Lactate Protects Microglia and Neurons from Oxygen-Glucose Deprivation/Reoxygenation.

Lactate has received attention as a potential therapeutic intervention for brain diseases, particularly those including energy deficit, exacerbated inflammation, and disrupted redox status, such as cerebral ischemia. However, lactate roles in metabolic or signaling pathways in neural cells remain elusive in the hypoxic and ischemic contexts. Here, we tested the effects of lactate on the survival of a microglial (BV-2) and a neuronal (SH-SY5Y) cell lines during oxygen and glucose deprivation (OGD) or OGD followed by reoxygenation (OGD/R). Lactate signaling was studied by using 3,5-DHBA, an exogenous agonist of lactate receptor GPR81. Inhibition of lactate dehydrogenase (LDH) or monocarboxylate transporters (MCT), using oxamate or 4-CIN, respectively, was performed to evaluate the impact of lactate metabolization and transport on cell viability. The OGD lasted 6 h and the reoxygenation lasted 24 h following OGD (OGD/R). Cell viability, extracellular lactate concentrations, microglial intracellular pH and TNF-ɑ release, and neurite elongation were evaluated. Lactate or 3,5-DHBA treatment during OGD increased microglial survival during reoxygenation. Inhibition of lactate metabolism and transport impaired microglial and neuronal viability. OGD led to intracellular acidification in BV-2 cells, and reoxygenation increased the release of TNF-ɑ, which was reverted by lactate and 3,5-DHBA treatment. Our results suggest that lactate plays a dual role in OGD, acting as a metabolic and a signaling molecule in BV-2 and SH-SY5Y cells. Lactate metabolism and transport are vital for cell survival during OGD. Moreover, lactate treatment and GPR81 activation during OGD promote long-term adaptations that potentially protect cells against secondary cell death during reoxygenation.

Therapeutic Polymer-Based Cannabidiol Formulation: Tackling Neuroinflammation Associated with Ischemic Events in the Brain.

Cannabidiol (CBD) is the most relevant nonpsychostimulant phytocompound found in Cannabis sativa. CBD has been extensively studied and has been proposed as a therapeutic candidate for neuroinflammation-related conditions. However, being a highly lipophilic drug, it has several drawbacks for pharmaceutical use, including low solubility and high permeability. Synthetic polymers can be used as drug delivery systems to improve CBD's stability, half-life, and biodistribution. Here, we propose using a synthetic polymer as a nanoparticulate vehicle for CBD (NPCBD) to overcome the pharmacological drawbacks of free drugs. We tested the NPCBD-engineered system in the context of ischemic events in a relevant oxygen and glucose deprivation (OGD) model in primary cortical cells (PCC). Moreover, we have characterized the inflammatory response of relevant cell types, such as THP-1 (human monocytes), HMC3 (human microglia), and PCC, to NPCBD and observed a shift in the inflammatory state of the treated cells after the ischemic event. In addition, NPCBD exhibited a promising ability to restore mitochondrial function after OGD insult in both HMC3 and PCC cells at low doses of 1 and 0.2 µM CBD. Taken together, these results suggest the potential for preclinical use.

Ginsenoside Rg1 attenuates cerebral ischemia-reperfusion injury through inhibiting the inflammatory activation of microglia.

It is recognized that the cerebral ischemia/reperfusion (I/R) injury triggers inflammatory activation of microglia and supports microglia-driven neuronal damage. Our previous studies have shown that ginsenoside Rg1 had a significant protective effect on focal cerebral I/R injury in middle cerebral artery occlusion (MCAO) rats. However, the mechanism still needs further clarification. Here, we firstly reported that ginsenoside Rg1 effectively suppressed the inflammatory activation of brain microglia cells under I/R conditions depending on the inhibition of Toll-likereceptor4 (TLR4) proteins. In vivo experiments showed that the ginsenoside Rg1 administration could significantly improve the cognitive function of MCAO rats, and in vitro experimental data showed that ginsenoside Rg1 significantly alleviated neuronal damage via inhibiting the inflammatory response in microglia cells co-cultured under oxygen and glucose deprivation/reoxygenation (OGD/R) condition in gradient dependent. The mechanism study showed that the effect of ginsenoside Rg1 depends on the suppression of TLR4/MyD88/NF-κB and TLR4/TRIF/IRF-3 pathways in microglia cells. In a word, our research shows that ginsenoside Rg1 has great application potential in attenuating the cerebral I/R injury by targeting TLR4 protein in the microglia cells.

Gypenoside A Protects Human Myocardial Cells from Ischemia/Reperfusion Injury via the circ_0010729/miR-370-3p/RUNX1 Axis.

Ischemia/reperfusion (I/R) injury is one of the major causes of cardiovascular disease. Gypenoside A (GP), the main active component of Gynostemma pentaphyllum, alleviates myocardial I/R injury. Circular RNAs (circRNAs) and microRNAs (miRNAs) are involved in the I/R injury. We explored the protective effect of GP on human cardiomyocytes (HCMs) via the circ_0010729/miR-370-3p/RUNX1 axis. Overexpression of circ_0010729 abolished the effects of GP on HMC, such as suppression of apoptosis and increase in cell viability and proliferation. Overexpression of miR-370-3p reversed the effect of circ_0010729 overexpression, resulting in the stimulation of HMC viability and proliferation and inhibition of apoptosis. The knockdown of miR-370-3p suppressed the effects of GP in HCMs. RUNX1 silencing counteracted the effect of miR-370-3p knockdown and maintained GP-induced suppression of apoptosis and stimulation of HMC viability and proliferation. The levels of RUNX1 mRNA and protein were reduced in cells expressing miR-370-3p. In conclusion, this study confirmed that GP alleviated the I/R injury of myocardial cell via the circ_0010729/miR-370-3p/RUNX1 axis.

α-Lipoic acid increases the viability of nephrocytes and elevates sulfane sulfur level in plasma of patients with chronic kidney disease.

BACKGROUND: Kidney diseases are a major global health problem affecting millions of people. Despite this, there is as yet no effective drug therapy improving outcome in patients with renal disease. The aim of this study was to examine the nephroprotective effect of α-lipoic acid (ALA) in vitro and to examine the effect of ALA administered in vivo on the production of reactive sulfur species (RSS), including hydrogen sulfide (H2S) and compounds containing sulfane sulfur. METHODS: The effect of ALA was studied in vitro by determining the viability of human embryonic kidney cells (HEK293) in normoxic and hypoxic conditions as well as in vivo in two groups of chronic kidney disease (CKD) patients: non-dialyzed (ND) and undergoing continuous ambulatory peritoneal dialysis (PD) after 30 days of ALA supplementation. RESULTS: The results revealed that the viability of HEK293 cells was significantly decreased by hypoxic conditions, while ALA administered during hypoxia increased the viability to the level observed in normoxic conditions. Studies performed in plasma of CKD patients after ALA supplementation suggested that ALA did not affect the parameters of oxidative stress, while significantly increased the level of reactive sulfane sulfur in both ND and PD patients suffering from CKD. The results suggest that ALA can exert nephroprotective effects which are related to sulfane sulfur production.

[Ginsenoside Rg_1 ameliorates OGD/R-induced PC12 cell injury by inhibiting autography via IRE1-JNK-CHOP pathway].

This study investigated the protective effect of ginsenoside Rg_1(GRg_1) on oxygen and glucose deprivation/reoxygenation(OGD/R)-injured rat adrenal pheochromocytoma(PC12) cells and whether the underlying mechanism was related to the regulation of inositol-requiring enzyme 1(IRE1)-c-Jun N-terminal kinase(JNK)-C/EBP homologous protein(CHOP) signaling pathway. An OGD/R model was established in PC12 cells, and PC12 cells were randomly classified into control, model, OGD/R+GRg_1(0.1, 1, 10 µmol·L~(-1)), OGD/R+GRg_1+rapamycin(autophagy agonist), OGD/R+GRg_1+3-methyladenine(3-MA,autophagy inhibitor), OGD/R+GRg_1+tunicamycin(endoplasmic reticulum stress agonist), OGD/R+GRg_1+4-phenylbutyric acid(4-PBA, endoplasmic reticulum stress inhibitor), and OGD/R+GRg_1+3,5-dibromosalicylaldehyde(DBSA, IRE1 inhibitor) groups. Except the control group, the other groups were subjected to OGD/R treatment, i.e., oxygen and glucose deprivation for 6 h followed by reoxygenation for 6 h. Cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT) assay. Apoptosis was detected by Hoechst 33342 staining, and the fluorescence intensity of autophagosomes by the monodansylcadaverine(MDC) assay. Western blot was employed to determine the expression of autophagy-related proteins(Beclin1, LC3-Ⅱ, and p62) and the pathway-related proteins [IRE1, p-IRE1, JNK, p-JNK, glucose-regulated protein 78(GRP78), and CHOP]. The results showed that GRg_1 dose-dependently increased the viability of PC12 cells and down-regulated the expression of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, compared with the model group. Furthermore, GRg_1 decreased the apoptosis rate and MDC fluorescence intensity and up-regulated the expression of p62 protein. Compared with the OGD/R+GRg_1(10 µmol·L~(-1)) group, OGD/R+GRg_1+rapamycin and OGD/R+GRg_1+tunicamycin groups showed increased apoptosis rate and MDC fluorescence intensity, up-regulated protein levels of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, decreased relative cell survival rate, and down-regulated protein level of p62. The 3-MA, 4-PBA, and DBSA groups exerted the opposite effects. Taken together, GRg_1 may ameliorate OGD/R-induced PC12 cell injury by inhibiting autophagy via the IRE1-JNK-CHOP pathway.

LSD1 regulates the expressions of core cardiogenic transcription factors and cardiac genes in oxygen and glucose deprivation injured mice fibroblasts in vitro.

Cardiac reprogramming has emerged as a novel therapeutic approach to regenerating the damaged heart by directly converting endogenous cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs). Cardiac reprogramming requires the activation of the cardiogenic transcriptional program in concert with the repression of the fibroblastic transcriptional program. Lysine-specific demethylase 1 (LSD1) plays an instrumental role in many physiological processes such as cell growth, differentiation and metabolism. The epigenetic modifications of histones are essential for the accurate expression of genes in cardiomyocytes and the normal functioning of the heart. However, the effect of LSD1 in regulating the cardiogenic transcriptional program under myocardial ischemia/reperfusion (I/R) injury remains unclear. Thus, mice I/R injury was induced by 4 and 24 h reperfusion after 1-h occlusion of the left anterior descending coronary artery. The primary CFs and CMs were exposed under oxygen and glucose deprivation (OGD) to mimic I/R injury. The expression of LSD1 significantly decreased in I/R injured heart tissue and OGD-injured primary CFs and CM, and methylated histone presented a notable increase in OGD-injured primary CFs. Overexpression of LSD1 inhibited the injury of primary CFs induced by OGD, but showed limited inhibition on injured primary CMs. Under the OGD condition, LSD1 overexpression significantly increased cell viability, decreased cell apoptosis and reactive oxygen species (ROS) production of primary CFs. The expression of core cardiogenic transcription factors and cardiac genes were significantly decreased in OGD injured primary CFs, whereas LSD1 overexpression reversed the decrease of transcription factors and cardiac genes under the OGD condition. In conclusion, the overexpression of LSD1 has a protective role in I/R injury by inhibiting the histone methylation of primary CFs and regulates the expressions of core cardiogenic transcription factors and cardiac genes, which can prove to be a potential approach for direct cardiac reprogramming.

Calycosin attenuates the inflammatory damage of microglia induced by oxygen and glucose deprivation through the HMGB1/TLR4/NF-κB signaling pathway.

Stroke seriously threatens human life and health worldwide, but only very few effective stroke medicines are currently available. Our previous studies have indicated that the phytoestrogen calycosin exerts neuroprotective effects in cerebral ischemia and reperfusion injury rats. Therefore, the objective of this study is to further explore the protective effect of calycosin on inflammatory injury in microglia after oxygen-glucose deprivation/reoxygenation (OGD/R) and to clarify whether its protective effect is related to the HMGB1/TLR4/NF-κB signaling pathway. Here, the OGD/R model of rodent microglia is established in vitro to simulate cerebral ischemia-reperfusion injury. Through the CCK-8 test, ELISA, qRT-PCR, and western blot analysis, we find that the activity of microglia is decreased, the expressions of HMGB1 and TLR4 and the phosphorylation of NF-κB (p-NF-κB) are increased, and the releases of the inflammatory factors IL-6, IL-1ß, and TNF-α are increased after OGD/R. Pretreatment with calycosin could ameliorate these states, increase cell viability, reduce HMGB1, TLR4 and p-NF-κB expression, and reduce inflammatory cytokine production. In addition, the effect of calycosin is similar to that of TAK-242 (an inhibitor of TLR4), and the effect of the combined treatment is better than that of the single treatment. The results indicate that calycosin protects microglia from OGD/R injury and reduces the inflammatory response. Calycosin might alleviate cerebral ischemia-reperfusion injury by inhibiting the HMGB1/TLR4/NF-κB pathway.

A relatively low glucose promotes the proliferation of vascular endothelial cells by suppressing VEGFR2 O-GlcNAcylation and its proteasome degradation.

PURPOSE: Vascular endothelial growth factor receptors (VEGFRs) have been demonstrated to play a critical role in ischemic retinal diseases, as VEGFRs mediate hypoxia-induced neovascularization. Not only hypoxia, ischemia also induces the deficiency of glucose, yet its effects on VEGFR signal and neovascularization have seldom been studied. Bioinformatics analysis predicted that VEGFRs may be regulated by O-GlcNAcylation, while glucose deficiency influences the O-GlcNAcylation. METHODS: In this study, we treated human retinal microvascular endothelial cells with low glucose (LG) alone or in combination with low oxygen (oxygen and glucose deprivation, OGD). Cell viability and apoptosis rate were used to evaluate cell growth characters. RESULTS: LG (2.8 mmol/L) treatment induced mRNA and protein levels of VEGFR1, 2, 3 even in the presence of the protein synthesis inhibitor, cycloheximide (CHX), suggesting that the increase in VEGFR proteins is partially associated with post-translational modifications. Immunoprecipitation analysis showed that O-GlcNAc level was decreased by LG in both VEGFR1, 2, but a de-O-GlcNAc glycosylase inhibitor restored the O-GlcNAc levels. This inhibitor also abolished the LG-induced increase in VEGFR2 protein, whereas this effect was not disappeared in the presence of the proteasome inhibitor, MG132. Similar results were also observed under OGD condition. VEGFR2 knockdown more significantly retarded the growth of hRMECs and HUVECs than VEGFR1, 3 knockdown under LG and OGD conditions. CONCLUSIONS: A relatively low glucose suppressed O-GlcNAcylation in VEGFR2, whereby inhibiting its proteasome degradation; up-regulated VEGFR2 promoted the proliferation of vascular endothelial cells under ischemic condition.

In Vitro Model for Ischemic Stroke: Functional Analysis of Vascular Smooth Muscle Cells.

The Neurovascular Unit (NVU) is formed by vascular and neural cells controlling the cerebral hyperaemia. All the components are anatomically and functionally linked to each other, resulting in a highly efficient regulation of the cerebral blood flow, which, when interrupted, can lead to stroke. An ischemic stroke (IS) is the most common type of stroke with high rates of morbidity, mortality and disability. Therefore, it is of extreme importance to protect the functional and structural integrity of the NVU in patients with IS, understanding the mechanisms involved and how it affects each component of the NVU. Thus, the aim of this work is to analyse how the vascular smooth muscle cells from the rat middle cerebral artery function/react after an ischemic event. To mimic this event, primary cortical cultures were challenged to oxygen and glucose deprivation (OGD) for 4 h and 6 h, and the smooth muscle cells (SMCs) contractility was analysed after exposure to different media previously conditioned by the cortical cultures upon reperfusion. The results show a dual effect on the SMCs response to the vasorelaxant agent, only for cells exposed to the reperfusion media conditioned by neuron-glia cultures challenged by OGD, leading to increased relaxation of the SMCs for OGD 4 h, whereas for OGD 6 h the effect is reversed leading to contraction of the SMCs. These differences demonstrate that the astrocytes mediate the vasoactive response of vascular smooth muscle by releasing factors into the reperfusion medium, and the hypoxia time is fundamental for a beneficial/harmful response by the vascular smooth muscle.

Lysosomal calcium is modulated by STIM1/TRPML1 interaction which participates to neuronal survival during ischemic preconditioning.

A robust activity of the lysosomal Ca2+ channel TRPML1 is sufficient to correct cellular defects in neurodegeneration. Importantly, lysosomes are refilled by the endoplasmic reticulum (ER). However, it is unclear how TRPML1 function could be modulated by the ER. Here, we deal with this issue in rat primary cortical neurons exposed to different oxygen conditions affecting neuronal survival. Under normoxic conditions, TRPML1: (1) showed a wide distribution within soma and along neuronal processes; (2) was stimulated by the synthetic agonist ML-SA1 and the analog of its endogenous modulator, PI(3,5)P2 diC8; (3) its knockdown by siRNA strategy produced an ER Ca2+ accumulation; (4) co-localized and co-immunoprecipitated with the ER-located Ca2+ sensor stromal interacting molecule 1 (STIM1). In cortical neurons lacking STIM1, ML-SA1 and PI(3,5)P2 diC8 failed to induce Ca2+ release and, more deeply, they induced a negligible Ca2+ passage through the channel in neurons transfected with the genetically encoded Ca2+ indicator GCaMP3-ML1. Moreover, TRPML1/STIM1 interplay changed at low-oxygen conditions: both proteins were downregulated during the ischemic preconditioning (IPC) while during IPC followed by 1 hour of normoxia, at which STIM1 is upregulated, TRPML1 protein was reduced. However, during oxygen and glucose deprivation (OGD) followed by reoxygenation, TRPML1 and STIM1 proteins peaked at 8 hours of reoxygenation, when the proteins were co-immunoprecipitated and reactive oxygen species (ROS) hyperproduction was measured in cortical neurons. This may lead to a persistent TRPML1 Ca2+ release and lysosomal Ca2+ loss. Collectively, we showed a new modulation exerted by STIM1 on TRPML1 activity that may differently intervene during hypoxia to regulate organellar Ca2+ homeostasis.

Delavatine A Attenuates OGD/R-Caused PC12 Cell Injury and Apoptosis through Suppressing the MKK7/JNK Signaling Pathway.

Delavatine A (DA) is an unusual isoquinoline alkaloid with a novel skeleton isolated from Chinese folk medicine Incarvillea delavayi. Studies conducted in our lab have demonstrated that DA has potential anti-inflammatory activity in lipopolysaccharide (LPS)-treated BV-2 cells. DA, however, has not been studied for its protective effect on neuronal cells yet. Thus, to explore whether DA can protect neurons, oxygen and glucose deprivation/reperfusion (OGD/R)-injured PC12 cell and middle cerebral artery occlusion/reperfusion (MCAO/R) rat model were used to assess the protective efficacy of DA against OGD/R damaged PC12 cells and MCAO/R injured rats. Our results demonstrated that DA pretreatment (0.31-2.5 µM) dose-dependently increased cell survival and mitochondrial membrane potential (MMP), whereas it lowered the leakage of lactate dehydrogenase (LDH), intracellular cumulation of Ca2+, and overproduction of reactive oxygen species (ROS), and inhibited the apoptosis rate in OGD/R-injured PC12 cells. Western blot demonstrated that DA pretreatment lowered the expression of apoptotic proteins and repressed the activation of the mitogen-activated protein kinase kinase 7 (MKK7)/c-Jun N-terminal kinase (JNK) pathway. It was also found that the neuroprotective efficacy of DA was significantly reversed by co-treatment with the JNK agonist anisomycin, suggesting that DA reduced PC12 cell injury and apoptosis by suppressing the MKK7/JNK pathway. Furthermore, DA oral administration greatly alleviated the neurological dysfunction and reduced the infarct volume of MCAO/R rats. Taken together, DA could ameliorate OGD/R-caused PC12 cell injury and improve brain ischemia/reperfusion (I/R) damage in MCAO/R rats, and its neuroprotection might be attributed to suppressing the MKK7/JNK signaling pathway.

Coffee-Derived Phenolic Compounds Activate Nrf2 Antioxidant Pathway in I/R Injury In Vitro Model: A Nutritional Approach Preventing Age Related-Damages.

Age-related injuries are often connected to alterations in redox homeostasis. The imbalance between free radical oxygen species and endogenous antioxidants defenses could be associated with a growing risk of transient ischemic attack and stroke. In this context, a daily supply of dietary antioxidants could counteract oxidative stress occurring during ischemia/reperfusion injury (I/R), preventing brain damage. Here we investigated the potential antioxidant properties of coffee-derived circulating metabolites and a coffee pulp phytoextract, testing their efficacy as ROS scavengers in an in vitro model of ischemia. Indeed, the coffee fruit is an important source of phenolic compounds, such as chlorogenic acids, present both in the brewed seed and in the discarded pulp. Therefore, rat brain endothelial cells, subjected to oxygen and glucose deprivation (OGD) and recovery (ogR) to mimic reperfusion, were pretreated or not with coffee by-products. The results indicate that, under OGD/ogR, the ROS accumulation was reduced by coffee by-product. Additionally, the coffee extract activated the Nrf2 antioxidant pathway via Erk and Akt kinases phosphorylation, as shown by increased Nrf2 and HO-1 protein levels. The data indicate that the daily intake of coffee by-products as a dietary food supplement represents a potential nutritional strategy to counteract aging.

Therapeutic Potential of Highly Selective A3 Adenosine Receptor Ligands in the Central and Peripheral Nervous System.

Ligands of the Gi protein-coupled adenosine A3 receptor (A3R) are receiving increasing interest as attractive therapeutic tools for the treatment of a number of pathological conditions of the central and peripheral nervous systems (CNS and PNS, respectively). Their safe pharmacological profiles emerging from clinical trials on different pathologies (e.g., rheumatoid arthritis, psoriasis and fatty liver diseases) confer a realistic translational potential to these compounds, thus encouraging the investigation of highly selective agonists and antagonists of A3R. The present review summarizes information on the effect of latest-generation A3R ligands, not yet available in commerce, obtained by using different in vitro and in vivo models of various PNS- or CNS-related disorders. This review places particular focus on brain ischemia insults and colitis, where the prototypical A3R agonist, Cl-IB-MECA, and antagonist, MRS1523, have been used in research studies as reference compounds to explore the effects of latest-generation ligands on this receptor. The advantages and weaknesses of these compounds in terms of therapeutic potential are discussed.

[Mitophagy mediated by ligustilide relieves OGD/R-induced injury in HT22 cells].

Mitochondrion, as the main energy-supply organelle, is the key target region that determines neuronal survival and death during ischemia. When an ischemic stroke occurs, timely removal of damaged mitochondria is very important for improving mitochondrial function and repairing nerve damage. This study investigated the effect of ligustilide(LIG), an active ingredient of Chinese medicine, on mitochondrial function and mitophagy based on the oxygen and glucose deprivation/reperfusion(OGD/R)-induced injury model in HT22 cells. By OGD/R-induced injury model was induced in vitro, HT22 cells were pre-treated with LIG for 3 h, and the cell viability was detected by the CCK-8 assay. Immunofluorescence and flow cytometry were used to detect indicators related to mitochondrial function, such as mitochondrial membrane potential, calcium overload, and reactive oxygen species(ROS). Western blot was used to detect the expression of dynamin-related protein 1(Drp1, mitochondrial fission protein) and cleaved caspase-3(apoptotic protein). Immunofluorescence was used to observe the co-localization of the translocase of outer mitochondrial membrane 20(TOMM20, mitochondrial marker) and lysosome-associated membrane protein 2(LAMP2, autophagy marker). The results showed that LIG increased the cell viability of HT22 cells as compared with the conditions in the model group. Furthermore, LIG also inhibited the ROS release, calcium overload, and the decrease in mitochondrial membrane potential in HT22 cells after OGD/R-induced injury, facilitated Drp1 expression, and promoted the co-localization of TOMM20 and LAMP2. The findings indicate that LIG can improve the mitochondrial function after OGD/R-induced injury and promote mitophagy. When mitophagy inhibitor mdivi-1 was administered, the expression of apoptotic protein increased, suggesting that the neuroprotective effect of LIG may be related to the promotion of mitophagy.

Neuroprotective effects of astaxanthin against oxygen and glucose deprivation damage via the PI3K/Akt/GSK3ß/Nrf2 signalling pathway in vitro.

Astaxanthin (ATX), which is the most abundant flavonoid in propolis, has previously shown neuroprotective properties against cerebral ischaemia-induced apoptosis. However, the mechanisms by which ATX mediates its therapeutic effects are unclear. At present, we explored the underlying mechanisms involved in the protective effects of ATX via the phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase 3 beta (GSK3ß)/nuclear factor erythroid 2-related factor 2 (Nrf2) signalling pathway in SH-SY5Y cells. The PI3K/Akt inhibitor LY294002 and GSK3ß inhibitor LiCl were employed in this study. Pre-treatment with ATX for 24 hours significantly decreased the oxygen and glucose deprivation (OGD)-induced viability loss, reduced the proportion of apoptosis and regulated OGD-mediated reactive oxygen species (ROS) production. Furthermore, ATX suppressed OGD-caused mitochondrial membrane potential and decomposition of caspase-3 to cleaved caspase-3, and heightened the B-cell lymphoma 2 (Bcl-2)/Bax ratio. PI3K/Akt/GSK3ß/Nrf2 signalling pathway activation in SH-SY5Y cells was verified by Western blot. ATX and LiCl treatment raised the protein levels of p-Akt, p-GSK3ß, nucleus Nrf2 and haeme oxygenase 1 (HO-1). However, these protein expression levels decreased by treatment of LY294002. The above in vitro data indicate that ATX can confer neuroprotection against OGD-induced apoptosis via the PI3K/Akt/GSK3ß/Nrf2 signalling pathway.

MicroRNA-132 protects H9c2 cells against oxygen and glucose deprivation-evoked injury by targeting FOXO3A.

Myocardial ischemia/reperfusion injury (MIRI) is a clinically familiar disease, which possesses a great negative impact on human health. But, the effective treatment is still absent. MicroRNAs (miRNAs) have been testified to play a momentous role in MIRI. The purpose of the study aimed to probe the functions of miR-132 in oxygen and glucose deprivation (OGD)-evoked injury in H9c2 cells. miR-132 expression in H9c2 cells accompanied by OGD disposition was evaluated via real-time quantitative polymerase chain reaction. After miR-132 mimic and inhibitor transfections, the impacts of miR-132 on OGD-affected H9c2 cell viability, apoptosis, cell cycle, and the interrelated factors were appraised by exploiting cell counting kit-8, flow cytometry, and western blot analysis. FOXO3A expression was estimated in above-transfected cells, meanwhile, the correlation between miR-132 and FOXO3A was probed by dual-luciferase report assay. Ultimately, above mentioned cell processes were reassessed in H9c2 cells after preprocessing OGD administration and transfection with si-FOXO3A and si-NC plasmids. We got that OGD disposition obviously enhanced miR-132 expression in H9c2 cells. Overexpressed miR-132 evidently reversed OGD-evoked cell viability repression and apoptosis induction in H9c2 cells. In addition, overexpressed miR-132 mitigated OGD-evoked G0/G1 cell arrest by mediating p21, p27, and cyclin D1 expression. Repression of FOXO3A was observed in miR-132 mimic-transfected cells, which was also predicated as a direct gene of miR-132. We discovered that silenced FOXO3A alleviated OGD-evoked cell injury in H9c2 cells via facilitating cell viability, hindering apoptosis and restraining cell arrest at G0/G1 phase. In conclusion, these investigations corroborated that miR-132 exhibited the protective impacts on H9c2 cells against OGD-evoked injury via targeting FOXO3A.

Low doses of Perampanel protect striatal and hippocampal neurons against in vitro ischemia by reversing the ischemia-induced alteration of AMPA receptor subunit composition.

Energy depletion caused by ischemic brain insults may result in persistent neuronal depolarization accompanied by hyper-stimulation of ionotropic glutamate receptors and excitotoxic phenomena, possibly leading to cell death. The use of glutamate receptor antagonists, such as the AMPARs antagonist Perampanel (PER), might be a pharmacological approach to counteract the excessive over-activation of glutamate receptors providing neuroprotective effects. Using electrophysiological and molecular analyses, we investigated the effect of PER against in vitro ischemia obtained by oxygen and glucose deprivation (OGD) in rat slices of two brain structures particularly sensitive to ischemic insults, the nucleus striatum and the hippocampus. We found that in these regions PER was able to avoid the OGD-induced neuronal suffering, at low doses not reducing basal excitatory synaptic transmission and not altering long-term potentiation (LTP) induction. Furthermore, in both the analysed regions, PER blocked a pathological form of LTP, namely ischemic LTP (iLTP). Finally, we hypothesized that the protective effect of PER against OGD was due to its capability to normalize the altered synaptic localization and function of AMPAR subunits, occuring after an ischemic insult. Taken together these findings support the idea that PER is a drug potentially effective to counteract ischemic damage.

The function of RNase L and its degradation mechanism in cardiac acute ischemic injury.

RNase L is generally thought to play a key role in antiviral defenses. Although RNase L protein and mRNA are known to be highly expressed in myocardial tissue, there are few studies of the potential functions of RNase L in myocardial tissue. In this study, we tested the hypothesis that RNase L may be involved in the pathological process of cardiac ischemic injury. RNase L-overexpressing and RNase L knockdown H9c2 cell lines were subjected to the oxygen and glucose deprivation (OGD) model, and RNase L knockout mice were subjected to acute myocardial infarction surgical procedures to investigate the function of RNase L in ischemic heart injury. OGD induced abnormal aggregation of double-stranded RNA in H9c2 cells, activated RNase L within 6 h of OGD initiation, and mediated apoptosis via the c-Jun N-terminal kinase pathway. In addition, RNase L knockout mice were more tolerant of myocardial infarction, and this knockout protected heart function and prevented pathological ventricular remodeling. Notably, both in in vivo and in vitro experiments, RNase L was gradually diminished during prolonged ischemic injury, which we speculate is an adaptive protective response serving to reduce myocardial ischemic damage. These results suggest that RNase L plays a role in the pathological process of cardiac acute ischemic injury. It is first activated by ischemic injury, causing cardiomyocyte death, but gradually diminishes to protect the heart from further damage.

RSK3 mediates necroptosis by regulating phosphorylation of RIP3 in rat retinal ganglion cells.

Receptor-interacting protein 3 (RIP3) plays an important role in the necroptosis signaling pathway. Our previous studies have shown that the RIP3/mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis occurs in retinal ganglion cell line 5 (RGC-5) following oxygen-glucose deprivation (OGD). However, upstream regulatory pathways of RIP3 are yet to be uncovered. The purpose of the present study was to investigate the role of p90 ribosomal protein S6 kinase 3 (RSK3) in the phosphorylation of RIP3 in RGC-5 cell necroptosis following OGD. Our results showed that expression of RSK3, RIP3, and MLKL was upregulated in necroptosis of RGC-5 after OGD. A computer simulation based on our preliminary results indicated that RSK3 might interact with RIP3, which was subsequently confirmed by co-immunoprecipitation. Further, we found that the application of a specific RSK inhibitor, LJH685, or rsk3 small interfering RNA (siRNA), downregulated the phosphorylation of RIP3. However, the overexpression of rip3 did not affect the expression of RSK3, thereby indicating that RSK3 could be a possible upstream regulator of RIP3 phosphorylation in OGD-induced necroptosis of RGC-5 cells. Moreover, our in vivo results showed that pretreatment with LJH685 before acute high intraocular pressure episodes could reduce the necroptosis of retinal neurons and improve recovery of impaired visual function. Taken together, our findings suggested that RSK3 might work as an upstream regulator of RIP3 phosphorylation during RGC-5 necroptosis.