OSW-1 triggers necroptosis in colorectal cancer cells through the RIPK1/RIPK3/MLKL signaling pathway facilitated by the RIPK1-p62/SQSTM1 complex.

BACKGROUND: Necroptosis has emerged as a novel molecular pathway that can be targeted by chemotherapy agents in the treatment of cancer. OSW-1, which is derived from the bulbs of Ornithogalum saundersiae Baker, exerts a wide range of pharmacological effects. AIM: To explore whether OSW-1 can induce necroptosis in colorectal cancer (CRC) cells, thereby expanding its range of clinical applications. METHODS: We performed a sequence of functional experiments, including Cell Counting Kit-8 assays and flow cytometry analysis, to assess the inhibitory effect of OSW-1 on CRC cells. We utilized quantitative proteomics, employing tandem mass tag labeling combined with liquid chromatography-tandem mass spectrometry, to analyze changes in protein expression. Subsequent bioinformatic analysis was conducted to elucidate the biological processes associated with the identified proteins. Transmission electron microscopy (TEM) and immunofluorescence studies were also performed to examine the effects of OSW-1 on necroptosis. Finally, western blotting, siRNA experiments, and immunoprecipitation were employed to evaluate protein interactions within CRC cells. RESULTS: The results revealed that OSW-1 exerted a strong inhibitory effect on CRC cells, and this effect was accompanied by a necroptosis-like morphology that was observable via TEM. OSW-1 was shown to trigger necroptosis via activation of the RIPK1/RIPK3/MLKL pathway. Furthermore, the accumulation of p62/SQSTM1 was shown to mediate OSW-1-induced necroptosis through its interaction with RIPK1. CONCLUSION: We propose that OSW-1 can induce necroptosis through the RIPK1/RIPK3/MLKL signaling pathway, and that this effect is mediated by the RIPK1-p62/SQSTM1 complex, in CRC cells. These results provide a theoretical foundation for the use of OSW-1 in the clinical treatment of CRC.

Involvement of necroptotic cell death in macrophages in progression of bleomycin and LPS-induced acute exacerbation of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the severe form of interstitial pneumonias. Acute exacerbation (AE) of IPF is characterized by progressive lung fibrosis with the irreversible lung function decline and inflammation, and is often fatal with poor prognosis. However, the physiological and molecular mechanisms in AE of IPF are still not fully understood. In this study, we investigated the mechanism underlying AE of IPF, using bleomycin (BLM) and lipopolysaccharide (LPS) (BLM + LPS)-treated mice. The mice were treated with a single dose of 1.5 mg/kg BLM (on day 0) and/or 0.5 mg/kg LPS (on day 14), and maintained for another 7 days (total 21 days). Administration of BLM + LPS more severely aggravated the respiratory function, fibrosis, and inflammation in the lungs, together with the elevated interleukin-6 level in bronchoalveolar lavage fluid, than the control or BLM alone-treated mice. Moreover, the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay demonstrated that subsequent treatment with LPS elevated cell death in the lungs of BLM-administered mice. Furthermore, the expression levels of mixed lineage kinase domain-like protein (MLKL), a marker of necroptotic cell death, and CD68-positive macrophages were increased, and most of them were co-stained in the lungs of BLM + LPS-treated mice. These results, taken together, indicate that BLM + LPS treatment showed more exacerbated the respiratory function with extensive fibrosis and inflammation than treatment with BLM alone in mice. Fibrosis and inflammation in AE of IPF seen in BLM + LPS-administered mice included an increase in macrophages and their necroptotic cell death.

Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

Elevated intracellular Ca2+ functions downstream of mitodysfunction to induce Wallerian-like degeneration and necroptosis in organophosphorus-induced delayed neuropathy.

Neurotoxic organophosphorus compounds can induce a type of delayed neuropathy in humans and sensitive animals, known as organophosphorus-induced delayed neuropathy (OPIDN). OPIDN is characterized by axonal degeneration akin to Wallerian-like degeneration, which is thought to be caused by increased intra-axonal Ca2+ concentrations. This study was designed to investigate that deregulated cytosolic Ca2+ may function downstream of mitodysfunction in activating Wallerian-like degeneration and necroptosis in OPIDN. Adult hens were administrated a single dosage of 750 mg/kg tri-ortho-cresyl phosphate (TOCP), and then sacrificed at 1 day, 5 day, 10 day and 21 day post-exposure, respectively. Sciatic nerves and spinal cords were examined for pathological changes and proteins expression related to Wallerian-like degeneration and necroptosis. In vitro experiments using differentiated neuro-2a (N2a) cells were conducted to investigate the relationship among mitochondrial dysfunction, Ca2+ influx, axonal degeneration, and necroptosis. The cells were co-administered with the Ca2+-chelator BAPTA-AM, the TRPA1 channel inhibitor HC030031, the RIPK1 inhibitor Necrostatin-1, and the mitochondrial-targeted antioxidant MitoQ along with TOCP. Results demonstrated an increase in cytosolic calcium concentration and key proteins associated with Wallerian degeneration and necroptosis in both in vivo and in vitro models after TOCP exposure. Moreover, co-administration with BATPA-AM or HC030031 significantly attenuated the loss of NMNAT2 and STMN2 in N2a cells, as well as the upregulation of SARM1, RIPK1 and p-MLKL. In contrast, Necrostatin-1 treatment only inhibited the TOCP-induced elevation of p-MLKL. Notably, pharmacological protection of mitochondrial function with MitoQ effectively alleviated the increase in intracellular Ca2+ following TOCP and mitigated axonal degeneration and necroptosis in N2a cells, supporting mitochondrial dysfunction as an upstream event of the intracellular Ca2+ imbalance and neuronal damage in OPIDN. These findings suggest that mitochondrial dysfunction post-TOCP intoxication leads to an elevated intracellular Ca2+ concentration, which plays a pivotal role in the initiation and development of OPIDN through inducing SARM1-mediated axonal degeneration and activating the necroptotic signaling pathway.

Hydrogen sulfide regulates macrophage polarization and necroptosis to accelerate diabetic skin wound healing.

Hydrogen sulfide (H2S), recognized as the third gasotransmitter, plays a pivotal role in the pathophysiological processes of various diseases. Cystathionine γ-lyase (CSE) is the main enzyme for H2S production in the skin. However, effects and mechanisms of H2S in diabetic skin wound healing remain unclear. Our findings revealed a decrease in plasma H2S content in diabetic patients with skin wounds. CSE knockout (KO) diabetic mice resulted in delayed wound healing, reduced blood perfusion, and CD31 expression around the wounds. It also led to increased infiltration of inflammatory cells and M1-type macrophages, decreased collagen levels, α-smooth muscle actin (α-SMA), and proliferating cell nuclear antigen (PCNA) expression. Additionally, there were enhanced expressions of necroptosis related proteins, including receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain like protein (MLKL). In comparison, sodium hydrosulfide (NaHS), H2S donor, accelerated skin wound healing in leptin receptor deficiency (db/db) mice. This acceleration was accompanied by increased blood perfusion and CD31 expression, reduced infiltration of inflammatory cells and M1-type macrophages, elevated collagen levels, α-SMA, and PCNA expressions, and decreased necroptosis-related protein expressions together with nuclear factor-κB (NF-κB) p65 phosphorylation. In conclusion, H2S regulates macrophage polarization and necroptosis, contributing to the acceleration of diabetic skin wound healing. These findings offer a novel strategy for the treatment of diabetic skin wounds.

Inhibition of mitochondrial ROS-mediated necroptosis by Dendrobium nobile Lindl. alkaloids in carbon tetrachloride induced acute liver injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Dendrobium nobile Lindl. (DNL) is a well-known traditional Chinese medicine that has been recorded in the Chinese Pharmacopoeia (2020 edition). The previous data showed that Dendrobium nobile Lindl. alkaloids (DNLA) protect against CCl4-induced liver damage via oxidative stress reduction and mitochondrial function improvement, yet the exact regulatory signaling pathways remain undefined. AIM OF THE STUDY: The aim of the present study was to investigate the role of necroptosis in the mode of CCl4-induced liver injury and determine whether DNLA protects against CCl4-induced acute liver injury (ALI) by inhibiting mitochondrial ROS (mtROS)-mediated necroptosis. MATERIALS AND METHODS: DNLA was extracted from DNL, and the content was determined using liquid chromatograph mass spectrometer (LC-MS). In vivo experiments were conducted in C57BL/6J mice. Animals were administrated with DNLA (20 mg/kg/day, ig) for 7 days, and then challenged with CCl4 (20 µL/kg, ip). CCl4-induced liver injury in mice was evaluated through the assessment of biochemical indicators in mouse serum and histopathological examination of hepatic tissue using hematoxylin and eosin (H&E) staining. The protein and gene expressions were determined with western blotting and quantitative real-time PCR (RT-qPCR). Reactive oxygen species (ROS) production was detected using the fluorescent probe DCFH-DA, and mitochondrial membrane potential was evaluated using a fluorescent probe JC-1. The mtROS level was assessed using a fluorescence probe MitoSOX. RESULTS: DNLA lessened CCl4-induced liver injury, evident by reduced AST and ALT levels and improved liver pathology. DNLA suppressed necroptosis by decreasing RIPK1, RIPK3, and MLKL phosphorylation, concurrently enhancing mitochondrial function. It also broke the positive feedback loop between mtROS and RIPK1/RIPK3/MLKL activation. Similar findings were observed with resveratrol and mitochondrial SOD2 overexpression, both mitigating mtROS and necroptosis. Further mechanistic studies found that DNLA inhibited the oxidation of RIPK1 and reduced its phosphorylation level, whereby lowering the phosphorylation of RIPK3 and MLKL, blocking necroptosis, and alleviating liver injury. CONCLUSIONS: This study demonstrates that DNLA inhibits the necroptosis signaling pathway by reducing mtROS mediated oxidation of RIPK1, thereby reducing the phosphorylation of RIPK1, RIPK3, and MLKL, and protecting against liver injury.

The cGAS/STING signaling pathway is involved in sevoflurane induced neuronal necroptosis via regulating microglia M1 polarization.

OBJECTIVE: The specific mechanisms of sevoflurane-induced neurotoxicity are still undetermined. The aim of the current study was to investigate the role of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway in sevoflurane-induced neuronal necroptosis. METHODS: BV2 microglial cells were divided into a control group and a 4% sevoflurane exposure group. Western blotting was used to detect expression of the M1 polarization marker inducible nitric oxide synthase (iNOS). RNA was collected for RNA sequencing analysis. After STING knockdown in microglia, western blotting was performed to examine expression of the pro-inflammatory markers CD16 and CD32. The tumor necrosis factor-α (TNF-α) level in media was detected using an enzyme-linked immunosorbent assay. BV2 microglia conditioned media was collected to incubate HT22 neuronal cells, and their cell activity was measured using a CCK8 assay. Calcium was observed by fluorescence. Western blotting was performed to evaluate receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) expression. Neuronal necroptosis rate were detected using flow cytometry. RESULTS: Sevoflurane exposure promoted microglial M1 polarization. The cGAS/STING pathway was screened and identified by RNA sequencing analysis of sevoflurane-exposed microglia and the control group. Compared with the control group, STING knockdown in microglia rescued the amoeboid morphology, inhibited TNF-α release, and significantly decreased iNOS, CD16, and CD32 expression. Moreover, calcium ions and necroptosis within neurons were decreased, and RIPK1, RIPK3, and p-MLKL expression was markedly decreased in microglia media culture with STING knockdown. CONCLUSION: These results suggest that sevoflurane can regulate microglial M1 polarization by activating the cGAS/STING signaling pathway and increasing immune factor release, thus accelerating the neuronal necroptosis induced by calcium overload.

Selected Flavonols Targeting Cell Death Pathways in Cancer Therapy: The Latest Achievements in Research on Apoptosis, Autophagy, Necroptosis, Pyroptosis, Ferroptosis, and Cuproptosis.

The complex and multi-stage processes of carcinogenesis are accompanied by a number of phenomena related to the potential involvement of various chemopreventive factors, which include, among others, compounds of natural origin such as flavonols. The use of flavonols is not only promising but also a recognized strategy for cancer treatment. The chemopreventive impact of flavonols on cancer arises from their ability to act as antioxidants, impede proliferation, promote cell death, inhibit angiogenesis, and regulate the immune system through involvement in diverse forms of cellular death. So far, the molecular mechanisms underlying the regulation of apoptosis, autophagy, necroptosis, pyroptosis, ferroptosis, and cuproptosis occurring with the participation of flavonols have remained incompletely elucidated, and the results of the studies carried out so far are ambiguous. For this reason, one of the therapeutic goals is to initiate the death of altered cells through the use of quercetin, kaempferol, myricetin, isorhamnetin, galangin, fisetin, and morin. This article offers an extensive overview of recent research on these compounds, focusing particularly on their role in combating cancer and elucidating the molecular mechanisms governing apoptosis, autophagy, necroptosis, pyroptosis, ferroptosis, and cuproptosis. Assessment of the mechanisms underlying the anticancer effects of compounds in therapy targeting various types of cell death pathways may prove useful in developing new therapeutic regimens and counteracting resistance to previously used treatments.

Itaconate inhibits corticosterone-induced necroptosis and neuroinflammation via up-regulating menin in HT22 cells.

Corticosterone (CORT) damages hippocampal neurons as well as induces neuroinflammation. The tricarboxylic acid cycle metabolite itaconate has an anti-inflammatory role. Necroptosis is a form of programmed cell death, also known as inflammatory cell death. Menin is a multifunctional scaffold protein, which deficiency aggravates neuroinflammation. In this study, we explored whether itaconate inhibits CORT-induced neuroinflammation as well as necroptosis and further investigated the mediatory role of Menin in this protective effect of itaconate by using an exposure of CORT to HT22 cells (a hippocampal neuronal cell line). The viability of HT22 cells was examined by the cell counting kit 8 (CCK-8). The morphology of HT22 cells was observed by transmission electron microscope (TEM). The expressions of necroptosis-related proteins (p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL) were evaluated by western blotting. The contents of inflammatory factors were detected by an enzyme-linked immunosorbent assay (ELISA) kit. Our results showed that CORT increases the contents of pro-inflammatory factors (IL-1ß, TNF-α) as well as decreases the contents of anti-inflammatory factors (IL-4, IL-10) in HT22 cells. We also found that CORT increases the expressions of necroptosis-related proteins (p-RIP1/RIP1, p-RIP3/RIP3, and p-MLKL/MLKL) and decreases the cell viability in HT22 cells, indicating that CORT induces necroptosis in HT22 cells. Itaconate improves CORT-induced neuroinflammation and necroptosis. Furthermore, itaconate upregulates the expression of Menin in CORT-exposed HT22 cells. Importantly, silencing Menin abolishes the antagonistic effect of itaconate on CORT-induced necroptosis and neuroinflammation. In brief, these results indicated that itaconate protects HT22 cells against CORT-induced neuroinflammation and necroptosis via upregulating Menin.

Dexmedetomidine attenuates lipopolysaccharide-induced renal cell fibrotic phenotypic changes by inhibiting necroinflammation via activating α2-adrenoceptor: A combined randomised animal and in vitro study.

BACKGROUND: Acute kidney injury (AKI) was reported to be one of the initiators of chronic kidney disease (CKD) development. Necroinflammation may contribute to the progression from AKI to CKD. Dexmedetomidine (Dex), a highly selective α2-adrenoreceptor (AR) agonist, has cytoprotective and "anti-" inflammation effects. This study was designed to investigate the anti-fibrotic properties of Dex in sepsis models. METHODS: C57BL/6 mice were randomly treated with an i.p. injection of lipopolysaccharides (LPS) (10 mg/kg) alone, LPS with Dex (25 µg/kg), or LPS, Dex and Atipamezole (Atip, an α2-adrenoreceptor antagonist) (500 µg/kg) (n=5/group). Human proximal tubular epithelial cells (HK2) were also cultured and then exposed to LPS (1 µg/ml) alone, LPS and Dex (1 µM), transforming growth factor-beta 1 (TGF-ß1) (5 ng/ml) alone, TGF-ß1 and Dex, with or without Atip (100 µM) in culture media. Epithelial-mesenchymal transition (EMT), cell necrosis, necroptosis and pyroptosis, and c-Jun N-terminal kinase (JNK) phosphorylation were then determined. RESULTS: Dex treatment significantly alleviated LPS-induced AKI, myofibroblast activation, NLRP3 inflammasome activation, and necroptosis in mice. Atip counteracted its protective effects. Dex attenuated LPS or TGF-ß1 induced EMT and also prevented necrosis, necroptosis, and pyroptosis in response to LPS stimulation in the HK2 cells. The anti-EMT effects of Dex were associated with JNK phosphorylation. CONCLUSIONS: Dex reduced EMT following LPS stimulation whilst simultaneously inhibiting pyroptosis and necroptosis via α2-AR activation in the renal tubular cells. The "anti-fibrotic" and cytoprotective properties and its clinical use of Dex need to be further studied.

RIPK1 inhibitors: A key to unlocking the potential of necroptosis in drug development.

Within the field of medical science, there is a great deal of interest in investigating cell death pathways in the hopes of discovering new drugs. Over the past two decades, pharmacological research has focused on necroptosis, a cell death process that has just been discovered. Receptor-interacting protein kinase 1 (RIPK1), an essential regulator in the cell death receptor signalling pathway, has been shown to be involved in the regulation of important events, including necrosis, inflammation, and apoptosis. Therefore, researching necroptosis inhibitors offers novel ways to treat a variety of disorders that are not well-treated by the therapeutic medications now on the market. The research and medicinal potential of RIPK1 inhibitors, a promising class of drugs, are thoroughly examined in this study. The journey from the discovery of Necrostatin-1 (Nec-1) to the recent advancements in RIPK1 inhibitors is marked by significant progress, highlighting the integration of traditional medicinal chemistry approaches with modern technologies like high-throughput screening and DNA-encoded library technology. This review presents a thorough exploration of the development and therapeutic potential of RIPK1 inhibitors, a promising class of compounds. Simultaneously, this review highlights the complex roles of RIPK1 in various pathological conditions and discusses potential inhibitors discovered through diverse pathways, emphasizing their efficacy against multiple disease models, providing significant guidance for the expansion of knowledge about RIPK1 and its inhibitors to develop more selective, potent, and safe therapeutic agents.

PROTACs Targeting MLKL Protect Cells from Necroptosis.

Mixed Lineage Kinase domain-Like pseudokinase (MLKL) is implicated in a broad range of diseases due to its role as the ultimate effector of necroptosis and has therefore emerged as an attractive drug target. Here, we describe the development of PROteolysis TArgeting Chimeras (PROTACs) as a novel approach to knock down MLKL through chemical means. A series of candidate degraders were synthesized from a high-affinity pyrazole carboxamide-based MLKL ligand leading to the identification of a PROTAC molecule that effectively degraded MLKL and completely abrogated cell death in a TSZ model of necroptosis. By leveraging the innate ability of these PROTACs to degrade MLKL in a dose-dependent manner, the quantitative relationship between MLKL levels and necroptosis was interrogated. This work demonstrates the feasibility of targeting MLKL using a PROTAC approach and provides a powerful tool to further our understanding of the role of MLKL within the necroptotic pathway.

Identification of Pyrido[3,4-d]pyrimidine derivatives as RIPK3-Mediated necroptosis inhibitors.

Necroptosis executed by RIPK3-mediated phosphorylation of MLKL is a programmed necrotic cell death and implicated with various diseases such as sterile inflammation. We designed and synthesized pyrido[3,4-d]pyrimidine derivatives as novel necroptosis inhibitors capable of suppressing the phosphorylation of MLKL. Our SAR studies reveal that 20 possesses comparable inhibitory activity against RIPK3-mediated pMLKL in HT-29 cells relative to GSK872 (2), a representative selective RIPK3 inhibitor. Based on biochemical kinase assay results, 20 is comparable to GSK872 (2) with regard to activity against RIPK3 and less potent against RIPK1 than GSK872, indicating selectivity of 20 towards RIPK3 over RIPK1 is higher than that of GSK872. In HT-29 cells, 20 inhibits necroptosis via MLKL oligomerization impediment. Moreover, 20 suppresses migration and invasion of AsPC-1 cells by necroptosis induced- CXCL5 secretion downregulation. Significantly, 20 could relieve the TNFα-induced systemic inflammatory response syndrome in vivo. Taken together, this study would provide a useful insight into the design of novel necroptosis inhibitors possessing RIPK3-mediated pMLKL inhibitory activity.

Scaffold hopping derived novel benzoxazepinone RIPK1 inhibitors as anti-necroptosis agents.

Receptor-interacting protein kinase 1 (RIPK1)-mediated necroptosis is believed to have a significant role in contributing to inflammatory diseases. Inhibiting RIPK1 has shown promise in effectively alleviating the inflammation process. In our current study, we employed scaffold hopping to develop a series of novel benzoxazepinone derivatives. Among these derivatives, compound o1 displayed the most potent antinecroptosis activity (EC50=16.17±1.878nM) in cellular assays and exhibited the strongest binding affinity to the target site. Molecular docking analyses further elucidated the mechanism of action of o1, revealing its ability to fully occupy the protein pocket and form hydrogen bonds with the amino acid residue Asp156. Our findings highlight that o1 specifically inhibits necroptosis, rather than apoptosis, by impeding the RIPK1/Receptor-interacting protein kinase 3 (RIPK3)/mixed-lineage kinase domain-like (MLKL) pathway's phosphorylation, triggered by TNFα, Smac mimetic, and z-VAD (TSZ). Additionally, o1 demonstrated dose-dependent improvements in the survival rate of mice with Systemic Inflammatory Response Syndrome (SIRS), surpassing the protective effect observed with GSK'772.

Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.

3,4-Dimethoxychalcone, a caloric restriction mimetic, enhances TFEB-mediated autophagy and alleviates pyroptosis and necroptosis after spinal cord injury.

Background: Caloric restriction mimetics (CRMs) mimic the favourable effects of caloric restriction (CR) and have been shown to have therapeutic effects in neuroinflammatory disease. However, whether CRMs improve the functional recovery from spinal cord injury (SCI) and the underlying mechanism of action remain unclear. In this study, we used the CRMs 3,4-dimethoxychalcone (3,4-DC) to evaluate the therapeutic value of CRMs for SCI. Methods: HE, Masson and Nissl staining; footprint analysis; and the Basso mouse scale were used to determine the functional recovery from SCI after 3,4-DC treatment. RNA sequencing was used to identify the mechanisms of 3,4-DC in SCI. Western blotting, qPCR and immunofluorescence were used to detect the levels of pyroptosis, necroptosis, autophagy and the AMPK-TRPML1-calcineurin signalling pathway. We employed a dual-luciferase reporter assay in vitro and applied AAV vectors to inhibit TFEB in vivo to explore the mechanism of 3,4-DC. Results: 3,4-DC reduced glial scar area and motor neuron death and improved functional recovery after SCI. RNA-sequencing results indicated that oxidative stress, pyroptosis, necroptosis, and autophagy may be involved in the ability of 3,4-DC to improve functional recovery. Furthermore, 3,4-DC inhibited pyroptosis and necroptosis by enhancing autophagy. We also found that 3,4-DC enhances autophagy by promoting TFEB activity. A decrease in the TFEB level abolished the protective effect of 3,4-DC. In addition, 3,4-DC partially regulated TFEB activity through the AMPK-TRPML1-calcineurin signalling pathway. Conclusions: 3,4-DC promotes functional recovery by upregulating TFEB-mediated autophagy and inhibiting pyroptosis and necroptosis after SCI, which may have potential clinical application value.

Targeting Necroptosis: A Novel Therapeutic Option for Retinal Degenerative Diseases.

The discovery of the necroptosis, a form of regulated necrosis that is mediated by receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like pseudokinase (MLKL), represents a major breakthrough that has dramatically altered the conception of necrosis - traditionally thought of as uncontrolled cell death - in various human diseases. Retinal cell death is a leading cause of blindness and has been identified in most retinal diseases, e.g., age-related macular degeneration, glaucoma, retinal detachment, retinitis pigmentosa, etc. Increasing evidence demonstrates that retinal degenerative diseases also share a common mechanism in necroptosis. Exacerbated necroptotic cell death hinders the treatment for retinal degenerative diseases. In this review, we highlight recent advances in identifying retinal necroptosis, summarize the underlying mechanisms of necroptosis in retinal degenerative diseases, and discuss potential anti-necroptosis strategies, such as selective inhibitors and chemical agents, for treating retinal degenerative diseases.

HIV silencing and cell survival signatures in infected T cell reservoirs.

Rare CD4 T cells that contain HIV under antiretroviral therapy represent an important barrier to HIV cure1-3, but the infeasibility of isolating and characterizing these cells in their natural state has led to uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit. Here we address this challenge using a microfluidic technology that isolates the transcriptomes of HIV-infected cells based solely on the detection of HIV DNA. HIV-DNA+ memory CD4 T cells in the blood from people receiving antiretroviral therapy showed inhibition of six transcriptomic pathways, including death receptor signalling, necroptosis signalling and antiproliferative Gα12/13 signalling. Moreover, two groups of genes identified by network co-expression analysis were significantly associated with HIV-DNA+ cells. These genes (n = 145) accounted for just 0.81% of the measured transcriptome and included negative regulators of HIV transcription that were higher in HIV-DNA+ cells, positive regulators of HIV transcription that were lower in HIV-DNA+ cells, and other genes involved in RNA processing, negative regulation of mRNA translation, and regulation of cell state and fate. These findings reveal that HIV-infected memory CD4 T cells under antiretroviral therapy are a distinctive population with host gene expression patterns that favour HIV silencing, cell survival and cell proliferation, with important implications for the development of HIV cure strategies.

Profiling of the chemical space on the phenyl group of substituted benzothiazole RIPK3 inhibitors.

Necroptosis is confirmed as a precisely programmed cell death that is activated in caspase-deficient conditions. Receptor-interacting protein kinase 1 (RIPK1), RIPK3 and mixed-lineage kinase domain-like pseudokinase (MLKL) are the key regulators involved in the signaling pathway. However, accumulating evidence suggests that RIPK1 also works in apoptosis and inflammation pathways independent of necroptosis. Differently, RIPK3 signals necroptosis independent of RIPK1. Thus, identification of specific RIPK3 inhibitors is of great importance for the drug development associated with necroptosis. The benzothiazole carboxamide is a privileged scaffold as RIPK3 inhibitors developed by our group recently. In this study, we work on the phenyl group in-between of benzothiazole and carboxamide to profile the chemical space. Finally, a chlorinated derivative XY-1-127 was found to specifically inhibit necroptosis rather than apoptosis with an EC50 value of 676.8 nM and target RIPK3 with a Kd of 420 nM rather than RIPK1 (Kd = 4300 nM). It was also confirmed to block the formation of necrosome by inhibiting RIPK3 phosphorylation at 1 µM in necroptosis cells. This work discovers the chemical space insights on the phenyl group of the substituted benzothiazole RIPK3 inhibitors and provides a new lead compound for further development.

Apigetrin Promotes TNFα-Induced Apoptosis, Necroptosis, G2/M Phase Cell Cycle Arrest, and ROS Generation through Inhibition of NF-κB Pathway in Hep3B Liver Cancer Cells.

Apigetrin (7-(ß-D-glucopyranosyloxy)-4',5-dihydroxyflavone), a glycoside bioactive dietary flavonoid derived from Taraxacum officinale and Teucrium gnaphalodes, is known to possess anticancer, antioxidant, and anti-inflammatory effects on numerous cancers. In the present study, we examined the effect of apigetrin in Hep3B hepatocellular cancer cell line (HCC). Apigetrin inhibited cell growth and proliferation of Hep3B cells, as confirmed by MTT and colony formation assay. We used apigetrin at concentrations of 0, 50, and 100 µM for later experiments. Of these concentrations, 100 µM of apigetrin showed a significant effect on cell inhibition. In apigetrin-treated Hep3B cells, cell cycle arrest occurred at the G2/M phase. Apoptosis and necroptosis of Hep3B cells treated with apigetrin were confirmed by Annexin V/propidium iodide (PI) staining and flow cytometry results. Morphological observation through 4',6-diamidino-2-phenylindole (DAPI) staining showed intense blue fluorescence representing chromatin condensation. Hematoxylin staining showed necroptotic features such as formation of vacuoles and swelling of organelles. Apigetrin increased reactive oxygen species (ROS) levels in cells, based on fluorescence imaging. Furthermore, the underlying mechanism involved in the apoptosis and necroptosis was elucidated through western blotting. Apigetrin up-regulated TNFα, but down-regulated phosphorylation of p-p65, and IκB. Apigetrin inhibited the expression of Bcl-xl but increased Bax levels. Up-regulation of cleaved PARP and cleaved caspase 3 confirmed the induction of apoptosis in apigetrin-treated Hep3B cells. Additionally, necroptosis markers RIP3, p-RIP3, and p-MLKL were significantly elevated by apigetrin dose-dependently, suggesting necroptotic cell death. Taken together, our findings strongly imply that apigetrin can induce apoptosis and necroptosis of Hep3B hepatocellular cancer cells. Thus, apigetrin as a natural compound might have potential for treating liver cancer.