Dihydromyricetin Attenuates Cerebral Ischemia Reperfusion Injury by Inhibiting SPHK1/mTOR Signaling and Targeting Ferroptosis.

Background: Dihydromyricetin (DHM) exerts protective effects in various brain diseases. The aim of this research was to investigate the biological role of DHM in cerebral ischemia reperfusion (I/R) injury. Methods: We generated a rat model of cerebral I/R injury by performing middle cerebral artery occlusion/reperfusion (MCAO/R). The neurological score and brain water content of the experimental rats was then evaluated. The infarct volume and extent of apoptosis in brain tissues was then assessed by 2,3,5-triphenyltetrazolium (TTC) and TdT-mediated dUTP nick end labeling (TUNEL) staining. Hippocampal neuronal cells (HT22) were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) and cell counting kit-8 (CCK-8) assays and flow cytometry were performed to detect cell viability and apoptosis. The levels of lipid reactive oxygen species (ROS) and iron were detected and the expression levels of key proteins were assessed by Western blotting. Results: DHM obviously reduced neurological deficits, brain water content, infarct volume and cell apoptosis in the brain tissues of MCAO/R rats. DHM repressed ferroptosis and inhibited the sphingosine kinase 1 (SPHK1)/mammalian target of rapamycin (mTOR) pathway in MCAO/R rats. In addition, DHM promoted cell viability and repressed apoptosis in OGD/R-treated HT22 cells. DHM also suppressed the levels of lipid ROS and intracellular iron in OGD/R-treated HT22 cells. The expression levels of glutathione peroxidase 4 (GPX4) was enhanced while the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) and phosphatidylethanolamine binding protein 1 (PEBP1) were reduced in OGD/R-treated HT22 cells in the presence of DHM. Moreover, the influence conferred by DHM was abrogated by the overexpression of SPHK1 or treatment with MHY1485 (an activator of mTOR). Conclusion: This research demonstrated that DHM repressed ferroptosis by inhibiting the SPHK1/mTOR signaling pathway, thereby alleviating cerebral I/R injury. Our findings suggest that DHM may be a candidate drug for cerebral I/R injury treatment.

Activation of RKIP Binding ASC Attenuates Neuronal Pyroptosis and Brain Injury via Caspase-1/GSDMD Signaling Pathway After Intracerebral Hemorrhage in Mice.

Pyroptosis has been proven to be responsible for secondary brain injury after intracerebral hemorrhage (ICH). A recent study reported that Raf kinase inhibitor protein (RKIP) inhibited assembly and activation of inflammasome in macrophages. Our present study aimed to investigate the effects of RKIP on inflammasome-mediated neuronal pyroptosis and underlying neuroprotective mechanisms in experimental ICH. Here, we showed that RKIP expression was decreased both in cerebrospinal fluid (CSF) samples from patients with ICH and in the peri-hematoma tissues after experimental ICH. In mouse ICH model, activation of RKIP remarkably improved neurological deficits, reduced brain water content and BBB disruption, and promoted hematoma absorption at 24 h after ICH, as well as alleviated neuronal degeneration, reduced membrane pore formation, and downregulated pyroptotic molecules NLRP3, caspase-1 P20, GSDMD-N, and mature IL-1ß. Besides, RKIP activation decreased the number of caspase-1 P20-positive neurons after ICH. However, RKIP inhibitor reserved the neuroprotective effects of RKIP at 24 h following ICH. Moreover, RKIP could bind with ASC, then interrupt the assembly of NLRP3 inflammasome. Mechanistically, inhibiting the caspase-1 by VX-765 attenuated brain injury and suppressed neuronal pyroptosis after RKIP inhibitor-pretreated ICH. In conclusion, our findings indicated that activation of RKIP could attenuate neuronal pyroptosis and brain injury after ICH, to some extent, through ASC/Caspase-1/GSDMD pathway. Thus, RKIP may be a potential target to attenuate brain injury via its anti-pyroptosis effect after ICH.

PEBP balances apoptosis and autophagy in whitefly upon arbovirus infection.

Apoptosis and autophagy are two common forms of programmed cell death (PCD) used by host organisms to fight against virus infection. PCD in arthropod vectors can be manipulated by arboviruses, leading to arbovirus-vector coexistence, although the underlying mechanism is largely unknown. In this study, we find that coat protein (CP) of an insect-borne plant virus TYLCV directly interacts with a phosphatidylethanolamine-binding protein (PEBP) in its vector whitefly to downregulate MAPK signaling cascade. As a result, apoptosis is activated in the whitefly increasing viral load. Simultaneously, the PEBP4-CP interaction releases ATG8, a hallmark of autophagy initiation, which reduces arbovirus levels. Furthermore, apoptosis-promoted virus amplification is prevented by agonist-induced autophagy, whereas the autophagy-suppressed virus load is unaffected by manipulating apoptosis, suggesting that the viral load is predominantly determined by autophagy rather than by apoptosis. Our results demonstrate that a mild intracellular immune response including balanced apoptosis and autophagy might facilitate arbovirus preservation within its whitefly insect vector.

NeiyiKangfu tablets control the progression of endometriosis through inhibiting RAF/MEK/ERK signal pathway by targeting RKIP.

BACKGROUND: NeiyiKangfu tablets (NYKF) are widely used clinically for the treatment of endometriosis (EMS), whose mechanism of action has been extensively studied. Researchers have found that NYKF may control the development of ectopic lesions by inhibiting angiogenesis and inflammatory cytokine secretion. Nevertheless, NYKF's mechanism of action remains unclear. METHODS: In the present study, the function of NYKF in the progression of EMS and the associated underlying mechanism was investigated by in vivo and in vitro experiments. EMS model mice were treated with NYKF and the pro-inflammatory factors and apoptosis of ectopic endometrium as well as RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling activation were assessed. In addition, human endometriosis-derived immortalized entopic stromal (hEM15A) cells transfected with or without RAF kinase inhibitor protein (RKIP)-small-interfering RNA (siRNA) were also treated with NYKF and the proliferation, migration, apoptosis, and RAF/MEK/ERK signaling activation were measured by Cell Counting Kit-8 (CCK-8), flow cytometry, Transwell, and western blot, respectively. RESULTS: Results showed that NYKF increased the expression of RKIP, inhibited RAF/MEK/ERK signaling activation, and induced apoptosis while inhibiting proliferation and migration both in EMS mice and hEM15A cells. RKIP knockdown could inhibit the effect of NYKF treatment, leading to the activation of RAF/MEK/ERK signaling and the proliferation and migration of hEM15A cells. CONCLUSIONS: In conclusion, these results suggest that NYKF treatment promotes apoptosis and inhibits proliferation and migration in EMS by inhibiting the RAF/MEK/ERK signaling pathway by targeting RKIP.

Synthesis, secretion, and antifungal mechanism of a phosphatidylethanolamine-binding protein from the silk gland of the silkworm Bombyx mori.

A silkworm cocoon contains several antimicrobial proteins such as protease inhibitors and seroins to provide protection for the enclosed pupa. In this study, we identified a new Bombyx mori phosphatidylethanolamine-binding protein (BmPEBP) with antimicrobial activity in the cocoon silk using semi-quantitative and quantitative RT-PCR, western blotting, and immunofluorescence. The results indicated that BmPEBP was synthesized in the middle silk gland and secreted into the sericin layer of the cocoon silk. Functional analysis showed that BmPEBP could inhibit the spore growth of four types of fungi, Candida albicans, Saccharomyces cerevisiae, Beauveriabassiana, and Aspergillus fumigates, by binding to the fungal cell membrane. Investigation of the interaction of BmPEBP with membrane phospholipids revealed that the protein showed a strong binding affinity to phosphatidylethanolamine, weak affinity to phosphatidylinositol, and no affinity to phosphatidylserine or phosphatidylcholine. Circular dichroism spectroscopy showed that binding to phosphatidylethanolamine caused conformational changes in the BmPEBP molecule by reducing ß-sheet formation and inducing the appearance of an α-helix motif. We speculate that BmPEBP performs antifungal function in the cocoon silk through interaction with phosphatidylethanolamine in the fungal membrane.

Molecular Underpinnings Governing Genetic Complexity of ETS-Fusion-Negative Prostate Cancer.

Inter- and intra-patient molecular heterogeneity of primary and metastatic prostate cancer (PCa) confers variable clinical outcome and poses a formidable challenge in disease management. High-throughput integrative genomics and functional approaches have untangled the complexity involved in this disease and revealed a spectrum of diverse aberrations prevalent in various molecular subtypes, including ETS fusion negative. Emerging evidence indicates that SPINK1 upregulation, mutations in epigenetic regulators or chromatin modifiers, and SPOP are associated with the ETS-fusion negative subtype. Additionally, patients with defects in a DNA-repair pathway respond to poly-(ADP-ribose)-polymerase (PARP) inhibition therapies. Furthermore, a new class of immunogenic subtype defined by CDK12 biallelic loss has also been identified in ETS-fusion-negative cases. This review focuses on the emerging molecular underpinnings driving key oncogenic aberrations and advancements in therapeutic strategies of this disease.

NF-κB Regulation of c-FLIP Promotes TNFα-Mediated RAF Inhibitor Resistance in Melanoma.

Targeted inhibitors elicit heterogeneous clinical responses in genetically stratified groups of patients. Although most studies focus on tumor intrinsic properties, factors in the tumor microenvironment were recently found to modulate the response to inhibitors. Here, we show that in cutaneous BRAF V600E melanoma, the cytokine tumor necrosis factor-α (TNFα) blocks RAF inhibitor-induced apoptosis via activation of NF-κB. Several NF-κB-dependent factors are upregulated following TNFα and RAF inhibitor treatment. Of these factors, we show that death receptor inhibitor cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) is required for TNFα-induced protection against RAF inhibitor. Overexpression of c-FLIP_S or c-FLIP_L isoform decreased RAF inhibitor-induced apoptosis in the absence of TNFα. Importantly, targeting NF-κB enhances response to RAF inhibitor in vitro and in vivo. Together, our results show mechanistic evidence for cytokine-mediated resistance to RAF inhibitor and provide a preclinical rationale for the strategy of cotargeting the RAF/MEK/ERK1/2 pathway and the TNFα/NF-κB axis to treat mutant BRAF melanomas.

The role of Raf-1 kinase inhibitor protein in the regulation of pancreatic beta cell proliferation in mice.

AIMS/HYPOTHESIS: Manoeuvres aimed at increasing beta cell mass have been proposed as regenerative medicine strategies for diabetes treatment. Raf-1 kinase inhibitor protein 1 (RKIP1) is a common regulatory node of the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways and therefore may be involved in regulation of beta cell homeostasis. The aim of this study was to investigate the involvement of RKIP1 in the control of beta cell mass and function. METHODS: Rkip1 (also known as Pebp1) knockout (Rkip1 (-/-)) mice were characterised in terms of pancreatic and glucose homeostasis, including morphological and functional analysis. Glucose tolerance and insulin sensitivity were examined, followed by assessment of glucose-induced insulin secretion in isolated islets and beta cell mass quantification through morphometry. Further characterisation included determination of endocrine and exocrine proliferation, apoptosis, MAPK activation and whole genome gene expression assays. Capacity to reverse a diabetic phenotype was assessed in adult Rkip1 (-/-) mice after streptozotocin treatment. RESULTS: Rkip1 (-/-) mice exhibit a moderately larger pancreas and increased beta cell mass and pancreatic insulin content, which correlate with an overall improvement in whole body glucose tolerance. This phenotype is established in young postnatal stages and involves enhanced cellular proliferation without significant alterations in cell death. Importantly, adult Rkip1 (-/-) mice exhibit rapid reversal of streptozotocin-induced diabetes compared with control mice. CONCLUSIONS/INTERPRETATION: These data implicate RKIP1 in the regulation of pancreatic growth and beta cell expansion, thus revealing RKIP1 as a potential pharmacological target to promote beta cell regeneration.

Raf kinase inhibitor protein positively regulates cell-substratum adhesion while negatively regulating cell-cell adhesion.

Raf kinase inhibitor protein (RKIP) regulates a number of cellular processes, including cell migration. Exploring the role of RKIP in cell adhesion, we found that overexpression of RKIP in Madin-Darby canine kidney (MDCK) epithelial cells increases adhesion to the substratum, while decreasing adhesion of the cells to one another. The level of the adherens junction protein E-cadherin declines profoundly, and there is loss of normal localization of the tight junction protein ZO-1, while expression of the cell-substratum adhesion protein beta1 integrin dramatically increases. The cells also display increased adhesion and spreading on multiple substrata, including collagen, gelatin, fibronectin and laminin. In three-dimensional culture, RKIP overexpression leads to marked cell elongation and extension of long membrane protrusions into the surrounding matrix, and the cells do not form hollow cysts. RKIP-overexpressing cells generate considerably more contractile traction force than do control cells. In contrast, RNA interference-based silencing of RKIP expression results in decreased cell-substratum adhesion in both MDCK and MCF7 human breast adenocarcinoma cells. Treatment of MDCK and MCF7 cells with locostatin, a direct inhibitor of RKIP and cell migration, also reduces cell-substratum adhesion. Silencing of RKIP expression in MCF7 cells leads to a reduction in the rate of wound closure in a scratch-wound assay, although not as pronounced as that previously reported for RKIP-knockdown MDCK cells. These results suggest that RKIP has important roles in the regulation of cell adhesion, positively controlling cell-substratum adhesion while negatively controlling cell-cell adhesion, and underscore the complex functions of RKIP in cell physiology.

Identification and characterization of PEBP as a calpain substrate.

Calpains are calcium- and thiol-dependent proteases whose dysregulation has been implicated in a number of diseases and conditions such as cardiovascular dysfunction, ischemic stroke, and Alzheimer's disease (AD). While the effects of calpain activity are evident, the precise mechanism(s) by which dysregulated calpain activity results in cellular degeneration are less clear. In order to determine the impact of calpain activity, there is a need to identify the range of specific calpain substrates. Using an in vitro proteomics approach we confirmed that phosphatidylethanolamine-binding protein (PEBP) as a novel in vitro and in situ calpain substrate. We also observed PEBP proteolysis in a model of brain injury in which calpain is clearly activated. In addition, with evidence of calpain dysregulation in AD, we quantitated protein levels of PEBP in postmortem brain samples from the hippocampus of AD and age-matched controls and found that PEBP levels were approximately 20% greater in AD. Finally, with previous evidence that PEBP may act as a serine protease inhibitor, we tested PEBP as an inhibitor of the proteasome and found that PEBP inhibited the chymostrypsin-like activity of the proteasome by approximately 30%. Together these data identify PEBP as a potential in vivo calpain substrate and indicate that increased PEBP levels may contribute to impaired proteasome function.