Neuropathic pain is a chronic debilitating symptom characterized by spontaneous pain and mechanical allodynia. It occurs in distinct forms, including brush-evoked dynamic and filament-evoked punctate mechanical allodynia. Potassium channel 2.1 (Kir2.1), which exhibits strong inward rectification, is and regulates the activity of lamina I projection neurons. However, the relationship between Kir2.1 channels and mechanical allodynia is still unclear. In this study, we first found that pretreatment with ML133, a selective Kir2.1 inhibitor, by intrathecal administration, preferentially inhibited dynamic, but not punctate, allodynia in mice with spared nerve injury (SNI). Intrathecal injection of low doses of strychnine, a glycine receptor inhibitor, selectively induced dynamic, but not punctate allodynia, not only in naïve but also in ML133-pretreated mice. In contrast, bicuculline, a GABA receptor antagonist, induced only punctate, but not dynamic, allodynia. These results indicated the involvement of glycinergic transmission in the development of dynamic allodynia. We further found that SNI significantly suppressed the frequency, but not the amplitude, of the glycinergic spontaneous inhibitory postsynaptic currents (gly-sIPSCs) in neurons on the lamina II-III border of the spinal dorsal horn, and pretreatment with ML133 prevented the SNI-induced gly-sIPSC reduction. Furthermore, 5 days after SNI, ML133, either by intrathecal administration or acute bath perfusion, and strychnine sensitively reversed the SNI-induced dynamic, but not punctate, allodynia and the gly-sIPSC reduction in lamina IIi neurons, respectively. In conclusion, our results suggest that blockade of Kir2.1 channels in the spinal dorsal horn selectively inhibits dynamic, but not punctate, mechanical allodynia by enhancing glycinergic inhibitory transmission.
Animals ; Bicuculline ; pharmacology ; Disease Models, Animal ; Glycine ; metabolism ; Hyperalgesia ; drug therapy ; etiology ; metabolism ; Imidazoles ; pharmacology ; Inhibitory Postsynaptic Potentials ; drug effects ; physiology ; Male ; Mice, Inbred C57BL ; Neurons ; drug effects ; metabolism ; Neurotransmitter Agents ; pharmacology ; Peripheral Nerve Injuries ; drug therapy ; metabolism ; Phenanthrolines ; pharmacology ; Potassium Channels, Inwardly Rectifying ; antagonists & inhibitors ; metabolism ; Receptors, GABA-A ; metabolism ; Receptors, Glycine ; metabolism ; Strychnine ; pharmacology ; Synaptic Transmission ; drug effects ; physiology ; Tissue Culture Techniques ; Touch

To investigate the role of p38MAPK signal pathway in spinal cord and dorsal root ganglion (DRG) in rats with phantom limb pain and the effects of specific inhibitors.
 Methods: Healthy adult male SD rats (n=48) were cut off one side of the sciatic under anesthesia to establish a model of phantom limb pain. In addition, the healthy rats were taken as a sham group (group S, n=24). The animals were scored by observing the action of chewing (0=no chewing, 13=the worst chewing) after the operation and were sacrificed on the following day after the operation. The successful model of phantom limb pain were randomly divided into 2 groups: a phantom limb pain group (group P, n=24) and a phantom limb pain plus inhibitor group (group P+I, n=24). SB203580 was given to the rat at 0.8 mg/kg on every Monday until the rats were sacrificed, the rest of the rats received an equal amount of saline. Eight rats from each group were randomly taken for the determination of levels of P-p38MAPK in spinal cord and DRG before administration and on the 4th, 6th, 8th weekend following the administration, respectively.
 Results: In the sham group, no animal developed chewing. Meanwhile, rats in successful model of phantom limb pain group began chewing from the 2nd day after operation with scores at eight to eleven. The chewing scores in the P+I group were reduced after the treatment. Compared with group S, P-p38MAPK levels were elevated in groups of P and P+I (P<0.05 or P<0.01). Compared with group P, P-p38MAPK level was decreased in the group P+I (P<0.05 or P<0.01).
 Conclusion: P38MAPK signal pathway involves in the development of phantom limb pain.
Animals ; Disease Models, Animal ; Enzyme Inhibitors ; pharmacology ; Ganglia, Spinal ; enzymology ; Imidazoles ; pharmacology ; Male ; Mastication ; physiology ; Phantom Limb ; enzymology ; etiology ; physiopathology ; Pyridines ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; injuries ; Self Mutilation ; enzymology ; physiopathology ; Signal Transduction ; Spinal Cord ; enzymology ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

Cancer stem cells (CSCs) have tumor initiation, self-renewal, metastasis and chemo-resistance properties in various tumors including colorectal cancer. Targeting of CSCs may be essential to prevent relapse of tumors after chemotherapy. Phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) signals are central regulators of cell growth, proliferation, differentiation, and apoptosis. These pathways are related to colorectal tumorigenesis. This study focused on PI3K and mTOR pathways by inhibition which initiate differentiation of SW620 derived CSCs and investigated its effect on tumor progression. By using rapamycin, LY294002, and NVP-BEZ235, respectively, PI3K and mTOR signals were blocked independently or dually in colorectal CSCs. Colorectal CSCs gained their differentiation property and lost their stemness properties most significantly in dual-blocked CSCs. After treated with anti-cancer drug (paclitaxel) on the differentiated CSCs cell viability, self-renewal ability and differentiation status were analyzed. As a result dual-blocking group has most enhanced sensitivity for anti-cancer drug. Xenograft tumorigenesis assay by using immunodeficiency mice also shows that dual-inhibited group more effectively increased drug sensitivity and suppressed tumor growth compared to single-inhibited groups. Therefore it could have potent anti-cancer effects that dual-blocking of PI3K and mTOR induces differentiation and improves chemotherapeutic effects on SW620 human colorectal CSCs.
AC133 Antigen/genetics/metabolism ; Animals ; Antineoplastic Agents/pharmacology/therapeutic use ; Cell Differentiation/*drug effects ; Cell Line, Tumor ; Cell Survival/drug effects ; Chromones/pharmacology/therapeutic use ; Colorectal Neoplasms/drug therapy/metabolism/pathology ; Humans ; Imidazoles/pharmacology/therapeutic use ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Morpholines/pharmacology/therapeutic use ; Neoplastic Stem Cells/cytology/drug effects/metabolism ; Paclitaxel/pharmacology/therapeutic use ; Phosphatidylinositol 3-Kinases/*antagonists & inhibitors/metabolism ; Quinolines/pharmacology/therapeutic use ; SOXB1 Transcription Factors/genetics/metabolism ; Signal Transduction/*drug effects ; Sirolimus/pharmacology/therapeutic use ; TOR Serine-Threonine Kinases/*antagonists & inhibitors/metabolism ; Xenograft Model Antitumor Assays

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

OBJECTIVE: To determine the effect of p38 MAPK inhibitor (SB203580) on TNF-α -induced high mobility group protein B1 (HMGB1) expression in microglial cells.
 METHODS: Microglial cells were treated with TNF-α (25 ng/mL, TNF-α group), TNF-α plus SB203580 (10 μmol/L, TNF-α+SB203580 group), SB203580 (SB203580 group) or serum-free medium (control group). After 16 h of incubation, the protein levels of p-p38 MAPK and HMGB1, and mRNA levels of HMGB1 were examined by ELISA, Western Blot and RT-PCR, respectively.
 RESULTS: There was a significant increase in p-p38 MAPK and HMGB1 levels in TNF-α-treated microglia cells (P<0.01). The TNF-α-induced HMGB1 protein and mRNA expression was suppressed by SB203580.
 CONCLUSION TNF-α up-regulates HMGB1 expression in microglial cells through activation of the p38 MAPK pathway.
Blotting, Western ; HMGB1 Protein ; metabolism ; Humans ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; Microglia ; drug effects ; metabolism ; Pyridines ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

The aim of the present study is to investigate the expressions of ATP-sensitive K(+) channels (KATP) in pulmonary artery smooth muscle cells (PASMCs) and the relationship with p38 MAPK signal pathway in rats. Male SD rat PASMCs were cultured in vitro, and a model of hypoxia and hypercapnia was reconstructed. PASMCs were divided to normal (N), hypoxia-hypercapnia (H), hypoxia-hypercapnia+DMSO incubation (HD), hypoxia-hypercapnia+SB203580 (inhibitor of p38 MAPK pathway) incubation (HS) and hypoxia-hypercapnia+Anisomycin (agonist of p38 MAPK pathway) incubation (HA) groups. Western blot was used to detect the protein expression of SUR2B and Kir6.1; semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of SUR2B and Kir6.1. The results demonstrated that: (1) Compared with N, H, HD and HS groups, the expressions of Kir6.1 mRNA and protein in PASMCs of HA group were decreased significantly (P < 0.01), but there were no differences among N, H, HD and HS groups (P > 0.05); (2) Compared with N group, the expressions of SUR2B mRNA and protein in H, HD, HS and HA groups were increased significantly (P < 0.05), but there were no differences among H, HD, HS and HA groups (P > 0.05). The results imply that: (1) Hypoxia-hypercapnia, SB203580 didn't change the expressions of Kir6.1 mRNA and protein in PASMCs, but Anisomycin decreased the expressions of Kir6.1 mRNA and protein, so Kir6.1 may be regulated by the other subfamily of MAPK pathway; (2) Hypoxia-hypercapnia raised SUR2B mRNA and protein expressions in PASMCs, but SB203580 and Anisomycin did not affect the changes, so the increasing of SUR2B mRNA and protein induced by hypoxia-hypercapnia may be not depend on p38 MAPK pathway.
Animals ; Anisomycin ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Hypercapnia ; Imidazoles ; pharmacology ; KATP Channels ; metabolism ; MAP Kinase Signaling System ; Male ; Myocytes, Smooth Muscle ; metabolism ; Pulmonary Artery ; cytology ; Pyridines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Sulfonylurea Receptors ; metabolism ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

Curcumin, as a main pharmacological component in the traditional Chinese medicine-turmeric, has shown anti-inflammatory, anti-oxidation, anti-tumor and anti-fibrotic effects. This study aimed to investigate the possible underlying signaling pathway which was involved in the inhibition of LDL-induced proliferation of mesangial cells and matrix by curcumin. Rat mesangial cells in vitro were incubated with low-density lipoprotein (LDL) and different concentrations of curcumin (0, 6.25, 12.5, 25.0 μmol/L) or p38 MAPK inhibitor, SB203580 (10 μmol/L). Under LDL incubation, mesangial cells proliferated, the expression of MMP-2 mRNA and protein was decreased, the expression of COX-2 mRNA and protein was increased, reactive oxygen species (ROS) generation was increased and p38 MAPK was activated significantly (P<0.05). When LDL-induced cells were treated with curcumin in the concentration of 12.5 or 25.0 μmol/L, LDL-induced proliferation of mesangial cells was suppressed, the expression of MMP-2 mRNA and protein increased, the expression of COX-2 mRNA and protein downregulated, the production of ROS inhibited and p38 MAPK inactivated (P<0.05). In conclusion, curcumin can inhibit the LDL-induced proliferation of mesangial cells and up-regulate the expression of MMP-2, which may be related with the inhibitory effect of curcumin on COX-2 expression, ROS production and p38 MAPK.
Animals ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; Blotting, Western ; Cell Proliferation ; drug effects ; Cells, Cultured ; Curcumin ; pharmacology ; Cyclooxygenase 2 ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Down-Regulation ; Enzyme Inhibitors ; pharmacology ; Extracellular Matrix ; drug effects ; metabolism ; Gene Expression ; drug effects ; Imidazoles ; pharmacology ; Lipoproteins, LDL ; pharmacology ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Mesangial Cells ; drug effects ; metabolism ; Pyridines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

The present study was to investigate the effects of exogenous insulin-like growth factor binding protein 7 (IGFBP7) on the proliferation of human breast cancer cell line MDA-MB-453 and its possible mechanism. By means of MTT method in vitro, the results showed exogenous IGFBP7 inhibited the growth of MDA-MB-453 cells (IC50 of IGFBP7 = 8.49 μg/mL) in time- and concentration-dependent manner. SB203580, p38(MAPK) inhibitor, blocked the anti-proliferative effect of exogenous IGFBP7. The flow cytometry assay showed that exogenous IGFBP7 remarkably induced G0/G1 arrest in MDA-MB-453 cells. The Western blot showed that exogenous IGFBP7 promoted phosphorylation of p38(MAPK), up-regulated expression of p21(CIP1/WAF1), and inhibited phosphorylation of Rb. SB203580 restrained exogenous IGFBP7-induced regulation of p21(CIP1/WAF1) and p-Rb in MDA-MB-453 cells. In conclusion, the present study suggests that exogenous IGFBP7 could activate the p38(MAPK) signaling pathway, upregulate p21(CIP1/WAF1) expression, inhibit phosphorylation of Rb, and finally induce G0/G1 arrest in MDA-MB-453 cells.
Breast Neoplasms ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Female ; Humans ; Imidazoles ; pharmacology ; Insulin-Like Growth Factor Binding Proteins ; pharmacology ; Phosphorylation ; Pyridines ; pharmacology ; Signal Transduction ; Somatomedins ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

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

OBJECTIVETo examine the effects of niacin on lysophosphatidylcholine (LPC)-induced intercellular adhesion molecule-1 (ICAM-1), and gained insight to the mechanisms.

METHODHuman umbilical vein endothelial cell line was cultured using Medium 200 medium in incubator at 37 °C and 5% CO2 condition.Experimental groups:(1) the negative control group:medium; (2) LPC different time groups:the medium added with 20 µmol/L final concentration of LPC, were cultured for 10 min and 8 h, 24 h; (3) LPC+ p38-mitogen-activated protein kinase (p38MAPK) inhibitor (SB203580) group:the medium added with 10 µmol/L p38MAPK inhibitor (SB203580) was cultured for 1 h, then human umbilical vein endothelial cells (HUVECs) added with the LPC were cultured for 10 min, 8 h and 24 h.(4) LPC+different niacin dose group:after separately adding with 0.25, 0.5, 1 mmol/L niacin, the cells were cultured for 18 h, then HUVECs added with the LPC were cultured for 10 min, 8 h and 24 h. Cell concentration in each group was 5×10(5)/ml, inoculated in 6-well plates, each well 1 ml. Detected by Western blot analysis of pp38MAPK, ICAM-1 protein content, real-time quantitative PCR to detect endothelial cell ICAM-1 mRNA expression, cell immunofluorescence to detect LPC-induced ICAM-1 protein expression.

RESULTIn LPC 24 h group, the expression of ICAM-1 protein was significantly increased 0.786 ± 0.02, the LPC+niacin group, ICAM-1 protein levels (0.487 ± 0.015) was significantly lower than the LPC 24 h group (P < 0.01), in LPC+SB203580 intervention group, ICAM-1 protein levels (0.461 ± 0.011) was significantly lower than that of the LPC 24 h group (P < 0.01), but did not reach the level of the control group. Adding LPC to culture for 10 min, phosphorylation of p38MAPK (pp38MAPK) reached its peak (0.47 ± 0.02), niacin could reduce the pp38MAPK (0.07 ± 0.02), SB203580 could also reduce its activity (0.11 ± 0.02). Adding LPC to culture for 8 h, ICAM-1 mRNA expression (8.16 ± 0.15) compared with the control group (1.00 ± 0.02) had a significant increase (t = 24.34, P < 0.01). Compared with the LPC 8 h, niacin reduced LPC-induced ICAM-1 mRNA expression (3.85 ± 0.14), and showed a dose-dependent manner (F = 8.06, P < 0.01), while SB203580 could not effectively reduce the ICAM-1 mRNA (8.09 ± 0.11).

CONCLUSIONNiacin prevented LPC-induced endothelial dysfunction by reducing expression of ICAM-1. These mechanisms appeared to be at least partly mediated by suppression of the pp38MAPK in endothelial cells. These pleiotropic effects of niacin may potentially contribute to the beneficial effects of risk reduction for atherosclerotic disease.

Atherosclerosis ; metabolism ; prevention & control ; Cell Adhesion ; drug effects ; Cells, Cultured ; Enzyme Inhibitors ; administration & dosage ; pharmacology ; Gene Expression Regulation ; drug effects ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Imidazoles ; administration & dosage ; pharmacology ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Lysophosphatidylcholines ; administration & dosage ; pharmacology ; Niacin ; administration & dosage ; pharmacology ; Pyridines ; administration & dosage ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism