Identification of Potential Core Genes for the Rupture of Intracranial Aneurysms by a Bioinformatics Analysis.

Background: Previous studies, using autopsy and angiography, have shown that 3.6-6% of the population have intracranial aneurysms, and the rupture of aneurysm can lead to brain dysfunction or even death in patients. Methods: To explore potential preventional target genes for the ruptured of aneurysm, we analyze three gene expression datasets (GSE13353, GSE15629 and GSE54083) derived from the GEO database. We confirm DEGs associated with the unrupture of aneurysms by R package. DAVID version provides functional classification and annotation analyses of associated genes, including GO and KEGG pathway. PPI of these DEGs is analyzed based on the string database and visualized by Cytoscape software. DEGs are verified by qRT-PCR using samples isolated from the patients. Results: 249 overlapping DEGs, including 96 up-regulated genes and 153 down-regulated genes are screened using the Venn diagram webtool. The GO term and KEGG pathways analysis results indicate that these DEGs are mainly enriched in protein phosphorylation, apoptotic process and inflammatory response in the BP term and focal adhesion, thyroid hormone signaling pathway, ErbB signaling pathway, cytokine-cytokine receptor interaction and some disease processes in the KEGG pathways. 6 candidates are confirmed by Cytoscape software and qRT-PCR, including APP, JUN, GSK3B, ErbB2, PPBP and THBS1. Conclusions: Our data and previous studies show that ErbB2 and THBS1 are crucial to prevent aneurysm rupture, while APP, JUN, GSK3B and PPBP performs the opposite role, and further experiments are needed to verify these findings.

Sodium ozagrel and atorvastatin for type 2 diabetes patients with lacunar cerebral infarction.

BACKGROUND: The main pathological factor of cerebral infarction is atherosclerosis, which is the pathological process of chronic inflammatory diseases such as vascular smooth muscle hyperplasia, inflammatory cell infiltration, extracellular matrix increase, and thrombosis. At present, the focus of clinical treatment is anti-platelet aggregation and improving blood status, and current research is limited to improving symptoms only. AIM: To observe the effect of sodium ozagrel and atorvastatin on type 2 diabetes patients with lacunar cerebral infarction. METHODS: Eighty-two patients with type 2 diabetes and lacunar cerebral infarction admitted to our hospital from January 2018 to February 2020 were equally categorized into two groups according to their treatment method. The control group was administered atorvastatin, and the observation group was administered sodium ozagrel combined with atorvastatin. The National Institutes of Health stroke scale (NIHSS) score, activities of daily living (ADL) score, blood glucose, lipid levels, inflammatory factors, high-mobility group box 1 (HMGB1) levels, paraoxonase-1 (PON-1) levels, erythrocyte sedimentation rate (ESR), and macrophage migration inhibitory factor (MIF) levels were recorded before and after treatment. The total effective rate and adverse reaction rate of the two groups were analyzed. RESULTS: The total effective rate of the observation group (94.00%) was significantly higher than that of the control group (80.00%) (χ 2 = 3.998; P = 0.046). The blood glucose indexes, total cholesterol levels, triglyceride levels, low-density lipoprotein cholesterol levels, high-sensitivity C-reactive protein levels, interleukin-1ß levels, tumor necrosis factor-α levels, HMGB1 Levels, ESR, MIF levels, platelet aggregation rates, and plasma viscosity of the two groups decreased after treatment; however, high-density lipoprotein cholesterol and PON-1 Levels increased after treatment. After treatment, the blood glucose indexes; blood lipid indexes; inflammatory factors; HMGB1, PON-1, and MIF levels; ESR; platelet aggregation rate; and plasma viscosity of the observation group were better than those of the control group (P < 0.05). After treatment, all patients in the observation group had higher ADL scores and lower NIHSS scores than those in the control group (P < 0.05). CONCLUSION: Sodium ozagrel with atorvastatin can reduce inflammatory reactions; regulate ESR and HMGB1, PON-1, and MIF levels; control blood glucose and lipid indexes; and alleviate nerve injury without increasing adverse effects of atorvastatin alone.

Targeted profiling of amino acid metabolome in serum by a liquid chromatography-mass spectrometry method: application to identify potential markers for diet-induced hyperlipidemia.

To better understand the mechanism of hyperlipidemia and discover potential biomarkers, we have used targeted metabolomics to analyze eight amino acid profiles of control and hyperlipidemia rats by a liquid chromatography-mass spectrometry method. With high fat diet, the concentrations of serum of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (ApoB) were increased by 666.7%, 99.0%, 61.7% and 51.0%, whereas the concentrations of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (ApoA-I) were decreased by 46.3% and 58.9%. The concentrations of alanine, arginine, lysine, methionine, serine, tyrosine and valine in hyperlipidemia rats were significantly decreased by 21.8%, 19.72%, 26.5%, 19.6%, 48.7%, 19.8% and 24.91%, while there was no striking change in threonine. Combined with experimental results and previous literature, we inferred that alanine and serine were gradually disordered and subsequently generated abundant acetyl-CoA through pyruvate, which resulted in energy metabolism deficiency. Furthermore, Spearman correlation analysis shows that TC was negatively associated with methionine (r = -0.640, p < 0.05), suggesting that the lowered level of methionine caused by the homocysteine pathway enhances absorption and synthesis of TC. Meanwhile, the reduction of tyrosine demonstrated that rapid metabolism of cholesterol in vivo was caused by high levels of exogenous cholesterol. Furthermore, the observed ApoB and lysine changes indicated that lysine was largely incorporated into ApoB particles during the disease process. In addition, the levels of arginine, SOD and MDA reflected the behavior of oxidative stress. Finally, the metabolism fluctuation of valine demonstrated that abnormal lipid metabolism could cause abnormal glucose metabolism. In general, disordered energy metabolism, lipid metabolism, glucose metabolism and elevated oxidative stress were important characteristics of metabolic perturbations in hyperlipidemia. Herein, the discovery of biomarkers and the biological explanations mentioned above could be used to analyze the pathogenesis of hyperlipidemia through metabolic pathways, and these results could play an important role in assisting the clinical diagnosis of hyperlipidemia.

Comparative pharmacokinetics study of leonurine and stachydrine in normal rats and rats with cold-stagnation and blood-stasis primary dysmenorrhoea after the administration of Leonurus japonicus houtt electuary.

Leonurus japonicus houtt, a well-known herb of traditional Chinese medicine, is widely used to treat gynaecological diseases. In this study, a rapid and sensitive liquid chromatography with tandem mass spectrometry method for simultaneously quantifying leonurine and stachydrine, the two main bioactive components in Leonurus japonicus houtt, was developed and validated. Plasma samples were prepared by protein precipitation with acetonitrile and separation by a Hewlett Packard XDB-C8 column (150 × 4.6 mm, id, 5 µm) equipped with a gradient elution system containing methanol-water and 0.1% formic acid at a flow-rate of 0.4 mL/min. Components were then detected by a mass spectrometer in positive electrospray ionization mode. This method showed good linearity, precision, accuracy, recovery, stability, and negligible matrix effects, which were within acceptable ranges. The method was successfully applied to compare the pharmacokinetics in normal rats and rats with cold-stagnation and blood-stasis primary dysmenorrhoea treated with Leonurus japonicus houtt electuary. The result showed significant differences (p < 0.05) in the pharmacokinetic parameters between the primary dysmenorrhoea and normal groups. This result implied that Leonurus japonicus houtt electuary remained longer and was absorbed slower in rats with primary dysmenorrhoea and exhibited higher bioavailability and peak concentration.

Characterization of the complete chloroplast genome sequence of wetland macrophyte Typha orientalis (Typhaceae).

Typha orientalis is an important wetland macrophyte native to the eastern parts of Asia and Oceania. Herein, the complete chloroplast genome of this species was assembled and characterized using whole-genome next-generation sequencing. The complete chloroplast genome showed a circular genome of 160,969 bp size with 36.6% GC content. The genome is of typical structure and contains a pair of inverted repeat (IR) regions with 26,691 bp, separated by one large single-copy (LSC) with 89,118 bp, and one small single-copy (SSC) regions with 18,469 bp. The genome contained 132 genes, including 86 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. A phylogenetic tree reconstructed based on 15 chloroplast genomes reveals that T. orientalis is most related to Typha latifolia.

A feed-forward regulation of endothelin receptors by c-Jun in human non-pigmented ciliary epithelial cells and retinal ganglion cells.

c-Jun, c-Jun N-terminal kinase(JNK) and endothelin B (ETB) receptor have been shown to contribute to the pathogenesis of glaucoma. Previously, we reported that an increase of c-Jun and CCAAT/enhancer binding protein ß (C/EBPß) immunohistostaining is associated with upregulation of the ETB receptor within the ganglion cell layer of rats with elevated intraocular pressure (IOP). In addition, both transcription factors regulate the expression of the ETB receptor in human non-pigmented ciliary epithelial cells (HNPE). The current study addressed the mechanisms by which ET-1 produced upregulation of ET receptors in primary rat retinal ganglion cells (RGCs) and HNPE cells. Treatment of ET-1 and ET-3 increased the immunocytochemical staining of c-Jun and C/EBPß in primary rat RGCs and co-localization of both transcription factors was observed. A marked increase in DNA binding activity of AP-1 and C/EBPß as well as elevated protein levels of c-Jun and c-Jun-N-terminal kinase (JNK) were detected following ET-1 treatment in HNPE cells. Overexpression of ETA or ETB receptor promoted the upregulation of c-Jun and also elevated its promoter activity. In addition, upregulation of C/EBPß augmented DNA binding and mRNA expression of c-Jun, and furthermore, the interaction of c-Jun and C/EBPß was confirmed using co-immunoprecipitation. Apoptosis of HNPE cells was identified following ET-1 treatment, and overexpression of the ETA or ETB receptor produced enhanced apoptosis. ET-1 mediated upregulation of c-Jun and C/EBPß and their interaction may represent a novel mechanism contributing to the regulation of endothelin receptor expression. Reciprocally, c-Jun was also found to regulate the ET receptors and C/EBPß appeared to play a regulatory role in promoting expression of c-Jun. Taken together, the data suggests that ET-1 triggers the upregulation of c-Jun through both ETA and ETB receptors, and conversely c-Jun also upregulates endothelin receptor expression, thereby generating a positive feed-forward loop of endothelin receptor activation and expression. This feed-forward regulation may contribute to RGC death and astrocyte proliferation following ET-1 treatment.

Neural stem cells over-expressing brain-derived neurotrophic factor promote neuronal survival and cytoskeletal protein expression in traumatic brain injury sites.

Cytoskeletal proteins are involved in neuronal survival. Brain-derived neurotrophic factor can increase expression of cytoskeletal proteins during regeneration after axonal injury. However, the effect of neural stem cells genetically modified by brain-derived neurotrophic factor transplantation on neuronal survival in the injury site still remains unclear. To examine this, we established a rat model of traumatic brain injury by controlled cortical impact. At 72 hours after injury, 2 × 107 cells/mL neural stem cells overexpressing brain-derived neurotrophic factor or naive neural stem cells (3 mL) were injected into the injured cortex. At 1-3 weeks after transplantation, expression of neurofilament 200, microtubule-associated protein 2, actin, calmodulin, and beta-catenin were remarkably increased in the injury sites. These findings confirm that brain-derived neurotrophic factor-transfected neural stem cells contribute to neuronal survival, growth, and differentiation in the injury sites. The underlying mechanisms may be associated with increased expression of cytoskeletal proteins and the Wnt/ß-catenin signaling pathway.

Bleomycin Suppresses the Proliferation and the Mobility of Human Gastric Cancer Cells Through the Smad Signaling Pathway.

BACKGROUND/AIMS: Extensive studies have demonstrated that Bleomycin (BLM) is a glycopeptide antibiotic that has been used as an anticancer chemotherapeutic reagent. It can induce both single- and double-strand DNA damage, inhibit synthesis of DNA, suppress proliferation, and induce apoptosis in cancer cells. Smad signaling transducers are considered as important molecules in tumor development and progression, and may closely be related to the biological behaviors of some malignant carcinomas, including gastric cancer. METHODS: The effects of different concentrations of BLM on the proliferation, cell cycle, apoptosis, migration, and invasion on gastric cancer cell lines MKN45 and AGS were assayed by using CCK-8 assay, Annexin V/PI double staining, PI staining, and transwell assay. Western blot and Immunohistochemistry were applied to analyze the potential mechanism(s). RESULTS: BLM treatment resulted in a low proliferation, high apoptosis, low migration and invasion in MKN45 and AGS cells. Furthermore, the possible mechanisms underlying that Smad3 activity could be changed after binding with BLM, and subsequently the Smad signaling pathway had a cascade response. CONCLUSION: These results highlight BLM as an exciting theme for gastric cancer treatment, which may represent an effective clinical therapeutic reagent for gastric cancer patients.

AMPA receptor desensitization is the determinant of AMPA receptor mediated excitotoxicity in purified retinal ganglion cells.

The ionotropic glutamate receptors (iGLuR) have been hypothesized to play a role in neuronal pathogenesis by mediating excitotoxic death. Previous studies on iGluR in the retina have focused on two broad classes of receptors: NMDA and non-NMDA receptors including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and kainate receptor. In this study, we examined the role of receptor desensitization on the specific excitotoxic effects of AMPAR activation on primary retinal ganglion cells (RGCs). Purified rat RGCs were isolated from postnatal day 4-7 Sprague-Dawley rats. Calcium imaging was used to identify the functionality of the AMPARs and selectivity of the s-AMPA agonist. Phosphorylated CREB and ERK1/2 expression were performed following s-AMPA treatment. s-AMPA excitotoxicity was determined by JC-1 mitochondrial membrane depolarization assay, caspase 3/7 luciferase activity assay, immunoblot analysis for α-fodrin, and Live (calcein AM)/Dead (ethidium homodimer-1) assay. RGC cultures of 98% purity, lacking Iba1 and GFAP expression were used for the present studies. Isolated prenatal RGCs expressed calcium permeable AMPAR and s-AMPA (100 µM) treatment of cultured RGCs significantly increased phosphorylation of CREB but not that of ERK1/2. A prolonged (6 h) AMPAR activation in purified RGCs using s-AMPA (100 µM) did not depolarize the RGC mitochondrial membrane potential. In addition, treatment of cultured RGCs with s-AMPA, both in the presence and absence of trophic factors (BDNF and CNTF), did not increase caspase 3/7 activities or the cleavage of α-fodrin (neuronal apoptosis marker), as compared to untreated controls. Lastly, a significant increase in cell survival of RGCs was observed after s-AMPA treatment as compared to control untreated RGCs. However, preventing the desensitization of AMPAR with the treatment with either kainic acid (100 µM) or the combination of s-AMPA and cyclothiazide (50 µM) significantly reduced cell survivability. Activation of the AMPAR in RGCs does not appear to activate a signaling cascade to apoptosis, suggesting that RGCs in vitro are not susceptible to AMPA excitotoxicity as previously hypothesized. Conversely, preventing AMPAR desensitization through differential agonist activation caused AMPAR mediated excitotoxicity. Activation of the AMPAR in increasing CREB phosphorylation was dependent on the presence of calcium, which may help explain this action in increasing RGC survival.

Neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma.

PURPOSE: Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP), leading to optic nerve head (ONH) cupping, axon loss, and apoptosis of retinal ganglion cells (RGCs), which could ultimately result in blindness. Brn3b is a class-4 POU domain transcription factor that plays a key role in RGC development, axon outgrowth, and pathfinding. Previous studies suggest that a decrease in Brn3b levels occurs in animal models of glaucoma. The goal of this study was to determine if adeno-associated virus (AAV)-directed overexpression of the Brn3b protein could have neuroprotective effects following elevated IOP-mediated neurodegeneration. METHODS: Intraocular pressure was elevated in one eye of Brown Norway rats (Rattus norvegicus), following which the IOP-elevated eyes were intravitreally injected with AAV constructs encoding either the GFP (rAAV-CMV-GFP and rAAV-hsyn-GFP) or Brn3b (rAAV-CMV-Brn3b and rAAV-hsyn-Brn3b). Retina sections through the ONH were stained for synaptic plasticity markers and neuroprotection was assessed by RGC counts and visual acuity tests. RESULTS: Adeno-associated virus-mediated expression of the Brn3b protein in IOP-elevated rat eyes promoted an upregulation of growth associated protein-43 (GAP-43), actin binding LIM protein (abLIM) and acetylated α-tubulin (ac-Tuba) both posterior to the ONH and in RGCs. The RGC survival as well as axon integrity score were significantly improved in IOP-elevated rAAV-hsyn-Brn3b-injected rats compared with those of the IOP-elevated rAAV-hsyn-GFP- injected rats. Additionally, intravitreal rAAV-hsyn-Brn3b administration significantly restored the visual optomotor response in IOP-elevated rat eyes. CONCLUSIONS: Adeno-associated virus-mediated Brn3b protein expression may be a suitable approach for promoting neuroprotection in animal models of glaucoma.

Flow Cytometry Method Analysis of Apoptosis: No Significant Difference Between EDTA and EDTA-free Trypsin Treatment Procedure.

Flow cytometry method (FCM) is a generally accepted tool to analyze apoptosis. Although apoptosis assay kit was applied by many companies, the manufacturers were not consistent with whether using Trypsin with EDTA to collect the adherent cells. In another words, the influence of EDTA on apoptotic ratio is not clear. In this work, we compared the proportion of apoptotic cells with EDTA or EDTA-free Trypsin treatment by FCM. We concluded that Trypsin with or without EDTA has little influence on the proportion of apoptotic cells. In addition, we found that the ratio of necrosis and apoptosis was different in cells collected by scraping. WAVE2 protein was analyzed as a typical example for movement related protein. WAVE2 expression is elevated in the EDTA Trypsin treated group, compared with EDTA-free Trypsin treatment and scrapping group.

Sigma-1 receptor stimulation protects retinal ganglion cells from ischemia-like insult through the activation of extracellular-signal-regulated kinases 1/2.

Sigma-1 receptor (σ-1) activation and mitogen-activated protein kinases (MAPKs) have been shown to protect retinal ganglion cells (RGCs) from cell death. The purpose of this study was to determine if σ-1 receptor stimulation with pentazocine could promote neuroprotection under conditions of an ischemia-like insult (oxygen glucose deprivation (OGD)) through the phosphorylation of extracellular signal regulated kinase (pERK)1/2. Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using Thy1.1 antibodies. RGCs were cultured for 7 days before subjecting the cells to an OGD insult (0.5% oxygen in glucose-free medium) for 6 h. During the OGD, RGCs were treated with pentazocine (σ-1 receptor agonist) with or without BD 1047 (σ-1 receptor antagonist). In other experiments, primary RGCs were treated with pentazocine in the presence or absence of an MEK1/2 inhibitor, PD098059. Cell survival/death was assessed by staining with the calcein-AM/ethidium homodimer reagent. Levels of pERK1/2, total ERK1/2, and beta tubulin expression were determined by immunoblotting and immunofluorescence staining. RGCs subjected to OGD for 6 h induced 50% cell death in primary RGCs (p < 0.001) and inhibited pERK1/2 expression by 65% (p < 0.001). Cell death was attenuated when RGCs were treated with pentazocine under OGD (p < 0.001) and pERK1/2 expression was increased by 1.6 fold (p < 0.05) compared to OGD treated RGCs without pentazocine treatment. The co-treatment of PD098059 (MEK1/2 inhibitor) with pentazocine significantly abolished the protective effects of pentazocine on the RGCs during this OGD insult. Activation of the σ-1 receptor is a neuroprotective target that can protect RGCs from an ischemia-like insult. These results also established a direct relationship between σ-1 receptor stimulation and the neuroprotective effects of the ERK1/2 pathway in purified RGCs subjected to OGD. These findings suggest that activation of the σ-1 receptor may be a therapeutic target for neuroprotection particularly relevant to ocular neurodegenerative diseases that effect RGCs.

Herb-drug interaction prediction based on the high specific inhibition of andrographolide derivatives towards UDP-glucuronosyltransferase (UGT) 2B7.

Herb-drug interaction strongly limits the clinical application of herbs and drugs, and the inhibition of herbal components towards important drug-metabolizing enzymes (DMEs) has been regarded as one of the most important reasons. The present study aims to investigate the inhibition potential of andrographolide derivatives towards one of the most important phase II DMEs UDP-glucuronosyltransferases (UGTs). Recombinant UGT isoforms (except UGT1A4)-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction and UGT1A4-catalyzed trifluoperazine (TFP) glucuronidation were employed to firstly screen the andrographolide derivatives' inhibition potential. High specific inhibition of andrographolide derivatives towards UGT2B7 was observed. The inhibition type and parameters (Ki) were determined for the compounds exhibiting strong inhibition capability towards UGT2B7, and human liver microsome (HLMs)-catalyzed zidovudine (AZT) glucuronidation probe reaction was used to furtherly confirm the inhibition behavior. In combination of inhibition parameters (Ki) and in vivo concentration of andrographolide and dehydroandrographolide, the potential in vivo inhibition magnitude was predicted. Additionally, both the in vitro inhibition data and computational modeling results provide important information for the modification of andrographolide derivatives as selective inhibitors of UGT2B7. Taken together, data obtained from the present study indicated the potential herb-drug interaction between Andrographis paniculata and the drugs mainly undergoing UGT2B7-catalyzed metabolic elimination, and the andrographolide derivatives as potential candidates for the selective inhibitors of UGT2B7.

Involvement of AP-1 and C/EBPß in upregulation of endothelin B (ETB) receptor expression in a rodent model of glaucoma.

Previous studies showed that the endothelin B receptor (ETB) expression was upregulated and played a key role in neurodegeneration in rodent models of glaucoma. However, the mechanisms underlying upregulation of ETB receptor expression remain largely unknown. Using promoter-reporter assays, the 1258 bp upstream the human ETB promoter region was found to be essential for constitutive expression of ETB receptor gene in human non-pigmented ciliary epithelial cells (HNPE). The -300 to -1 bp and -1258 to -600 bp upstream promoter regions of the ETB receptor appeared to be the key binding regions for transcription factors. In addition, the crucial AP-1 binding site located at -615 to -624 bp upstream promoter was confirmed by luciferase assays and CHIP assays which were performed following overexpression of c-Jun in HNPE cells. Overexpression of either c-Jun or C/EBPß enhanced the ETB receptor promoter activity, which was reflected in increased mRNA and protein levels of ETB receptor. Furthermore, knock-down of either c-Jun or C/EBPß in HNPE cells was significantly correlated to decreased mRNA levels of both ETB and ETA receptor. These observations suggest that c-Jun and C/EBPß are important for regulated expression of the ETB receptor in HNPE cells. In separate experiments, intraocular pressure (IOP) was elevated in one eye of Brown Norway rats while the corresponding contralateral eye served as control. Two weeks of IOP elevation produced increased expression of c-Jun and C/EBPß in the retinal ganglion cell (RGC) layer from IOP-elevated eyes. The mRNA levels of c-Jun, ETA and ETB receptor were upregulated by 2.2-, 3.1- and 4.4-fold in RGC layers obtained by laser capture microdissection from retinas of eyes with elevated IOP, compared to those from contralateral eyes. Taken together, these data suggest that transcription factor AP-1 plays a key role in elevation of ETB receptor in a rodent model of ocular hypertension.

Apoptosis and G2/M arrest induced by Allium ursinum (ramson) watery extract in an AGS gastric cancer cell line.

BACKGROUND: The present study was designed to determine whether Allium ursinum L (ramson) could inhibit the proliferation of human AGS gastric cancer cells. Furthermore, we attempted to determine whether this inhibition could occur by targeting regulatory elements of the cell cycle. METHODS: Flow cytometry was used to observe apoptosis and the cell cycle in AGS cell lines treated or not treated with ramson watery extract. Proteins related to the cell cycle were detected by Western blotting. Caspase activity was measured using a colorimetric assay kit according to the manufacturer's instructions. RESULTS: Ramson watery extract induced apoptosis and G2/M phase arrest in AGS cells. Western blotting showed that cyclin B was inhibited by ramson watery extract. However, G1 phase-related proteins remain unchanged after treatment. CONCLUSION: Our results indicate that ramson effectively sup pressed proliferation and induced apoptosis and G2/M arrest in AGS cells by regulating elements of the cell cycle.

Sigma-1 receptor stimulation attenuates calcium influx through activated L-type Voltage Gated Calcium Channels in purified retinal ganglion cells.

Sigma-1 receptors (σ-1rs) exert neuroprotective effects on retinal ganglion cells (RGCs) both in vivo and in vitro. This receptor has unique properties through its actions on several voltage-gated and ligand-gated channels. The purpose of this study was to investigate the role that σ-1rs play in regulating cell calcium dynamics through activated L-type Voltage Gated Calcium Channels (L-type VGCCs) in purified RGCs. RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy1.1 antibody. Calcium imaging was used to measure changes in intracellular calcium after depolarizing the cells with potassium chloride (KCl) in the presence or absence of two σ-1r agonists [(+)-SKF10047 and (+)-Pentazocine], one σ-1r antagonist (BD1047), and one L-type VGCC antagonist (Verapamil). Finally, co-localization studies were completed to assess the proximity of σ-1r with L-type VGCCs in purified RGCs. VGCCs were activated using KCl (20 mM). Pre-treatment with a known L-type VGCC blocker demonstrated a 57% decrease of calcium ion influx through activated VGCCs. Calcium imaging results also demonstrated that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Antagonist treatment using BD1047 demonstrated a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs, implying that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in purified RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in purified RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in purified RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in purified RGCs implied that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs.

Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma.

Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP) characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1) is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET(B)) receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET(B) receptors in the retina, mainly in retinal ganglion cells (RGCs), nerve fiber layer (NFL), and also in the inner plexiform layer (IPL) and inner nuclear layer (INL). To determine the role of ET(B) receptors in neurodegeneration, Wistar-Kyoto wild type (WT) and ET(B) receptor-deficient (KO) rats were subjected to retrograde labeling with Fluoro-Gold (FG), following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET(B) receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.

Three new triterpenoids from Panax ginseng exhibit cytotoxicity against human A549 and Hep-3B cell lines.

Three new triterpenoid derivatives, 3-O-ß-D-glucopyranosyl-20(S)-protopanaxtriol (1), 3-formyloxy-20-O-ß-D-glucopyranosyl-20(S)-protopanaxtriol (2) and 26-hydroxyl-24(E)-20(S)-protopanaxtriol (3), along with six known ginsenosides, were isolated from leaves of Panax ginseng. Their structures were established on the basis of spectral analysis (IR, 1D and 2D NMR, HRESI-MS). Compounds 1-3 exhibited various degree of cytotoxicity towards human A549 pulmonary carcinoma cells and Hep3B hepatoma cells.

An effective and safe supplement for stem cells expansion ex vivo: cord blood serum.

Mesenchymal stem cells (MSCs) are potential and optimal stem cells in clinical cell therapy, and fetal bovine serum (FBS) is widely used for expansion of MSCs, despite the risks of infectious disease transmission and immunological reaction of the xenogenic origin. This study was designed to compare human four blood group cord blood serum (CBS) with FBS in culturing human placenta-derived mesenchymal stem cells (hPDMSCs), which were derived from four blood group donors. The expansion medium included: 10% FBS, 10% A-CBS, 10% B-CBS, 10% O-CBS, and 10% AB-CBS. Cumulative population doubling, generation time, fold expansion rates and differentiation capacity, cell cycle, and immunophenotype were also assessed. The results showed that fold expansion rate and cumulative population doubling of hPDMSCs significantly increased during long-term MSC expansion in CBS medium, but the generation time decreased compared with FBS. CBS might be an effective supplement for stem cells expand rapidly ex vivo. Cell cycle and differentiation assays showed that most of the hPDMSCs expanding in the presence of CBS were in stationary phase, which was the characteristic of stem cells, and they retained their ability to differentiate into chondrogenic and endothelial cells. By comparing these four blood groups of CBS, we found that there was no significant difference among different blood groups in culturing hPDMSCs, which were isolated from different blood group donors. So CBS may be an optimal replacement to avoid the risks of FBS application in expansion of stem cell for clinical cell therapy and tissue engineering.

[Protective effects of tianji capsule on vascular endothelial cells from oxidative injury induced by hydrogen peroxide in vitro].

OBJECTIVE: To study the protective effects of Tianji capsule (TJ) on vascular endothelial cells from oxidative injury induced by hydrogen peroxide and its possible mechanism of anti-oxidation. METHODS: The effect of TJ on the proliferation of normal human umbilical vascular endothelial cells (HUVECs) as well as its cytotoxicity was evaluated with methylthiazolyl tetrazolium (MTT) assay. After the establishment of oxidative injury model of HUVECs, control, oxidative injury model, TJ and CoQ10 treatment groups were set up. HUVECs were incubated with 37.5, 75, 150 and 300 microg/mL TJ or 100 microg/mL CoQ10 for 24 h, and 0.1 mmol/L H2O2 (final concentration) was added to HUVECs in each groups for 30 min. Then collected the cells for proliferation detection with MTT assay, and the levels of MDA and NO, the activities of SOD, GSH-Px and NOS, as well as the releasing rate of LDH in HUVECs were also determined. RESULTS: No cytotoxicity was observed in HUVECs with less than 400 microg/ mL TJ incubated for 48 h, but increased proliferation rates were noticed. Pretreated with TJ (37.5, 75, 150 and 300 microg/mL), increased proliferation rate, the activities of SOD, GSH-Px, NOS were observed, but the decreased level of MDA and releasing rate of LDH were also found. CONCLUSION: TJ could protect HUVECs against oxidative injury induced by H2O2.