ABSTRACT
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.
Subject(s)
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Chloride Channels/antagonists & inhibitors , NF-kappa B/antagonists & inhibitors , Peritonitis/drug therapy , Toll-Like Receptor 4/antagonists & inhibitors , Animals , Chloride Channels/genetics , Chloride Channels/immunology , Female , Gene Expression Regulation , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Lipopolysaccharides , Male , Mice , NF-kappa B/genetics , NF-kappa B/immunology , Peritonitis/chemically induced , Peritonitis/genetics , Peritonitis/pathology , RAW 264.7 Cells , RNA, Small Interfering/genetics , RNA, Small Interfering/immunology , Signal Transduction , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/immunologyABSTRACT
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.
Subject(s)
Benzofurans/therapeutic use , Disseminated Intravascular Coagulation/chemically induced , Disseminated Intravascular Coagulation/drug therapy , Lipopolysaccharides/toxicity , Animals , Disseminated Intravascular Coagulation/complications , Disseminated Intravascular Coagulation/pathology , Gene Expression Regulation/drug effects , Kidney Diseases/drug therapy , Kidney Diseases/etiology , Liver Diseases/drug therapy , Liver Diseases/etiology , Male , Rabbits , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
OBJECTIVE: To investigate the role of chloride channels in the apoptosis of poorly differentiated nasopharyngeal carcinoma CNE-2Z cells induced by gambogic acid (GA). METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Subject(s)
Apoptosis/drug effects , Chloride Channels/physiology , Nasopharyngeal Neoplasms/pathology , Xanthones/pharmacology , Cell Line, Tumor , Chloride Channels/drug effects , Humans , Patch-Clamp TechniquesABSTRACT
AIM: To explore the the effect of gastrodine on hepatitic cell line L02 injury induced by ethanol. METHODS: 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. RESULTS: 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 (P<0.01). Gastrodine obviously inhibited cell damage, reduced ROS level, and increased mitochondrial membrane potential and ATP content in cells. CONCLUSION: 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.
Subject(s)
Benzyl Alcohols/pharmacology , Ethanol/toxicity , Glucosides/pharmacology , Liver/drug effects , Cell Line , Cell Proliferation/drug effects , Humans , Liver/cytology , Liver/metabolism , Membrane Potential, Mitochondrial/drug effects , Reactive Oxygen Species/metabolismABSTRACT
OBJECTIVE: To construct the eukaryotic expression vectors of human cyclin D1 gene and express them in poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells). METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Subject(s)
Cyclin D1/biosynthesis , Nasopharyngeal Neoplasms/pathology , Cell Line, Tumor , Cloning, Molecular , Cyclin D1/genetics , Genetic Vectors/genetics , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Humans , Nasopharyngeal Neoplasms/metabolism , Plasmids/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , TransfectionABSTRACT
AIM: To explore the expression of ER and PR mRNAs in endometrium with endometriosis. METHODS: 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). RESULTS: The expression of ER and PR mRNAs in ectopic endometrium was significantly lower than that in eutopic and normal endometrium (P < 0.01). But no difference was observed between eutopic and normal endometrium (P > 0.05). Ratio of ER/PR mRNA in ectopic endometrium was larger than that in eutopic and in normal endometrium (P < 0.01). CONCLUSION: The result illuminates that the increased ER plays a vital role in the onset of endometriosis.
