OBJECTIVE: To investigate the regulatory role of miRNA-26a in vascular smooth muscle cell (VSMC) calcification by regulating connective tissue growth factor (CTGF). METHODS: Rat thoracic aorta VSMCs (A7r5 cells) with induced calcification were treated with AR234960 agonist or transfected with miR-26a mimic, or with both treatments. Alizarin red staining was used to determine calcium deposition, and phosphatase (ALP) activity in the cells was measured. The mRNA and protein expressions of miR-26a, OPG, OPN, BMP-2 and collagen Ⅱ were detected using qPCR and Western blotting. The binding of miR-26a to CTGF was verified using dual luciferase reporter gene assay. RESULTS: After induced calcification, A7r5 cells showed gradually decreased miR-26a expression (P < 0.05) and progressively increased CTGF expression (P < 0.05) with the extension of induction time. Treatment of the cells with AR234960 obviously increased calcification in the cells, while transfection with miR-26a mimic significantly reduced cell calcification. The calcifying cells showed significantly increased ALP activity and expressions of OPN, BMP-2 and collagen Ⅱ (P < 0.05) and lowered OPG expression (P < 0.05), and treatment with AR234960 did not produce obvious effects on these changes (P > 0.05). Transfection with miR-26a mimic resulted in significantly decreased ALP activity and expressions OPN, BMP-2 and collagen Ⅱ expression (P < 0.05) and increased OPG expression (P < 0.05) in the calcifying cells. These effects of miR-26a mimic was significantly attenuated by treatment of the cells with AR234960 (P < 0.05). The result of luciferase reporter gene assay confirmed the binding of miR-26a to CTGF. CONCLUSION miRNA-26a can effectively alleviate vascular calcification by lowering the level of CTGF, reducing ALP activity and the expressions of OPN, BMP-2 and collagen Ⅱ, and increasing the expression of OPG.
Animals ; Calcium/metabolism* ; Cells, Cultured ; Connective Tissue Growth Factor/pharmacology* ; MicroRNAs/metabolism* ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle ; Phosphoric Monoester Hydrolases/pharmacology* ; RNA, Messenger/metabolism* ; Rats ; Sulfones ; Vascular Calcification

OBJECTIVETo investigate the effects of polyunsaturated fatty acids (PUFA) ω-3 and ω-6, and their middle metabolites PGE2 and PGE3 on angiogenesis formation of gastric cancer, and to explore associated mechanism.

METHODSThe effects of ω-3, ω-6, PGE2, PGE3 on the proliferation and migration of human umbilical vein endothelial cell (HUVEC) were measured by proliferation and migration assay respectively. The angiogenesis assay in vivo was used to measure the effects of ω-3, ω-6, PGE2 and PGE3 on neovascularization. In all the assays, groups without ω-3, ω-6, PGE2 and PGE3 were designed as the control.

RESULTSWith the increased concentration of ω-6 from 1 μmol/L to 10 μmol/L, the proliferation ability of HUVECs enhanced, and the number of migration cells also increased from 28.2±3.0 to 32.8±2.1, which was higher than control group (21.2±3.2) respectively (both P<0.05). With the increased concentration of ω-3 from 1 μmol/L to 10 μmol/L, the proliferation ability of HUVECs was inhibited, and the number of migration cells decreased from 15.8±2.0 to 11.0±2.1, which was lower than control group (22.1±3.0) respectively (both P<0.05). In the angiogenesis assay, compared with control group (standard number: 43 721±4 654), the angiogenesis ability of HUVECs was significantly enhanced by ω-6 in concentration-dependent manner (1 μmol/L group: 63 238±4 795, 10 μmol/L group: 78 166±6 123, all P<0.01). Meanwhile, with the increased concentration of ω-3 from 1 μmol/L to 10 μmol/L, the angiogenesis ability was significantly decreased from 30 129±3 102 to 20 012±1 541(all P<0.01). The proliferation and migration ability of HUVECs were significantly promoted by ω-6 metabolites PGE2 (P<0.05) in a concentration-dependent manner. In contrast, ω-3 metabolites PGE3 significantly inhibited the proliferation and migration ability of HUVECs in a concentration-dependent manner (all P<0.05). After rofecoxib (a COX-2 specific inhibitor) inhibited the expression of COX-2, the expression level of PGE2 was significantly decreased in a dose-dependent manner. In co-culture system, whose gastric cancer cells expressed positive COX-2, ω-6 could increase angiogenesis of gastric cancer cells(P<0.01), but ω-3 could inhibit such angiogenesis(P<0.01). In co-culture system, whose gastric cancer cells did not express COX-2, ω-3 could inhibit the angiogenesis of gastric cancer cells (P<0.05), but ω-6 had no effect on angiogenesis.

CONCLUSIONSThe PUFA ω-6 can enhance the angiogenesis via the promotion of proliferation and migration of HUVECs, and COX-2 and PGE2 may play an important role in this process, whereas, the ω-3 can inhibit the angiogenesis through its middle metabolites PGE3 to inhibit the proliferation and migration of HUVECs. Results of this experiment may provide a new approach to inhibit and prevent the spread of gastric cancer.

Alprostadil ; analogs & derivatives ; pharmacology ; Angiogenesis Inducing Agents ; metabolism ; pharmacology ; Angiogenesis Inhibitors ; pharmacology ; Cell Count ; methods ; Cell Line, Tumor ; drug effects ; physiology ; Cell Migration Assays ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Coculture Techniques ; Cyclooxygenase 2 ; pharmacology ; Dinoprostone ; metabolism ; pharmacology ; Fatty Acids, Omega-3 ; pharmacology ; Fatty Acids, Omega-6 ; metabolism ; pharmacology ; Fatty Acids, Unsaturated ; pharmacology ; Human Umbilical Vein Endothelial Cells ; drug effects ; physiology ; Humans ; Lactones ; pharmacology ; Neovascularization, Pathologic ; physiopathology ; Stomach Neoplasms ; physiopathology ; Sulfones ; pharmacology

OBJECTIVETo investigate the effect of Euphorbia fischeriana extract on latent HIV reactivation and the pathway involved in this process and discuss the value of Euphorbia fischeriana extract in eliminating HIV.

METHODSFresh tissues of Euphorbia fischeriana root were crushed into powder after quick freezing with liquid nitrogen and extracted with acetone followed by a three-day vacuum freeze-drying for dehydration of the extract. The extract (EFE) was separated using RP-C18 column with high-performance liquid chromatography (HPLC) and identified with mass spectrometry (MS). The activity of reactivated latent HIV was analyzed by fluorescence-activated cell sorting in a J-Lat 10.6 cell model treated with EFE (50 µg/mL) for 24 h, using TNF-α (10 ng/mL) as the positive control. The effect of a NF-κB pathway inhibitor (Bay 11-7082) on EFE activity was tested. The changes in P65 expression in the cell nuclei within 2 h and HIV protein p24 expression within 24 h were analyzed by Western blotting in cells treated with EFE.

RESULTSEFE was obtained by one-step acetone extraction, and the concentration of prostratin in the extract was around 0.53 mmol/L. About 50% of the cells showed HIV reactivation after treatment with 50 µg/mL EFE for 24 h accompanied by a significantly increased p24 expression. The activity of EFE in reactivating latent HIV was inhibited by Bay 11-7082 in a concentration-dependent manner, and p65 accumulation was detected in the cell nuclei within 2 h.

CONCLUSIONEFE we obtained contains the active compounds of prostratin and its analogues and shows a strong capacity to reactivate latent HIV through classical NF-κB pathway.

Euphorbia ; chemistry ; Flow Cytometry ; HIV ; drug effects ; HIV Infections ; Humans ; NF-kappa B ; metabolism ; Nitriles ; Phorbol Esters ; chemistry ; Plant Extracts ; pharmacology ; Signal Transduction ; Sulfones ; Tumor Necrosis Factor-alpha ; Virus Latency ; drug effects

cAMP-specific phosphodiesterase type 4 (PDE4) is one of the hot targets for treatment of inflammatory diseases. PDE4 inhibitors can suppress inflammation by increasing the concentration of cAMP in inflammatory cells. The efficacy and safety evaluations of several PDE4 inhibitors are currently carried on in clinical trials, for example GSK256066 in asthma, roflumilast and GSK256066 in chronic obstructive pulmonary disease, tetomilast in inflammatory bowel disease, and apremilast in dermatitis and arthritis etc. This article reviews the recent progress on PDE4-targeted therapy for inflammatory diseases.
Aminopyridines ; pharmacology ; Aminoquinolines ; pharmacology ; Arthritis ; drug therapy ; Asthma ; drug therapy ; Benzamides ; pharmacology ; Cyclopropanes ; pharmacology ; Dermatitis ; drug therapy ; Humans ; Inflammation ; drug therapy ; Inflammatory Bowel Diseases ; drug therapy ; Phosphodiesterase 4 Inhibitors ; pharmacology ; Pulmonary Disease, Chronic Obstructive ; drug therapy ; Sulfones ; pharmacology ; Thalidomide ; analogs & derivatives ; pharmacology ; Thiazoles ; pharmacology

OBJECTIVETo explore the effect of ataxia telangiectasia mutated and RAD3 related protein (ATR) expression and ATR kinase activity on the sensitivity to cisplatin in ovarian cancer SKOV3 cells.

METHODSSiRNA targeting ATR was transfected into SKOV3 cells for 48 h to reduce the ATR protein level, and ATR kinase inhibitor VE-821 was used for 12 h to inhibit the ATR pathway activity. The alteration of cell viability was examined by CCk-8 assay. Expression levels of ATR, p-ATR and γ-H2AX proteins were detected by Western blot. The DNA double strand breaks (DSB) marker γ-H2AX and homologous recombination repair key protein RAD51 and their co-localization in the cells were examined under the confocal microscope. The status of DNA double strand breaks (DSB) in single cells was visualized by alkaline comet assay. Finally, the cell cycle distribution was assessed using flow cytometry.

RESULTSDDP caused evident DNA double strands breaks and activated ATR kinase pathway. ATR-siRNA notably reduced ATR protein level, the 48 h IC(50) value of DDP was 72.12 µmol/L and 41.25 µmol/L, respectively, in the NC-siRNA and ATR-siRNA groups (P < 0.05). Confocal microscopic assay presented decreased recruitment of RAD51 at the DSB loci and comet assay showed enhanced DSB in the cells after ATR knocking down. After the inhibition of ATR kinase by VE-821, the 48 h IC(50) value of DDP was 75.32 µmol/L and 45.64 µmol/L, respectively, in the DMSO and VE-821 groups (P < 0.05 for both), confocal microscopic assay demonstrated reduced RAD51 recruitment, and comet assay showed increased DSB in cells after ATR kinase inhibition. Flow cytometry showed that percentage of cells distributed in G(0)/G(1), S and G(2)/M phases was 71.2%, 13.4% and 15.4%, repectively, after 40 µmol/L DDP treatment for 24 h. Compared with that of control group (G(0)/G(1): 54.2%, S: 21.3% and G(2)/M: 24.4%), DDP induced G(0)/G(1) phase arrest. DDP intervention resulted in the cell cycle status (G(0)/G(1): 43.2%, S: 20.4%, G(2)/M: 36.4%) in the ATR-siRNA group and (G(0)/G(1): 40.2%, S: 22.5%, G(2)/M: 37.3%) in the VE-821 group, indicating that the inhibition of ATR or ATR kinase could abrogate the effect of G(0)/G(1) phase arrest induced by DDP.

CONCLUSIONSSuppression of ATR can affect the homologous recombination repair in ovarian cancer cells, leading to accumulation of DNA double strand breaks in the cell nuclei as well as reduction of DDP-caused G(0)/G(1) phase arrest, finally enhances the sensitivity to cisplatin in the ovarian cancer SKOV3 cells.

Antineoplastic Agents ; pharmacology ; Cell Cycle ; Cell Line, Tumor ; Cisplatin ; pharmacology ; DNA Repair ; Female ; Humans ; Ovarian Neoplasms ; Pyrazines ; RNA, Small Interfering ; Sulfones ; Transfection

OBJECTIVETo examine whether calcineurin/NFAT signaling pathway mediates endothelin-1 (ET-1)-induced proliferation of pulmonary artery smooth muscle cells (PASMCs) by regulating phosphodiesterase-5 (PDE5) and the effect of the selective calcineurin inhibitor cyclosporine A and PDE5 inhibitor sildenafil on ET-1-induced PASMC proliferation.

METHODSPASMCs were treated with ET-1 to stimulate their proliferation with or without prior treatment of the cells with CsA or sildenafil. Calcineurin activity in the cells was measured using a calcineurin activity assay kit, PDE5 expression examined using immunoblotting, and cGMP level detected using a cGMP direct immunoassay kit. PASMC proliferation following the treatments was determined using [(3)H]thymidine incorporation assay.

RESULTSET-1 caused a 2.05-fold increase in the cellular calcineurin activity, a 1.80-fold increase in PDE5 expression, and a 3.20-fold increase in the DNA synthesis rate, and reduced the cGMP level by 67%. Pretreatment of the cells with Cyclosporine blocked the effects of ET-1, and PDE5 inhibition by sildenafil pretreatment also abolished ET-1-induced reduction of cGMP level in the cells. Both Cyclosporine and sildenafil suppressed ET-1-stimulated PASMC proliferation.

CONCLUSIONActivation of calcineurin/NFAT signaling pathway mediates ET-1-induced PASMC proliferation by stimulating PDE5 expression, which further degrades cGMP. Both Cyclosporine and sildenafil can suppress ET-1-stimulated PASMC proliferation in vitro.

Animals ; Calcineurin ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclic GMP ; metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; metabolism ; Cyclosporine ; DNA ; biosynthesis ; Endothelin-1 ; pharmacology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; enzymology ; NFATC Transcription Factors ; metabolism ; Piperazines ; Pulmonary Artery ; cytology ; Purines ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Sildenafil Citrate ; Sulfones

This study was purposed to explore the changes of possible angiogenetic factors other than VEGF after inhibition of NHE1 and their related mechanisms. The K562 cells were treated by NHE1 specific inhibitor cariporide, the angiogenesis factors after inhibition of NHE1 were screened by using protein chip, the IL-8 expression level after cariporide treatment was detected by real-time quantitative PCR; the K562 cells with stable interference of NHE1 were constructed, the IL-8 expression level after interference of NHE1 was detected by real-time quantitative PCR; the p38 phosphorylation level in K562 cells treated with cariporide was detected by Western blot. After treatment of K562 cells with p38 inhibitor SB203580, the IL-8 expression level was decreased by real-time quantitative PCR. The results of protein chip showed that IL-8 expression decreased after cariporide treatment. Real-time quantitative PCR confirmed this inhibitory effect. The p38 phosphorylation level increased after cariporide treatment. The down-regulation of IL-8 expression induced by cariporide treatment was partially restored after K562 cells were treated with p38 inhibitor SB203580. It is concluded that the inhibition of NHE1 can inhibit IL-8 expression through up-regulation of p38 phosphorylation.
Cation Transport Proteins ; antagonists & inhibitors ; Down-Regulation ; Guanidines ; pharmacology ; Humans ; Imidazoles ; pharmacology ; Interleukin-8 ; metabolism ; K562 Cells ; Phosphorylation ; drug effects ; Pyridines ; pharmacology ; Sodium-Hydrogen Exchanger 1 ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors ; Sulfones ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

BACKGROUNDSildenafil is one of the selective phosphodiesterase 5 inhibitors that has been proven by many investigators to suppress growth factor stimulated (e.g. platelet-derived growth factor (PDGF) or epidermal growth factor (EGF)) proliferation and hypertrophy of pulmonary artery smooth muscle cells (PASMCs) via cGMP/cGKIa pathway. Serotonin promotes cell cycle progression leading to cell mitogenesis and plays a key role in the pathogenesis of pulmonary artery hypertension. The role of sildenafil in proliferation of PASMCs induced by serotonin has not been investigated so far. In this study we explored the underlying mechanism of the effect of sildenafil on serotonin induced proliferation of porcine PASMCs.

METHODSPASMCs were cells from primary cultures by the explant method from the pulmonary artery of swine and cells at passage 3 - 5 were used in this study. MTT colorimetric assay and flow cytometry analysis were used to evaluate the cell proliferation and alterations in cell cycle progression respectively. Western blotting analysis was applied to determine the expression of phosphorylated extracellular signal-regulated kinase (ERK), proliferating cell nuclear antigen (PCNA) and mitogen activated protein kinase (MAPK) phosphatase-1 (MKP-1).

RESULTSSerotonin (10 µmol/L) induced the upregulation of phosphorylation of ERK1/ERK2 and PCNA, an increase in the percentage of cells in S phase and subsequent cell proliferation. Pretreatment with 1 µmol/L sildenafil potentiated the phosphorylation of ERK1/ERK2, an increase in the percentage of cells in S phase and cell proliferation, compared with serotonin stimulation alone (P < 0.05). Furthermore, 30-minute pretreatment with 10 µmol/L U0126, specific antagonist for ERK kinase (MEK) prevented the increase in phosphorylation of ERK1/ERK2 and abolished cell cycle progression and the proliferation of PASMCs induced by sildenafil.

CONCLUSIONThis study shows that sildenafil potentiated the proliferative effect of serotonin on PASMCs via phosphorylation of ERK1/ERK2.

Animals ; Blotting, Western ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dual Specificity Phosphatase 1 ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Myocytes, Smooth Muscle ; cytology ; Phosphorylation ; drug effects ; Piperazines ; pharmacology ; Proliferating Cell Nuclear Antigen ; metabolism ; Pulmonary Artery ; cytology ; Purines ; pharmacology ; Serotonin ; pharmacology ; Sildenafil Citrate ; Sulfones ; pharmacology ; Swine

This experimental study was designed to explore the possible mechanisms of Cariporide, a kind of Na+/H+ exchanger inhibitor, for protecting the lung from warm ischemia/reperfusion injury (WI/RI) of isolated rat lung model. Thirty isolated rat lungs were established on the Langendorff apparatus and randomly divided to three groups (n = 10, each): control group (C group), ischemia/reperfusion group (IR group) and Cariporide group (CP group). Mean pulmonary artery pressure (MPAP) and peak airway pressure (pAwP) were monitored continuously. At the end of reperfusion, right bronchoalveolar lavage was performed, bronchoalveolar lavage fluid (BALF) recovery rate (BALFRR) was recorded, and protein content in BALF was measured. Lung water content (LWC), malondialdehyde (MDA) and superoxide dismutase (SOD)of left lung tissue were measured; histomorphology evaluation was performed under light microscope and transmission electron microscope. In comparison with the data from IR group, BALF protein concentration, LWC, MDA content and MPAP content of reperfusion were significantly decreased, but SOD activity was increased in CP group. Histomorphologic feature also showed that pathological change significantly reduced in CP group. In this rat WI/RI model, the mechanism by which the selective Na+/H+ exchanger inhibitor (Cariporide) attenuates lipid peroxidation induced by WI/IR may be: preventing Ca2+ overload via inhibiting the transport of Na+/H2 exchanger-1 (NHE1) in the context of the coupled exchanger, thereby reducing the activation of xanthine oxidase pathway and oxygen free radical liberation which is dependent on certain intracellular Ca2+ concentration, and lastly promoting the endogenous antioxidative mechanism.
Animals ; Antioxidants ; pharmacology ; Guanidines ; pharmacology ; In Vitro Techniques ; Ischemic Preconditioning ; Lung ; blood supply ; Male ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; etiology ; prevention & control ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors ; Sulfones ; pharmacology ; Superoxide Dismutase ; metabolism ; Warm Ischemia

Sildenafil increases the cyclic guanosine monophosphate (cGMP) by inhibition of a phosphodiesterase 5, thereby leading to an antinociceptive effect. The increased cGMP may exert the effect on an L-type calcium channel through the activation of protein kinase G (PKG). The purpose of this study was to examine the possible involvement of a PKG-L-type calcium channel on the effect of sildenafil at the spinal level. Catheters were inserted into the intrathecal space of male SD rats. Pain was induced by applying 50 microliter of a 5% formalin solution to the hindpaw. The sildenafil-induced effect was examined after an intrathecal pretreatment of a PKG inhibitor (KT 5823), or a L-type calcium channel activator (FPL 64176). Intrathecal sildenafil produced an antinociceptive effect during phase 1 (0~10 min interval) and phase 2 (10~60 min interval) in the formalin test. Intrathecal KT 5823 and FPL 64176 attenuated the antinociceptive effect of sildenafil during both phases. Sildenafil is effective against both acute pain and the facilitated pain state at the spinal level. In addition, the inhibition of an L-type calcium channel by activation of the PKG may contribute to the antinocieptive mechanism of sildenafil in the spinal cord.
Animals ; Calcium Channel Agonists/pharmacology ; Calcium Channels, L-Type/*physiology ; Carbazoles/pharmacology ; Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors/*physiology ; Dose-Response Relationship, Drug ; Male ; Pain/drug therapy/*physiopathology ; Pain Measurement ; Piperazines/*pharmacology/therapeutic use ; Protein Kinase Inhibitors/pharmacology ; Purines/pharmacology/therapeutic use ; Pyrroles/pharmacology ; Rats ; Rats, Sprague-Dawley ; Sulfones/*pharmacology/therapeutic use