Resveratrol inhibits necroptosis by mediating the TNF-α/RIP1/RIP3/MLKL pathway in myocardial hypoxia/reoxygenation injury.

Resveratrol (RES) protects myocardial cells from hypoxia/reoxygenation (H/R)-caused injury. However, the mechanism of this effect has not been clarified. Thus, in this study, we aimed to determine whether RES attenuates H/R-induced cell necroptosis by inhibiting the tumor necrosis factor-alpha (TNF-α)/receptor-interacting protein kinase 1 (RIP1)/RIP3/mixed-lineage kinase domain-like (MLKL) signaling pathway. Rat myocardial ischemia/reperfusion (I/R) models and H/R-injured cell models were constructed. Our study showed that myocardial H/R injury significantly increased the levels of TNF-α, RIP1, RIP3, and p-MLKL/MLKL by western blot analysis. Cell viability assay and 4,6-dianmidino-2-phenylindole (DAPI)-propidium iodide staining showed that the cell viability was decreased, and necroptosis was increased after myocardial H/R injury. The expressions of TNF-α, RIP1, RIP3, and p-MLKL/MLKL in H/R myocardial cells treated with different concentrations of RES were significantly downregulated. In addition, we also found that the cell viability was increased and necroptosis was decreased in dose-dependent manners when H/R-injured cells were treated with RES. In addition, the enhanced effect of TNF-α on necroptosis in myocardial H/R-injured cells was improved by RES, and the effect of RES was confirmed in vivo in I/R rats. This study also showed that RES suppresses necroptosis in H9c2 cells, which may occur through the inhibition of the TNF-α/RIP1/RIP3/MLKL signaling pathway. Our data suggest that necroptosis is a promising therapeutic target and may be a promising therapeutic target for the treatment of myocardial I/R injury.

Hu Y ，Pan H ，Peng J ，He J ，Tang M ，Yan S ，Rong J ，Li J ，Zheng Z ，Wang H ，Liu Y ，Zhong X ... -  《-》

PI3K mediates tumor necrosis factor induced-necroptosis through initiating RIP1-RIP3-MLKL signaling pathway activation.

Necroptosis is a recently identified programmed cell death, which is initiated by receptor-interacting serine/threonine-protein kinase 1 (RIP1), RIP3 and mixed-lineage kinase domain-like protein (MLKL). It has been reported that necroptosis induced by tumor necrosis factor (TNF) was inhibited by the inhibitor of phosphatidylinositol-3-kinase (PI3K) and its substrate protein AKT, indicating that PI3K-AKT signaling pathway was involved in mediating TNF-induced necroptosis, whereas it is unclear how PI3K initiates necroptosis. In this study, we found that TNF-induced necroptosis was inhibited by chemical inhibition or genetic deletion of PI3K. Moreover, knockdown of p110α, the catalytic subunit of PI3K, significantly suppressed the phosphorylation of PI3K substrate protein AKT, and TNF-induced necroptosis was blocked by AKT inhibitors. Furthermore, we found that p110α knockdown also suppressed the phosphorylation and oligomerization of RIP1, RIP3 and MLKL in response to TNF stimulation. In addition to the critical role in mediating TNF-induced necrosome formation, p110α was also essential for the spontaneous phosphorylation of RIP1 and RIP3. Finally, we found that p110α bound to RIP3, but not RIP1, to form protein complex in the process of TNF-induced necroptosis, and mediated TNF-induced necroptosis in the absence of RIP1. Our results demonstrate that PI3K is essential for TNF-induced necroptosis, which may act as the partner of RIP3 to initiate the activation of RIP1-RIP3-MLKL signal pathway and the subsequent necroptosis.

Hu S ，Chang X ，Zhu H ，Wang D ，Chen G ... -  《-》

RIP1/RIP3/MLKL-mediated necroptosis contributes to vinblastine-induced myocardial damage.

Zhou H ，Liu L ，Ma X ，Wang J ，Yang J ，Zhou X ，Yang Y ，Liu H ... -  《-》

RIP1, RIP3, and MLKL Contribute to Cell Death Caused by Clostridium perfringens Enterotoxin.

Clostridium perfringens type F strains cause gastrointestinal disease when they produce a pore-forming toxin named C. perfringens enterotoxin (CPE). In human enterocyte-like Caco-2 cells, low CPE concentrations cause caspase-3-dependent apoptosis, while high CPE concentrations cause necrosis. Since necrosis or apoptosis sometimes involves receptor-interacting serine/threonine-protein kinase-1 or 3 (RIP1 or RIP3), this study examined whether those kinases are important for CPE-induced apoptosis or necrosis. Highly specific RIP1 or RIP3 inhibitors reduced both CPE-induced apoptosis and necrosis in Caco-2 cells. Those findings suggested that the form of necrosis induced by treating Caco-2 cells with high CPE concentrations involves necroptosis, which was confirmed when high, but not low, CPE concentrations were shown to induce oligomerization of mixed-lineage kinase domain-like pseudokinase (MLKL), a key late step in necroptosis. Furthermore, an MLKL oligomerization inhibitor reduced cell death caused by high, but not low, CPE concentrations. Supporting RIP1 and RIP3 involvement in CPE-induced necroptosis, inhibitors of those kinases also reduced MLKL oligomerization during treatment with high CPE concentrations. Calpain inhibitors similarly blocked MLKL oligomerization induced by high CPE concentrations, implicating calpain activation as a key intermediate in initiating CPE-induced necroptosis. In two other CPE-sensitive cell lines, i.e., Vero cells and human enterocyte-like T84 cells, low CPE concentrations also caused primarily apoptosis/late apoptosis, while high CPE concentrations mainly induced necroptosis. Collectively, these results establish that high, but not low, CPE concentrations cause necroptosis and suggest that RIP1, RIP3, MLKL, or calpain inhibitors can be explored as potential therapeutics against CPE effects in vivoIMPORTANCEC. perfringens type F strains are a common cause of food poisoning and antibiotic-associated diarrhea. Type F strain virulence requires production of C. perfringens enterotoxin (CPE). In Caco-2 cells, high CPE concentrations cause necrosis while low enterotoxin concentrations induce apoptosis. The current study determined that receptor-interacting serine/threonine-protein kinases 1 and 3 are involved in both CPE-induced apoptosis and necrosis in Caco-2 cells, while mixed-lineage kinase domain-like pseudokinase (MLKL) oligomerization is involved in CPE-induced necrosis, thereby indicating that this form of CPE-induced cell death involves necroptosis. High CPE concentrations also caused necroptosis in T84 and Vero cells. Calpain activation was identified as a key intermediate for CPE-induced necroptosis. These results suggest inhibitors of RIP1, RIP3, MLKL oligomerization, or calpain are useful therapeutics against CPE-mediated diseases.

Shrestha A ，Mehdizadeh Gohari I ，McClane BA 《mBio》

Melatonin attenuates chronic pain related myocardial ischemic susceptibility through inhibiting RIP3-MLKL/CaMKII dependent necroptosis.

Chronic pain aggravates cardiovascular injury via incompletely understood mechanisms. While melatonin may participate in the pathophysiological process of chronic pain, its cardiovascular effects under chronic pain states remains unknown. In this study, chronic pain was induced by spared nerve injury model (SNI) for 4 weeks. We showed decreased the ipsilateral hind paw withdrawal mechanical threshold (PWMT) in SNI mice. High dose melatonin treatment (60 mg/kg, i.p.) could reversed nociceptive threshold in SNI mice. To verify the effect of chronic pain on the cardiac tolerance to ischemic stress, mice were subjected to myocardial ischemia-reperfusion (MI/R) in vivo. SNI mice showed exaggerated MI/R-induced detrimental effects and myocardial necroptosis compared with control group (P < .05). Mechanically, an increased level of tumor necrosis factor-α (TNF-α) was found in SNI group following by a robust interaction of RIP1/RIP3. RIP3-induced phosphor-MLKL and CaMKII more significantly in SNI mice (P < .05). We found that RIP3 deficiency provided a comparable protection against MI/R-induced necroptosis under chronic pain conditions. More importantly, low dose melatonin (20 mg/kg, i.p.) treatment 10 min before reperfusion decreased the level of TNF-α following with a negatively regulating the RIP3 induced phosphor-MLKL/CaMKII signaling, thus significantly reduced ROS production and cardiomyocyte necroptosis and ameliorated cardiac function. In summarize, our results demonstrated that chronic pain sensitizes heart to MI/R injury and myocardial necrosis plays an important role in this pathophysiological process. We also define melatonin acted as triple cardioprotective effects: ameliorating TNF-α level, suppressing RIP3-MLKL/CaMKII signaling induced necroptosis and exerting analgesia effect.

Yang Z ，Li C ，Wang Y ，Yang J ，Yin Y ，Liu M ，Shi Z ，Mu N ，Yu L ，Ma H ... -  《-》