Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Anthracenes ; pharmacology ; Chloride Channels ; metabolism ; Chlorides ; agonists ; antagonists & inhibitors ; metabolism ; Culture Media ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; metabolism ; Humans ; Hypotonic Solutions ; metabolism ; pharmacology ; Indoles ; pharmacology ; Ion Transport ; drug effects ; Maleimides ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; pharmacology ; Primary Cell Culture ; Protein Kinase C ; metabolism

BACKGROUNDAn acute respiratory distress syndrome (ARDS) is still one of the major challenges in critically ill patients. This study aimed to investigate the effect of inhibiting c-Jun N-terminal kinase (JNK) on ARDS in a lipopolysaccharide (LPS)-induced ARDS rat model.

METHODSThirty-six rats were randomized into three groups: control, LPS, and LPS + JNK inhibitor. Rats were sacrificed 8 h after LPS treatment. The lung edema was observed by measuring the wet-to-dry weight (W/D) ratio of the lung. The severity of pulmonary inflammation was observed by measuring myeloperoxidase (MPO) activity of lung tissue. Moreover, the neutrophils in bronchoalveolar lavage fluid (BALF) were counted to observe the airway inflammation. In addition, lung collagen accumulation was quantified by Sircol Collagen Assay. At the same time, the pulmonary histologic examination was performed, and lung injury score was achieved in all three groups.

RESULTSMPO activity in lung tissue was found increased in rats treated with LPS comparing with that in control (1.26 ± 0.15 U in LPS vs. 0.77 ± 0.27 U in control, P < 0.05). Inhibiting JNK attenuated LPS-induced MPO activity upregulation (0.52 ± 0.12 U in LPS + JNK inhibitor vs. 1.26 ± 0.15 U in LPS, P < 0.05). Neutrophils in BALF were also found to be increased with LPS treatment, and inhibiting JNK attenuated LPS-induced neutrophils increase in BALF (255.0 ± 164.4 in LPS vs. 53 (44.5-103) in control vs. 127.0 ± 44.3 in LPS + JNK inhibitor, P < 0.05). At the same time, the lung injury score showed a reduction in LPS + JNK inhibitor group comparing with that in LPS group (13.42 ± 4.82 vs. 7.00 ± 1.83, P = 0.001). However, the lung W/D ratio and the collagen in BALF did not show any differences between LPS and LPS + JNK inhibitor group.

CONCLUSIONSInhibiting JNK alleviated LPS-induced acute lung inflammation and had no effects on pulmonary edema and fibrosis. JNK inhibitor might be a potential therapeutic medication in ARDS, in the context of reducing lung inflammatory.

Animals ; Anthracenes ; therapeutic use ; Collagen ; metabolism ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism ; Lipopolysaccharides ; toxicity ; Lung ; drug effects ; metabolism ; pathology ; Male ; Rats ; Respiratory Distress Syndrome, Adult ; chemically induced ; drug therapy ; Signal Transduction ; drug effects

BACKGROUNDCathepsin L (CatL) is a cysteine protease with strong matrix degradation activity that contributes to photoaging. Mannose phosphate-independent sorting pathways mediate ultraviolet A (UVA)-induced alternate trafficking of CatL. Little is known about signaling pathways involved in the regulation of UVA-induced CatL expression and activity. This study aims to investigate whether a single UVA irradiation affects CatL expression and activity and whether mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway is involved in the regulation of UVA-induced CatL expression and activity in human dermal fibroblasts (HDFs).

METHODSPrimary HDFs were exposed to UVA. Cell proliferation was determined by a cell counting kit. UVA-induced CatL production and activity were studied with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and fluorimetric assay in cell lysates collected on three consecutive days after irradiation. Time courses of UVA-activated JNK and p38MAPK signaling were examined by Western blotting. Effects of MAPK inhibitors and knockdown of Jun and Fos on UVA-induced CatL expression and activity were investigated by RT-PCR, Western blotting, and fluorimetric assay. Data were analyzed by one-way analysis of variance.

RESULTSUVA significantly increased CatL gene expression, protein abundance, and enzymatic activity for three consecutive days after irradiation (F = 83.11, 56.14, and 71.19, respectively; all P < 0.05). Further investigation demonstrated phosphorylation of JNK and p38MAPK activated by UVA. Importantly, inactivation of JNK pathway significantly decreased UVA-induced CatL expression and activity, which were not affected by p38MAPK inhibition. Moreover, knockdown of Jun and Fos significantly attenuated basal and UVA-induced CatL expression and activity.

CONCLUSIONSUVA enhances CatL production and activity in HDFs, probably by activating JNK and downstreaming AP-1. These findings provide a new possible molecular approach for antiphotoaging therapy.

Anthracenes ; pharmacology ; Cathepsin L ; metabolism ; Cells, Cultured ; Child ; Child, Preschool ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Fibroblasts ; cytology ; drug effects ; metabolism ; radiation effects ; Humans ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; drug effects ; radiation effects ; Oncogene Proteins v-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-jun ; genetics ; metabolism ; Pyridines ; pharmacology ; Skin ; cytology ; Ultraviolet Rays

OBJECTIVETo study the effect of histone deacetylase inhibitors trichostatin A (TSA) and LBH589 on the growth of human renal cell carcinoma OS-RC-2 cells in vitro and explore the underlying molecular mechanism.

METHODSOS-RC-2 cells were treated with LBH589 or TSA with or without SP600125 pretreatment, and the cell viability was measured by MTT assay. The changes of cell cycle distribution and apoptosis of OS-RC-2 cells were examined by flow cytometry, and the expressions of c-Jun, p-c-Jun, Bcl-2, and Bax were quantified by Western blotting.

RESULTSTSA and LBH589 both inhibited the growth of OS-RC-2 cells in a dose- and time-dependent manner. TSA at 1 µnmol/L and LBH589 at 50 nmol/L caused obvious cell cycle arrest in G2/M phase and cell apoptosis, and significantly increased the protein levels of phosphorylated c-Jun. TSA treatment obviously increased Bax expression but decreased Bcl2 expression in the cells. The growth inhibitory effect of TSA was attenuated by the JNK inhibitor SP600125 in OS-RC-2 cells. TSA-induced phosphorylation of c-Jun and Bax upregulation was partially counteracted by SP600125.

CONCLUSIONTSA and LBH589 can cause cell cycle arrest and induce apoptosis in OS-RC-2 cells, in which process P-JNK pathway plays an important role.

Anthracenes ; pharmacology ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Renal Cell ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Survival ; Dose-Response Relationship, Drug ; Histone Deacetylase Inhibitors ; administration & dosage ; pharmacology ; Humans ; Hydroxamic Acids ; administration & dosage ; pharmacology ; Indoles ; administration & dosage ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Kidney Neoplasms ; metabolism ; pathology ; MAP Kinase Signaling System ; drug effects ; Phosphorylation ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

BACKGROUNDC-Jun N-terminal kinase (JNK) signaling pathway and ankylosis gene (ANK) play a critical role in endplate chondrocytes degeneration. The purpose of this study was to investigate whether the expression levels of ANK was associated with the activation of JNK.

METHODSCartilage endplates of 49 patients were divided into the control group (n = 19) and the experimental group (n = 30). The patients in the control group were graded 0 and those in the experimental group were graded I-III according to Miller's classification. Endplate chondrocytes were isolated by enzyme digestion and cultured in vitro. The inverted phase contrast microscope, teluidine blue staining, HE staining, real time RT-PCR, and MTT were used to observe morphological appearances, biological characteristics, and growth curve of endplate chondrocytes from the cartilage endplate of the two groups. Real time RT-PCR and Western blotting were used to analyze the mRNA and protein expression levels of associated factors in the degeneration process in the cultured endplate chondrocytes with or without subjected SP600125.

RESULTSThe expression levels of type II collagen, aggrecan, and ANK in endplate chondrocytes of experimental group were lower than that of control group and phosphorylation level of JNK in the experimental group which was higher than that in the control group. Application of JNK phosphorylation inhibitor to degeneration chondrocytes resulted in a marked decrease in the phosphorylation level of JNK and a significant increase in the expression levels of type II collagen, aggrecan, and ANK.

CONCLUSIONThe degeneration of the human cervical endplate chondrocytes might be promoted by JNK phosphorylation by down-regulating the expression of ANK.

Adult ; Aged ; Anthracenes ; pharmacology ; Cells, Cultured ; Cervical Vertebrae ; metabolism ; pathology ; Chondrocytes ; metabolism ; pathology ; Down-Regulation ; Female ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Male ; Middle Aged ; Phosphate Transport Proteins ; genetics ; physiology ; Phosphorylation

OBJECTIVETo study the role of c-jun N-terminal kinase (JNK) signaling pathway in chondrocyte apoptosis induced by nitric oxide (NO) using NO donor sodium nitroprusside (SNP) and JNK inhibitor SP600125.

METHODSArticular chondrocytes were separated from New Zealand rabbits aged 3 weeks by mechanical digestion and enzyme digestion and identified by toluidine blue staining, and then the chondrocytes were treated with SNP and SP600125 for 24 h. The cell apoptosis was evaluated by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL), and the expression levels of nuclear factor-kappa B (NF-κB) p65 and p53 were measured by western blot.

RESULTSCompared with those in control group, the early apoptotic rate of SNP-treated chondrocytes increased as the concentration of SNProse, exhibiting a concentration dependency (P < 0.05), and the expression levels of NF-κB p65 and p53 also increased (P < 0.05); JNK inhibitor SP600125 inhibited these increases (P < 0.05).

CONCLUSIONJNK signaling pathway plays an important role in NO-induced chondrocyte apoptosis. JNK inhibitor SP600125 can reduce NO-induced apoptosis and expression of NF-κB p65 and p53 in articular chondrocytes of rabbits in a concentration-dependent manner.

Animals ; Anthracenes ; pharmacology ; Apoptosis ; drug effects ; Cells, Cultured ; Chondrocytes ; drug effects ; metabolism ; pathology ; MAP Kinase Signaling System ; drug effects ; NF-kappa B ; metabolism ; Nitric Oxide ; pharmacology ; Rabbits ; Transcription Factor RelA ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVE: To investigate the extracellular release of high mobility group box 1 (HMGB1) in laryngeal Hep-2 carcinoma cells induced by hypoxia and its possible mechanism. METHOD: The changes of HMGB1 concentration in the culture medium as well as HMGB1 protein and mRNA expression in Hep-2 cells were investigated after the cells were cultured with 1% O2 for different durations. Inhibitory effects of MAPK pathway inhibitors (PD98059. SP600125, and SB202190) and nuclear NF-kappaB pathway inhibitor (PDTC) with various concentrations on extracellular HMGB1 release were observed in hypoxia-induced Hep-2 cells. The HMGB1 concentration and HMGB1 protein expression were measured by enzyme-linked immunosorbent assay (ELISA) and western blot, respectively. The HMGB1 mRNA expression was determined by real-time quantitative PCR(RT-PCR). RESULT: The HMGB1 concentration in the culture medium and the HMGB1 protein expression in Hep-2 cells increased after the cells were subjected to hypoxia culture for 12 h in a time-dependent manner. The level of HMGB1 mRNA expression in Hep-2 cells increased after the cells were induced by hypoxia for 6h PD98059 and SP600125 with 20 micromol/ L and PDTC with 50 mg/L partly inhibited extracellular release of HMGB1 in hypoxia-cultured Hcp-2 cells. CONCLUSION Hypoxia induces laryngeal carcinoma cells to release HMGH1. which may be related to MAPK/NF-kappaB signaling pathway.
Anthracenes ; Cell Hypoxia ; Cell Line, Tumor ; Flavonoids ; HMGB1 Protein ; metabolism ; Humans ; Imidazoles ; MAP Kinase Signaling System ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Protein Kinase Inhibitors ; Pyridines ; Pyrrolidines ; RNA, Messenger ; genetics ; Thiocarbamates

OBJECTIVETo study the effect and potential mechanism of expression of c-jun N-terminal kinase (JNK) signal pathway on neuron autophagy after diffuse brain injury (DBI).

METHODSMale Sprague Dawley rats (n = 216) were randomly divided into four groups: DBI group (n = 54), SP600125 intervene group (n = 54), DMSO group (n = 54) and sham operation group (n = 54). DBI rat model was established according to the description of Marmarou DBI. At different time points (1, 6, 12, 24, 48 and 72 h) after operation, the histopathologic changes of neurons in cortex were observed by HE staining method; The expression of p-JNK, p-P53, DRAM and Beclin-1 were detected by Western blot and immunohistochemistry.

RESULTSThe results showed that under light microscope degenerated and necrotic neurons were observed to be scattered in cortex at 6 h after operation in DBI group, but these changes were low in SP600125 intervene group. Compared with SP600125 intervene group, the expression of p-JNK in DBI group were enhanced obviously at 6, 12 and 24 h (F = 17.902, P < 0.05); the expression of p-P53 in DBI group were enhanced obviously at 12, 24, 48 and 72 h (F = 7.107, P < 0.05); the expression of DRAM in DBI group were enhanced obviously at 6, 12, 24, 48 and 72 h (F = 15.455, P < 0.05); the expression of Beclin-1 in DBI group were enhanced obviously at 6, 12, 24, 48 and 72 h (F = 11.517, P < 0.05). Compared with DBI group, the expression of p-JNK, p-P53, DRAM and Beclin-1 in DMSO group were similar at 1, 6, 12, 24, 48 and 72 h (F = 1.509, P > 0.05).

CONCLUSIONSThe present results indicate that SP600125 can dramatically improve trauma brain injury from autophagy after DBI and the molecular mechanism is related to the modulation of JNK signal pathway following DBI, while it measures the neuron autophagy by means of intervening JNK signal pathway.

Animals ; Anthracenes ; pharmacology ; Autophagy ; Brain Injuries ; metabolism ; pathology ; Disease Models, Animal ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Male ; Neurons ; pathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the protective effects and mechanism of SP600125-specificity inhibitor of c-Jun N-terminal kinase (JNK)on lung ischemia /reperfusion injury in rats.

METHODSThe unilateral lung ischemia/reperfusion model was replicated in vivo. Rats were randomly divided into three groups (n = 10): control group, ischemia/reperfusion group ( I/R group) and ischemia/reperfusion + SP600125 group (SP600125 group). The lung tissues sampled at the end of each experiment were assayed for wet/dry weight ratio (W/D),the injured alveoli rate (IAR), the expression of phosphorylation JNK (p-JNK) and JNK protein were detected by Western blot, the expression of Bcl-2, Bax, Caspase3 protein were detected by immunocytochemistry techniques, the pneumocyte apoptosis index (AI) was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end abeling(TUNEL), the ultrastructure changes were observed under electron microscope.

RESULTSCompared to I/R group, the expression of p-JNK, Bcl-2, Bax and caspase-3 protein were markedly decreased (all P < 0.01), the expression of Bcl-2 protein and the ratio of Bcl-2/Bax were markedly increased in SP600125 group(all P < 0.01). The value of AI, W/D, IAR showed significantly lower than those in I/R group (all P <0.01). Meanwhile, light morphological and ultrastructure injury were found in SP600125 group.

CONCLUSIONSP600125 can suppress JNK signal pathway, up-regulate the ratio of Bcl-2/Bax to inhibit Caspase-3 dependent apoptosis, so that it protects lung tissue from ischemia/reperfusion injury.

Animals ; Anthracenes ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Lung ; blood supply ; metabolism ; pathology ; MAP Kinase Signaling System ; Phosphorylation ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism ; pathology ; bcl-2-Associated X Protein ; metabolism

NMDA-induced excitotoxicity cause severe neuronal damage including apoptosis and necrosis. The present study was aimed to evaluate the proportion of NMDA-induced apoptosis of rat cortical neurons and discover signal transduction mechanism. Caspase inhibitor and lactate dehydrogenase (LDH) assay were used to study the NMDA-induced apoptosis. To explore the involved signal pathways, the primary culture of rat cortical neurons were pretreated by the inhibitors of three MAPK pathways, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. With 2 h of NMDA treatment, cellular apoptosis was measured by caspase-3 activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and Annexin V staining. The results showed that: (1) Caspase-dependent apoptosis accounted for 22.49% in NMDA-induced neuronal death; (2) Pretreatment with p38 MAPK inhibitor SB203580 (10 μmol/L) significantly decreased NMDA-mediated caspase-3 activity by 30.43% (P < 0.05). However, ERK inhibitor PD98059 (20 μmol/L) or JNK inhibitor SP600125 (20 μmol/L) did not influence caspase-3 activity; (3) Pretreatment with SB203580 significantly reduced the number of NMDA-induced TUNEL-positive cells by 33.10% (P < 0.05). PD98059 (20 μmol/L) or SP600125 (20 μmol/L) did not show obvious effect; (4) Pretreatment with SB203580 (10 μmol/L) significantly reduced the number of NMDA-induced early apoptotic neurons by 55.56% (P < 0.05). Also, SP600125 (20 μmol/L) significantly decreased the amount of late apoptotic/dead cells by 67.59% (P < 0.05). There was no effect of PD98059 (20 μmol/L). These results indicate that: (1) NMDA induces neuronal apoptosis besides necrosis; (2) p38 MAPK, but not JNK and ERK, is involved in NMDA-induced neuronal apoptosis, and inhibition of the apoptotic signaling pathway contributes to neuroprotection; (3) JNK activation might contribute to NMDA-induced neuronal necrosis rather than apoptosis.
Animals ; Anthracenes ; pharmacology ; Apoptosis ; Caspase 3 ; metabolism ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Imidazoles ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; MAP Kinase Signaling System ; N-Methylaspartate ; pharmacology ; Neurons ; cytology ; Primary Cell Culture ; Pyridines ; pharmacology ; Rats ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors