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The aim of the present study was to investigate the effects of salvianolic acid B on lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) in rabbits. Continuous infusion of LPS was used to induce a DIC model in rabbits. Treatment with salvianolic acid B (1, 3 or 6 mg/kg) was started simultaneously with LPS infusion (0.5 mg/kg LPS in 60 mL saline; 10 mL/h over a period of 6 h) through the contralateral marginal ear vein. Activated partial thromboplastin time (APTT), prothrombin time (PT), platelet count and fibrinogen concentration were determined, as were plasma levels of fibrin-fibrinogen degradation products (FDP), alanine aminotransferase (ALT), blood urea nitrogen (BUN), protein C activity, antithrombin III (ATIII) and tumour necrosis factor (TNF)-α concentration. The gradual impairment of haemostatic parameters was induced by continuous infusion of LPS. There were marked increases in APTT, PT, BUN, ALT and plasma TNF-α and marked decreases in the platelet count, fibrinogen, FDP, protein C and ATIII. The intravenous administration of 1, 3 or 6 mg/kg salvianolic acid B attenuated the increases in APTT, PT, BUN, ALT and plasma TNF-α and the decreases in fibrinogen, platelet, FDP, protein C and ATIII induced by LPS infusion. These observations indicate that salvianolic acid B has an effect against LPS-induced DIC in rabbits.

This study investigated the function of a chloride channel blocker, DIDS. Both in vitro and in vivo studies found that DIDS significantly inhibits lipopolysaccharide (LPS)-induced release of proin flammatory cytokines. Here, we show that DIDS inhibits LPS-induced inflammation, as shown by downregulation of inflammatory cytokines via inhibition of the TLR4/NF-κB pathway. Furthermore, we show that ClC-3siRNA transfection reduces LPS-induced pro-inflammation in Raw264.7 cells, indicating that ClC-3 is involved in the inhibitory effect of DIDS during LPS-induced cytokines release. In vivo, DIDS reduced LPS-induced mortality, decreased LPS-induced organic damage and down-regulated LPS-induced expression of inflammatory cytokines. In sum, we demonstrate that ClC-3 is a pro-inflammatory factor and that inhibition of ClC-3 inhibits inflammatory induction both in vitro and in vivo, suggesting that ClC-3 is a potential anti-inflammatory target.

Chloride channel activity is one of the critical factors responsible for cell apoptotic volume decrease (AVD). However, the roles of chloride channels in apoptosis have not been fully understood. In the current study, we assessed the role of chloride channels in hydrogen peroxide (H(2)O(2))-induced apoptosis of pheochromocytoma cells (PC12). Extracellular application of H(2)O(2) activated a chloride current and induced cell volume decrease in a few minutes. Incubation of cells with H(2)O(2) elevated significantly the membrane permeability to the DNA dye Hoechst 33258 in 1h and induced apoptosis of most PC12 cells tested in 24h. The chloride channel blocker NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) prevented appearance of H(2)O(2)-induced high membrane permeability and cell shrinkage, suppressed H(2)O(2)-activated chloride currents and protected PC12 cells from apoptosis induced by H(2)O(2). The results suggest that chloride channels may contribute to H(2)O(2)-induced apoptosis by ways of elevation of membrane permeability and AVD in PC12 cells.

NPPB3-(4-5-nitro-2-(3-phenylpropylamino) benzoic acid, as one of the most commonly used chloride channel blockers, is very essential for chloride channel studies. However, the role of NPPB in mediating apoptosis is uncertain. The purpose of this study was to investigate the apoptosis mechanism of NPPB in human cancer DU145 cell lines. Cell viability was examined by MTT assay. JC-1 fluorescent probe was used for detecting l membrane potential. Cell apoptosis was determined by flow method (FCM). The expression of apoptosis-related protein Caspase-3, Cleaved caspase-3, PARP, Cleaved PARP, Bcl-2, Mcl-1, Bcl-xl, Bcl-w and Bax was detected by western blotting. Cell proliferation rate was remarkably inhibited 2 h after treating with NPPB(50,100 μmol/L) (p

To investigate the role of chloride channels in the apoptosis of poorly differentiated nasopharyngeal carcinoma CNE-2Z cells induced by gambogic acid (GA).MTT assay was applied to detect the proliferation of CNE-2Z cells after GA treatment, and the cell apoptosis was detected by Hoechst 33342 staining. Whole-cell patch clamp technique was employed to record GA-activated Cl(-) currents in the cells.GA inhibited the cell proliferation in a time- and concentration-dependent manner with an IC(50) of 3.1 µmol/L for a 48-h treatment. The apoptosis-inducing effect of 8 µmol/L GA was attenuated by the chloride channel blocker NPPB (100 µmol/L) and tamoxifen (20 µmol/L). GA induced an outward-rectified Cl(-) current in the cells, which was significantly inhibited by NPPB.GA suppresses cell proliferation and induces apoptosis by activating Cl(-) channels in CNE-2Z cells, suggesting the important role of Cl(-) channels in GA-induced apoptosis.

To explore the expression of ER and PR mRNAs in endometrium with endometriosis.The rat model of endometriosis was established, and the expression of ER, PR mRNAs in the endometrium was examined by reverse transcription-polymerase chain reaction (RT-PCR).The expression of ER and PR mRNAs in ectopic endometrium was significantly lower than that in eutopic and normal endometrium (P0.01). But no difference was observed between eutopic and normal endometrium (P0.05). Ratio of ER/PR mRNA in ectopic endometrium was larger than that in eutopic and in normal endometrium (P0.01).The result illuminates that the increased ER plays a vital role in the onset of endometriosis.

To explore the the effect of gastrodine on hepatitic cell line L02 injury induced by ethanol.The growing L02 cells were treated with different concentrations of alcohol. For screening the proper concentration of alcohol, the proliferation of the cells were measured by MTT colorimetry. The level of reactive oxygen species (ROS) and change of mitochondrial membrane potential in cells were tested by flow cytometry. Reversed-phase high-performance liquid chromatography (RP-HPLC) was applied to assay ATP content in cells.The cells were injured when treated with 25 mL/L alcohol for 36 h. Compared with the control group , the ROS level in cells markedly increased while mitochondrial membrane potential and ATP content in cells significantly decreased (P0.01). Gastrodine obviously inhibited cell damage, reduced ROS level, and increased mitochondrial membrane potential and ATP content in cells.Above results suggest that gastrodine may suppress cellular injury and lipid peroxidation induced by ethanol, improve mitochondrial function and raise ATP level, which play an important role in protecting hepatocytes.

To construct the eukaryotic expression vectors of human cyclin D1 gene and express them in poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells).The full-length cyclin D1 was cloned from CNE-2Z cells by RT-PCR. The cDNA fragments were inserted into pIRES2-EGFP plasmids and pEGFP-C2 plasmids and confirmed by restriction enzyme digestion, PCR and sequencing. The recombinant vectors were transfected into CNE-2Z cells via Lipofectamine 2000, and the expression of cyclin D1 in the cells was examined by immunofluorescence and Western blotting.Agarose gel electrophoresis showed a 918 bp band of the RT-PCR products, which matched the expected size. Restriction enzyme digestion, PCR and sequencing demonstrated successful construction of the recombinant vectors. CNE-2Z cells transfected with the recombinant vectors expressed cyclin D1 protein or cyclin D1-GFP protein as were verified by immunofluorescence and Western blotting.We have cloned cyclin D1 gene and constructed its eukaryotic expression vectors that can be expressed in nasopharyngeal carcinoma cells, which may facilitate the study of the role of cyclin D1 in the development of nasopharyngeal carcinoma.

Lithium salts have been used as a class of mood stabilizing agents to treat bipolar disorders for over a century. Although lithium is known to affect cell proliferation, apoptosis and migration, the underlying mechanisms have not been well-explored. Emerging evidence indicates that cell volume regulation and volume-activated chloride channels are involved in cell proliferation, apoptosis and migration. To understand the mechanism of lithium's actions, we investigated the effect of lithium chloride on cell volume regulation and volume-activated chloride channels in the nasopharyngeal carcinoma cell line CNE-2Z. Our results show that lithium chloride attenuates regulatory volume decrease induced by 47% hypotonic challenges in a concentration-dependent manner with an IC(50) of 756 microM. Using the patch clamp techniques, we further show that lithium chloride concentration-dependently (IC(50)=440 microM) inhibits the volume-activated chloride current as well as the background chloride current. Furthermore, using a nanoscale atomic force microscope, we show that lithium chloride prevents the hypotonic challenge-induced changes in the ultrastructures of the cell membrane. These changes include an increase in the number and the size of the small holes, which are observed in the surface of the cell membrane under isotonic conditions. The lithium chloride-induced inhibition in cell volume regulation, in volume-activated chloride current and in the ultrastructures of the cell membrane may contribute to its effects on cell proliferation, apoptosis and migration.