The active ingredient (DSH-20) of Salvia miltiorrhiza flower reduces oxidative damage and apoptosis in cardiomyocytes by regulating miR-1.

BACKGROUND: DSH-20, the active ingredient of Salvia miltiorrhiza flower extract, is used to treat cardiovascular diseases. However, its mechanism of action remains unclear. Herein, we investigated the intervention of DSH-20 in H2O2-induced oxidative damage and apoptosis in cardiomyocytes. METHODS AND RESULTS: H2O2 was used to induce oxidative damage and apoptosis in H9c2 cardiomyocytes. Based on concentration gradient studies, we found that 62.5 µg/mL DSH-20 significantly reduced reactive oxygen species and lactate dehydrogenase levels and increased superoxide dismutase levels. DSH-20 also alleviated the apoptosis rate, the changes in mRNA of apoptosis-related genes (Bcl-2, BAX, and Caspase-3) and miR-1 expression. Moreover, transfection of miR-1 mimics aggravated oxidative damage and apoptosis, whereas DSH-20 alleviated these effects. CONCLUSIONS: DSH-20 reduced H2O2-induced oxidative damage and apoptosis in H9c2 cardiomyocytes likely by downregulating miR-1 expression.

Ligustrazine suppresses platelet aggregation through inhibiting the activities of calcium sensors

Abstract Ligustrazine is widely used for the treatment of cardiovascular diseases in traditional Chinese medication. It has been reported that Ligustrazine decreases the concentration of intracellular calcium ions (Ca2+); however, the underlying mechanism remains unknown. In the present study, the effect of Ligustrazine on adenosine diphosphate (ADP)-induced platelet aggregation was evaluated using a turbidimetric approach. The changes in concentration of intracellular Ca2+ stimulated by ADP was measured using fluo-4, a fluorescent Ca2+ indicator dye. The mRNA expression of stromal interaction molecule l (STIM1) and Orai1, calcium sensor, was determined using real-time PCR. In addition, the protein expression of STIM1, Orai1, and serum/glucocorticoid-regulated protein kinase 1 (SGK1) was determined using Western blot analysis. The data demonstrated that Ligustrazine significantly suppressed platelet aggregation in a dose-dependent manner and reduced the concentration of intracellular Ca2+ triggered by ADP. Our data showed that Ligustrazine treatment inhibited the expression of STIM1 and Orai1 induced by ADP at both mRNA and protein levels, and suppressed the protein expression of SGK1. Taken together, our data indicated that Ligustrazine suppressed platelet aggregation by partly inhibiting the activities of calcium sensors, thereby suggesting that Ligustrazine may be a promising candidate for the treatment of platelet aggregation.

miR-126 regulates angiogenesis in myocardial ischemia by targeting HIF-1α.

Promoting angiogenesis by targeting various angiogenic regulators has emerged as a new treatment strategy for myocardial ischemia (MI). MicroRNA-126 (miR-126) has been identified as the main regulator of compensatory angiogenesis; however, its role in MI is unclear. A rat MI model and an EA. hy926 endothelial cell hypoxia model were constructed and it was found that miR-126 was highly expressed in both models. The knockdown of HIF-1α expression in EA. hy926 cells in turn downregulated VEGF and CD34 expression and consequently inhibited angiogenesis. MiR-126 inhibitor inhibited EA. hy926 cell migration and tube formation as well as downregulated VEGF and CD34 expression, and these were reversed by transfection of miR-126 mimics. Rescue tests using miR-126 and HIF-1α demonstrated that miR-126-mediated regulation of angiogenesis was dependent on HIF-1α. In summary, miR-126 regulates the occurrence and progression of angiogenesis during MI via HIF-1α and may be a potential new therapeutic target.

Yiqi-Huoxue granule promotes angiogenesis of ischemic myocardium through miR-126/PI3K/Akt axis in endothelial cells.

BACKGROUND: Yiqi-Huoxue granule (YQHX), consisting of four kinds of traditional Chinese medicine, is an empirical prescription for the treatment of coronary heart disease. It is known to promote angiogenesis, but the mechanism is unknown. PURPOSE: This article investigates the possible mechanism of YQHX inducing angiogenesis in the ischemic myocardium. METHODS: EAhy.926 cells were treated with YQHX hypoxic cardiomyocyte-conditioned medium (YHMCM) and the levels of VEGF, CD34, and phosphorylation of PI3K/Akt were detected by western blotting. Also, the effects on endothelial tube formation and migration were observed. The level of miR-126 was detected by qRT-PCR. RESULTS: YQHX promoted tube formation and migration of EAhy.926 cells and upregulated VEGF, CD34, and the phosphorylation of PI3K/AKT via regulating miR-126 levels. However, these effects were inhibited by a miR-126 inhibitor. CONCLUSION: In summary, YQHX improves angiogenesis by regulating the miR-126/PI3K/Akt signaling pathway, which indicates that YQHX could be a promising therapeutic strategy for ischemic myocardium.

Effect of Yiqi Huoxue Granules on Platelet Activation Induced by Thrombin.

OBJECTIVE: To study the effects of Yiqi Huoxue (YQHX) granules on platelet activation and aggregation induced by thrombin. METHODS: The effect of YQHX on platelet aggregation rate was detected by platelet aggregation instrument; the effect of YQHX on thrombosis time was observed by the mouse mesentery thrombosis model. DAMI cells were induced to transform into platelet-like granules using PMA, and the effects of SCH (PAR-1 inhibitor) on thrombin-induced changes in platelet intracellular calcium concentration, PAR-1 protein expression, and phosphorylation of MAPK were examined. RESULTS: Compared with the control group, the platelet aggregation rate, PAR-1 protein expression, phosphorylation of ERK1/2, and p38 protein in the YQHX group decreased (P < 0.05), and there was no significant difference between the YQHX + SCH group and YQHX group (P > 0.05). CONCLUSION: YQHX suppresses the platelet activation induced by thrombin by inhibiting PAR-1 expression.

Epigenetic modulation of glycoprotein VI gene expression by DNA methylation.

AIMS: Glycoprotein VI (GPVI) is an important platelet membrane receptor. The expression of GPVI on platelet membranes is increased in patients with coronary heart disease (CHD). DNA methylation is one of the most common post-replication and pre-transcriptional modifications and plays a critical role in the regulation of gene expression. Here, we aimed to reveal how methylation regulates GPVI expression. MAIN METHODS: Pyrosequencing was used to determine whether the GPVI promoter region in leukocytes from CHD patients is hypomethylated. The expressions of GPVI in CHD patients were detected using qRT-PCR and Western blot. The effect of methylation of the GPVI promoter region on regulating its transcriptional activity was analyzed using in vitro luciferase assay. The expression of P-selectin in platelet-like particles was determined using flow cytometry, and SYK phosphorylation was observed using Western blot. KEY FINDINGS: We found that the GPVI promoter region in leukocytes from CHD patients was hypomethylated and the expression of GPVI at the mRNA and protein level was elevated in CHD patients. We also found that the hypermethylation of GPVI promoter region inhibited the expression of GPVI in the -322 to +75, -539 to +75, and -937 to +75 regions in Dami cells. Moreover, the data showed that the methylation or demethylation regulated the GPVI expression and platelet-like particle activation in Dami cells. SIGNIFICANCE: Taken together, these results indicate that DNA methylation regulates GPVI expression and that CpG methylation levels in the promoter region of the GPVI gene may be a biomarker of CHD.

Identification of key microRNAs in the carotid arteries of ApoE-/- mice exposed to disturbed flow.

BACKGROUND: Atherosclerosis (AS) is one of the main causes of cardiovascular disease. AS plaques often occur in blood vessels with oscillatory blood flow and their formation can be regulated by microRNAs (miRNAs). The aim of this study is to identify the key miRNAs and molecular pathways involved in this pathological process. METHODS: In this study, gene chip data obtained from the GEO database was analyzed using the LIMMA package to find differentially expressed miRNAs (DE miRNAs) in the carotid arteries of ApoE-/- mice exposed to different blood flow rates. Predicted targets of the DE miRNAs were identified using the TargetScan, miRDB, and DIANA databases respectively, and the potential target genes (PTGs) were found by analyzing the common results of three databases. The DAVID database was used to enrich the PTGs based on gene ontology (GO) and pathway (Kyoto Encyclopedia of Genes and Genomes, KEGG), and the STRING database was used to uncover any protein-protein interactions (PPI) of the PTGs. RESULTS: The networks of the DE miRNAs-PTGs, Pathway-PTGs-DE miRNAs, and PTGs PPI, were constructed using Cytoscape, and 11 up-regulated and 13 down-regulated DE miRNAs and 1479 PTGs were found. GO results showed that PTGs were significantly enriched in functions such as transcriptional regulation and DNA binding. KEGG results showed that PTGs were significantly enriched in inflammation-related mitogen-activated protein kinase (MAPK) and AS-related FOXO pathways. The PPI network revealed some key target genes in the PTGs. CONCLUSIONS: The analysis of key miRNAs and molecular pathways that regulate the formation of AS plaques induced by oscillatory blood flow will provide new ideas for AS treatment.

YiqiHuoxue Decoction and Its Ethanol Precipitation Show Anti-Platelet and Antithrombotic Effects by Suppressing Thromboxane B2 Formation.

BACKGROUND: YiqiHuoxue decoction (YHD) is frequently prescribed to prevent and treat cardiovascular diseases. YHD inhibits platelet aggregation, however the underlying mechanisms are unclear. METHODS: The in vitro and in vivo anti-platelet and antithrombotic effects of YHD and ethanol-precipitated YHD (EYHD) and underlying mechanisms were investigated. Forty-six Sprague-Dawley (SD) rats and 36 male Kunming mice were examined. Ten SD rats were used to assess the cytotoxicity of YHD and EYHD by releasing lactate dehydrogenase from treated platelets. The remaining 36 SD rats were divided into six groups (six per group), including control saline (5 mL/kg), aspirin (20 mg/kg), YHD low dosage (0.2 g/kg), YHD high dosage (2.0 g/kg), 75% EYHD low dosage (0.2 g/kg), and 75% EYHD high dosage (2.0 g/kg) groups to detect platelet aggregation; the 36 Kunming mice were divided into 6 groups to detect mesenteric arterial thrombosis induction. Thromboxane B2 (TXB2) levels were determined by enzyme immunoassay. RESULTS: YHD high dosage and 75% EYHD (low and high dosage) inhibited ADP-induced platelet aggregation. Moreover, collagen-induced platelet aggregation was significantly suppressed by YHD (high dosage), 75% EYHD (high dosage), and 75% EYHD (low dose). Rats given 75% EYHD (high dose) displayed a marked reduction in collagen-induced platelet aggregation at 2 h post-administration. YHD and EYHD markedly prolonged the onset of thrombosis causing loose attachment of the thrombus to the vascular endothelium, but bleeding and clotting times were not significantly changed. Finally, YHD and EYHD markedly reduced TXB2 levels. CONCLUSIONS: YHD and EYHD effectively inhibit platelet activation and thrombosis, presumably by suppressing TXB2.

Long non-coding RNA MEG3 attends to morphine-mediated autophagy of HT22 cells through modulating ERK pathway.

Context: Morphine is an alkaloid isolated from the poppy plants. The addiction of morphine is a very serious social issue. Some long non-coding RNAs (lncRNAs) have been proposed to engage in drug addiction. Objective: Whether lncRNA maternally expressed gene 3 (MEG3) attended to morphine-mediated autophagy of mouse hippocampal neuronal HT22 cells was probed. Materials and methods: HT22 cells were subjected to 10 µM morphine for 24 h. Cell autophagy was assessed by measuring LC3-II/LC3-I and Beclin-1 expression. qRT-PCR was carried out to measure MEG3 expression. SiRNA oligoribonucleotides targeting MEG3 (si-MEG3) was transfected to silence MEG3. The orexin1 receptor (OX1R), c-fos, p/t-ERK and p/t-PKC expressions were tested by western blotting. SCH772984 was used as an inhibitor of ERK pathway. Results: Morphine elevated OX1R (2.92 times), c-fos (2.06 times), p/t-ERK (2.04 times) and p/t-PKC (2.4 times), Beclin-1 (3.2 times) and LC3-II/LC3-I (3.96 times) expression in HT22 cells. Moreover, followed by morphine exposure, the MEG3 expression was also elevated in HT22 cells (3.03 times). The silence of MEG3 lowered the Beclin-1 (1.85 times), LC3-II/LC3-I (2.12 times), c-fos (1.39 times) and p/t-ERK (1.44 times) expressions in morphine-treated HT22 cells. Inhibitor of ERK pathway SCH772984 further promoted the influence of MEG3 silence on morphine-caused Beclin-1 (1.97 times) and LC3-II/LC3-I (1.92 times) expressions decreases. Conclusions: Up-regulation of MEG3 attended to the morphine-caused autophagy of HT22 cells might be through elevating c-fos expression and promoting ERK pathway activation. More experiments are also needed in the future to analyse the influence of other lncRNAs in drug addiction.

Yiqi-Huoxue Granule (YQHX) Downregulates Prothrombotic Factors by Modulating KLF2 and NF-κB in HUVECs following LPS Stimulation.

The Yiqi-Huoxue granule (YQHX) is a traditional Chinese medication widely used in the therapy of the traditional Chinese medicine diagnosis "Qi deficiency" or "blood stasis" in China. Both these symptoms are related to inflammation, but the mechanisms of YQHX against inflammation are largely unknown. Thus, our present study investigated the effects of YQHX on regulating inflammatory responses induced by lipopolysaccharides (LPS) in HUVECs. Our data found that YQHX remarkably inhibits the production of prothrombotic factors, plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF), while it upregulates the protein expression of Kruppel-like factor 2 (KLF2). The increase in PAI-1 and TF was significantly attenuated through a transgenic knockdown in KLF2 with a Lenti-shKLF2 vector. YQHX also decreases the phosphorylation of nuclear factor-κB (NF-κB) p65 and IκB following LPS stimulation, and it effectively suppresses PAI-1 and TF via a NF-κB-dependent mechanism. Taken together, our results suggest that YQHX provides a notable antithrombotic activity via regulating the KLF2 expression and NF-κB signaling pathway in HUVECs. The KLF2 and NF-κB may be potential therapeutic targets for interventions of inflammation associated with atherosclerosis.

Inhibitory effects of fucoidan on NMDA receptors and l-type Ca2+ channels regulating the Ca2+ responses in rat neurons.

CONTEXT: Fucoidan, a sulphated polysaccharide extracted from brown algae [Fucus vesiculosus Linn. (Fucaceae)], has multiple biological activities. OBJECTIVE: The effects of fucoidan on Ca2+ responses of rat neurons and its probable mechanisms with focus on glutamate receptors were examined. MATERIALS AND METHODS: The neurons isolated from the cortex and hippocampi of Wistar rats in postnatal day 1 were employed. The intracellular Ca2+ responses triggered by various stimuli were measured in vitro by Fura-2/AM. Fucoidan at 0.5 mg/mL or 1.5 mg/mL was applied for 3 min to determine its effects on Ca2+ responses. RT-PCR was used to determine the mRNA expression of neuron receptors treated with fucoidan at 0.5 mg/mL for 3 h. RESULTS: The Ca2+ responses induced by NMDA were 100% suppressed by fucoidan, and those induced by Bay K8644 90% in the cortical neurons. However, fucoidan has no significant effect on the Ca2+ responses of cortical neurons induced by AMPA or quisqualate. Meanwhile, the Ca2+ responses of hippocampal neurons induced by glutamate, ACPD or adrenaline, showed only a slight decrease following fucoidan treatment. RT-PCR assays of cortical and hippocampal neurons showed that fucoidan treatment significantly decreased the mRNA expression of NMDA-NR1 receptor and the primer pair for l-type Ca2+ channels, PR1/PR2. DISCUSSION AND CONCLUSIONS: Our data indicate that fucoidan suppresses the intracellular Ca2+ responses by selectively inhibiting NMDA receptors in cortical neurons and l-type Ca2+ channels in hippocampal neurons. A wide spectrum of fucoidan binding to cell membrane may be useful for designing a general purpose drug in future.

A Chinese 4-herb formula, Yiqi-Huoxue granule, alleviates H2O2-induced apoptosis by upregulating uncoupling protein 2 in H9c2 cells.

BACKGROUND: Although the protective effects of Yiqi-Huoxue granule (YQHX), a Chinese 4-herb formula, on patients with ischemic heart diseases are related to the attenuation of oxidative stress injury, the mechanism(s) underlying these actions remains poorly understood. PURPOSE: Our aim was to investigate the potential protective effects of YQHX treatment against oxidative stress induced by hydrogen peroxide (H2O2) in rat H9c2 cells. METHODS: H9c2 cells were treated with YQHX for 16 h before exposed to 200 µM H2O2 for 6 h. The apoptosis induced by H2O2 was measured using hoechst 33,342 staining and Annexin-V FITC/PI assay. The expression of uncoupling protein 2 (UCP2), Bcl-2, Bax, and caspase-3 were observed using western blot. The effects of UCP2 knockdown on cell apoptosis and intracellular ROS production were also investigated. RESULTS: H2O2 exposure led to significant activation of oxidative stress followed by increased apoptosis and ROS production, as well as decreased UCP2 expression in H9c2 cells. YQHX treatment at the concentration of 0.75 and 1.5 mg/ml remarkably reduced the expression of Bax and caspase-3, whereas increased the protein expression of Bcl-2 and UCP2. These changes were attenuated by transgenic knockdown of UCP2 with Lenti-shUCP2 vector. CONCLUSIONS: Taken together, our study demonstrated that YQHX attenuates H2O2-induced apoptosis by upregulating UCP2 expression in H9c2 Cells, suggesting that YQHX is a promising therapeutic approach for the treatment of I/R injury-mediated apoptosis.

GM1 Acupoint Injection Improves Mental Retardation in Children with Cerebral Palsy.

To study the clinical effectiveness and mechanism of GM1 acupoint injection therapy on mental retardation for children with cerebral palsy (CP). A total of 90 children with CP were divided into acupoint injection group (group A), subcutaneous injection group (group B), and control group (group C). Another 30 healthy children were set as a healthy control group (group D). The Mental Developmental Index (MDI), Psychomotor Developmental Index (PDI), and hemodynamic parameters in the cerebral arteries were measured before and after treatment. After three treatment courses, the MDI and PDI in groups A, B, and C were increased, and the increase in group A was most obvious (P < 0.05). Peak systolic velocity, mean velocity, and end-diastolic velocity were also elevated in group A, and after three treatment courses, resistance index decreased with a statistical significance (P < 0.05). However, there were no significant changes in groups B and C (P > 0.05). For all groups, neuron-specific enolase levels decreased and total superoxide dismutase increased after treatment. Acupoint injection therapy combined with conventional rehabilitation therapy demonstrated significant effects on cerebral hemodynamic conditions for children with CP.

Fucoidan inhibits Ca2+ responses induced by a wide spectrum of agonists for G­protein­coupled receptors.

Fucoidan, a sulfated polysaccharide extracted from brown seaweed, has been used in traditional Chinese herbal medicine to treat thyroid tumors for many years. Although a number of its cellular effects have been investigated, the role of fucoidan in molecular signaling, particularly in Ca2+ signaling, remains largely unknown. In the present study, the effects of fucoidan on Ca2+ responses in HeLa cells, human umbilical vein endothelial cells and astrocytes were investigated using a wide range of receptor agonists. Fucoidan inhibited the increase in intracellular free calcium concentration that was induced by histamine, ATP, compound 48/80 and acetylcholine. The responses induced by the same agonists in the absence of extracellular Ca2+ were also markedly suppressed by fucoidan. Reverse transcription­polymerase chain reaction demonstrated that 0.5 and 1.0 mg/ml fucoidan treatment for 3 h decreased histamine receptor 1 expression in HeLa cells. Similarly, the expressions of purinergic receptor P2Y, G­protein coupled (P2YR)1, P2YR2 and P2YR11 were significantly downregulated within cells pretreated with 1.0 mg/ml fucoidan for 3 h, and 0.5 mg/ml fucoidan significantly inhibited P2YR1 and P2YR11 expression. The results demonstrated that fucoidan may exert a wide spectrum of inhibitory effects on Ca2+ responses and that fucoidan may inhibit a number of different G­protein coupled receptors associated with Ca2+ dynamics.

[Effects of Guoshu acupoint pressure therapy on acute mastitis during lactation].

OBJECTIVE: To observe the clinical effect of Guoshu acupoint pressure therapy on acute mastitis during lactation. METHODS: Fifteen cases suffered from acute lactation mastitis were treated with Guoshu acupoint pressure therapy, that is, firstly with lifting and flicking reduction at "Taiji" and "Xuepen" point, whose intensity was varied from patient's physical fitness. Subsequently, the patients were treated with flame therapy induced by distillate spirit, once each day. RESULTS: After the treatment, all the patients were cured completely in from 1 to 5 days, with an average of 2.5 days. CONCLUSION: Guoshu acupoint pressure therapy is effective on acute mastitis during lactation.

Sex-specific effects of prenatal stress on neuronal development in the medial prefrontal cortex and the hippocampus.

Evidence suggests that maternal stress during gestation in humans and animals can cause emotional and cognitive dysfunction in the offspring. In the present study, we examined neurons of the hippocampus and the medial prefrontal cortex of adult rats exposed to prenatal stress. Using a revised Golgi-Cox staining method, we found decreases in dendritic length and complexity in area CA3 and the dentate gyrus of male rats exposed to prenatal stress compared with the controls, as well as decreased dendritic complexity in the prelimbic cortex. In contrast, we did not detect any changes in dendrites of female rats exposed to prenatal stress. Our results suggest that prenatal stress can induce long-lasting morphological changes in the medial prefrontal cortex and the hippocampus that are sex specific.

Fucoidan suppresses endocytosis in cultured HeLa cells.

OBJECTIVE: To evaluate the effects of fucoidan on endocytosis in cultured HeLa cells: in vitro using live cell imaging. METHODS: A confocal scanning system and an incubation imaging system were used to: observe the effects of fucoidan on the initial (6 h) stages of endocytosis using the fl uorescent probe FM1-43 and inorganic fl uorescent quantum dot (Q-dots). RESULTS: According to the time-lapse images, fucoidan inhibited the: formation of endocytic vesicles in HeLa cells, in which the FM1-43 dye was entrapped. Fucoidan also had an inhibitory effect on the uptake of the Q-dots by the cell membranes of HeLa cells. CONCLUSION: It was concluded: that fucoidan suppresses Ca(2+)-dependent endocytosis in HeLa cells, which may be caused by its inhibitory -effects on agonist-induced Ca(2+) responses.

Cocaine enhances resistance to extinction of responding for brain-stimulation reward in adult prenatally stressed rats.

The present experiment assessed whether prenatal stress (PS) can alter the ability of acute and chronic cocaine administration to increase and decrease the rewarding effectiveness of the medial forebrain bundle (MFB) using intracranial self-stimulation (ICSS), and also whether PS can affect the extinction of the MFB stimulation response. Adult male offspring of female rats that received PS or no PS (nPS) were implanted with MFB stimulating electrodes, and were then tested in ICSS paradigms. In both nPS and PS offspring, acute cocaine injection decreased ICSS thresholds dose-dependently. However, the threshold-lowering effects at any dose were not significantly different between groups. There was also no group-difference in the threshold-elevating effects of chronic cocaine administration. Nevertheless, chronically drug-administered PS rats exhibited a resistance to the extinguishing of the response for brain-stimulation reward when acutely treated with cocaine, as compared to extinction without cocaine treatment. The results suggest that PS may weaken the ability for response inhibition under cocaine loading in male adult offspring.

Intrathecal cocaine delivery enables long-access self-administration with binge-like behavior in mice.

RATIONALE: Long-access intravenous drug self-administration shows diurnal alterations in drug intake, with escalation and binge patterns, in rats. A similar long-access model in mice would allow the use of genetically modified animals to better understand the molecular mechanisms underlying drug addiction and relapse. However, attempts to transfer this model to mice have been less successful, mainly because of technical difficulties with long-term maintenance of the indwelling catheter implanted into small veins. OBJECTIVES: We devised an intrathecal probe implanted in the supracerebellar cistern as an alternative for intravenous drug administration to address this challenge and allow continuous, chronic drug self-administration in mice. RESULTS: We found that mice readily self-administered intrathecal infusions of cocaine as a drug reward, and, under daily 24-h access conditions, animals exhibited a binge-like behavior comparable to rats. CONCLUSIONS: This innovation enables a full analysis of long-access drug self-administration behavior in mice not possible with intravenous administration.