Multi-level differential characterization of polysaccharides and the cardiomyocyte protection simultaneously from three raw and processed ginseng varieties

BackgroundGrowth arrest and DNA-damage inducible protein 45 beta (Gadd45b) is serving as a neuronal activity sensor. Brain ischemia induces the expression of Gadd45b, which stimulates recovery after stroke and may play a protective role in cerebral ischemia. However, little is known of the molecular mechanisms of how Gadd45b expression regulated and the down-stream targets in brain ischemia. Here, using an oxygen-glucose deprivation and reperfusion (OGD/R) model, we identified Huwe1/Mule/ARF-BP1, a HECT domain containing ubiquitin ligase, involved in the control of Gadd45b protein level. In this study, we also investigated the role of Huwe1-Gadd45b mediated pathway in BDNF methylation.ResultsWe found that the depletion of Huwe1 by lentivirus shRNA mediated interference significantly increased the expression of Gadd45b and BDNF at 24 h after OGD. Moreover, treatment with Cycloheximide (CHX) inhibited endogenous expression of Gadd45b, and promoted expression of Gadd45b after co-treated with lentivirus shRNA-Huwe1. Inhibition of Gadd45b by lentivirus shRNA decreased the expression levels of brain derived neurotrophic factor (BDNF) and phosphorylated cAMP response element-binding protein (p-CREB) pathway, while inhibition of Huwe1 increased the expression levels of BDNF and p-CREB. Moreover, shRNA-Huwe1 treatment decreased the methylation level of the fifth CpG islands (123 bp apart from BDNF IXa), while shRNA-Gadd45b treatment increased the methylation level of the forth CpG islands (105 bp apart from BDNF IXa).ConclusionsThese findings suggested that Huwe1 involved in the regulation of Gadd45b expression under OGD/R, providing a novel route for neurons following cerebral ischemia-reperfusion injury. It also indicated that the methylation of BDNF IXa was affected by Gadd45b as well as Huwe1 in the OGD/R model.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0178-y) contains supplementary material, which is available to authorized users.

A quinolinyl resveratrol derivative alleviates acute ischemic stroke injury by promoting mitophagy for neuroprotection via targeting CK2α’

Nucleotide-binding oligomerization domain protein 1 enhances oxygen-glucose deprivation and reperfusion injury in cortical neurons via activation of endoplasmic reticulum stress-mediated autophagy

Inflammatory response contributes to brain injury after ischemia and reperfusion (I/R). Our previous literature has shown isoquercetin plays an important role in protecting against cerebral I/R injury. The present study was conducted to further investigate the effect of isoquercetin on inflammation-induced neuronal injury in I/R rats with the involvement of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and inhibitor of NF-κB (I-κB)/nuclear factor-kappa B (NF-κB) signaling pathway mediated by Toll-like receptor 4 (TLR4) and C5a receptor 1 (C5aR1). In vivo middle cerebral artery occlusion and reperfusion (MCAO/R) rat model and in vitro oxygen-glucose deprivation and reperfusion (OGD/R) neuron model were used. MCAO/R induced neurological deficits, cell apoptosis, and release of cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in ischemic brain in rats. Simultaneously, the expression of TLR4 and C5aR1 was significantly up-regulated in both MCAO/R rats and OGD/R neurons, accompanied with the inhibition of cAMP/PKA signaling and activation of I-κB/NF-κB signaling in the cortex of MCAO/R rats. Over-expression of C5aR1 in neurons induced decrease of cell viability, exerting similar effects with OGD/R injury. Isoquercetin acted as a neuroprotective agent against I/R brain injury to suppress inflammatory response and improve cell recovery by inhibiting TLR4 and C5aR1 expression, promoting cAMP/PKA activation, and inhibiting I-κB/NF-κB activation and Caspase 3 expression. TLR4 and C5aR1 contributed to inflammation and apoptosis via activating cAMP/PKA/I-κB/NF-κB signaling during cerebral I/R, suggesting that this signaling pathway may be a potent therapeutic target in ischemic stroke. Isoquercetin was identified as a neuroprotective agent, which maybe a promising therapeutic agent used for the treatment of ischemic stroke and related diseases.

Hydrogen-rich saline protects rat from oxygen glucose deprivation and reperusion-induced apoptosis through VDAC1 via Bcl-2

Heart failure (HF) has become one of the severe public health problems. The detailed role of mitochondrial function in HF was still unclear. Benzoylaconine (BAC) is a traditional Chinese medicine, but its role in HF still needs to be explored. In this study, oxygen-glucose deprivation and reperfusion (OGD/R) was executed to mimic the injury of H9C2 cells in HF. The viability of H9C2 cells was assessed via MTT assay. OGD/R treatment markedly decreased the viability of H9C2 cells, but BAC treatment evidently increased the viability of OGD/R-treated H9C2 cells. The apoptosis of H9C2 was enhanced by OGD/R treatment but suppressed by BAC treatment. The mitochondrial membrane potential was evaluated via JC-1 assay. BAC improved the mitochondrial function and suppressed oxidative stress in OGD/R-treated H9C2 cells. Moreover, Western blot analysis revealed that the protein expression of p-AMPK and PGC-1α were reduced in OGD/R-treated H9C2 cells, which was reversed by BAC. Rescue assays indicated that AMPK attenuation reversed the BAC-mediated protective effect on OGD/R-treated cardiomyocytes. Moreover, BAC alleviated myocardial injury in vivo. In a word, BAC modulated the mitochondrial function in OGD/R-induced cardiomyocyte injury by activation of the AMPK/PGC-1 axis. The findings might provide support for the application of BAC in the treatment of HF.

Emerging studies show that circRNA catenin beta 1 (circCTNNB1) plays a critical role in cancer. However, the expression and function of circCTNNB1 in cerebral ischemia/reperfusion injury (IRI) have not been reported. The present study discovered that circCTNNB1 and scavenger receptor class B type 1 (SRB1) expression levels were significantly down-regulated in mouse astrocytes (mAS) treated with oxygen glucose deprivation and reperfusion (OGD/R), and similar results were observed in a mouse middle cerebral artery occlusion model. Overexpression of circCTNNB1 alleviated cell apoptosis, oxidative stress and the inflammatory response induced by OGD/R in vitro. Up-regulation of circCTNNB1 increased SRB1 expression levels to protect mAS cells from OGD/R-induced damage. CircCTNNB1 and SRB1 interacted with miR-96-5p, and the overexpression of miR-96-5p efficiently reversed the function of circCTNNB1 in OGD/R-treated mAS cells. CircCTNNB1 protected against cerebral ischemia-reperfusion injury by up-regulating SRB1 in vivo. In conclusion, our findings suggest that circCTNNB1 acts as a competitive endogenous RNA for miR-96-5p to alleviate cerebral IRI, which provides novel evidence that circCTNNB1 and SRB1 may be biomarkers and therapeutic targets for cerebral IRI.

Investigation on property differences of ginseng and American ginseng by spatial metabolomics of neurochemicals with desorption electrospray ionization mass spectrometry imaging

Tissue pharmacokinetics is crucial for druggability assessment and plays a vital role in identifying the core bioactive components of traditional Chinese medicines (TCMs). This study aims to develop an integrated analytical approach combining ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) to systematically characterize the temporal and spatial brain exposure profiles of Corydalis Decumbentis Rhizoma (CDR) in rats. Using UPLC-quadrupole time of flight mass spectrometry (UPLC-QTOF), a total of 35 drug-derived constituents were identified from rat brain after oral administration of CDR. Then, a rapid and sensitive UPLC-triple-stage quadrupole mass spectrometry (UPLC-TSQ) method was developed and validated for the quantification of 15 major alkaloids in rat brain tissue. The pharmacokinetic results indicated that most compounds reached peak concentrations within 4 h and were rapidly eliminated within 12 h. Tetrahydropalmatine and protopine exhibited notably high brain exposure levels (AUC0-t > 100 ng·h·g⁻1), suggesting their potential as key active constituents responsible for the anti-ischemic effects of CDR. DESI-MSI further revealed the spatial distribution of these compounds, with most alkaloids predominantly localized in the telencephalon. The spatial distribution of CDR constituents furnished a scientific basis for its traditional application against cerebral ischemic injury. This study provides a scientific basis for the anti-stroke effects of CDR and a reference for investigating the material basis of other TCMs.

MicroRNA-212 has been found to play an important role in several types of diseases, but the functional and potential mechanisms of microRNA-212 in ischemic brain injury are still unclear. The aims of this study were to investigate the potential role of microRNA-212 in ischemic brain injury and to reveal potential molecular mechanisms. The rat oxygen-glucose deprivation and simulated reperfusion model was established to study the role of microRNA-212 in ischemic brain injury. The expression of microRNA-212 in oxygen-glucose deprivation and simulated reperfusion model and its effect on cell proliferation were measured by quantitative reverse transcription PCR and Cell Counting Kit-8 assay, respectively. The relationships between microRNA-212 and sirtuin 2 were confirmed by luciferase-reporter assay. We observed that microRNA-212 was downregulated after oxygen-glucose deprivation and simulated reperfusion treatment. Besides, the cells viabilities were increased/decreased in oxygen-glucose deprivation and simulated reperfusion model after transfection with microRNA-212 agomir (agonist of microRNA-212 action) and microRNA-212 antagomir (inhibitor of microRNA-212 action). In addition, luciferase and western blot experiments showed that microRNA-212 directly regulated sirtuin 2 changes. Furthermore, promotion of neuronal survival by microRNA-212 was blocked by overexpression of sirtuin 2, whereas the neuronal death induced by microRNA-212 inhibition was rescued by sirtuin 2 inhibition. Taken together, our study revealed that the role of miR-212 in the modulation of ischemic brain injury might be achieved by regulating sirtuin 2, which provides potential biomarkers and candidates for the treatment of cerebral ischemia.

This study aimed to explore whether USP18 regulates cerebral ischemia-reperfusion (I/R) injury via fat mass and obesity-associated proteins (FTO)-mediated NCOA4. Middle cerebral artery occlusion (MCAO) models were established in mice, and PC-12 cells treated with oxygen-glucose deprivation and reperfusion (OGD/R) were used as in vitro models. The USP18 lentiviral vector was transfected into cells in vitro and MCAO mice to observe its effect on ferroptosis. The relationship between USP18 and FTO was assessed using Co-IP and western blot. The effect of FTO on NCOA4 m6A modification was also elucidated. Overexpression of USP18 in MCAO models decreased cerebral infarct size and attenuated pathological conditions in mouse brain tissues. Moreover, USP18 reduced iron content, MDA, ROS, and LDH release, increased GSH levels and cell viability in both MCAO models and OGD/R cells, and promoted LC3 expression and autophagy flux. In vitro experiments on neurons showed that USP18 maintained FTO stability. The presence of FTO-m6A-YTFDH1-NCOA4 was also verified in neurons. Both in vivo and in vitro experiments showed that FTO and NCOA4 abrogated the protective effects of USP18 against ferritinophagy-mediated ferroptosis. Notably, USP18 maintains FTO stability, contributing to the removal of NCOA4 m6A modification and the suppression of NCOA4 translation, which consequently inhibits ferritinophagy-mediated ferroptosis to attenuate cerebral I/R injury.

This study aimed to explore the expression and mechanism of transforming growth factor β1 (TGF-β1) in oxygen glucose deprivation reperfusion (OGD/R)-induced ischemia/reperfusion (I/R) injury. An OGD/R model was established in cardiomyocytes H9c2, resulting in upregulation of Beclin-1 and LC3II/LC3I expression. Upon overexpression of TGF-β1, the viability of OGD/R-induced H9c2 cells was enhanced, while apoptosis was suppressed by downregulating Bax and upregulating Bcl-2. Additionally, TGF-β1 overexpression promoted autophagy in OGD/R-induced H9c2 cells by further upregulating the levels of Beclin-1 and LC3II/LC3I. Importantly, treatments with 3-methyladenine (3-MA), an autophagy inhibitor, and U0126, an extracellular signal-related kinases 1 and 2 (ERK1/2) inhibitor, significantly inhibited cell viability, increased intracellular reactive oxygen species levels, promoted cell apoptosis (by upregulating Bax and downregulating Bcl-2), and inhibited cell autophagy (by downregulating Beclin-1 and LC3II/LC3I) in OGD/R-induced H9c2 cells with TGF-β1 overexpression. Additionally, OGD/R induction significantly increased the levels of p-ERK, p-P38, and p-JNK, which were further enhanced by TGF-β1 overexpression. U0126 treatments significantly downregulated the p-ERK compared to OGD/R-induced H9c2 cells with TGF-β1 overexpression. Our study suggests that TGF-β1 could inhibit the growth of cardiomyocytes H9c2 by regulating autophagy and ERK pathways, providing a new theoretical basis for the treatment and prevention of OGD/R in clinical practice.

To investigate the effects of Notch signal on hypoxic induction factor (HIF-1α) and autophagy-associated genes Beclin1, LC3I, LC3II in oxygen-glucose deprivation (OGD) induced myocardial cell injury. The OGD model was established using hypoxic culture box and hypoglycemic DMEM medium. The cells were divided into normal control group, OGD group, OGD + NC siRNA group, OGD + Notch1 siRNA group and OGD + HIF-1α siRNA group. Western blot was used to detect the interference effects of HIF-1α siRNA and Notch1 siRNA. The effects of Notch1 siRNA and HIF-1α siRNA on the activity of myocardial cells in OGD model were detected by the CCK-8 assay. The effects of Notch1 siRNA and HIF-1α siRNA on autophage-associated genes Beclin1, LC3I and LC3II expression were detected by Western blot. The results of Western blot showed that HIF-1α siRNA could effectively knock down the expression of HIF-1α in myocardial cells in OGD model, and Notch1 siRNA could effectively knock down the expression of Notch1 and HIF-1α in myocardial cells in OGD model. The result of CCK-8 assay showed that Notch1 siRNA and HIF-1α siRNA reduced the activity of myocardial cells in OGD model, and there was no statistical difference between the two groups. Western blot results showed that Notch1 siRNA and HIF-1α siRNA could reduce the expressions of the autophagy-associated genes Beclin1, LC3I and LC3II, and reduce the ratio of LC3II to LC3I at mRNA level. Notch1 plays a role in myocardial protection by regulating the expression of HIF-1α to regulate the autophagy in OGD model cells.

Processibility of LDPE is known to be sensitive to processing history. Significant changes in processibility and product properties can occur through repeated extrusion without detectable changes in molecular weight and molecular weight distribution. Such changes of LDPE rheology are simulated with DSM micro-compounder and characterized with the small angle oscillatory tests and the SER uni-axial extensional flow with a K-BKZ analysis. Both the linear and the nonlinear rheology data suggest the major mechanism in the extrusion with the DSM micro-compounder is the reduction of the effects of long chain branching. The linear rheology analysis, along with GPC and NMR characterization confirms that the changes in the extrusion are not due to chemical crosslinking, though an increase in backbone molecular weight at extreme conditions is perceivable through the minor increase in molecular weight distribution, the increase in zero-shear viscosity and the decrease of melt index. The extensional rheology results, on the other hand, reveal the depression of the strain hardening with prolonged extrusion at 190 degree C or higher extrusion temperature. The stress growth data were evaluated with a K-BKZ constitutive with exponential damping function. The depression of strain hardening is well characterized with the strain hardening factor and the non-linear exponent beta as function of the extrusion conditions. The damping factor β is observed to be especially effective as the parameter to follow the depression of the strain hardening due to microcompounder extrusion. The microcompounder extrusion and the SER analysis with the K-BKZ analysis prove to be a new useful tool for simulating the shear modification of the LDPE.

Calf-thymus (CT-DNA) is widely used as a binding agent. The commercial samples are known to be "highly polymerized DNA" samples. CT-DNA is known to be fragile in particular upon ultrasonic wave irradiation. Degradation products could have dramatic consequences on its bio-sensing activity, and an accurate determination of the molecular weight distribution and stability of commercial samples is highly demanded. We investigated the sensitivity of charge detection mass spectrometry (CDMS), a single-molecule MS method, both with single-pass and ion trap CDMS ("Benner" trap) modes to the determination of the composition and stability (under multiphoton IR irradiation) of calf-thymus DNAs. We also investigated the changes in molecular weight distributions in the course of sonication by irradiating ultrasonic waves to CT-DNA. We report, for the first time, the direct molecular weight (MW) distribution of DNA sodium salt from calf-thymus revealing two populations at high (~10MDa) and low (~3MDa) molecular weights. We evidence a transition between the high-MW to the low-MW distribution, confirming that the low-MW distribution results from degradation of CT-DNA. Finally, we report also IRMPD experiments carried out on trapped single-stranded linear DNAs from calf-thymus allowing extraction of their activation energy for unimolecular dissociation. We show that single-pass CDMS is a direct, efficient and accurate MS-based approach to determine the composition of calf-thymus DNAs. Furthermore, ion trap CDMS allows us to evaluate the stability (both under multiphoton IR irradiation and in the course of sonication by irradiating ultrasonic wave) of calf-thymus DNAs.