Exploration of the mechanism of luteolin against ischemic stroke based on network pharmacology, molecular docking and experimental verification.

Stroke is a leading cause of morbidity and mortality worldwide. As the most common type of stroke cases, treatment effectiveness is still limited despite intensive research. Recently, traditional Chinese medicine has attracted attention because of potential benefits for stroke treatment. Among these, luteolin, a natural plant flavonoid compound, offers neuroprotection following against ischemic stroke, although the specific mechanisms are unknown. Here we used network pharmacology, molecular docking, and experimental verification to explore the mechanisms whereby luteolin can benefit stroke recovery. The pharmacological and molecular properties of luteolin were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. The potential targets of luteolin and ischemic stroke were collected from interrogating public databases. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed by Funrich and Database for Annotation, Visualization and Integrated Discovery respectively, a luteolin-target-pathway network constructed using Cytoscape, Autodock vina was used for molecular docking simulation with Discovery Studio was used to visualize and analyze the docked conformations. Lastly, we employed an in vitro model of stroke injury to evaluate the effects of luteolin on cell survival and expression of the putative targets. From 95 candidate luteolin target genes, our analysis identified six core targets . KEGG analysis of the candidate targets identified that luteolin provides therapeutic effects on stroke through TNF signaling and other pathways. Our experimental analyses confirmed the conclusions analyzed above. In summary, the molecular and pharmacological mechanisms of luteolin against stroke are indicated in our study from a systematic perspective.

Sex-Specific Differences in Autophagic Responses to Experimental Ischemic Stroke.

Ischemic stroke triggers a series of complex pathophysiological processes including autophagy. Differential activation of autophagy occurs in neurons derived from males versus females after stressors such as nutrient deprivation. Whether autophagy displays sexual dimorphism after ischemic stroke is unknown. We used a cerebral ischemia mouse model (middle cerebral artery occlusion, MCAO) to evaluate the effects of inhibiting autophagy in ischemic brain pathology. We observed that inhibiting autophagy reduced infarct volume in males and ovariectomized females. However, autophagy inhibition enhanced infarct size in females and in ovariectomized females supplemented with estrogen compared to control mice. We also observed that males had increased levels of Beclin1 and LC3 and decreased levels of pULK1 and p62 at 24 h, while females had decreased levels of Beclin1 and increased levels of ATG7. Furthermore, the levels of autophagy markers were increased under basal conditions and after oxygen and glucose deprivation in male neurons compared with female neurons in vitro. E2 supplementation significantly inhibited autophagy only in male neurons, and was beneficial for cell survival only in female neurons. This study shows that autophagy in the ischemic brain differs between the sexes, and that autophagy regulators have different effects in a sex-dependent manner in neurons.

Morphological Analysis of the Hindbrain Glucose Sensor-Hypothalamic Neural Pathway Activated by Hindbrain Glucoprivation.

Lowered glucose availability, sensed by the hindbrain, has been suggested to enhance gluconeogenesis and food intake as well as suppress reproductive function. In fact, our previous histological and in vitro studies suggest that hindbrain ependymal cells function as a glucose sensor. The present study aimed to clarify the hindbrain glucose sensor-hypothalamic neural pathway activated in response to hindbrain glucoprivation to mediate counterregulatory physiological responses. Administration of 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, into the fourth ventricle (4V) of male rats for 0.5 hour induced messenger RNA (mRNA) expression of c-fos, a marker for cellular activation, in ependymal cells in the 4V, but not in the lateral ventricle, the third ventricle or the central canal without a significant change in blood glucose and testosterone levels. Administration of 2DG into the 4V for 1 hour significantly increased blood glucose levels, food intake, and decreased blood testosterone levels. Simultaneously, the expression of c-Fos protein was detected in the 4V ependymal cells; dopamine ß-hydroxylase-immunoreactive cells in the C1, C2, and A6 regions; neuropeptide Y (NPY) mRNA-positive cells in the C2; corticotropin-releasing hormone (CRH) mRNA-positive cells in the hypothalamic paraventricular nucleus (PVN); and NPY mRNA-positive cells in the arcuate nucleus (ARC). Taken together, these results suggest that lowered glucose availability, sensed by 4V ependymal cells, activates hindbrain catecholaminergic and/or NPY neurons followed by CRH neurons in the PVN and NPY neurons in the ARC, thereby leading to counterregulatory responses, such as an enhancement of gluconeogenesis, increased food intake, and suppression of sex steroid secretion.

Therapeutically reprogrammed nutrient signalling enhances nanoparticulate albumin bound drug uptake and efficacy in KRAS-mutant cancer.

Nanoparticulate albumin bound paclitaxel (nab-paclitaxel, nab-PTX) is among the most widely prescribed nanomedicines in clinical use, yet it remains unclear how nanoformulation affects nab-PTX behaviour in the tumour microenvironment. Here, we quantified the biodistribution of the albumin carrier and its chemotherapeutic payload in optically cleared tumours of genetically engineered mouse models, and compared the behaviour of nab-PTX with other clinically relevant nanoparticles. We found that nab-PTX uptake is profoundly and distinctly affected by cancer-cell autonomous RAS signalling, and RAS/RAF/MEK/ERK inhibition blocked its selective delivery and efficacy. In contrast, a targeted screen revealed that IGF1R kinase inhibitors enhance uptake and efficacy of nab-PTX by mimicking glucose deprivation and promoting macropinocytosis via AMPK, a nutrient sensor in cells. This study thus shows how nanoparticulate albumin bound drug efficacy can be therapeutically improved by reprogramming nutrient signalling and enhancing macropinocytosis in cancer cells.

Gastrodin Alleviates Cerebral Ischaemia/Reperfusion Injury by Inhibiting Pyroptosis by Regulating the lncRNA NEAT1/miR-22-3p Axis.

Cerebral ischaemia/reperfusion (I/R) injury-induced irreversible brain injury is a major cause of mortality and functional impairment in ageing people. Gastrodin (GAS), derived from the traditional Chinese herbal medicine Tianma, has been reported to inhibit the progression of stroke, but the mechanism whereby GAS modulates the progression of cerebral I/R remains unclear. The middle cerebral artery occlusion method was used as a model of I/R in vivo. Rats were pretreated with GAS by intraperitoneal injection 7 days before I/R surgery and were then treated with GAS for 7 days after I/R surgery. Additionally, an oxygen-glucose deprivation/reoxygenation model using neuronal cells was established in vitro to simulate I/R injury. 2,3,5-Triphenyltetrazolium chloride and Nissl staining were used to evaluate infarct size and neuronal damage, respectively. Lactate dehydrogenase release and cell counting kit-8 assays were used to assess neuronal cell viability. Enzyme-linked immunosorbent assay, qPCR, flow cytometry and western blotting were performed to analyse the expression levels of inflammatory factors (IL-1ß, IL-18), lncRNA NEAT1, miR-22-3p, NLRP3 and cleaved caspase-1. Luciferase reporter experiments were performed to verify the association between lncRNA NEAT1 and miR-22-3p. The results indicated that GAS could significantly improve the neurological scores of rats and reduce the area of cerebral infarction. Meanwhile, GAS inhibited pyroptosis by downregulating NLRP3, inflammatory factors (IL-1ß, IL-18) and cleaved caspase-1. In addition, GAS attenuated I/R-induced inflammation in neuronal cells through the modulation of the lncRNA NEAT1/miR-22-3p axis. GAS significantly attenuated cerebral I/R injury via modulation of the lncRNA NEAT1/miR-22-3p axis. Thus, GAS might serve as a new agent for the treatment of cerebral I/R injury.

Elongation factor eEF2 kinase and autophagy jointly promote survival of cancer cells.

Cells within solid tumours can become deprived of nutrients; in order to survive, they need to invoke mechanisms to conserve these resources. Using cancer cells in culture in the absence of key nutrients, we have explored the roles of two potential survival mechanisms, autophagy and elongation factor 2 kinase (eEF2K), which, when activated, inhibits the resource-intensive elongation stage of protein synthesis. Both processes are regulated through the nutrient-sensitive AMP-activated protein kinase and mechanistic target of rapamycin complex 1 signalling pathways. We find that disabling both autophagy and eEF2K strongly compromises the survival of nutrient-deprived lung and breast cancer cells, whereas, for example, knocking out eEF2K alone has little effect. Contrary to some earlier reports, we find no evidence that eEF2K regulates autophagy. Unexpectedly, eEF2K does not facilitate survival of prostate cancer PC3 cells. Thus, eEF2K and autophagy enable survival of certain cell-types in a mutually complementary manner. To explore this further, we generated, by selection, cells which were able to survive nutrient starvation even when autophagy and eEF2K were disabled. Proteome profiling using mass spectrometry revealed that these 'resistant' cells showed lower levels of diverse proteins which are required for energy-consuming processes such as protein and fatty acid synthesis, although different clones of 'resistant cells' appear to adapt in dissimilar ways. Our data provide further information of the ways that human cells cope with nutrient limitation and to understanding of the utility of eEF2K as a potential target in oncology.

Yiqi Huoxue Recipe inhibits cardiomyocyte apoptosis caused by heart failure through Keap1/Nrf2/HIF-1α signaling pathway.

Yiqi Huoxue Recipe (YHR) is commonly used in China to treat diseases such as heart failure (HF). It has been reported that YHR can treat HF and has a certain protective effect on myocardial cell damage. The purpose of this study is to determine the cardioprotective effects of YHR on HF-induced apoptosis and to clarify its mechanism of action. Oxygen glucose deprivation/recovery (OGD/R) induces H9C2 cell apoptosis model. Ligation of the left anterior descending artery (LAD) coronary artery can induce an animal model of HF. We found that YHR protected H9C2 cells from OGD/R-induced apoptosis, reduced the level of reactive oxygen species (ROS) in H9C2 cells, and increased the mitochondrial membrane potential in H9C2 cells. The results of in vivo animal experiments showed that in the HF model, YHR could reduce infarct area of heart tissue and cardiomyocyte apoptosis rate. YHR regulated the expression of key apoptotic molecules, including increasing the ratio of Bcl-2 and Bax, and reducing the expression of Kelch-like ECH-associated protein 1 (Keap1) and caspase-3. Interestingly, YHR also regulates the expression of NF-E2-related factor 2 (Nrf2) in the nucleus. In summary, YHR may provide cardioprotective effects in heart failure through inhibiting the Keap1/Nrf2/HIF-1α apoptosis pathway.

Tectorigenin attenuates the OGD/R-induced HT-22 cell damage through regulation of the PI3K/AKT and the PPARγ/NF-κB pathways.

Tectorigenin (TEC) is an effective compound that derived from many plants, such as Iris unguicularis, Belamcanda chinensis and Pueraria thunbergiana Benth. Evidence suggested that TEC has anti-tumor, anti-oxidant activity, anti-bacterial and anti-inflammatory effects. In addition, there has some evidence indicated that TEC is a potential anti-stroke compound; however, its specific roles and associated mechanism have not yet been elucidated. In the present study, we aimed to investigate the anti-inflammatory, anti-oxidant activity and anti-apoptosis effects of TEC on oxygen-glucose deprivation/reperfusion (OGD/R)-induced HT-22 cells, and clarified the relevant mechanisms. Here, we observed that TEC significantly promoted cell survival, impeded cell apoptosis, inhibited ROS and inflammatory cytokines IL-1ß, IL-6, TNF-α production in OGD/R-induced HT-22 cells. Moreover, TEC activated PI3K/AKT signal pathway, increased PPARγ expression and inhibited NF-κB pathway activation in OGD/R-induced HT-22 cells. Further studies indicated that PPARγ inhibitor GW9662 activated NF-κB pathway after TEC treatment in OGD/R-induced HT-22 cells. Also, PI3K/AKT inhibitor LY294002, PPARγ inhibitor GW9662 and NF-κB activator LPS both reversed the effects of TEC on OGD/R-induced HT-22 cell biology. Taken together, this research confirmed that TEC benefit to HT-22 cell survival and against OGD/R damage through the PI3K/AKT and PPARγ/NF-κB pathways. These results indicated that TEC might be an effective compound in the treatment for ischemic brain injury.

The Growth Response to Beta-Hydroxybutyrate in SH-SY5Y Neuroblastoma Cells is Suppressed by Glucose and Pyruvate Supplementation.

Neuroblastoma (NB) is a childhood malignancy of the sympathetic nervous system and is commonly studied using the SH-SY5Y cell line. Its neoplastic and neurodevelopmental manifestations are characterised by a high glucose demand which maintains its high proliferative capacity. This metabolic phenotype may be utilised in dietary therapies such as the ketone diet which alter substrate availability and thus starve NB cells of their preferred biosynthetic requirements. However, the effects of ketone metabolism on cancer growth remain poorly understood due to the involvement of other metabolic substrates in experimental paradigms and complexities underlying the Warburg effect. We investigated how the primary ketone body beta-hydroxybutyrate (ßOHB) affects the growth of SH-SY5Y NB cells in the presence or absence of culture metabolic substrates. We demonstrated that while glucose deprivation reduced the growth and viability of SH-SY5Y cells, they proliferated and were initially unaffected by the addition of ßOHB. However, a growth response to ßOHB was subsequently revealed in media containing low levels of glucose, as well as in glucose and pyruvate deprived conditions. These data shed light on the roles of metabolic substrate availability as key determinants of the responses of SH-SY5Y NB cells to ketone supplementation.

Cottonseed Oil Alleviates Ischemic Stroke-Induced Oxidative Stress Injury Via Activating the Nrf2 Signaling Pathway.

Oxidative stress is believed to be one of the primary causes in ischemic stroke injury, and the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway is the most important endogenous antioxidative stress damage pathway. Cottonseed oil (CSO), which is used mostly as a solvent for lipid-soluble drugs, has been shown to exert antioxidative effects against peripheral tissue injury. However, the effects and mechanisms of CSO on ischemic stroke-induced oxidative stress injury and the Nrf2 signaling pathway remain largely unknown. In this study, we investigated the potential of CSO in regulating oxidative stress injury induced by middle cerebral artery occlusion and reperfusion (MCAO-R), or oxygen and glucose deprivation and reperfusion (OGD-R). We found that 1.3 mL/kg CSO treatment of male rats with a subcutaneous injection once every other day for 3 weeks significantly improved neurological deficit; reduced infarction volume; alleviated neuronal injuries; reduced the content of ROS and MDA; increased the activity of SOD, GSH, and GSH-PX; and markedly increased the expression of Nrf2. Furthermore, treatment with 10-9 µL/mL CSO to a neuron cell line (HT-22) for 24 h significantly increased cell viability and decreased cell apoptosis after OGD-R injury; significantly reduced the levels of ROS and MDA; increased the activity of SOD, GSH, and GSH-PX; and induced an increase in Nrf2 nuclear translocation. Based on our findings, we conclude that CSO treatment alleviates ischemic stroke injury-induced oxidative stress via activating the Nrf2 signaling pathway, highlighting the potential that CSO has as a therapeutic for ischemic strokes.

Neuroprotective effects of Rosmarinus officinalis L. extract in oxygen glucose deprivation (OGD)-injured human neural-like cells.

Rosmarinus officinalis L. (RO), an aromatic plant used as food condiment and in traditional medicine, exerts numerous beneficial properties including antioxidant, analgesic and neuroprotective effects. Onset and progression of homeostatic imbalances observed in the early phases of a number of neurodegenerative diseases, have been associated with a gap junction (GJ)-dependent increased membrane permeability and alterations of connexins (Cxs), including Cx43. Here, we evaluate spray-dried RO extract (SDROE)-mediated effects on cell viability, apoptosis and Cx43-based intercellular communication using human SH-SY5Y neuron-like and human A-172 glial-like cells in an in vitro model of oxygen glucose deprivation (OGD) injury. We found that SDROE exerts a protective action in OGD-injured cells, increasing cell viability and metabolic turnover and decreasing Cx43-based cell coupling. These data suggest that SDROE-mediated Cx43 reduction may be the molecular basis for its beneficial effects to be exploited for preventive treatment against the risk of some neurodegenerative disorders.

Protocatechualdehyde protects oxygen-glucose deprivation/reoxygenation-induced myocardial injury via inhibiting PERK/ATF6α/IRE1α pathway.

Endoplasmic reticulum (ER) stress has been considered as a promising strategy in developing novel therapeutic agents for cardiovascular diseases through inhibiting cardiomyocyte apoptosis. Protocatechualdehyde (PCA) is a natural phenolic compound from medicinal plant Salvia miltiorrhiza with cardiomyocyte protection. However, the potential mechanism of PCA on cardiovascular ischemic injury is largely unexplored. Here, we found that PCA exerted markedly anti-apoptotic effect in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced H9c2 cells (Rat embryonic ventricular H9c2 cardiomyocytes), which was detected by 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), lactate dehydrogenase (LDH), Hoechst 33258 and acridine orange/ethidium bromide (AO/EB) assays. PCA also obviously protected cardiomyocytes in myocardial fibrosis model of mice, which was determined by hematoxylin-eosin (HE) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining. Transcriptomics coupled with bioinformatics analysis revealed a complex pharmacological signaling network especially for PCA-mediated ER stress on cardiomyocytes. Further mechanism study suggested that PCA suppressed ER stress via inhibiting protein kinase R-like ER kinase (PERK), inositol-requiring enzyme1α (IRE1α), and transcription factor 6α (ATF6α) signaling pathway through Western blot, DIOC6 and ER-Tracker Red staining, leading to a protective effect against ER stress-mediated cardiomyocyte apoptosis. Taken together, our observations suggest that PCA is a major component from Salvia miltiorrhiza against cardiovascular ischemic injury by suppressing ER stress-associated PERK, IRE1α and ATF6α signaling pathways.

Ginsenoside Rb1 attenuates microglia activation to improve spinal cord injury via microRNA-130b-5p/TLR4/NF-κB axis.

Ginsenoside Rb1 (GRb1), a major ingredient of ginseng, has been found to be a potential protective agent in spinal cord injury (SCI) and in activated microglia-induced neuronal injury. This study discovered that GRb1 could facilitate miR-130b-5p expression in SCI rats and Toll-like receptor 4 (TLR4; a crucial player in inflammation) was a potential target of miR-130b-5p. Hence, we further investigated whether GRb1 could relieve SCI by reducing microglia-mediated inflammatory responses and neuronal injury via miR-130b-5p/TLR4 pathways. The results showed that GRb1 alleviated SCI through inhibiting neuronal apoptosis and proinflammatory factor expression via increasing miR-130b-5p.GRb1 weakened the damage of activated microglia to neurons through upregulating miR-130b-5p. miR-130b-5p attenuated activated microglia-induced neuron injury via targeting TLR4. GRb1 inactivated TLR4/nuclear factor-κB (NF-κB) activation and inhibited proinflammatory cytokine secretion by increasing miR-130b-5p in activated microglia. As a conclusion, GRb1 alleviated SCI through reducing activated microglia-induced neuronal injury via miR-130b-5p/TLR4/NF-κB axis, providing a deep insight into the molecular basis of GRb1 in the treatment of SCI.

Garcinol protects against cerebral ischemia-reperfusion injury in vivo and in vitro by inhibiting inflammation and oxidative stress.

Garcinol, a polyisoprenylated benzophenone derivative, is isolated from fruit rind of Garcinia indica. It is known to exert potent anti-inflammatory and anti-oxidative properties. In the present study, we tried to investigate the neuroprotective effects of garcinol on a rat model with middle cerebral artery occlusion/reperfusion (MCAO/R) and a cell model subjected to oxygen glucose deprivation and reperfusion (OGD/R). In vivo, we found that the rats with garcinol treatment showed a lower neurological deficit score and a smaller infarct size compared with the rats with ischemia-reperfusion (I/R) injury alone. We further found that garcinol treatment decreased cerebral I/R-induced inflammatory cytokines and oxidative stress, including inhibiting the production of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α (TNF-α), decreasing the levels of malonaldehyde (MDA) and nitric oxide (NO), and suppressing the decreased superoxide dismutase (SOD) activity. Moreover, the suppression of toll-like receptor (TLR) 4 and nuclear NF-κB (p65) expression by garcinol was found both in vivo and in vitro. In addition, NF-κB activator or TLR4 overexpression was employed to investigate its involvement in the effects of garcinol. The results showed that NF-κB activator or TLR4 overexpression at least in part reversed the anti-inflammatory and anti-oxidative properties of garcinol in vitro. Taken together, the data suggest that garcinol could protect against cerebral I/R injury through attenuating inflammation and oxidative stress, and improving neurological function. The molecular mechanism might be related to its suppression of TLR4/NF-ĸB signal pathway.

Salvianolate reduces neuronal apoptosis by suppressing OGD-induced microglial activation.

AIMS: The aim of this study was to investigate the mechanism of pro-inflammatory phenotype transformation of microglia induced by oxygen-glucose deprivation (OGD), and how salvianolate regulates the polarization of microglia to exert neuroprotective effects. MAIN METHODS: The immunofluorescence and western blot experiments were used to verify the injury effect on neuronal cells after inflammatory polarization of microglia. Secondly, immunofluorescence staining and western blot were analyzed inflammatory phenotype of microglia and TLR4 signaling pathway after salvianolate treatment. RT-qPCR and ELISA assays were showed the levels of RNA and proteins of inflammatory factors in microglia. Finally, flow cytometry and western blot assay proved that salvianolate had a certain protective effect on neuronal injury after inhibiting the phenotype of microglia. KEY FINDINGS: The OGD condition could promote inflammation and activate of TLR4 signal pathway in microglia, and the polarization of microglia triggered caspase-3 signal pathway of neuronal cell. The optimal concentrations of salvianolate were incubated with microglia under OGD condition, which could reduce the reactive oxygen species (ROS) expression (P = 0.002) and also regulate the activity of SOD, CAT and GSH-px enzymes (P < 0.05). Moreover, salvianolate treatment could inhibit TLR4 signal pathway (P = 0.012), suppress the pro-inflammatory phenotype of microglia in OGD condition (P = 0.018), and reduce the expression of IL-6 and TNF-α (P < 0.05). Finally, neuronal damage induced by microglia under OGD condition was reversed after administration of the microglia supernatant after salvianolate treatment. SIGNIFICANCE: Salvianolate, as an antioxidant, plays a neuroprotective role by inhibiting the pro-inflammatory phenotype and decreasing the expression of ROS in microglia.

Sex differences in glucoprivic regulation of glycogen metabolism in hypothalamic primary astrocyte cultures: Role of estrogen receptor signaling.

Hypoglycemia causes sex-reliant changes in hypothalamic astrocyte glycogen metabolism in vivo. The role of nuclear versus membrane astrocyte estrogen receptors (ER) in glucoprivic regulation of glycogen is unclear. Here, primary hypothalamic astrocyte cultures were treated with selective ER antagonists during glucoprivation to investigate the hypothesis that ER mediate sex-specific glycogen responses to glucoprivation. Results show that glucoprivic down-regulation of glycogen synthase expression is mediated by transmembrane G protein-coupled ER-1 (GPER) signaling in each sex and estrogen receptor (ER)-beta (ERß) activity in females. Glucoprivic inhibition of glycogen phosphorylase involves GPER and ERß in females, but ER-independent mechanisms in males. GPER, ERß, and ER-alpha (ERα) inhibit or stimulate AMPK protein expression in male versus female astrocytes, respectively. Glucoprivic augmentation of phospho-AMPK profiles in male glia was opposed by GPER activation, whereas GPER and ERß suppress this protein in females. Astrocyte ERα and GPER content was down-regulated in each sex during glucose deficiency, whereas ERß levels was unaltered (males) or increased (females). Glucoprivation correspondingly elevated or diminished male versus female astrocyte glycogen content; ER antagonism reversed this response in males, but not females. Results identify distinctive ER variants involved in sex-similar versus sex-specific astrocyte protein responses to withdrawal of this substrate fuel. Notably, glucoprivation elicits a directional switch or gain-of-effect of GPER and ERß on specific glial protein profiles. Outcomes infer that ERs are crucial for glucoprivic regulation of astrocyte glycogen accumulation in males. Alternatively, estradiol may act independently of ER signaling to disassemble this reserve in females.

EGb761 Protects Brain Microvascular Endothelial Cells Against Oxygen-Glucose Deprivation-Induced Injury Through lncRNA Rmst/miR-150 Axis.

In the present study, we aimed to illustrate the roles and working mechanisms of long non-coding RNA (lncRNA) rhabdomyosarcoma 2-associated transcript (Rmst) and EGb761 in oxygen-glucose deprivation (OGD)-induced brain microvascular endothelial cells (BMECs). OGD exposure augmented the level of Rmst while reduced the expression of miR-150 in bEnd.3 cells. MiR-150 could directly bind to Rmst in bEnd.3 cells. Rmst silencing abrogated the inhibitory influences on the proliferation and migration and the promoting impact on the apoptosis of bEnd.3 cells caused by OGD exposure. Rmst overexpression intensified OGD-induced injury in bEnd.3 cells. OGD induced the injury of bEnd.3 cells through Rmst/miR-150 axis. EGb761 attenuated the damage in bEnd.3 cells induced by OGD through targeting Rmst/miR-150 axis. EGb761 might be an effective therapeutic agent to protect brain microvascular endothelial cells from hypoxia-ischemia induced injury.

ROS Mediate xCT-Dependent Cell Death in Human Breast Cancer Cells under Glucose Deprivation.

xCT, also known as solute carrier family 7 member 11 (SLC7A11), the light chain of the cystine/glutamate antiporter, is positively correlated with cancer progression due to antioxidant function. During glucose deprivation, the overexpression of xCT does not protect cancer cells but instead promotes cell death. Further understanding the mechanism of glucose deprivation-induced cell death is important for developing anticancer treatments targeting the glucose metabolism. In this study, we found that breast cancer cells with a high expression of xCT demonstrated increased levels of reactive oxygen species (ROS) and were more sensitive to glucose deprivation than the cells with a low expression of xCT. However, AMP-activated protein kinase (AMPK) did not significantly affect glucose-deprivation-induced cell death. The antioxidant N-acetyl-cysteine prevented glucose-deprivation-induced cell death, and the glutathione biosynthesis inhibitor L-buthionine-S, R-sulfoximine enhanced glucose-deprivation-induced cell death. The inhibition of xCT by sulfasalazine or a knockdown of xCT reduced the glucose-deprivation-increased ROS levels and glucose-deprivation-induced cell death. Glucose deprivation reduced the intracellular glutamate, and supplementation with α-ketoglutarate prevented the glucose-deprivation-increased ROS levels and rescued cell death. The knockdown of sirtuin-3 (SIRT3) further enhanced the ROS levels, and promoted xCT-related cell death after glucose deprivation. In conclusion, our results suggested that ROS play a critical role in xCT-dependent cell death in breast cancer cells under glucose deprivation.

Crotonylation at serine 46 impairs p53 activity.

Post-translational modifications (PTMs) play pivotal roles in controlling the stability and activity of the tumor suppressor p53 in response to distinct stressors. Here we report an unexpected finding of a short chain fatty acid modification of p53 in human cells. Crotonic acid (CA) treatment induces p53 crotonylation, but surprisingly reduces its protein, but not mRNA level, leading to inhibition of p53 activity in a dose dependent fashion. Surprisingly this crotonylation targets serine 46, instead of any predicted lysine residues, of p53, as detected in TCEP-probe labeled crotonylation and anti-crotonylated peptide antibody reaction assays. This is further confirmed by substitution of serine 46 with alanine, which abolishes p53 crotonylation in vitro and in cells. CA increases p53-dependent glycolytic activity, and augments cancer cell proliferation in response to metabolic or DNA damage stress. Since serine 46 is only found in human p53, our studies unveil an unconventional PTM unique for human p53, impairing its activity in response to CA. Because CA is likely produced by the gut microbiome, our results also predict that this type of PTM might play a role in early human colorectal neoplasia development by negating p53 activity without mutation of this tumor suppressor gene.

Mu-Xiang-You-Fang protects PC12 cells against OGD/R-induced autophagy via the AMPK/mTOR signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Mu-Xiang-You-Fang (MXYF) is a classic prescription of Hui medicine. It is composed of five herbs and has been used to treat ischemic stroke for many years. However, the potential pharmacological mechanisms of MXYF remain unclear. The present research is aimed to investigate the protective effect and possible mechanisms of MXYF treatment in an in vitro model of cerebral ischemia-reperfusion injury. MATERIALS AND METHODS: An oxygen-glucose deprivation and reperfusion (OGD/R) model of PC12 cells was established. The effect of MXYF on the cell viability after OGD/R injury was determined using a cell counting kit (CCK-8) assay. The colorimetric method was used to determine the lactate dehydrogenase (LDH) leakage rate. The calcium concentration was determined by the chemical fluorescence method, and mitochondrial membrane potential was determined using flow cytometry. Monodansylcadaverine (MDC) staining and electron microscopic analysis were then conducted to detect autophagy after oxygen-glucose deprivation and reperfusion in PC12 cells. Immunofluorescence and western blot analyses were used to detect the expression of proteins associated with autophagy. RESULTS: It was found that MXYF (1, 2, 4 µg/mL) could significantly increase cell viability and mitochondrial membrane potential and decrease the calcium concentration and LDH release rate in PC12 cells. After OGD/R injury in PC12 cells, the number of autophagosomes and autophagolysosome significantly increased. MXYF (4 µg/mL) inhibited the autophagy induced by OGD/R and inhibited the expression of LC3, beclin1, p-AMPK, and ULK1. In contrast, the expression of p-mTOR, p-p70s6k, and p62 was significantly enhanced. CONCLUSIONS: These findings suggest that MXYF inhibits autophagy after OGD/R-induced PC12 cell injury through the AMPK-mTOR pathway. Thus, MXYF might have therapeutic potential in treating ischemic stroke.