Oxygen-glucose Deprivation Followed By Reperfusion Research Articles

Tetramethylpyrazine Analogue T-006 Protects Neuronal and Endothelial Cells Against Oxidative Stress via PI3K/AKT/mTOR and Nrf2 Signaling.

T-006, a novel neuroprotective derivative of tetramethylpyrazine (TMP), exhibits multifunctional neuroprotective properties. T-006 has been shown to improve neurological and behavioral functions in animal models of ischemic stroke and neurodegenerative diseases. The present study aims to further elucidate the mechanisms underlying the protective effects of T-006 against oxidative injuries induced by glutamate or hypoxia. Mouse hippocampal HT22 cells were used to evaluate the neuroprotective effects of T-006 against glutamate-induced injuries, while mouse brain endothelial bEnd.3 cells were used to evaluate the cerebrovascular protective effects of T-006 against oxygen-glucose deprivation followed by reperfusion (OGD/R)-induced injuries. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were used to measure cell viability and oxidative stress. Western blot and immunofluorescence analyses of protein expression were used to study cell signaling pathways. T-006 exhibited significant protective effects in both oxidative injury models. In HT22 cells, T-006 reduced cell death and enhanced antioxidant capacity by upregulating mTOR and nuclear factor erythroid 2-related factor 2/Heme oxygenase-1 (Nrf2/HO-1) signaling. Similarly, in bEnd.3 cells, T-006 reduced oxidative injuries and preserved tight junction integrity through Nrf2/HO-1 upregulation. These effects were inhibited by LY294002, a Phosphoinositide 3-kinase (PI3K) inhibitor. T-006 may exert its neuroprotective and cerebrovascular protective effects via the regulation of PI3K/AKT-mediated pathways, which facilitate downstream mTOR and Nrf2 signaling, leading to improved cell survival and antioxidant defenses.

Folic Acid Deficiency Enhances the Tyr705 and Ser727 Phosphorylation of Mitochondrial STAT3 in In Vivo and In Vitro Models of Ischemic Stroke.

Ischemic stroke remains one of the most common causes of death and disability worldwide. The stroke patients with an inadequate intake of folic acid tend to have increased brain injury and poorer prognosis. However, the precise mechanisms underlying the harmful effects of folic acid deficiency (FD) in ischemic stroke is still elusive. Here, we aimed to test the hypothesis that mitochondrial localized STAT3 (mitoSTAT3) expression may be involved in the process of neuronal damage induced by FD in in vivo and in vitro models of ischemic stroke. Our results exhibited that FD increased infarct size and aggravated the damage of mitochondrial ultrastructure in ischemic brains. Meanwhile, FD upregulated the phosphorylation levels of mitoSTAT3 at Tyr705 (Y705) and Ser727 (S727) sites in the rat middle cerebral artery occlusion/reperfusion (MCAO/R) model and oxygen-glucose deprivation followed by reperfusion (OGD/R) N2a cells. Furthermore, the inhibition of JAK2 by AG490 led to a significant decrease in FD-induced phosphorylation of Y705, while S727 phosphorylation was unaffected. Conversely, U0126 and LY294002, which respectively inhibited phosphorylation of ERK1/2 and Akt, partially prevented S727 phosphorylation, but had limited effects on the level of pY705, suggesting that phosphorylation of Y705 and S727 is regulated via independent mechanisms in FD-treated brains.

Tomatidine protects against ischemic neuronal injury by improving lysosomal function

Cerebral ischemia is a severe neurological disorder with limited therapy. Autophagy refers to the intracellular degradation process via an autophagosome-lysosome pathway. Emerging studies indicated the neuroprotective effects of autophagy against ischemic neuronal injury, suggesting the potential neuroprotection of autophagy-inducing compounds. Tomatidine is a gut microbiota-derived metabolite from unripe tomatoes. Tomatidine activates autophagy either in mammal cells or C elegans. However, potential neuroprotection of tomatidine against ischemic neuronal injury has not been determined. In the present investigation, N2a cells and primary cultured mice cortical neurons were subjected to oxygen-glucose deprivation followed by reperfusion (OGD/R). Cell injury was determined by MTT and lactate dehydrogenase release. Autophagosomes and autolysosomes were visualized by transfecting mCherry-GFP-tandem fluorescent LC3. The protein levels of LC3, Cathepsin D, Cathepsin B, and transcription factor EB (TFEB) were detected by Western blot. Lysosomes were stained with LysoTracker Red and dequenched-bovine serum albumin (DQ-BSA red). Tomatidine alleviated OGD/R-induced injury in N2a cells and neurons. Interestingly, tomatidine treatment attenuated, rather than reinforced, the OGD/R-elevated LC3-II, which can be reversed by lysosome inhibitor. These results indicated enhanced lysosomal activity rather than autophagosome generation with tomatidine treatment in our models. Indeed, tomatidine increased the lysosome number, proteolytic activities, as well as the expression of Cathepsin D and Cathepsin B. In addition, tomatidine increased the expression and nucleus translocation of (TFEB). Besides, lysosomal inhibitors chloroquine and bafilomycin, but not wortmannin, abolished the protection of tomatidine. In conclusion, the present study revealed the neuroprotection of tomatidine against ischemic injury by promoting lysosomal activity, possibly with the involvement of TFEB-related mechanisms.

Blockade of HCN2 Channels Provides\xa0Neuroprotection\xa0Against Ischemic Injury via Accelerating Autophagic\xa0Degradation in Hippocampal Neurons

In the central nervous system, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential to maintain normal neuronal function. Recent studies have shown that HCN channels may be involved in the pathological process of ischemic brain injury, but the mechanisms remain unclear. Autophagy is activated in cerebral ischemia, but its role in cell death/survival remains controversial. In this study, our results showed that the HCN channel blocker ZD7288 remarkably decreased the percentage of apoptotic neurons and corrected the excessive autophagy induced by oxygen-glucose deprivation followed by reperfusion (OGD/R) in hippocampal HT22 neurons. Furthermore, in the OGD/R group, p-mTOR, p-ULK1 (Ser757), and p62 were significantly decreased, while p-ULK1 (Ser317), atg5, and beclin1 were remarkably increased. ZD7288 did not change the expression of p-ULK1 (Ser757), ULK1 (Ser317), p62, Beclin1, and atg5, which are involved in regulating autophagosome formation. Besides, we found that OGD/R induced a significant increase in Cathepsin D expression, but not LAMP-1. Treatment with ZD7288 at 10 μmol/L in the OGD/R group did not change the expression of cathepsin D and LAMP-1. However, chloroquine (CQ), which decreases autophagosome-lysosome fusion, eliminated the correction of excessive autophagy and neuroprotection by ZD7288. Besides, shRNA knockdown of HCN2 channels significantly reduced the accumulation of LC3-II and increased neuron survival in the OGD/R and transient global cerebral ischemia (TGCI) models, and CQ also eliminated the effects of HCN2-shRNA. Furthermore, we found that the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes decreased in Con-shRNA-transfected HT22 neurons exposed to OGD/R or CQ. In HCN2-shRNA-transfected HT22 neurons, the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes increased under OGD/R; however, the percentage was significantly decreased by the addition of CQ to HCN2-shRNA-transfected HT22 neurons. The present results demonstrated that blockade of HCN2 channels provides neuroprotection against OGD/R and TGCI by accelerating autophagic degradation attributable to the promotion of autophagosome and lysosome fusion.

REDD1 knockdown protects H9c2 cells against myocardial ischemia/reperfusion injury through Akt/mTORC1/Nrf2 pathway-ameliorated oxidative stress: An in vitro study

Oxidative stress plays a significant role involved in myocardial ischemia/reperfusion (MI/R) injury. The regulated in development and DNA damage response 1 (REDD1) is an mTORC1 inhibitor participating in response to hypoxia and oxidative stress. However, whether and how REDD1 is associated with MI/R injury are unclear. By investigating an in vitro model, we reveal that REDD1 is induced by HIF-1α in H9c2 cells subjected to oxygen/glucose deprivation followed by reperfusion (OGD/R). Further, cells depleted of REDD1 exhibit less OGD/R-induced injury, as evidenced by reduced lactate dehydrogenase (LDH) release and decreased apoptosis. Moreover, Nrf2 silencing abrogates REDD1 depletion-reduced reactive oxygen species (ROS) level and OGD/R-induced injury, indicating that the REDD1 depletion-mediated cellular protection is dependent on Nrf2-eliminated oxidative stress. Lastly, REDD1 depletion activates Akt/mTORC1 pathway following OGD/R treatment, and inhibition of this pathway using both LY294002 and rapamycin abrogates REDD1 effects. Altogether, these results suggest that REDD1 depletion protects H9c2 cells against OGD/R-induced injury through ameliorating oxidative stress, which is modulated by Akt/mTORC1/Nrf2 signaling. Our study may also reveal REDD1 as a potential therapeutic target for improving cardioprotection in MI/R injury treatment.

Madecassoside protects BV2 microglial cells from oxygen-glucose deprivation/reperfusion-induced injury via inhibition of the toll-like receptor 4 signaling pathway

In a previous study, the authors reported that madecassoside (MA) exerted a potent neuroprotective effect against cerebral ischemia-reperfusion (I/R) injury in rats, mediated by anti-oxidative, anti-inflammatory, and anti-apoptotic mechanisms. However, the cellular and molecular bases for its neuroprotective effects have not been fully elucidated. In this study, an in vitro ischemic model of oxygen-glucose deprivation followed by reperfusion (OGD/R) was used to investigate the role of the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB) pathway in the neuroprotective and anti-inflammatory effects of MA. BV2 microglia viability after OGD/R, treated with or without MA, was measured using the MTT assay. Messenger RNA and protein expression of pro-inflammatory cytokines (tumor necrosis factor α [TNF-α], interleukin-1β [IL-1β], interleukin-6 [IL-6]) were measured using real-time polymerase chain reaction (RT-PCR) and ELISA after OGD/R or lipopolysaccharide treatment. Expression of TLR4/MyD88 and NF-κB p65 were measured using RT-PCR, Western blotting, and immunofluorescence analysis. MA significantly rescued OGD/R-induced cytotoxicity in BV2 microglia. Meanwhile, MA suppressed the secretion of pro-inflammatory mediators, including TNF-α, IL-1β, and IL-6, induced by OGD/R or lipopolysaccharide in BV2 microglia. The mechanism of its neuroprotection and anti-inflammation from OGD/R may involve the inhibition of activation of TLR4 and MyD88 in BV2 microglia, and the blockage of NF-κB p65 nuclear translocation. MA exhibited a significant neuroprotective effect against I/R injury in both in vivo and in vitro experiments by attenuating microglia-mediated neuroinflammation via inhibition of the TLR4/MyD88/NF-κB signaling pathway.

Protective Effect of Salvianolic Acid A on Brain Endothelial Cells after Treatment with Deprivation and Reperfusion of Oxygen-glucose

ObjectiveSalvianolic acid A (SAA) has a significant protective effect on ischemia/reperfusion injury of brain. However, it is not clear for SAA to exert its cerebral protection by targeting at the microvascular endothelial cells of blood brain barrier (BBB). Our previous study demonstrated that SAA could hardly pass through the BBB. This present study was therefore designed to investigate the protective effect of SAA on brain microvascular endothelial cells (BMECs) induced by deprivation and reperfusion with oxygen-glucose. MethodsRat BMECs were treated with oxygen glucose deprivation (OGD), followed by reperfusion (OGD/R). Cell viability was assessed by MTT and the content of reactive oxygen species (ROS) in cells after OGD/R in the absence or presence of SAA. GC-MS based metabolomic platform was applied to evaluate the regulation of SAA on the cellular metabolic perturbation induced by OGD/R. ResultsOGD/R significantly increased the production of intracellular reactive oxygen species (ROS), and decreased the activity of cells. SAA significantly reduced ROS and improve the cell viability. Metabolomic study revealed distinct perturbation of metabolic pathways of energy metabolism in the BMEC induced by OGD/R, while SAA significantly regulated the perturbed metabolism involved in energy metabolism pathways, especially for intermediates in TCA cycle. ConclusionSAA shows protective effects on BMECs involved in central nervous system.

Down-regulation of lncRNA KCNQ1OT1 protects against myocardial ischemia/reperfusion injury following acute myocardial infarction

This study aimed to investigate the protective effects of long non-coding RNA KCNQ1OT1 against myocardial ischemia/reperfusion (I/R) injury following acute myocardial infarction, as well as its regulatory mechanism. We used the cardiac muscle H9c2 cells under condition of oxygen glucose deprivation followed by reperfusion (OGD/R) to induce myocardial I/R injury. Then H9C2 cells were transfected with si-NC, si-KCNQ1OT1, pc-NC, pc-KCNQ1OT1, si-AdipoR1 and si-AdipoR2, respectively. The myocardial cell viability and apoptosis were respectively detected. In addition, the expression levels of inflammatory factors, apoptosis-related proteins and p38 MAPK/NF-κB pathway-related proteins were detected. Besides, an inhibitor of p38 MAPK/NF-κB pathway SB203580 was used to treat cells to verify the relationship between KCNQ1OT1 and p38 MAPK/NF-κB pathway. The expression of KCNQ1OT1 was significantly up-regulated in OGD/R-induced myocardial H9C2 cells. The OGD/R-induced decreased cell viability and AdipoR1 expression could be reversed after suppression of KCNQ1OT1. In addition, suppression of KCNQ1OT1 reduced OGD/R-induced increased expressions of TNF-α, IL-6 and IL-1β and OGD/R-induced increased cell apoptosis, which were reversed after knockdown of AdipoR1. Besides, suppression of KCNQ1OT1 significantly down-regulated the OGD/R-induced increased expression of p-p38 and p-NF-κB, which were also reversed after knockdown of AdipoR1. Moreover, SB203580, an inhibitor of p38 MAPK/NF-κB signal pathway, could further enhance the inhibitory effects of KCNQ1OT1 suppression on the expression of p-p38, TNF-α, IL-6, IL-1β and p-NF-κB in OGD/R-induced myocardial H9C2 cells. Suppression of KCNQ1OT1 may prevent myocardial I/R injury following acute myocardial infarction via regulating AdipoR1 and involving in p38 MAPK/NF-κB signal pathway.

Tanshinone IIA Protects Hippocampal Neuronal Cells from Reactive Oxygen Species Through Changes in Autophagy and Activation of Phosphatidylinositol 3-Kinase, Protein Kinas B, and Mechanistic Target of Rapamycin Pathways.

Background:Tanshinone IIA is a key active ingredient of danshen, which is derived from the dried root or rhizome of Salviae miltiorrhizae Bge. The tanshinone IIA has protective effects against the focal cerebral ischemic injury. However, the underlying mechanisms remain unclear.Methods:An in vitro model of cerebral ischemia was established by subjecting cultures of hippocampal neuronal cells to oxygen-glucose deprivation followed by reperfusion (OGD/R). The probes of 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and 5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine,iodide (JC-1) were used to determine the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production. Western-blot was used to detect the expression of proteins in HT-22 cells.Results:The results of cell proliferative assays showed that the tanshinone IIA attenuated OGD/R-mediated neuronal cell death, with the evidence of increased cell viability. In addition, OGD/R exposure led to increase the levels of intracellular reactive oxygen species (ROS), which were significantly suppressed by tanshinone IIA treatment. Furthermore, tanshinone IIA treatment inhibited elevations in MMP and autophagy following exposure to OGD/R. Additionally, OGD/R promoted cell death with concomitant inhibiting phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of Rapamycin (mTOR) pathway, which was reversed by tanshinone IIA.Conclusion:These results suggest that the tanshinone IIA protects against OGD/R-mediated cell death in HT-22 cells, in part, due to activating PI3K/Akt/mTOR pathway.

Britanin Ameliorates Cerebral Ischemia-Reperfusion Injury by Inducing the Nrf2 Protective Pathway.

Oxidative stress is considered the major cause of tissue injury after cerebral ischemia. The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is one of the most important defensive mechanisms against oxidative stresses and has been confirmed as a target for stroke treatment. Thus, we desired to find new Nrf2 activators and test their neuronal protective activity both in vivo and in vitro. The herb-derived compound, Britanin, is a potent inducer of the Nrf2 system. Britanin can induce the expression of protective enzymes and reverse oxygen-glucose deprivation, followed by reperfusion (OGD-R)-induced neuronal injury in primary cortical neurons in vitro. Furthermore, the administration of Britanin significantly ameliorated middle cerebral artery occlusion-reperfusion (MCAO-R) insult in vivo. We report here the crystal structure of the complex of Britanin and the BTB domain of Keap1. Britanin selectively binds to a conserved cysteine residue, cysteine 151, of Keap1 and inhibits Keap1-mediated ubiquitination of Nrf2, leading to induction of the Nrf2 pathway. Britanin is a potent inducer of Nrf2. The complex crystal structure of Britanin and the BTB domain of Keap1 help clarify the mechanism of Nrf2 induction. Britanin was proven to protect primary cortical neurons against OGD-R-induced injury in an Nrf2-dependant way. Additionally, Britanin had excellent cerebroprotective effect in an MCAO-R model. Our results demonstrate that the natural product Britanin with potent Nrf2-activating and neural protective activities both in vitro and in vivo could be developed into a cerebroprotective therapeutic agent. Antioxid. Redox Signal. 27, 754-768.

Neuroprotective effects of syringic acid against OGD/R-induced injury in cultured hippocampal neuronal cells

Cerebral ischemic injury and treatment are important topics in neurological science. In the present study, an invitro model of cerebral ischemia was established by subjecting primary cultures of hippocampal neuronal cells to oxygen-glucose deprivation followed by reperfusion(OGD/R), in order to evaluate the possible neuroprotective role of syringic acid(SA). The results of 3-(4,5-dimethylthiazol‑2-yl)-2,5-diphenyltetrazolium bromide(MTT) and lactate dehydrogenase(LDH) assays showed that pre-treatment with SA(0.1, 1, 10, and 20µM) attenuated OGD/R-induced neuronal injury in a dose-dependent manner, with evidence of increased cell viability and decreased LDH leakage. In addition, oxidative stress markers were evaluated using commercial kits, and the results demonstrated that OGD/R exposure induced distinct oxidative stress, accompanied by elevated levels of intracellular reactive oxygen species(ROS) and malondialdehyde(MDA) production, and reduced activity of the antioxidant enzyme superoxide dismutase(SOD), which were dose-dependently restored by pre-treatment with SA. In addition, the concentration of intracellular free calcium [Ca2+]i and mitochondrial membrane potential(MMP or Δψm) were determined in order to evaluate the degree of neuronal damage by performing flow cytometric analysis and observing the cells under a fluorescence microscope, respectively. We demonstrated that pre-treatment with SA inhibited elevations in [Ca2+]i, whereas it increased the MMP dose-dependently following exposure to OGD/R. Western blot analysis revealed that OGD/R promoted cell apoptosis with concomitant increases in Bax and caspase-3 expression, and reduced Bcl-2 expression, which was reversed by pre‑treatment with SA in a dose-dependent manner. Moreover, these effects were mediated through the JNK and p38 pathways, as pre‑treatment with SA inhibited the OGD/R-induced increase in phosphorylated(p-)JNK and p-p38 expression. Taken together, these results suggested that SA exerted strong neuroprotective effects in hippocampal neuronal cells, which may be attributed to the attenuation of OGD/R-induced cell injury through the JNK and p38 signaling pathways.

HspB8 mediates neuroprotection against OGD/R in N2A cells through the phosphoinositide 3-kinase/Akt pathway

In a previous study, we found that Heat shock protein B8 (HspB8) overexpression could prevent the apoptosis and reduced cell viability induced by OGD/R and showed that the neuroprotective effect of HspB8 was mediated by inhibition of the mitochondrial apoptotic pathway. In recent study, HspB8 has been shown to protect the heart against ischemia/reperfusion (I/R) injury via activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, whether this protective effect applied to brain I/R injury remained unexplored. To further test the mechanism of HspB8's effects in brain, we used oxygen-glucose deprivation followed by reperfusion (OGD/R), an in vitro model of ischemia to examine the involvement of PI3K/Akt signaling by treating mouse neuroblastoma cells (N2A cells) (untransfected or transfected with an HspB8 expression vector) with the PI3K inhibitor LY294002 before OGD/R. Our results revealed that the apoptosis-suppressing effect of HspB8 was mediated by the PI3K/Akt pathway. Therefore, HspB8 protected the N2A cells against OGD/R insult, possibly by activating the PI3K/Akt signaling pathway.

CIC-3 chloride channel blockade protects mouse photoreceptor-derived 661W cells against ischemia-reperfusion-induced injury in vitro

Exposure to ischemia/reperfusion leads to the development and progression of retinal degenerative diseases. However, the exact mechanisms are not fully understood. In this article, the role of CIC-3 chloride channel in OGD-R (oxygen-glucose deprivation followed by reperfusion)-induced retinal damage was examined. Mouse photoreceptor-derived 661W cells were treated with the CIC-3 antisense oligonucleotide before exposure to OGD-R. Cell viability, mitochondrial membrane potential, cytochrome-c level, DNA fragmentation, caspase activity and protein expression were detected. Pretreatment of 661W cells with CIC-3 antisense oligonucleotide significantly decreased OGD-R-mediated toxicity. In addition, apoptosis-related biochemical indicators showed that pre-incubation of CIC-3 antisense oligonucleotide would elevate the mitochondrial membrane potential, decrease the release of cytochrome-c as well as formation of DNA fragmentation, and inhibit activities of caspase-3 and caspase- 9 in exogenous OGD-R-treated 661W cells. Moreover, treatment with CIC-3 antisense oligonucleotide changed the expression of apoptosis-related protein. These results suggest that CIC-3 chloride channel mediates OGD-R-induced apoptosis, at least partially through mitochondrial membrane potential pathway and increasing the levels of proapoptotic molecules in 661W cells. CIC-3 chloride channel blockade may provide a new therapeutic approach for preventing ischemia/reperfusion-induced retinal neural damage.

Cardioprotective effect of propofol against oxygen glucose deprivation and reperfusion injury in H9c2 cells.

Background. The intravenous anesthetic propofol is reported to be a cardioprotective agent against ischemic-reperfusion injury in the heart. However, the regulatory mechanism still remains unclear. Methods. In this study, we used H9c2 cell line under condition of oxygen glucose deprivation (OGD) followed by reperfusion (OGD/R) to induce in vitro cardiomyocytes ischemia-reperfusion injury. Propofol (5, 10, and 20 μM) was added to the cell cultures before and during the OGD/R phases to investigate the underlying mechanism. Results. Our data showed that OGD/R decreased cell viability, and increased lactate dehydrogenase leakage, and reactive oxygen species and malondialdehyde production in H9c2 cells, all of which were significantly reversed by propofol. Moreover, we found that propofol increased both the activities and protein expressions of superoxide dismutase and catalase. In addition, propofol increased FoxO1 expression in a dose-dependent manner and inhibited p-AMPK formation significantly. Conclusions. These results indicate that the propofol might exert its antioxidative effect through FoxO1 in H9c2 cells, and it has a potential therapeutic effect on cardiac disorders involved in oxidative stress.

Mulberroside a protects against ischemic impairment in primary culture of rat cortical neurons after oxygen-glucose deprivation followed by reperfusion

Mulberroside A is a natural polyhydroxylated stilbene compound present at relatively high abundance in the roots and twigs of Morus alba L. It is known for its nephroprotective, hypoglycemic, and antidiabetic effects. Because its metabolite, oxyresveratrol, possessed purported anti-inflammatory and neuroprotective effects, we proposed that mulberroside A may elicit neuroprotective effects that can be used in the treatment of brain ischemic injury. Therefore, we decided to investigate the pharmacological properties of mulberroside A in primary culture of rat cortical neurons after oxygen-glucose deprivation followed by reperfusion (OGD/R), evaluating its ability to counteract the hypoxia-ischemia impairment. The results showed that mulberroside A elicited neuroprotective effects comparable to nimodipine. The mechanistic studies showed that mulberroside A decreased the expressions of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 and inhibited the activation of NALP3, caspase-1, and nuclear factor-κB and the phosphorylation of extracellular signal-regulated protein kinases, the c-Jun N-terminal kinase, and p38, exhibiting anti-inflammatory antiapoptotic effects. Our results also further demonstrate that the proinflammatory cytokines of IL-1β, IL-6, and TNF-α are promising targets for treatment of cerebral ischemic injury. Although further investigation is required for its development, all of these findings led us to speculate that mulberroside A is a candidate for the treatment of ischemic stroke, which would act as a multifactorial neuroprotectant.

PP1γ functionally augments the alternative splicing of CaMKIIδ through interaction with ASF

Protein phosphatase 1 (PP1) and Ca2+/calmodulin-dependent protein kinase δ (CaMKIIδ) are upregulated in heart disorders. Alternative splicing factor (ASF), a major splice factor for CaMKIIδ splicing, can be regulated by both protein kinase and phosphatase. Here we determine the role of PP1 isoforms in ASF-mediated splicing of CaMKIIδ in cells. We found that 1) PP1γ, but not α or β isoform, enhanced the splicing of CaMKIIδ in HEK293T cells; 2) PP1γ promoted the function of ASF, evidenced by the existence of ASF-PP1γ association as well as the PP1γ overexpression- or silencing-mediated change in CaMKIIδ splicing in ASF-transfected HEK293T cells; 3) CaMKIIδ splicing was promoted by overexpression of PP1γ and impaired by application of PP1 inhibitor 1 (I1PP1) or pharmacological inhibitor tautomycetin in primary cardiomyocytes; 4) CaMKIIδ splicing and enhancement of ASF-PP1γ association induced by oxygen-glucose deprivation followed by reperfusion (OGD/R) were potentiated by overexpression of PP1γ and suppressed by inhibition of PP1γ with I1PP1 or tautomycetin in primary cardiomyocytes; 5) functionally, overexpression and inhibition of PP1γ, respectively, potentiated or suppressed the apoptosis and Bax/Bcl-2 ratio, which were associated with the enhanced activity of CaMKII in OGD/R-stimulated cardiomyocytes; and 6) CaMKII was required for the OGD/R induced- and PP1γ exacerbated-apoptosis of cardiomyocytes, evidenced by a specific inhibitor of CaMKII KN93, but not its structural analog KN92, attenuating the apoptosis and Bax/Bcl-2 ratio in OGD/R and PP1γ-treated cells. In conclusion, our results show that PP1γ promotes the alternative splicing of CaMKIIδ through its interacting with ASF, exacerbating OGD/R-triggered apoptosis in primary cardiomyocytes.

Isoquercetin protects cortical neurons from oxygen–glucose deprivation–reperfusion induced injury via suppression of TLR4–NF-кB signal pathway

In the present study, oxygen–glucose deprivation followed by reperfusion (OGD/R), an in vitro model of ischemia, was used to evaluate the neuroprotective effect of isoquercetin in primary culture of rat cortical neuronal cells. It was found that isoquercetin administered prior to the insult could prevent OGD/R-induced intracellular calcium concentrations ([Ca2+]i) increase, lactate dehydrogenase (LDH) release and cell viability decrease. For the first time, isoquercetin is described as a neuroprotective agent that potentially explains the alleviation and prevention from OGD/R-induced injury in neurons. Mechanistic studies showed that the neuroprotective effect of isoquercetin was carried out by anti-inflammatory signaling pathway of inhibiting protein expression of toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB), and mRNA expression of TNF-α and IL-6, accompanied by the anti-apoptotic signaling pathway of deactivation of extracellular-regulated kinase (ERK), Jun kinase (JNK) and p38, and inhibition of activity of caspase-3. Therefore, these studies highlighted the confirmation of isoquercetin, a flavonoid compound, as an anti-inflammation and anti-apoptosis factor which might be used as a therapeutic strategy for the ischemia/reperfusion (I/R) brain injury and related diseases.

4-Hydroxybenzyl Alcohol Confers Neuroprotection Through Up-Regulation of Antioxidant Protein Expression

An herb-derived phenolic compound, 4-hydroxybenzyl alcohol (4-HBA), exhibits beneficial effects in cerebral ischemic injury. However, the molecular mechanisms underlying this observation remain unclear. Here we used an in vitro ischemic model of oxygen-glucose deprivation followed by reperfusion (OGD/R) and an in vivo ischemic model of middle cerebral artery occlusion to investigate the relevant neuroprotective mechanisms. We demonstrated that 4-HBA reduced the neuronal injury, LDH release, and up-regulation of 8-hydroxydeoxyguanosine (8-OHdG) induced by OGD/R. Furthermore, 4-HBA reduced the cerebral infarct size and improved the behavioral parameters after cerebral ischemia. These neuroprotective effects may be conferred by the 4-HBA mediated upregulation of the transcription factor nuclear factor E2-related factor 2 (Nrf2), peroxiredoxin 6 (Prdx6) and protein disulfide isomerase (PDI) by the use of 4-HBA. Interestingly, LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocked the increase in phosphorylation of Akt and abolished the neuroprotection associated with 4-HBA. Our results suggested that 4-HBA protects neurons against cerebral ischemic injury, and this neuroprotection may occur through upregulation of Nrf2, Prdx6, and PDI expression via the PI3K/Akt pathway.

Hyperoside protects cortical neurons from oxygen–glucose deprivation–reperfusion induced injury via nitric oxide signal pathway

Hyperoside is a flavonoid compound and widely used in clinic to relieve pain and improve cardiovascular functions. However, the effects of hyperoside on ischemic neurons and the molecular mechanisms remain unclear. Here, we used an in vitro ischemic model of oxygen–glucose deprivation followed by reperfusion (OGD-R) to investigate the protective effects of hyperoside on ischemic neuron injury and further explore the possible related mechanisms. Our results demonstrated that hyperoside protected cultured cortical neurons from OGD-R injury, it also relieved glutamate-induced neuronal injury and NMDA-induced [Ca2+]i elevation. As for the mechanisms, hyperoside firstly attenuated the phosphorylation of CaMKII caused by OGD-R lesions. Meanwhile, hyperoside lessened iNOS expression induced by OGD-R via inhibition of NF-κB activation. Furthermore, ameliorating of ERK, JNK and Bcl-2 family-related apoptotic signaling pathways were also involved in the neuroprotection of hyperoside. Taken together, these studies revealed that hyperoside had protective effects on neuronal ischemia-reperfusion impairment, which was related to the regulation of nitric oxide signaling pathway.

Protection by borneol on cortical neurons against oxygen-glucose deprivation/reperfusion: involvement of anti-oxidation and anti-inflammation through nuclear transcription factor κappaB signaling pathway

Borneol, a terpene and bicyclic organic compound found in several species, can easily penetrate the blood-brain barrier (BBB) and helps the absorption of many agents through BBB in the brain, but there has been no study about its direct action on neurons in the CNS. In the present study, we used an in vitro ischemic model of oxygen-glucose deprivation followed by reperfusion (OGD/R) to investigate the neuroprotective effects of borneol and the related mechanisms. We demonstrated that borneol reversed OGD/R-induced neuronal injury, nuclear condensation, intracellular reactive oxygen species (ROS) generation, and mitochondrial membrane potential dissipation. The elevation of nitric oxide (NO), the increase of inducible nitric oxide synthase (iNOS) enzymatic activity and the upregulation of iNOS expression were also attenuated by borneol. The inhibition of caspase-related apoptotic signaling pathway was consistently involved in the neuroprotection afforded by borneol. Meanwhile, borneol inhibited proinflammatory factor release and IκBα degradation, and blocked nuclear transcription factor κappaB (NF-κB) p65 nuclear translocation induced by OGD/R. On the other hand, borneol did not show obvious effect on the inhibition of phospho-IKKα activation. Furthermore, it failed to affect the OGD/R-induced enhanced level of phospho-SAPK/JNK. In conclusion, our study indicated that borneol protects against cerebral ischemia/reperfusion injury through multifunctional cytoprotective pathways. The mechanisms of this reversal from OGD/R may be involved in the alleviation of intracellular ROS and iNOS/NO pathway, inhibition of inflammatory factor release and depression of caspase-related apoptosis. Among these effects, the inhibition of IκBα-NF-κB and translocation signaling pathway might play a significant role in the neuroprotection of borneol.