Neuroprotective Effect of Kinase Inhibition in Ischemic Factor Modeling In Vitro

The contribution of many neuronal kinases to the adaptation of nerve cells to ischemic damage and their effect on functional neural network activity has not yet been studied. The aim of this work is to study the role of the four kinases belonging to different metabolic cascades (SRC, Ikkb, eEF2K, and FLT4) in the adaptive potential of the neuron-glial network for modeling the key factors of ischemic damage. We carried out a comprehensive study on the effects of kinases blockade on the viability and network functional calcium activity of nerve cells under ischemic factor modeling in vitro. Ischemic factor modelling was performed on day 14 of culturing primary hippocampal cells obtained from mouse embryos (E18). The most significant neuroprotective effect was shown in the blockade of FLT4 kinase in the simulation of hypoxia. The studies performed revealed the role of FLT4 in the development of functional dysfunction in cerebrovascular accidents and created new opportunities for the study of this enzyme and its blockers in the formation of new therapeutic strategies.     

Death Processes in Bovine Theca and Granulosa Cells Modelled and Analysed Using a Systems Biology Approach

In this paper, newly discovered mechanisms of atresia and cell death processes in bovine ovarian follicles are investigated. For this purpose the mRNA expression of receptor interacting protein kinases 1 and 3 (RIPK1 and RIPK3) of the granulosa and theca cells derived from healthy and atretic follicles are studied. The follicles were assigned as either healthy or atretic based on the estradiol to progesterone ratio. A statistically significant difference was recorded for the mRNA expression of a RIPK1 and RIPK3 between granulosa cells from healthy and atretic follicles. To further investigate this result a systems biology approach was used. The genes playing roles in necroptosis, apoptosis and atresia were chosen and a network was created based on human genes annotated by the IMEx database in Cytoscape to identify hubs and bottle-necks. Moreover, correlation networks were built in the Cluepedia plug-in. The networks were created separately for terms describing apoptosis and programmed cell death. We demonstrate that necroptosis (RIPK—dependent cell death pathway) is an alternative mechanism responsible for death of bovine granulosa and theca cells. We conclude that both apoptosis and necroptosis occur in the granulosa cells of dominant follicles undergoing luteinisation and in the theca cells from newly selected follicles.     

Zearalenone Induces MLKL-Dependent Necroptosis in Goat Endometrial Stromal Cells via the Calcium Overload/ROS Pathway

Zearalenone (ZEA) is a fungal mycotoxin known to exert strong reproductive toxicity in animals. As a newly identified type of programmed cell death, necroptosis is regulated by receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and mixed-lineage kinase domain-like pseudokinase (MLKL). However, the role and mechanism of necroptosis in ZEA toxicity remain unclear. In this study, we confirmed the involvement of necroptosis in ZEA-induced cell death in goat endometrial stromal cells (gESCs). The release of lactate dehydrogenase (LDH) and the production of PI-positive cells markedly increased. At the same time, the expression of RIPK1 and RIPK3 mRNAs and P-RIPK3 and P-MLKL proteins were significantly upregulated in ZEA-treated gESCs. Importantly, the MLKL inhibitor necrosulfonamide (NSA) dramatically attenuated gESCs necroptosis and powerfully blocked ZEA-induced reactive oxygen species (ROS) generation and mitochondrial dysfunction. The reactive oxygen species (ROS) scavengers and N-acetylcysteine (NAC) inhibited ZEA-induced cell death. In addition, the inhibition of MLKL alleviated the intracellular Ca²⁺ overload caused by ZEA. The calcium chelator BAPTA-AM markedly suppressed ROS production and mitochondrial damage, thus inhibiting ZEA-induced necroptosis. Therefore, our results revealed the mechanism by which ZEA triggers gESCs necroptosis, which may provide a new therapeutic strategy for ZEA poisoning.     

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.     

Necrostatin-1 Attenuates Ischemia Injury Induced Cell Death in Rat Tubular Cell Line NRK-52E through Decreased Drp1 Expression

Necrostatin-1 (Nec-1) inhibits necroptosis and is usually regarded as having no effect on other cell deaths. Here, this study explored whether the addition of Nec-1 has an effect on cell death induced by simulated ischemia injury in rat tubular cell line NRK-52E. In addition, we also investigated the mechanism of Nec-1 attenuates cell death in this renal ischemia model. The NRK-52E cells were incubated with TNF-α + antimycinA (TA) for 24 h with or without Nec-1. Cell death was observed under fluorescent microscope and quantified by flow cytometry. Cell viabilities were detected by MTT assay. The protein expression of dynamin-related protein 1 (Drp1) was detected by Western blotting and immunofluorescence assay. Increased cell death in simulated ischemia injury of NRK-52E cells were markedly attenuated in the Nec-1 pretreated ischemia injury group. Meanwhile, cell viability was significantly improved after using Nec-1. In addition, we also observed that the protein expression of Drp1, a mediator of mitochondrial fission, was significantly increased in simulated ischemia injury group. Increased Drp1 expression in the ischemia injury group can be abolished by Nec-1 or Drp1-knock down, accompanied with decreased cell death and improved cell viabilities. These results suggest that Nec-1 may inhibit cell death induced by simulated ischemia injury in the rat tubular cell line NRK-52E through decreased Drp1 expression.     

Mitochondrial Mechanisms of Necroptosis in Liver Diseases

Cell death represents a basic biological paradigm that governs outcomes and long-term sequelae in almost every hepatic disease. Necroptosis is a common form of programmed cell death in the liver. Necroptosis can be activated by ligands of death receptors, which then interact with receptor-interactive protein kinases 1 (RIPK1). RIPK1 mediates receptor interacting receptor-interactive protein kinases 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) and necrosome formation. Regarding the molecular mechanisms of mitochondrial-mediated necroptosis, the RIPK1/RIPK3/MLKL necrosome complex can enhance oxidative respiration and generate reactive oxygen species, which can be a crucial factor in the susceptibility of cells to necroptosis. The necrosome complex is also linked to mitochondrial components such as phosphoglycerate mutase family member 5 (PGAM5), metabolic enzymes in the mitochondrial matrix, mitochondrial permeability protein, and cyclophilin D. In this review, we focus on the role of mitochondria-mediated cell necroptosis in acute liver injury, chronic liver diseases, and hepatocellular carcinoma, and its possible translation into clinical applications.     

Necroptosis Inhibition by Hydrogen Sulfide Alleviated Hypoxia-Induced Cardiac Fibroblasts Proliferation via Sirtuin 3

Myocardial ischemia or hypoxia can induce myocardial fibroblast proliferation and myocardial fibrosis. Hydrogen sulfide (H₂S) is a gasotransmitter with multiple physiological functions. In our present study, primary cardiac fibroblasts were incubated with H₂S donor sodium hydrosulfide (NaHS, 50 μM) for 4 h followed by hypoxia stimulation (containing 5% CO₂ and 1% O₂) for 4 h. Then, the preventive effects on cardiac fibroblast proliferation and the possible mechanisms were investigated. Our results showed that NaHS reduced the cardiac fibroblast number, decreased the hydroxyproline content; inhibited the EdU positive ratio; and down-regulated the expressions of α-smooth muscle actin (α-SMA), the antigen identified by monoclonal antibody Ki67 (Ki67), proliferating cell nuclear antigen (PCNA), collagen I, and collagen III, suggesting that hypoxia-induced cardiac fibroblasts proliferation was suppressed by NaHS. NaHS improved the mitochondrial membrane potential and attenuated oxidative stress, and inhibited dynamin-related protein 1 (DRP1), but enhanced optic atrophy protein 1 (OPA1) expression. NaHS down-regulated receptor interacting protein kinase 1 (RIPK1) and RIPK3 expression, suggesting that necroptosis was alleviated. NaHS increased the sirtuin 3 (SIRT3) expressions in hypoxia-induced cardiac fibroblasts. Moreover, after SIRT3 siRNA transfection, the inhibitory effects on cardiac fibroblast proliferation, oxidative stress, and necroptosis were weakened. In summary, necroptosis inhibition by exogenous H₂S alleviated hypoxia-induced cardiac fibroblast proliferation via SIRT3.     

RIPK1 and TRADD Regulate TNF-Induced Signaling and Ripoptosome Formation

TNF is a proinflammatory cytokine that is critical for the coordination of tissue homeostasis. RIPK1 and TRADD are the main participants in the transduction of TNF signaling. However, data on the cell fate-controlling functions of both molecules are quite controversial. Here, we address the functions of RIPK1 and TRADD in TNF signaling by generating RIPK1- or TRADD-deficient human cell lines. We demonstrate that RIPK1 is relevant for TNF-induced apoptosis and necroptosis in conditions with depleted IAPs. In addition, TRADD is dispensable for necroptosis but required for apoptosis. We reveal a new possible function of TRADD as a negative regulator of NIK stabilization and subsequent ripoptosome formation. Furthermore, we show that RIPK1 and TRADD do not appear to be essential for the activation of MAPK signaling. Moreover, partially repressing NF-κB activation in both RIPK1 and TRADD KO cells does not result in sensitization to TNF alone due to the absence of NIK stabilization. Importantly, we demonstrate that RIPK1 is essential for preventing TRADD from undergoing TNF-induced ubiquitination and degradation. Taken together, our findings provide further insights into the specific functions of RIPK1 and TRADD in the regulation of TNF-dependent signaling, which controls the balance between cell death and survival.     

The E3 Ubiquitin-Protein Ligase RNF4 Promotes TNF-α-Induced Cell Death Triggered by RIPK1

Receptor-interacting protein kinase 1 (RIPK1) is a key component of the tumor necrosis factor (TNF) receptor signaling complex that regulates both pro- and anti-apoptotic signaling. The reciprocal functions of RIPK1 in TNF signaling are determined by the state of the posttranslational modifications (PTMs) of RIPK1. However, the underlying mechanisms associated with the PTMs of RIPK1 are unclear. In this study, we found that RING finger protein 4 (RNF4), a RING finger E3 ubiquitin ligase, is required for the RIPK1 autophosphorylation and subsequent cell death. It has been reported that RNF4 negatively regulates TNF-α-induced activation of the nuclear factor-κB (NF-κB) through downregulation of transforming growth factor β-activated kinase 1 (TAK1) activity, indicating the possibility that RNF4-mediated TAK1 suppression results in enhanced sensitivity to cell death. However, interestingly, RNF4 was needed to induce RIPK1-mediated cell death even in the absence of TAK1, suggesting that RNF4 can promote RIPK1-mediated cell death without suppressing the TAK1 activity. Thus, these observations reveal the existence of a novel mechanism whereby RNF4 promotes the autophosphorylation of RIPK1, which provides a novel insight into the molecular basis for the PTMs of RIPK1.     

Nec-1 Enhances Shikonin-Induced Apoptosis in Leukemia Cells by Inhibition of RIP-1 and ERK1/2

Necrostatin-1 (Nec-1) inhibits necroptosis by allosterically inhibiting the kinase activity of receptor-interacting protein 1 (RIP1), which plays a critical role in necroptosis. RIP1 is a crucial adaptor kinase involved in the activation of NF-κB, production of reactive oxygen species (ROS) and the phosphorylation of mitogen activated protein kinases (MAPKs). NF-κB, ROS and MAPKs all play important roles in apoptotic signaling. Nec-1 was regarded as having no effect on apoptosis. Here, we report that Nec-1 increased the rate of nuclear condensation and caspases activation induced by a low concentration of shikonin (SHK) in HL60, K562 and primary leukemia cells. siRNA-mediated knockdown of RIP1 significantly enhanced shikonin-induced apoptosis in K562 and HL60 cells. Shikonin treatment alone could slightly inhibit the phosphorylation of ERK1/2 in leukemia cells, and the inhibitory effect on ERK1/2 was significantly augmented by Nec-1. We also found that Nec-1 could inhibit NF-κB p65 translocation to the nucleus at a later stage of SHK treatment. In conclusion, we found that Nec-1 can promote shikonin-induced apoptosis in leukemia cells. The mechanism by which Nec-1 sensitizes shikonin-induced apoptosis appears to be the inhibition of RIP1 kinase-dependent phosphorylation of ERK1/2. To our knowledge, this is the first study to document Nec-1 sensitizes cancer cells to apoptosis.     

BAG3 Attenuates Ischemia-Induced Skeletal Muscle Necroptosis in Diabetic Experimental Peripheral Artery Disease

Peripheral artery disease (PAD) is characterized by impaired blood flow to the lower extremities, resulting in ischemic limb injuries. Individuals with diabetes and PAD typically have more severe ischemic limb injuries and limb amputations, but the mechanisms involved are poorly understood. Previously, we identified BAG3 as a gene within a mouse genetic locus termed limb salvage QTL1 on mouse chromosome 7 that determined the extent of limb necrosis following ischemic injury in C57Bl/6 mice. Whether BAG3 deficiency plays a role in the severe ischemic injury observed in diabetic PAD is not known. In vitro, we found simulated ischemia enhanced BAG3 expression in primary human skeletal muscle cells, whereas BAG3 knockdown increased necroptosis markers and decreased cell viability. In vivo, ischemic skeletal muscles from hind limbs of high-fat diet (HFD)-fed mice showed poor BAG3 expression compared to normal chow diet (NCD)-fed mice, and this was associated with increased limb amputations. BAG3 overexpression in ischemic skeletal muscles from hind limbs of HFD mice rescued limb amputation and improved autophagy, necroptosis, skeletal muscle function and regeneration. Therefore, BAG3 deficiency in ischemic skeletal muscles contributes to the severity of ischemic limb injury in diabetic PAD, likely through autophagy and necroptosis pathways.     

Cyclophilin D Regulates the Nuclear Translocation of AIF,Cardiac Endothelial Cell Necroptosis and Murine Cardiac Transplant Injury

Ischemia-reperfusion injury (IRI) is an inevitable consequence of organ transplant procedure and associated with acute and chronic organ rejection in transplantation. IRI leads to various forms of programmed cell death, which worsens tissue damage and accelerates transplant rejection. We recently demonstrated that necroptosis participates in murine cardiac microvascular endothelial cell (MVEC) death and murine cardiac transplant rejection. However, MVEC death under a more complex IRI model has not been studied. In this study, we found that simulating IRI conditions in vitro by hypoxia, reoxygenation and treatment with inflammatory cytokines induced necroptosis in MVECs. Interestingly, the apoptosis-inducing factor (AIF) translocated to the nucleus during MVEC necroptosis, which is regulated by the mitochondrial permeability molecule cyclophilin D (CypD). Furthermore, CypD deficiency in donor cardiac grafts inhibited AIF translocation and mitigated graft IRI and rejection (n = 7; p = 0.002). Our studies indicate that CypD and AIF play significant roles in MVEC necroptosis and cardiac transplant rejection following IRI. Targeting CypD and its downstream AIF may be a plausible approach to inhibit IRI-caused cardiac damage and improve transplant survival.     

Neuroprotective Effects of Pre-Treament with l-Carnitine and Acetyl-l-Carnitine on Ischemic Injury In Vivo and In Vitro

The therapeutic effect of stroke is hampered by the lack of neuroprotective drugs against ischemic insults beyond the acute phase. Carnitine plays important roles in mitochondrial metabolism and in modulating the ratio of coenzyme A (CoA)/acyl-CoA. Here, we investigate the neuroprotective effects of l-carnitine (LC) and Acetyl-l-carnitine (ALC) pre-treatment on ischemic insults under the same experimental conditions. We used a transient middle cerebral artery occlusion (MCAO) model to evaluate the protective roles of LC and ALC in acute focal cerebral ischemia in vivo and to understand the possible mechanisms using model of PC12 cell cultures in vitro. Results showed that ALC, but not LC, decreased infarction size in SD rats after MCAO in vivo. However, both LC and ALC pretreatment reduced oxygen-glucose deprivation (OGD)-induced cell injury and decreased OGD-induced cell apoptosis and death in vitro; at the same time, both of them increased the activities of super oxide dismutase (SOD) and ATPase, and decreased the concentration of malondialdehyde (MDA) in vitro. Thus, our findings suggested that LC and ALC pre-treatment are highly effective in the prevention of neuronal cell against ischemic injury in vitro, however, only ALC has the protective effect on neuronal cell injury after ischemia in vivo.     

PD-L1 Inhibitors: Different Classes,Activities, and Mechanisms of Action

Targeting the programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) interaction has become an established strategy for cancer immunotherapy. Although hundreds of small-molecule, peptide, and peptidomimetic inhibitors have been proposed in recent years, only a limited number of drug candidates show good PD-1/PD-L1 blocking activity in cell-based assays. In this article, we compare representative molecules from different classes in terms of their PD-1/PD-L1 dissociation capacity measured by HTRF and in vitro bioactivity determined by the immune checkpoint blockade (ICB) co-culture assay. We point to recent discoveries that underscore important differences in the mechanisms of action of these molecules and also indicate one principal feature that needs to be considered, which is the eventual human PD-L1 specificity.     

Ischemia-Reperfusion under Hyperthermia Increases Heme Oxygenase-1 in Pyramidal Neurons and Astrocytes with Accelerating Neuronal Loss in Gerbil Hippocampus

It has been studied that the damage or death of neurons in the hippocampus is different according to hippocampal subregions, cornu ammonis 1–3 (CA1–3), after transient ischemia in the forebrain, showing that pyramidal neurons located in the subfield CA1 (CA1) are most vulnerable to this ischemia. Hyperthermia is a proven risk factor for brain ischemia and can develop more severe and extensive brain damage related with mortality rate. It is well known that heme oxygenase-1 (HO-1) activity and expression is increased by various stimuli in the brain, including hyperthermia. HO-1 can be either protective or deleterious in the central nervous system, and its roles depend on the expression levels of enzymes. In this study, we investigated the effects of hyperthermia during ischemia on HO-1 expression and neuronal damage/death in the hippocampus to examine the relationship between HO-1 and neuronal damage/death following 5-min transient ischemia in the forebrain using gerbils. Gerbils were assigned to four groups: (1) sham-operated gerbils with normothermia (Normo + sham group); (2) ischemia-operated gerbils with normothermia (Normo + ischemia group); (3) sham-operated gerbils with hyperthermia (39.5 ± 0.2 °C) during ischemia (Hyper + sham group); and (4) ischemia-operated gerbils with hyperthermia during ischemia (Hyper + ischemia group). HO-1 expression levels in CA1–3 of the Hyper + ischemia group were significantly higher than those in the Normo + ischemia group. HO-1 immunoreactivity in the Hyper + ischemia group was significantly increased in pyramidal neurons and astrocytes with time after ischemia, and the immunoreactivity was significantly higher than that in the Normo + ischemia group. In the Normo + Ischemia group, neuronal death was shown in pyramidal neurons located only in CA1 at 5 days after ischemia. However, in the Hyper + ischemia group, pyramidal neuronal death occurred in CA1–3 at 2 days after ischemia. Taken together, our findings showed that brain ischemic insult during hyperthermic condition brings up earlier and severer neuronal damage/death in the hippocampus, showing that HO-1 expression in neurons and astrocytes is different according to brain subregions and temperature condition. Based on these findings, we suggest that hyperthermia in patients with ischemic stroke must be taken into the consideration in the therapy.     

Involvement of Glutamate Transporter-1 in Neuroprotection against Global Brain Ischemia-Reperfusion Injury Induced by Postconditioning in Rats

Ischemic postconditioning refers to several transient reperfusion and ischemia cycles after an ischemic event and before a long duration of reperfusion. The procedure produces neuroprotective effects. The mechanisms underlying these neuroprotective effects are poorly understood. In this study, we found that most neurons in the CA1 region died after 10 minutes of ischemia and is followed by 72 hours of reperfusion. However, brain ischemic postconditioning (six cycles of 10 s/10 s reperfusion/re-occlusion) significantly reduced neuronal death. Significant up-regulation of Glutamate transporter-1 was found after 3, 6, 24, 72 hours of reperfusion. The present study showed that ischemic postconditioning decreases cell death and that upregulation of GLT-1 expression may play an important role on this effect.     

Obstructive Sleep Apnea Syndrome In Vitro Model: Controlled Intermittent Hypoxia Stimulation of Human Stem Cells-Derived Cardiomyocytes

Cardiovascular morbidity is the leading cause of death of obstructive sleep apnea (OSA) syndrome patients. Nocturnal airway obstruction is associated with intermittent hypoxia (IH). In our previous work with cell lines, incubation with sera from OSA patients induced changes in cell morphology, NF-κB activation and decreased viability. A decrease in beating rate, contraction amplitude and a reduction in intracellular calcium signaling was also observed in human cardiomyocytes differentiated from human embryonic stem cells (hESC-CMs). We expanded these observations using a new controlled IH in vitro system on beating hESC-CMs. The Oxy-Cycler system was programed to generate IH cycles. Following IH, we detected the activation of Hif-1α as an indicator of hypoxia and nuclear NF-κB p65 and p50 subunits, representing pro-inflammatory activity. We also detected the secretion of inflammatory cytokines, such as MIF, PAI-1, MCP-1 and CXCL1, and demonstrated a decrease in beating rate of hESC-CMs following IH. IH induces the co-activation of inflammatory features together with cardiomyocyte alterations which are consistent with myocardial damage in OSA. This study provides an innovative approach for in vitro studies of OSA cardiovascular morbidity and supports the search for new pharmacological agents and molecular targets to improve diagnosis and treatment of patients.     

In Vivo and In Vitro Evaluation of Urinary Biomarkers in Ischemia/Reperfusion-Induced Kidney Injury

Oxidative stress plays an important role in the pathophysiology of acute kidney injury (AKI). Previously, we reported that vanin-1, which is involved in oxidative stress, is associated with renal tubular injury. This study was aimed to determine whether urinary vanin-1 is a biomarker for the early diagnosis of AKI in two experimental models: in vivo and in vitro. In a rat model of AKI, ischemic AKI was induced in uninephrectomized rats by clamping the left renal artery for 45 min and then reperfusing the kidney. On Day 1 after renal ischemia/reperfusion (I/R), serum creatinine (SCr) in I/R rats was higher than in sham-operated rats, but this did not reach significance. Urinary N-acetyl-β-D-glucosaminidase (NAG) exhibited a significant increase but decreased on Day 2 in I/R rats. In contrast, urinary vanin-1 significantly increased on Day 1 and remained at a significant high level on Day 2 in I/R rats. Renal vanin-1 protein decreased on Days 1 and 3. In line with these findings, immunofluorescence staining demonstrated that vanin-1 was attenuated in the renal proximal tubules of I/R rats. Our in vitro results confirmed that the supernatant from HK-2 cells under hypoxia/reoxygenation included significantly higher levels of vanin-1 as well as KIM-1 and NGAL. In conclusion, our results suggest that urinary vanin-1 might be a potential novel biomarker of AKI induced by I/R.     

Using Small Molecules to Dissect Non‐apoptotic Programmed Cell Death: Necroptosis,Ferroptosis, and Pyroptosis

Genetically programmed cell death is a universal and fundamental cellular process in multicellular organisms. Apoptosis and necroptosis, two common forms of programmed cell death, play vital roles in maintenance of homeostasis in metazoans. Dysfunction of the regulatory machinery of these processes can lead to carcinogenesis or autoimmune diseases. Inappropriate death of essential cells can lead to organ dysfunction or even death; ischemia–reperfusion injury and neurodegenerative disorders are examples of this. Recently, novel forms of non‐apoptotic programmed cell death have been identified. Although these forms of cell death play significant roles in both physiological and pathological conditions, the detailed molecular mechanisms underlying them are still poorly understood. Here, we discuss progress in using small molecules to dissect three forms of non‐apoptotic programmed cell death: necroptosis, ferroptosis, and pyroptosis.