Effect of electroacupuncture on neuronal programmed necrosis by regulating RIP1/RIP3/MLKL pathway in rats with cerebral ischemia reperfusion injury. / 电针调节RIP1/RIP3/MLKLé€šè·¯å¯¹è„‘ç¼ºè¡€å†çŒæ³¨æŸä¼¤å¤§é¼ ç¥žç»å ƒç¨‹åºæ€§åæ­»çš„å½±å“.

OBJECTIVES: To investigate the neuroprotective effect of electroacupuncture (EA) at "Quchi"(LI11) and "Zusanli"(ST36) in the rats with cerebral ischemia reperfusion injury and its influence on programmed necrosis of cerebral cortical neurons. METHODS: Sixty male SD rats were randomly divided into sham-operation group, model group, EA group and inhibitor group, with 15 rats in each group. Left middle cerebral artery occlusion model was established using the modified thread embolism method. In the sham-operation group, the carotid artery was exposed and dissociated in each rat. EA was applied to "Quchi"(LI11) and "Zusanli"(ST36) on the right side for 30 min each time, once daily for 7 days in the rats of the EA group. The rats in the inhibitor group were intraperitoneally injected with norstatin-1 (0.6 mg/kg) for consecutive 7 days. The neurological deficit score of rats in each group was observed. HE staining was adopted to detect the degree of pathological damage of the cerebral cortex in the infarction area. Using TUNEL staining, the apoptosis of cortical neurons in the infarction area was determined；the contents of tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and IL-6 were detected by ELISA；the mRNA and protein expression of the receptor interacting protein-1 (RIP1), the receptor interacting protein-3 (RIP3) and the substrate mixed lineage kinase like protein (MLKL) were detected by fluorescence quantitative PCR and Western blot, respectively. RESULTS: In comparison with the sham-operation group, the neurological deficit score in the model group was higher(P<0.01)；HE staining showed that there was the pathological damage in the infarction area；the neuron apoptosis rate, the contents of TNF-α, IL-1ß and IL-6, and the mRNA and protein expressions of RIP1, RIP3 and MLKL increased(P<0.01) in the model group. In the EA group, the neurological deficit score was reduced(P<0.01)；HE staining showed that the pathological damage was ameliorated in the infarction area；the neuron apoptosis rate, the contents of TNF-α, IL-1ß and IL-6, and the mRNA and protein expressions of RIP1, RIP3, MLKL decreased(P<0.05, P<0.01) when compared with those in the model group. CONCLUSIONS: EA can attenuate cerebral ischemia reperfusion injury and display its neuroprotective effect probably through inhibiting programmed necrosis of cerebral cortical neurons in the rats.

Plasma membrane repair empowers the necrotic survivors as innate immune modulators.

The plasma membrane is crucial to the survival of animal cells, and damage to it can be lethal, often resulting in necrosis. However, cells possess multiple mechanisms for repairing the membrane, which allows them to maintain their integrity to some extent, and sometimes even survive. Interestingly, cells that survive a near-necrosis experience can recognize sub-lethal membrane damage and use it as a signal to secrete chemokines and cytokines, which activate the immune response. This review will present evidence of necrotic cell survival in both in vitro and in vivo systems, including in C. elegans, mouse models, and humans. We will also summarize the various membrane repair mechanisms cells use to maintain membrane integrity. Finally, we will propose a mathematical model to illustrate how near-death experiences can transform dying cells into innate immune modulators for their microenvironment. By utilizing their membrane repair activity, the biological effects of cell death can extend beyond the mere elimination of the cells.

Discriminating Between Apoptosis, Necrosis, Necroptosis, and Ferroptosis by Microscopy and Flow Cytometry.

Apoptosis is a mode of programmed cell death that plays important roles in tissue sculpting during development, in the maintenance of tissue homeostasis in the adult, and in the eradication of injured or infected cells during pathological processes. Numerous physiological as well as pathological stimuli trigger apoptosis, such as engagement of plasma-membrane-associated Fas, TRAIL, or TNF receptors, growth factor deprivation, hypoxia, radiation, and exposure to diverse cytotoxic drugs. Apoptosis is coordinated by members of the caspase family of cysteine proteases, which, upon activation, trigger a series of dramatic morphological and biochemical changes including retraction from the substratum, cell shrinkage, extensive and protracted plasma membrane blebbing, chromatin condensation, DNA hydrolysis, nuclear fragmentation, and proteolytic cleavage of numerous caspase substrates. These dramatic structural and biochemical alterations result not only in the controlled dismantling of the cell, but also in the rapid recognition and removal of apoptotic cells by phagocytes through the cell surface display of phagocytotic triggers such as phosphatidylserine. Necrosis, which is typically nonprogrammed or imposed upon the cell by overwhelming membrane or organelle damage, is characterized by high-amplitude cell swelling, followed by rapid plasma membrane rupture and release of cellular contents into the extracellular space. Necrosis is often provoked by infectious agents or severe departure from physiological conditions due to toxins, temperature extremes, or physical injury. However, forms of programmed necrosis (necroptosis, pyroptosis, ferroptosis) can also occur in specific circumstances. Nonprogrammed and programmed necrosis can be distinguished from apoptosis by morphological features, based on the rapid uptake of vital dyes, and through the application of specific inhibitors of key molecules associated with the latter modes of cell death. This unit describes protocols for the measurement of apoptosis and necrosis and for distinguishing apoptosis from programmed as well as conventional necrosis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Analysis of cell morphology by phase-contrast microscopy Alternative Protocol 1: Assessment of morphological changes using eosin-methylene blue staining Alternative Protocol 2: Analysis of nuclear morphology by fluorescence microscopy Support Protocol: Preparation of cytospins Basic Protocol 2: Measurement of plasma membrane composition with annexin V and propidium iodide Basic Protocol 3: Measurement of DNA fragmentation by flow cytometry Alternative Protocol 3: Analysis of DNA fragmentation by the TUNEL assay Basic Protocol 4: Measurement of caspase activation by flow cytometry Basic Protocol 5: Discriminating between apoptosis, necrosis, necroptosis, and ferroptosis.

Augmented Concentration of Isopentyl-Deoxynyboquinone in Tumors Selectively Kills NAD(P)H Quinone Oxidoreductase 1-Positive Cancer Cells through Programmed Necrotic and Apoptotic Mechanisms.

Lung and breast cancers rank as two of the most common and lethal tumors, accounting for a substantial number of cancer-related deaths worldwide. While the past two decades have witnessed promising progress in tumor therapy, developing targeted tumor therapies continues to pose a significant challenge. NAD(P)H quinone oxidoreductase 1 (NQO1), a two-electron reductase, has been reported as a promising therapeutic target across various solid tumors. ß-Lapachone (ß-Lap) and deoxynyboquinone (DNQ) are two NQO1 bioactivatable drugs that have demonstrated potent antitumor effects. However, their curative efficacy has been constrained by adverse effects and moderate lethality. To enhance the curative potential of NQO1 bioactivatable drugs, we developed a novel DNQ derivative termed isopentyl-deoxynyboquinone (IP-DNQ). Our study revealed that IP-DNQ treatment significantly increased reactive oxygen species generation, leading to double-strand break (DSB) formation, PARP1 hyperactivation, and catastrophic energy loss. Notably, we discovered that this novel drug induced both apoptosis and programmed necrosis events, which makes it entirely distinct from other NQO1 bioactivatable drugs. Furthermore, IP-DNQ monotherapy demonstrated significant antitumor efficacy and extended mice survival in A549 orthotopic xenograft models. Lastly, we identified that in mice IP-DNQ levels were significantly elevated in the plasma and tumor compared with IB-DNQ levels. This study provides novel preclinical evidence supporting IP-DNQ efficacy in NQO1+ NSCLC and breast cancer cells.

Antagonistic effect of selenium on programmed necrosis of testicular Leydig cells caused by cadmium through endoplasmic reticulum stress in chicken.

Cadmium (Cd) is a widely distributed environmental contaminant that is highly toxic to animals and humans. However, detailed reports on Cd-induced programmed necrosis have not been seen in chicken testicular Leydig cells. Selenium (Se) is a trace element in the human body that has cytoprotective effects in a variety of pathological damages caused by heavy metals. This study investigated the potential mechanisms of Cd-induced programmed cell necrosis and the antagonistic effect of Se on Cd toxicity. Chicken testis Leydig cells were divided into six groups, namely, control, Se (5 µmol/L Na2SeO3), Cd (20 µmol/L CdCl2), Se + Cd (5 µmol/L Na2SeO3 and 20 µmol/L CdCl2), 4-phenylbutyric acid (4-PBA) + Cd (10 mmol/L 4-phenylbutyric acid and 20 µmol/L CdCl2), and Necrostatin-1 (Nec-1) + Cd (60 µmol/L Necrostatin-1 and 20 µmol/L CdCl2). The results showed that Cd exposure decreased the activity of CAT, GSH-Px, and SOD and the concentration of GSH, and increased the concentration of MDA and the content of ROS. Relative mRNA and protein expression of GRP78, PERK, ATF6, IRE1, CHOP, and JNK increased in the Cd group, and mRNA and protein expression of TNF-α, TNFR1, RIP1, RIP3, MLKL, and PARP1 significantly increased in the Cd group, while Caspase-8 mRNA and protein expression significantly decreased. The abnormal expression of endoplasmic reticulum stress-related proteins was significantly reduced by 4-PBA pretreatment; the increased expression of TNF-α, TNFR1, RIP1, RIP3, MLKL, and PARP1 caused by Cd toxicity was alleviated; and the expression of caspase-8 was upregulated. Conversely, the increased mRNA and protein expression of endoplasmic reticulum stress marker genes (GRP78, ATF6, PERK, IRE1, CHOP, JNK) caused by Cd was not affected after pretreatment with Nec-1. We also found that these Cd-induced changes were significantly attenuated in the Se + Cd group. We clarified that Cd can cause programmed necrosis of chicken testicular Leydig cells through endoplasmic reticulum stress, and Se can antagonize Cd-induced programmed necrosis of chicken testicular Leydig cells.

LncRNA NR_003508 Suppresses Mycobacterium tuberculosis-Induced Programmed Necrosis via Sponging miR-346-3p to Regulate RIPK1.

Emerging evidence suggests that long non-coding RNAs (LncRNAs) are involved in Mtb-induced programmed necrosis. Among these LncRNAs, LncRNA NR_003508 is associated with LPS-induced acute respiratory distress syndrome. However, whether LncRNA NR_003508 contributes to Mtb-induced programmed necrosis remains undocumented. Firstly, the expression of LncRNA NR_003508 was determined using RT-qPCR and FISH. The protein expression of RIPK1, p-RIPK1, RIPK3, p-RIPK3, MLKL, and p-MLKL was measured by Western blot in RAW264.7 and mouse lung tissues. Furthermore, luciferase reporter assays and bioinformatics were used to predict specific miRNA (miR-346-3p) and mRNA (RIPK1) regulated by LncRNA NR_003508. In addition, RT-qPCR was used to detect the RIPK1 expression in TB patients and healthy peripheral blood. The flow cytometry assay was performed to detect cell necrosis rates. Here we show that BCG infection-induced cell necrosis and increased LncRNA NR_003508 expression. si-NR_003508 inhibited BCG/H37Rv-induced programmed necrosis in vitro or in vivo. Functionally, LncRNA NR_003508 has been verified as a ceRNA for absorbing miR-346-3p, which targets RIPK1. Moreover, RIPK1 expression was elevated in the peripheral blood of TB patients compared with healthy people. Knockdown of LncRNA NR_003508 or miR-346-3p overexpression suppresses cell necrosis rate and ROS accumulation in RAW264.7 cells. In conclusion, LncRNA NR_003508 functions as a positive regulator of Mtb-induced programmed necrosis via sponging miR-346-3p to regulate RIPK1. Our findings may provide a promising therapeutic target for tuberculosis.

The web of death: the expanding complexity of necroptotic signaling.

The past decade has seen the emergence of the necroptosis programmed cell death pathway as an important contributor to the pathophysiology of myriad diseases. The receptor interacting protein kinase (RIPK)1 and RIPK3, and the pseudokinase executioner protein, mixed lineage kinase domain-like (MLKL), have grown to prominence as the core pathway components. Depending on cellular context, these proteins also serve as integrators of signals, such as post-translational modifications and protein or metabolite interactions, adding layers of complexity to pathway regulation. Here, we describe the emerging picture of the web of proteins that tune necroptotic signal transduction and how these events have diverged across species, presumably owing to selective pressures of pathogens upon the RIPK3-MLKL protein pair.

Selenium alleviates cadmium-induced oxidative stress, endoplasmic reticulum stress and programmed necrosis in chicken testes.

Cadmium (Cd) is a common heavy metal pollutant, and one of the important target organs of its toxicity is the testis. Selenium (Se) has the ability to antagonize the toxicity of Cd. However, the mechanism of the alleviating effects of Se on Cd in chicken testis injury through oxidative stress, endoplasmic reticulum stress (ERS), and programmed necrosis remained unclear. To explore this, 80 7-day-old chickens were divided into the Control group, the Se group (1.00 mg/kg Se), the Cd group (150.00 mg/kg Cd), and the CdSe group. On the 30th and 60th days, serum and chicken testis tissue samples were collected for testing. The results showed that Cd exposure resulted in swelling and deformation of seminiferous tubules, and thinning of the seminiferous epithelium. The ROS and MDA increased, and the SOD, CAT, GSH, GSH-Px decreased. The expression of GRP78, PERK, IRE1, ATF6, CHOP, and JNK in the Cd group increased. The expression of TNF-α, TNFR1, RIP1, RIP3, MLKL, and PARP1 increased, while the expression of Caspase-8 decreased. Histopathological changes, oxidative stress, ERS, and programmed necrosis were improved after CdSe treatment. In conclusion, Se antagonized the toxicity of Cd, and Se could alleviate Cd-induced oxidative stress, ERS, and programmed necrosis in chicken testis.

[Hsp90 participates in the necroptosis of mouse neural cells induced by aluminum through the RIP1/RIP3/MLKL pathway].

Objective: To investigate whether heat shock protein 90 (HSP90) participates in the necroptosis of C57BL/6 mouse neurons and spatial memory impairment induced by Aluminum maltol [Al (mal) (3)] through RIP1/RIP3/MLKL pathway. Methods: In March 2022, Thirty-two C57 mice were randomly divided into control group, Low dose group, a medium dose group, and a high-dose group, with 8 mice in each group, and injected intraperitoneally with physiological saline, 20, 40, and 80, respectivelyµmol/kgAl (mal) (3) was administered, it was injected 5 days a week and discontinued 2 days for 60 days. Morris water maze test was used to test the spatial learning and memory ability of mice. Nissl staining was used to observe the pathological changes of brain tissue. The protein expression levels of RIP1, RIP3, MLKL and HSP90 in hippocampus were determined by Western blotting. Results: In the water maze experiment, compared with the control group, the number of mice crossing the platform decreased in each dose group, the difference was statistically significant (H=9.50, P=0.023), and the number of mice crossing the platform was statistically significant among each dose group (P <0.05). Compared with the control group, the number of hippocampal nerve cells in each dose group decreased, the arrangement was disordered, and the Nissellite bodies decreased. Western blotting results showed that compared with the control group, the expression level of RIP1 protein in the hippocampus of mice in high-dose group was higher, and the difference was statistically significant (P <0.05). The expression levels of RIP3, MLKL and HSP90 in hippocampal tissue of mice in medium and high dose groups were increased, and the differences were statistically significant (P<0.05). After siRNA intervention decreased the expression of HSP90 protein, the expressions of HSP90, RIP1, RIP3 and MLKL in Al (mal) (3) groups were increased, and the differences were statistically significant (P<0.05) . Conclusion: Through RIP1/RIP3/MLKL pathway, HSP90 is involved in neuronal programmed necrosis and spatial memory impairment induced by maltol aluminum in C57 mice.

Advances in RIPK1 kinase inhibitors.

Programmed necrosis is a new modulated cell death mode with necrotizing morphological characteristics. Receptor interacting protein 1 (RIPK1) is a critical mediator of the programmed necrosis pathway that is involved in stroke, myocardial infarction, fatal systemic inflammatory response syndrome, Alzheimer's disease, and malignancy. At present, the reported inhibitors are divided into four categories. The first category is the type I ATP-competitive kinase inhibitors that targets the area occupied by the ATP adenylate ring; The second category is type Ⅱ ATP competitive kinase inhibitors targeting the DLG-out conformation of RIPK1; The third category is type Ⅲ kinase inhibitors that compete for binding to allosteric sites near ATP pockets; The last category is others. This paper reviews the structure, biological function, and recent research progress of receptor interaction protein-1 kinase inhibitors.

Paraquat induces apoptosis, programmed necrosis, and immune dysfunction in CIK cells via the PTEN/PI3K/AKT axis.

Paraquat (PQ) is a highly water-soluble, non-selective herbicide. Due to water pollution and lack of specific medicines, it is extremely harmful to humans and aquatic animals. Oxidative stress and apoptosis can affect the immune function of the body. However, the effects and mechanisms of PQ on the immune function, apoptosis and programmed necrosis on CIK cells are still unclear. Therefore, we constructed low (L, 50 µmol/L), medium (M, 100 µmol/L), and high (H, 150 µmol/L) dose models of PQ exposure on CIK cells. The expression of oxidative stress-related indexes (MDA, CAT, GSH-Px and SOD) and interrelated genes were examined by flow cytometry, qRT-PCR, and western blotting methods. Our data demonstrated that PQ treatment caused an increase in MDA content and the decreases in the activities of antioxidase and antioxidants (SOD, GSH-Px and CAT) on CIK cells (p < 0.05). We also discovered the PTEN/PI3K/AKT pathway was significantly activated in a dose dependent manner (p < 0.05). Furthermore, the proportion of programmed necrosis cells increased dramatically at PQ doses from 0 µmol/L to 150 µmol/L. Apoptosis and necrosis-related genes also showed dose-dependent changes (p < 0.05). Briefly, PQ exposure leads to apoptosis and programmed necrosis via the oxidative stress and PTEN/PI3K/AKT pathway, thereby causing immune dysfunction of CIK cells. This study enriches the toxic influences of PQ on the cells of aquatic organisms and provides a reference for comparative medicine.

DNA damage and reticular stress in cytotoxicity and oncotic cell death of MCF-7 cells treated with fluopsin C.

Fluopsin C is an antibiotic compound derived from secondary metabolism of different microorganisms, which possesses antitumor, antibacterial, and antifungal activity. Related to fluopsin C antiproliferative activity, the aim of this study was to examine the following parameters: cytotoxicity, genotoxicity, cell cycle arrest, cell death induction (apoptosis), mitochondrial membrane potential (MMP), colony formation, and mRNA expression of genes involved in adaptive stress responses and cellular death utilizing a monolayer. In addition, a three-dimensional cell culture was used to evaluate the effects on growth of tumor spheroids. Fluopsin C was cytotoxic (1) producing cell division arrest in the G1 phase, (2) elevating expression of mRNA of the CDKN1A gene and (3) decrease in expression of mRNA H2AFX gene. Further, fluopsin C enhanced DNA damage as evidenced by increased expression of mRNA of GADD45A and GPX1 genes, indicating that reactive oxygen species (ROS) may be involved in the observed genotoxic response. Reticulum stress was also detected as noted from activation of the ribonuclease inositol-requiring protein 1 (IRE1) pathway, since a rise in mRNA expression of the ERN1 and TRAF2 genes was observed. During the cell death process, an increase in mRNA expression of the BBC3 gene was noted, indicating participation of this antibiotic in oncotic (ischemic) cell death. Data thus demonstrated for the first time that fluopsin C interferes with the volume of tumor spheroids, in order to attenuate their growth. Our findings show that fluopsin C modulates essential molecular processes in response to stress and cell death.

TLR3 deletion inhibits programmed necrosis of brain cells in neonatal mice with sevoflurane-induced cognitive dysfunction.

This research aimed to explore the influence of TLR deletion on sevoflurane-induced postoperative cognitive dysfunction in neonatal mice. Herein, WT and TLR3 KO neonatal mice, each with 24, were randomly divided into control group, sevoflurane group, and TLR3-/-+sevoflurane group. The hippocampal neurons of WT, TLR3 KO and RIP3 KO neonatal mice in C group, SEV group, TLR3-/-+SEV group and RIP3-/-+SEV group were extracted for in vitro experiments. The results revealed the degeneration and necrosis of nerve cells in SEV group. Microscopic findings indicated that nerve cells showed shrinkage and nuclear hyperchromatism, along with lessening or even disappearance of nuclei and enlargement of cell spaces, and apoptotic cells in the brain tissues were evidently increased. Compared with SEV group, TLR3-/-+SEV group displayed reductions in these phenomena. Additionally, SEV group showed the reduced SHP2 expression and the increased expressions of proteins associated with TLR signaling pathway and apoptosis. Furthermore, there were no obvious differences in the expressions of such proteins in hippocampal neurons between RIP3-/-+SEV and TLR3-/-+SEV groups. The results confirmed that inhibiting RIP3 phosphorylation and suppressing TLR3 expressions exerted the same influence on the expressions of these proteins in the hippocampus of neonatal mice with sevoflurane-induced cognitive dysfunction. Based on these, it is speculated that TLR3 influences neonatal mice with sevoflurane-induced cognitive dysfunction probably by regulating RIP3 phosphorylation.

[Programmed Necrosis Inducers for Cancer Treatment].

Programmed necrosis,a mode of cell death independent of Caspase,is mainly mediated by receptor-interacting protein kinase-1 (RIPK1),receptor-interacting protein kinase-3 (RIPK3),and mixed lineage kinase domain-like protein (MLKL).Studies have demonstrated that programmed necrosis has the dual role of promoting and inhibiting tumor growth and thus we can control the development of tumor by regulating programmed necrosis.The drugs capable of inducing programmed necrosis show potential anti-tumor activity.In addition,inducing programmed necrosis is an effective way to overcome tumor resistance to apoptosis.This paper summarized the mechanisms of programmed necrosis and its relationship with tumors.We focused on the antitumor activity of programmed necrosis inducers including natural products,chemotherapeutic drugs,death receptor ligands,kinase inhibitors,inorganic salts,metal complexes,and metal nanoparticles.These agents will provide new therapeutic candidates for the treatment of tumors,especially the tumors acquiring resistance to apoptosis.

PM2.5-Induced Programmed Myocardial Cell Death via mPTP Opening Results in Deteriorated Cardiac Function in HFpEF Mice.

PM2.5 exposure can induce or exacerbate heart failure and is associated with an increased risk of heart failure hospitalization and mortality; however, the underlying mechanisms remain unclear. This study focuses on the potential mechanisms underlying PM2.5 induction of cardiomyocyte programmed necrosis as well as its promotion of cardiac function impairment in a mouse model of heart failure with preserved ejection fraction (HFpEF). HFpEF mice were exposed to concentrated ambient PM2.5 (CAP) (CAP group) or filtered air (FA) (FA group) for 6 weeks. Changes in myocardial pathology and cardiac function were documented for comparisons between the two groups. In vitro experiments were performed to measure oxidative stress and mitochondrial permeability transition pore (mPTP) dynamics in H9C2 cells following 24 h exposure to PM2.5. Additionally, co-immunoprecipitation was conducted to detect p53 and cyclophilin D (CypD) interactions. The results showed exposure to CAP promoted cardiac function impairment in HFpEF mice. Myocardial pathology analysis and in vitro experiments demonstrated that PM2.5 led to mitochondrial damage in cardiomyocytes and, eventually, their necrosis. Moreover, our experiments also suggested that PM2.5 increases mitochondrial reactive oxygen species (ROS), induces DNA oxidative damage, and decreases the inner mitochondrial membrane potential (ΔΨm). This indicates the presence of mPTP opening. Co-immunoprecipitation results showed a p53/CypD interaction in the myocardial tissue of HFpEF mice in the CAP group. Inhibition of CypD by cyclosporin A was found to reverse PM2.5-induced mPTP opening and H9C2 cell death. In conclusion, PM2.5 induces mPTP opening to stimulate mitochondria-mediated programmed necrosis of cardiomyocytes, and it might exacerbate cardiac function impairment in HFpEF mice.

Protective effect of C-phycocyanin and apo-phycocyanin subunit on programmed necrosis of GC-1 spg cells induced by H2 O2.

C-phycocyanin (C-PC) is an effective antioxidant and has an important value in medical research. Oxidative stress is considered to be one of the main underlying mechanisms of cell death, and reducing oxidative stress is one of the strategies to enhance germ cell viability. Herein, we investigated the protective effect and the mechanism of C-PC and apo-phycocyanin subunit on oxidative stress damage induced by H2 O2 in GC-1 spg cells. C-PC genes were cloned into the pGEX-4T-1 vectorand transformed into Escherichia coli BL21 to achieve the efficient expression of C-PC subunit. GC-1 spg cells were treated with 600 µM H2 O2 for 24 h to establish the oxidative stress damage model. Cell viability was detected by CCK-8. The degree of oxidative stress was detected by testing Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and glutathione (GSH) and Malondialdehyde (MDA) levels. Reactive oxygen species (ROS) was evaluated utilizingby 2', 7'-dichlorofluorescent-diacetate (DCFH-DA). Mitochondrial membrane potential was determined by JC-1. Cell necrosis rate was detected by Annexin V-FITC/PI. Expression of protein was detected by western blot. We found that C-PC and GST-CPC ß significantly inhibited H2 O2 -induced oxidative damage of GC-1 spg cells, improved the ability of antioxidation, reduced ROS overproduction, and mitochondrial membrane potential loss, and inhibited the RIP-1/RIP-3/ p-MLKL signaling pathway to reduce the necrosis rate. The results demonstrated that C-PC played a protective role against H2 O2 -induced cell damage, especially its ß subunit. This study provides a theoretical basis for C-PC as a potential protective agent of reproductive system.

COL-3-Induced Molecular and Ultrastructural Alterations in K562 Cells.

Tetracycline-3 (4-dedimethylamino sancycline, COL-3) is a non-antibiotic tetracycline derivative. COL-3 exerts potent anti-metalloproteinase activity and its antitumor effects have been reported both in vitro and in vivo. In this study, we investigated the mechanisms of COL-3-induced cytotoxicity in a chronic myeloid leukemia cell line, K562, characterized by the BCR-ABL fusion protein. COL-3 induced K562 cell death in a concentration-dependent manner with an IC50 of 10.8 µg/mL and exhibited features of both apoptosis and necrosis. However, flow cytometry analysis revealed that necrotic cells dominated over the early and late apoptotic cells upon treatment with COL-3. Transmission electron microscopy analysis in combination with Western blotting (WB) analysis revealed early mitochondrial swelling accompanied by the early release of cytochrome c and truncated apoptosis inducing factor (tAIF). In addition, ultrastructural changes were detected in the endoplasmic reticulum (ER). COL-3 affected the levels of glucose-regulated protein-94 (GRP94) and resulted in m-calpain activation. DNA double strand breaks as a signature for DNA damage was also confirmed using an antibody against γH2AX. WB analyses did not demonstrate caspase activation, while Bcl-xL protein remained unaffected. In conclusion, COL-3-induced cell death involves DNA damage as well as mitochondrial and ER perturbation with features of paraptosis and programmed necrosis.

Erratum: Advances in RIPK1 kinase inhibitors.

[This corrects the article DOI: 10.3389/fphar.2022.976435.].

Programmed Necrosis Inducers for Cancer Treatment / 中国医学科学院学报

Programmed necrosis,a mode of cell death independent of Caspase,is mainly mediated by receptor-interacting protein kinase-1 (RIPK1),receptor-interacting protein kinase-3 (RIPK3),and mixed lineage kinase domain-like protein (MLKL).Studies have demonstrated that programmed necrosis has the dual role of promoting and inhibiting tumor growth and thus we can control the development of tumor by regulating programmed necrosis.The drugs capable of inducing programmed necrosis show potential anti-tumor activity.In addition,inducing programmed necrosis is an effective way to overcome tumor resistance to apoptosis.This paper summarized the mechanisms of programmed necrosis and its relationship with tumors.We focused on the antitumor activity of programmed necrosis inducers including natural products,chemotherapeutic drugs,death receptor ligands,kinase inhibitors,inorganic salts,metal complexes,and metal nanoparticles.These agents will provide new therapeutic candidates for the treatment of tumors,especially the tumors acquiring resistance to apoptosis.

[RIP1/RIP3-MLKL signaling pathway correlates with occurrence, progression and prognosis of chronic heart failure].

OBJECTIVE: To detect plasma levels of receptor-interacting protein kinase 1 (RIP1), RIP3 and mixed lineage kinase domain-like protein (MLKL) in patients with chronic heart failure and explore the expression pattern of programmed necrosis signaling pathway RIP1/RIP3-MLKL in the progression of heart failure. METHODS: The patients with chronic heart failure (NYHA class Ⅱ-Ⅳ) admitted in our hospital between February, 2020 and March, 2021 were prospectively enrolled in this study, with 21 healthy volunteers as the control group. The enrolled patients included 20 with grade Ⅱ, 33 with grade Ⅲ, and 43 with grade Ⅳ cardiac function. Fasting venous blood was collected from all the participants for detecting plasma levels of RIP1, RIP3, and MLKL and protein expressions of RIP1/RIP3-MLKL pathway using enzyme-linked immunosorbent assay (ELISA) and Western blotting. The patients with grade Ⅳ cardiac function were followed up for 5 months to evaluate the clinical prognostic indicators. RESULTS: Compared with the healthy volunteers, the patients with grade Ⅱ, Ⅲ and Ⅳ cardiac function had significantly increased plasma levels of RIP1, RIP3, and MLKL (P < 0.01), and their levels were significantly higher in grade Ⅲ/Ⅳ patients than in those with grade Ⅱ cardiac function (P < 0.01); the plasma levels of RIP1 and MLKL were significantly higher in grade Ⅳ patients than in grade Ⅲ patients (P < 0.05). The results of Western blotting also showed increased expressions of the proteins in the RIP1/RIP3-MLKL pathway in patients with heart failure. Pearson correlation analysis suggested that in patients with heart failure, the expression levels of RIP1, RIP3, and MLKL were positively correlated with SCR, AST, LVEDD and NT-proBNP (P < 0.05). Follow-up study of the patients with grade Ⅳ cardiac function showed that higher expression levels of RIP1/RIP3-MLKL were associated with a poorer prognosis of the patients. CONCLUSION: The expressions of RIP1, RIP3 and MLKL are significantly upregulated in patients with heart failure in positive correlation with the severity of the disease condition, and the activation of the RIP1/RIP3-MLKL signaling pathway may contribute to the occurrence, development and prognosis of chronic heart failure.