrial Z-DNA and initiating RIPK3/MLKL-mediated necroptosis, contributing to the development of oxalate-induced AKI.

Genomic and Transcriptional Analysis of the Necroptosis Pathway Elements RIPK and MLKL in Sea Cucumber, Holothuria leucospilota.

Background: Receptor-interacting protein kinases (RIPKs) and mixed-lineage kinase domain-like protein (MLKL) are crucial in regulating innate immune responses and cell death signaling (necroptosis and apoptosis), and are potential candidates for genetic improvement in breeding programs. Knowledge about the RIPK family and MLKL in sea cucumber remains limited. Methods: We searched the genomes of sea cucumber Holothuria leucospilota for genes encoding RIPKs and MLKL, performed phylogenetic tree, motif and functional domain analyses, and examined tissue distribution and embryonic development patterns using qPCR. Results: RIPK5 (Hl-RIPK5), RIPK7 (Hl-RIPK7) and MLKL (Hl-MLKL) were identified in sea cucumber H. leucospilota. Hl-RIPK5 and Hl-RIPK7 were mainly expressed in coelomocytes, suggesting that they play a role in innate immunity, whereas Hl-MLKL exhibited relatively low expression across tissues. During embryonic development, Hl-MLKL was highly expressed from the 2-cell stage to the morula stage, while Hl-RIPK5 and Hl-RIPK7 were primarily expressed after the morula stage, indicating different roles in embryonic development. In primary coelomocytes, Hl-RIPK5 transcriptional activity was significantly depressed by LPS, poly(I:C), or pathogen Vibrio harveyi. Hl-RIPK7 expression levels were unchanged following the same challenges. Hl-MLKL mRNA levels were significantly decreased with poly(I:C) or V. harveyi, but did not change with LPS. Conclusions: These findings provide valuable insights into the evolutionary tree and characterization of RIPK and MLKL genes in sea cucumber, contributing to the broader understanding of the RIPK gene family and MLKL in ancient echinoderms.

The Evolution and Biological Activity of Metazoan Mixed Lineage Kinase Domain-Like Protein (MLKL).

In mammals, mixed lineage kinase domain-like protein (MLKL) is the executor of necroptosis. MLKL comprises an N-terminal domain (NTD), which alone suffices to trigger necroptosis by forming pores in the plasma membrane, and a C-terminal domain that inhibits the NTD activity. Evolutionarily, MLKL is poorly conserved in animals and not found in Protostomia. Although MLKL orthologs exist in invertebrate Deuterostomia, the biological activity of invertebrate MLKL is unknown. Herein, we examined 34 metazoan phyla and detected MLKL not only in Deuterostomia but also in Protostomia (Rotifera). The Rotifera MLKL exhibited low identities with non-Rotifera MLKL but shared relatively high identities with non-metazoan MLKL. In invertebrates, MLKL formed two phylogenetic clades, one of which was represented by Rotifera. In vertebrates, MLKL expression was tissue-specific and generally rich in immune organs. When expressed in human cells, the MLKL-NTD of Rotifera, Echinodermata, Urochordata, and Cephalochordata induced strong necroptosis. The necroptotic activity of Rotifera MLKL depended on a number of conserved residues. Together these findings provided new insights into the evolution of MLKL in Metazoa and revealed the biological activity of invertebrate MLKL.

Curcuminoids as natural modulators of necroptosis: therapeutic implications.

Necroptosis is an emerging form of programmed cell death characterized by necrosis, an inflammatory type of cell death. Necroptosis is primarily initiated by specific mediators that interact with receptor proteins, leading to the activation of protein kinases RIPK1 and RIPK3. These kinases transmit death signals and recruit and phosphorylate mixed lineage kinase domain-like protein (MLKL), which ultimately triggers cell death and necroptosis. Curcuminoids, natural compounds derived from turmeric, have been shown to possess various therapeutic benefits, including neuroprotective, anti-metabolic syndrome, anti-inflammatory, and anti-cancer effects. In this concise overview, we aim to explore the relationship between curcuminoids and the molecular mechanisms of the necroptosis pathway based on recent in vivo and in vitro studies. The available literature indicates that curcuminoids, mainly curcumin, can act as inhibitors of necroptosis in tissue damage scenarios while serving as a necroptosis inducer in cancer cells. Curcuminoids significantly influence key indicators of necroptosis, highlighting their potential to enhance disease treatment. Future studies should focus on further investigating this important component of turmeric to advance therapeutic approaches.

Prognostic significance and response to immune checkpoint inhibitors of RIPK3, MLKL and necroptosis in non-small cell lung cancer

Lung cancer remains the leading cause of cancer death. Treatment with immune checkpoint inhibitor (ICI) alone or combination with chemotherapy served as first-line therapy in non-small cell lung cancer (NSCLC). However, only 20–50% of NSCLC patients respond to ICI. Necroptosis, an inflammatory form of cell death plays an important role in the regulation of tumor immune microenvironment which may affect prognosis and ICI response but its clinical significance in NSCLC patients has remained largely unknown. Therefore, we aimed to analyze the correlation between key necroptotic proteins and necroptosis and clinical outcomes, the status of tumor-infiltrating immune cells, and response to ICI in NSCLC patients. The expression of receptor-interacting protein kinase 3 (RIPK3), mixed lineage kinase domain-like protein (MLKL) and phosphorylated MLKL (pMLKL) were immunolocalized in 125 surgically resected NSCLC patients and 23 NSCLC patients administered with ICI therapy. CD8 + and FOXp3 + T cells and CD163 + M2 macrophages were also immunolocalized. High RIPK3 status was positively correlated with survival of the patients and RIPK3 turned out an independent favorable prognostic factor of the patients. RIPK3 was negatively correlated with CD8 + T cells, while MLKL positively correlated with CD163 + M2 macrophages, suggesting the possible involvement of RIPK3 and MLKL in formulating immunosuppressive microenvironment. In addition, high RIPK3 status tended to be associated with clinical resistance to ICI therapy (P-value = 0.057). Furthermore, NSCLC cells-expressing RIPK3 suppressed T cells response to ICI therapy in vitro. Therefore, RIPK3 and MLKL could induce an immunosuppressive microenvironment, resulting in low response to ICI therapy in NSCLC.

Parkin deficiency exacerbates particulate matter-induced injury by enhancing airway epithelial necroptosis

Exposure to fine particulate matter (PM) disrupts the function of airway epithelial barriers causing cellular stress and damage. However, the precise mechanisms underlying PM-induced cellular injury and the associated molecular pathways remain incompletely understood. In this study, we used intratracheal instillation of PM in C57BL6 mice and PM treatment of the BEAS-2B cell line as in vivo and in vitro models, respectively, to simulate PM-induced cellular damage and inflammation. We collected lung tissues and bronchoalveolar lavage fluids to assess histopathological changes, necroptosis, and airway inflammation. Our findings reveal that PM exposure induces necroptosis in mouse airway epithelial cells. Importantly, concurrent administration of a receptor interacting protein kinases 3 (RIPK3) inhibitor or the deletion of the necroptosis effector mixed-lineage kinase domain-like protein (MLKL) effectively attenuated PM-induced airway inflammation. PM exposure dose-dependently induces the expression of Parkin, an E3 ligase we recently reported to play a pivotal role in necroptosis through regulating necrosome formation. Significantly, deletion of endogenous Parkin exacerbates inflammation by enhancing epithelial necroptosis. These results indicate that PM-induced Parkin expression plays a crucial role in suppressing epithelial necroptosis, thereby reducing airway inflammation. Overall, these findings offer valuable mechanistic insights into PM-induced airway injury and identify a potential target for clinical intervention.

Near-infrared laser diode mitigates Aβ1–42-induced neurodegeneration in cortical neurons

Alzheimer's disease, a prevalent neurodegenerative condition primarily affecting older adults, remains incurable. Its principle pathological hallmark is the accelerated accumulation of amyloid β (Aβ) protein. This study investigates the potential of photobiomodulation using near infrared light to counteract Aβ1–42-induced synaptic degeneration and neurotoxicity. We focused on the effect of 808 nm near-infrared laser diode (LD) on Aβ1–42 cytotoxicity in primary cultured cortical neurons. We assessed cell survival using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, observing substantial benefits from LD irradiation with a power of 10 mW and a dose of 30 J. Cells exposed to Aβ1–42 exhibited morphological changes indicative of synaptic damage and a significant decrease in the number of postsynaptic density protein-95 (PSD-95) contacts, which were significantly improved with near-infrared LD therapy. Furthermore, this therapy reduced Aβ and phosphorylated tau (P-tau) protein accumulation. Additionally, near-infrared LD irradiation substantially lessened the Aβ1–42–induced rise in glial fibrillary acid protein (GFAP) and ionized calcium-binding adaptor molecule 1 (IBA1) in astrocytes and microglia. Remarkably, near-infrared LD irradiation effectively inhibited phosphorylation of key proteins involved in Aβ1–42-induced necroptosis, namely Receptor-interacting protein kinase-3 (RIP3) and Mixed Lineage Kinase domain-Like protein (MLKL). Our findings suggest that near-infrared LD treatment significantly reduces neurodegeneration by reducing glial overactivation and neuronal necroptosis triggered by Aβ1–42. Thus, near-infrared LD treatment emerges as a promising approach for slowing or treating Alzheimer's disease, offering new avenues in its management.

Myo-inositol: A potential game-changer in preventing gill cell death and alleviating “gill rot” in grass carp (Ctenopharyngodon idellus)

Increasing evidence shows the potential threat of gill rot in freshwater fish culture. F. columnare is wide-spread in aquatic environments, which can cause fish gill rot and result in high mortality and losses of fish. This study investigated the effects of myo-inositol (MI) on the proliferation, structural integrity, and different death modes of grass carp (Ctenopharyngodon idella) gill epithelial cells, as well as its possible mechanism. 30 mg/L MI up-regulated CCK8 OD value and the protein level of solute carrier family 5A 3 (SLC5A3), and down-regulated the reactive oxygen species (ROS) content in gill cells and lactate dehydrogenase (LDH) release in the culture medium (P < 0.05). MI up-regulated the protein level of Beclin1, the protein level and fluorescence expression of microtubule-associated protein light chain 3B (LC3B) and down-regulated the protein level of sequestosome-1 (SQSTM1, also called p62) (P < 0.05). MI down-regulated the protein levels of Cysteine aspartate protease-1 (caspase-1), Gasdermin E (GSDME) and Cleaved interleukin 1 beta (IL-1β) (P < 0.05). MI up-regulated the protein level of caspase-8 (P < 0.05), but had no effect on apoptosis (P > 0.05). MI down-regulated the mRNA expressions and protein levels of tumor necrosis factor α (tnfα), TNF receptor 1 (tnfr1), receptor interacting protein 1 (ripk1), receptor interacting protein 3 (ripk3) and mixed lineage kinase domain-like protein (mlkl), and reduce the ratio of p-MLKL/MLKL (P < 0.05). The addition of MI or necrosulfonamide (NSA) alone, or the addition of MI after induction of necroptosis, significantly up-regulated the cell activity and the protein level of SLC5A3 in gill cells, and significantly reduced the LDH release in the culture medium and the intracellular ROS content, the number of necroptosis cells, the protein expression of TNFα, TNFR1 and RIPK1, and the ratio of p-RIPK3/RIPK3 and p-MLKL/MLKL (P < 0.05). It indicated MI induce autophagy may relate to Beclin1/LC3/p62 signaling pathway, inhibits pyroptosis may attribute to Caspase-1/GSDMD/IL-1β signaling pathway, and inhibits necroptosis via MLKL signaling pathway. However, MI had no effect on apoptosis.

The role of hydrogen sulfide in the regulation of necroptosis across various pathological processes.

Necroptosis is a programmed cell death form executed by receptor-interacting protein kinase (RIPK) 1, RIPK3 and mixed lineage kinase domain-like protein (MLKL), which assemble into an oligomer called necrosome. Accumulating evidence reveals that necroptosis participates in many types of pathological processes. Hence, clarifying the mechanism of necroptosis in pathological processes is particularly important for the prevention and treatment of various diseases. For over 300 years, hydrogen sulfide (H2S) has been widely known in the scientific community as a toxic and foul-smelling gas. However, after discovering the important physiological and pathological functions of H2S, human understanding of this small molecule changed, believing that H2S is the third gas signaling molecule after carbon monoxide (CO) and nitric oxide (NO). H2S plays an important role in various diseases, but the related mechanisms are not yet fully understood. In recent years, more and more studies have shown that H2S regulation of necroptosis is involved in various pathological processes. Herein, we focus on the recent progress on the role of H2S regulation of necroptosis in different pathological processes and profoundly analyze the related mechanisms.

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

PurposeBenzalkonium 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. MethodsHuman 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. ResultsIn 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. ConclusionsNecroptosis 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.

A kinase fusion protein from Aegilops longissima confers resistance to wheat powdery mildew

Many disease resistance genes have been introgressed into wheat from its wild relatives. However, reduced recombination within the introgressed segments hinders the cloning of the introgressed genes. Here, we have cloned the powdery mildew resistance gene Pm13, which is introgressed into wheat from Aegilops longissima, using a method that combines physical mapping with radiation-induced chromosomal aberrations and transcriptome sequencing analysis of ethyl methanesulfonate (EMS)-induced loss-of-function mutants. Pm13 encodes a kinase fusion protein, designated MLKL-K, with an N-terminal domain of mixed lineage kinase domain-like protein (MLKL_NTD domain) and a C-terminal serine/threonine kinase domain bridged by a brace. The resistance function of Pm13 is validated through transient and stable transgenic complementation assays. Transient over-expression analyses in Nicotiana benthamiana leaves and wheat protoplasts reveal that the fragment Brace-Kinase122-476 of MLKL-K is capable of inducing cell death, which is dependent on a functional kinase domain and the three α-helices in the brace region close to the N-terminus of the kinase domain.

Discovery of a Novel Benzimidazole Necroptosis Inhibitor from an In-House Compound Library

AbstractNecroptosis, a caspase-independent regulated cell death, is primarily mediated by the serine/threonine kinases RIPK1 and RIPK3, and the mixed lineage kinase domain-like protein (MLKL). Targeting necroptosis is a validated therapeutic strategy for various diseases. We screened compound 1, a novel benzimidazole-based necroptosis inhibitor, from our in-house compound library. We assessed its inhibitory roles and mechanisms in blocking HT-29 cell necroptosis. HT-29 cells were treated with pan caspase inhibitor Z-VAD-FMK + Smac mimetic (TSZ), or Z-VAD-FMK + cycloheximide (TCZ), then with tumor necrosis factor α (TNFα) to induce necroptosis in vitro. Prior to stimulation, cells were exposed to compound 1. GSK'843 served as a control drug. HT-29 cells were treated with TNFα + Smac mimetic (TS) or TNFα + cycloheximide (TC) to induce apoptosis in vitro. Cell viability, cell death, and necroptotic cells were evaluated by luminescence-based CellTiter-Lumi assay or flow cytometry. Western blots, immunoprecipitation, and KINOMEscan technology were used to assess RIPK1, RIPK3, and MLKL's involvement in compound 1's mechanisms. Compound 1's roles in mouse TNFα induced systemic inflammatory response syndrome (SIRS) in mice were also investigated by assessing body temperature, mouse survival rate, and interleukin (IL)-β and IL-6 levels in respective tissues. We found that necroptosis triggered by TSZ or TCZ was effectively mitigated by compound 1, showing a dose-responsive inhibition, and it could protect mice from TNF-induced SIRS. The mechanism study showed that compound 1 could interact with RIPK1, inhibiting RIPK1 phosphorylation activation to block necrosome formation in necroptotic cells. In summary, compound 1 is a promising lead compound for developing treatments targeting diseases associated with necroptosis.

Necroptosis plays a role in TL1A-induced airway inflammation and barrier damage in asthma

BackgroundAirway epithelial cell (AEC) necroptosis contributes to airway allergic inflammation and asthma exacerbation. Targeting the tumor necrosis factor-like ligand 1 A (TL1A)/death receptor 3 (DR3) axis has a therapeutic effect on asthmatic airway inflammation. The role of TL1A in mediating necroptosis of AECs challenged with ovalbumin (OVA) and its contribution to airway inflammation remains unclear.MethodsWe evaluated the expression of the receptor-interacting serine/threonine-protein kinase 3(RIPK3) and the mixed lineage kinase domain-like protein (MLKL) in human serum and lung, and histologically verified the level of MLKL phosphorylation in lung tissue from asthmatics and OVA-induced mice. Next, using MLKL knockout mice and the RIPK3 inhibitor GSK872, we investigated the effects of TL1A on airway inflammation and airway barrier function through the activation of necroptosis in experimental asthma.ResultsHigh expression of necroptosis marker proteins was observed in the serum of asthmatics, and necroptosis was activated in the airway epithelium of both asthmatics and OVA-induced mice. Blocking necroptosis through MLKL knockout or RIPK3 inhibition effectively attenuated parabronchial inflammation, mucus hypersecretion, and airway collagen fiber accumulation, while also suppressing type 2 inflammatory factors secretion. In addition, TL1A/ DR3 was shown to act as a death trigger for necroptosis in the absence of caspases by silencing or overexpressing TL1A in HBE cells. Furthermore, the recombinant TL1A protein was found to induce necroptosis in vivo, and knockout of MLKL partially reversed the pathological changes induced by TL1A. The necroptosis induced by TL1A disrupted the airway barrier function by decreasing the expression of tight junction proteins zonula occludens-1 (ZO-1) and occludin, possibly through the activation of the NF-κB signaling pathway.ConclusionsTL1A-induced airway epithelial necroptosis plays a significant role in promoting airway inflammation and barrier dysfunction in asthma. Inhibition of the TL1A-induced necroptosis pathway could be a promising therapeutic strategy.

Sirt1 Deficiency Promotes Age-Related AF Through Enhancing Atrial Necroptosis by Activation of RIPK1 Acetylation.

Aging is one of the most potent risk determinants for the onset of atrial fibrillation (AF). Sirts (sirtuins) have been implicated in the pathogenesis of cardiovascular disease, and their expression declines with aging. However, whether Sirts involved in age-related AF and its underlying mechanisms remain unknown. The present study aims to explore the role of Sirts in age-related AF and delineate the underlying molecular mechanisms. Sirt1 levels in the atria of both elderly individuals and aging rats were evaluated using quantitative real-time polymerase chain reaction and Western blot analysis. Mice were engineered to specifically knockout Sirt1 in the atria and right ventricle (Sirt1mef2c/mef2c). Various techniques, such as echocardiography, atrial electrophysiology, and protein acetylation modification omics were employed. Additionally, coimmunoprecipitation was utilized to substantiate the interaction between Sirt1 and RIPK1 (receptor-interacting protein kinase 1). We discerned that among the diverse subtypes of sirtuin proteins, only Sirt1 expression was significantly diminished in the atria of elderly people and aged rats. The Sirt1mef2c/mef2c mice exhibited an enlarged atrial diameter and heightened vulnerability to AF. Acetylated proteomics and cell experiments identified that Sirt1 deficiency activated atrial necroptosis through increasing RIPK1 acetylation and subsequent pseudokinase MLKL (mixed lineage kinase domain-like protein) phosphorylation. Consistently, necroptotic inhibitor necrosulfonamide mitigated atrial necroptosis and diminished both the atrial diameter and AF susceptibility of Sirt1mef2c/mef2c mice. Resveratrol prevented age-related AF in rats by activating atrial Sirt1 and inhibiting necroptosis. Our findings first demonstrated that Sirt1 exerts significant efficacy in countering age-related AF by impeding atrial necroptosis through regulation of RIPK1 acetylation, highlighting that the activation of Sirt1 or the inhibition of necroptosis could potentially serve as a therapeutic strategy for age-related AF.

MLKL deficiency alleviates acute alcoholic liver injury via inhibition of NLRP3 inflammasome

Mixed lineage kinase domain-like protein (MLKL) is identified as the terminal executor of necroptosis. However, its role in acute alcoholic liver injury remains unclear. This study elucidates that MLKL can contribute to acute alcoholic liver injury independently of necroptosis. Although the expression of MLKL was upregulated, no significant increase in its phosphorylation or membrane translocation was observed in the liver tissues of mice treated with ethanol. This finding confirms that alcohol intake does not induce necroptosis in mouse liver tissue. Additionally, the deletion of Mlkl resulted in the downregulation of NLRP3 expression, which subsequently inhibited the activation of the NLRP3 inflammasome and the ensuing inflammatory response, thereby effectively mitigating liver injury induced by acute alcohol consumption. The knockout of Nlrp3 did not affect the expression of MLKL, further confirming that MLKL acts upstream of NLRP3. Mechanistically, inhibiting the nuclear translocation of MLKL reduced the nuclear entry of p65, the principal transcriptional regulator of NLRP3, thereby limiting the transcription of Nlrp3 mRNA and subsequent NLRP3 expression. Overall, this study unveils a novel mechanism of MLKL regulates the activation of NLRP3 inflammasomes in a necroptosis independent way in acute alcoholic liver injury

DL-3-n-Butylphthalide Ameliorates Post-stroke Emotional Disorders by Suppressing Neuroinflammation and PANoptosis.

Post-stroke emotional disorders such as post-stroke anxiety and post-stroke depression are typical symptoms in patients with stroke. They are closely associated with poor prognosis and low quality of life. The State Food and Drug Administration of China has approved DL-3-n-butylphthalide (NBP) as a treatment for ischemic stroke (IS). Clinical research has shown that NBP alleviates anxiety and depressive symptoms in patients with IS. Therefore, this study explored the role and molecular mechanisms of NBP in cases of post-stroke emotional disorders using network pharmacology and experimental validation. The results showed that NBP treatment significantly increased the percentage of time spent in the center of the middle cerebral artery occlusion (MCAO) rats in the open field test and the percentage of sucrose consumption in the sucrose preference test. Network pharmacology results suggest that NBP may regulate neuroinflammation and cell death. Further experiments revealed that NBP inhibited the toll-like receptor 4/nuclear factor kappa B signaling pathway, decreased the level of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin-1β, and interleukin-6, and M1-type microglia markers (CD68, inducible nitric oxide synthase), and reduced the expression of PANoptosis-related molecules including caspase-1, caspase-3, caspase-8, gasdermin D, and mixed lineage kinase domain-like protein in the hippocampus of the MACO rats. These findings demonstrate that the mechanisms through which NBP ameliorates post-stroke emotional disorders in rats are associated with inhibiting neuroinflammation and PANoptosis, providing a new strategy and experimental basis for treating post-stroke emotional disorders.

An acidic pH environment converts necroptosis to apoptosis

Cardiac ischemia results in anaerobic metabolism and lactic acid accumulation and with time, intracellular and extracellular acidosis. Ischemia and subsequent reperfusion injury (IRI) lead to various forms of programmed cell death. Necroptosis is a major form of programmed necrosis that worsens cardiac function directly and also promotes inflammation by the release of cellular contents. Potential effects of increasing acidosis on programmed cell death and their specific components have not been well studied. While apoptosis is caspase-dependent, in contrast, necroptosis is mediated by the receptor-interacting protein kinases 1 and 3 (RIPK1/3). In our study, we observed that at physiological pH = 7.4, caspase-8 inhibition did not prevent TNFα-induced cell death in mouse cardiac vascular endothelial cells (MVECs) but promoted necroptotic cell death. As expected, necroptosis was blocked by RIPK1 inhibition. However, at pH = 6.5, TNFα induced an apoptosis-like pattern which was inhibited by caspase-8 inhibition. Interestingly phosphorylation of necroptotic molecules RIPK1, RIPK3, and mixed lineage kinase domain-like protein (MLKL) was enhanced in an acidic pH environment. However, RIPK3 and MLKL phosphorylation was self-limited which may have limited their participation in necroptosis. In addition, an acidic pH promoted apoptosis-inducing factor (AIF) cleavage and nuclear translocation. AIF RNA silencing inhibited cell death, supporting the role of AIF in this cell death. In summary, our study demonstrated that the pH of the micro-environment during inflammation can bias cell death pathways by altering the function of necroptosis-related molecules and promoting AIF-mediated cell death. Further insights into the mechanisms by which an acidic cellular micro-environment influences these and perhaps other forms of regulated cell death, may lead to therapeutic strategies to attenuate IRI.

In Vitro and In Silico Anti-Glioblastoma Activity of Hydroalcoholic Extracts of Artemisia annua L. and Artemisia vulgaris L.

Glioblastoma, the most aggressive and challenging brain tumor, is a key focus in neuro-oncology due to its rapid growth and poor prognosis. The C6 glioma cell line is often used as a glioblastoma model due to its close simulation of human glioma characteristics, including rapid expansion and invasiveness. Alongside, herbal medicine, particularly Artemisia spp., is gaining attention for its anticancer potential, offering mechanisms like apoptosis induction, cell cycle arrest, and the inhibition of angiogenesis. In this study, we optimized extraction conditions of polyphenols from Artemisia annua L. and Artemisia vulgaris L. herbs and investigated their anticancer effects in silico and in vitro. Molecular docking of the main phenolic compounds of A. annua and A. vulgaris and potential target proteins, including programmed cell death (apoptosis) pathway proteins proapoptotic Bax (PDB ID 6EB6), anti-apoptotic Bcl-2 (PDB ID G5M), and the necroptosis pathway protein (PDB ID 7MON), mixed lineage kinase domain-like protein (MLKL), in complex with receptor-interacting serine/threonine-protein kinase 3 (RIPK3), revealed the high probability of their interactions, highlighting the possible influence of chlorogenic acid in modulating necroptosis processes. The cell viability of rat C6 glioma cell line was assessed using a nuclear fluorescent double-staining assay with Hoechst 33342 and propidium iodide. The extracts from A. annua and A. vulgaris have demonstrated anticancer activity in the glioblastoma model, with the synergistic effects of their combined compounds surpassing the efficacy of any single compound. Our results suggest the potential of these extracts as a basis for developing more effective glioblastoma treatments, emphasizing the importance of further research into their mechanisms of action and therapeutic applications.

Hyperlipidemia-induced hematopoiesis is repressed by MLKL in endothelial cells of the splenic niche.

Dysregulation of the hematopoietic niche during hyperlipidemia facilitates pathologic leukocyte production, driving atherogenesis. Although definitive hematopoiesis occurs primarily in the bone marrow, during atherosclerosis this also occurs in the spleen. Cells of the bone marrow niche, particularly endothelial cells, have been studied in atherosclerosis, although little is known about how splenic endothelial cells respond to the atherogenic environment. Here we show unique dysregulated pathways in splenic compared to bone marrow endothelial cells during atherosclerosis, including perturbations of lipid metabolism and endocytic trafficking pathways. As part of this response, we identify the mixed lineage kinase domain-like (MLKL) protein as a repressor of splenic, but not bone marrow, myelopoiesis. Silencing MLKL in splenic endothelial cells results in inefficient endosomal trafficking and lipid accumulation, ultimately promoting the production of myeloid cells that participate in plaque development. These studies identify endocytic trafficking by MLKL as a key mechanism of splenic endothelial cell maintenance, splenic hematopoiesis and, subsequently, atherosclerosis.