OBJECTIVE: To explore the dynamic impacts of simulated microgravity (SM) on different vital brain regions of rats. METHODS: Microgravity was simulated for 7 and 21 days, respectively, using the tail-suspension rat model. Histomorphology, oxidative stress, inflammatory cytokines and the expression of some key proteins were determined in hippocampus, cerebral cortex and striatum. RESULTS: 21-day SM decreased brain derived neurotrophic factor and induced neuron atrophy in the cerebral cortex. Strong oxidative stress was triggered at day 7 and the oxidative status returned to physiological level at day 21. Inflammatory cytokines were gradually suppressed and in striatum, the suppression was regulated partially through c-Jun/c-Fos. CONCLUSION The results revealed that the significant impacts of SM on rat brain tissue depended on durations and regions, which might help to understand the health risk and to prevent brain damage for astronauts in space travel.
Animals ; Brain ; metabolism ; pathology ; Brain-Derived Neurotrophic Factor ; metabolism ; Cytokines ; metabolism ; Male ; Oxidative Stress ; Proto-Oncogene Proteins c-fos ; metabolism ; Proto-Oncogene Proteins c-jun ; metabolism ; Random Allocation ; Rats ; Weightlessness Simulation

OBJECTIVE: To investigate the mechanism by which doublecortin promotes the recovery of cytoskeleton in arginine vasopressin (AVP) neurons in rats with electrical lesions of the pituitary stalk (PEL). METHODS: Thirty-two SD rats were randomized into PEL group with electrical lesions of the pituitary stalk through the floor of the skull base (=25) and sham operation group (=7), and the daily water consumption (DWC), daily urine volume (DUV) and urine specific gravity (USG) of the rats were recorded. Four rats on day 1 and 7 rats on each of days 3, 7 and 14 after PEL as well as the sham-operated rats were sacrificed for detection of the expressions of β-Tubulin (Tuj1), doublecortin and caspase- 3 in the AVP neurons of the supraoptic nucleus using immunofluorescence assay and Western blotting. RESULTS: After PEL, the rats exhibited a typical triphasic pattern of diabetes insipidus, with the postoperative days 1-2 as the phase one, days 3-5 as the phase two, and days 6-14 as the phase three. Immunofluorescent results indicated the repair of the AVP neurons evidenced by significantly increased doublecortin expressions in the AVP neurons following PEL; similarly, the expression of Tuj1 also increased progressively after PEL, reaching the peak level on day 7 after PEL. The apoptotic rates of the AVP neurons exhibited a reverse pattern of variation, peaking on postoperative day 3 followed by progressive reduction till day 14. Western blotting showed that the expressions of c-Jun and p-c-Jun were up-regulated significantly on day 3 ( < 0.05) and 7 ( < 0.01) after PEL, while an upregulated p-JNK expression was detected only on day 3 ( < 0.05), as was consistent with the time-courses of neuronal recovery and apoptosis after PEL. CONCLUSIONS JNK/c-Jun pathway is activated after PEL to induce apoptosis of AVP neurons in the acute phase and to promote the repair of neuronal cytoskeleton by up-regulation of doublecortin and Tuj1 expressions.
Animals ; Apoptosis ; Arginine Vasopressin ; pharmacology ; Cytoskeleton ; metabolism ; MAP Kinase Signaling System ; Neurons ; cytology ; Pituitary Gland ; cytology ; injuries ; Proto-Oncogene Proteins c-jun ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Regeneration ; Tubulin ; metabolism

PURPOSE: In the gastric mucosa of Helicobacter pylori (H. pylori)-infected patients with gastritis or adenocarcinoma, proliferation of gastric epithelial cells is increased. Hyperproliferation is related to induction of oncogenes, such as β-catenin and c-myc. Even though transcription factors NF-κB and AP-1 are activated in H. pylori-infected cells, whether NF-κB or AP-1 regulates the expression of β-catenein or c-myc in H. pylori-infected cells has not been clarified. The present study was undertaken to investigate whether H. pylori-induced activation of NF-κB and AP-1 mediates the expression of oncogenes and hyperproliferation of gastric epithelial cells. MATERIALS AND METHODS: Gastric epithelial AGS cells were transiently transfected with mutant genes for IκBα (MAD3) and c-Jun (TAM67) or treated with a specific NF-κB inhibitor caffeic acid phenethyl ester (CAPE) or a selective AP-1 inhibitor SR-11302 to suppress activation of NF-κB or AP-1, respecively. As reference cells, the control vector pcDNA was transfected to the cells. Wild-type cells or transfected cells were cultured with or without H. pylori. RESULTS: H. pylori induced activation of NF-κB and AP-1, cell proliferation, and expression of oncogenes (β-catenein, c-myc) in AGS cells, which was inhibited by transfection of MAD3 and TAM67. Wild-type cells and the cells transfected with pcDNA showed similar activities of NF-κB and AP-1, proliferation, and oncogene expression regardless of treatment with H. pylori. Both CAPE and SR-11302 inhibited cell proliferation and expression of oncogenes in H. pylori-infected cells. CONCLUSION: H. pylori-induced activation of NF-κB and AP-1 regulates transcription of oncogenes and mediates hyperproliferation in gastric epithelial cells.
Blotting, Western ; Caffeic Acids ; Cell Line, Tumor ; Cell Proliferation ; DNA, Bacterial/analysis/genetics ; DNA-Binding Proteins/*metabolism ; Epithelial Cells/*metabolism ; Gastric Mucosa/*metabolism/pathology ; Gastritis/pathology ; Gene Expression Regulation, Bacterial ; Helicobacter Infections/metabolism/pathology/physiopathology ; Helicobacter pylori/pathogenicity/physiology ; Humans ; NF-kappa B/antagonists & inhibitors/*biosynthesis/metabolism ; Peptide Fragments ; Phenylethyl Alcohol/analogs & derivatives ; Proto-Oncogene Proteins c-jun ; Repressor Proteins ; Transcription Factor AP-1/*biosynthesis ; Transcription Factors/*metabolism ; beta Catenin/*metabolism

OBJECTIVETo investigate the effect of inhibiting and activating Wnt signalling pathway on monocyte differentiation of HL-60 cells induced with a new steroidal drug NSC67657 and its possible mechamism.

METHODSThe HL-60 cells were treated with 5, 10 and 20 µmol/L XAV-939 (inhibitor of Wnt signalling pathway) for 3 days, and with 10, 20 and 30 mmol/L LiCl (activator of Wnt signalling pathway) for 1 day; the expression levels of down-stream genes and proteins of Wnt signolling pathway were detected by RT-PCR and Western blot, respectively; the expression of cell surface differentiation antigen CD14 and early apoptosis of HL-60 cells was detected by flow cytometry, moreover the most suitable concentration of Wnt inhibitor and activator for HL-60 cells was determined. Then the HL-60 cells with inhibited and activated Wnt pathway were treated with NSC67657 of 10 µmol/L for 3 days; the expression levels of CD14 and down-stream target proteins of Wnt signalling pathway in blank control (culture mediam) group, simple NSC67657-treated group, NSC67657 combined with inhibitor group and NSC67657 combined activator group were compared and analyzed.

RESULTS20 µmol/L XAV-939 and 20 mmol/L LiCl could effectively inhibit and activate Wnt signalling pathway of HL-60 cells respectively, could significantly down- and up-regulate the expression of cyclinD1, TCF1 and c-Jun genes (P < 0.05) and proteins (P < 0.05); moreover, the number of CD10(+) HL-60 cells in these conditions was below 1%, no early apoptosis of HL-60 cells was found. In the simple NSC67657-treated groups, the expression of cyclinD1, TCF1 and c-Jun proteins was down-regulated (P < 0.05), and the percentage of CD14(+) HL-60 cells accounted for 62.13 ± 9.44; after the HL-60 cells were treated with XAV-939, the NSC67657 could more significantly down-regulate the expression of cyclinD1, TCF1 and c-Jun proteins and the percentage of CD14(+) HL-60 cell accounted for 84.17 ± 5.39%, as compared with simple NSC67657-treated group; as compared with blank controls group, the expression of cyclinD1, TCF1 and c-Jun proteins was more obviously down-regulated and the percentage of CD14(+) HL-60 cells decreased to 33.99 ± 8.37% in NSC67657 combined LiC1 streated group, but which were higher than those in simple NSC67657-treated group (P < 0.05).

CONCLUSION20 µmol/L XAV-939 and 20 mmol/L LiCl as effective inhabitor and activator of Wnt signalling pathway respectively can significantly down- and up-regulate the expression of Wnt down-stream pathway target genes and proteins. The influence of XAV-939 and LiC1 on differentiation of HL-60 cells induced by NSC67657 suggests that Wnt signalling pathway plays a key role in monocyte differentiction of HL-60 cells induced by NSC67657.

Apoptosis ; Cell Differentiation ; Cyclin D1 ; metabolism ; Flow Cytometry ; HL-60 Cells ; Hepatocyte Nuclear Factor 1-alpha ; metabolism ; Humans ; Lipopolysaccharide Receptors ; metabolism ; Mesylates ; pharmacology ; Monocytes ; cytology ; Proto-Oncogene Proteins c-jun ; metabolism ; Steroids ; pharmacology ; Wnt Signaling Pathway

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 observe the effect of solar infrared ray (IR) radiation on the expressions of c-Jun and collagens I and III in cultured human skin fibroblasts (HSFs) and explore the molecular mechanism by which IR radiation causes aging of the skin.

METHODSPrimarily cultured HSFs exposed to IR radiation were examined for changes of the cell viability with MTT assay. The mRNA and protein expressions of c-Jun and collagens I and III was detected with real-time quantitative PCR and immunocytochemistry.

RESULTSMTT assay showed that IR irradiation caused inhibition of cell proliferation compared with the control cells. The mRNA and protein expression of collagen I was decreased significantly by IR irradiation with the increase of the irradiation dose (P<0.01). HSFs irradiated by IR for 12 h showed a dose-dependent reduction of the expression of collagen type III mRNA and protein (P<0.05, P<0.01), but the expression increased dose-dependently in response to IR exposure for 24 h (P<0.05 or 0.01). IR irradiation enhanced the mRNA and protein expression of c-Jun in a dose-dependence manner (P<0.05 or 0.01).

CONCLUSIONSIR irradiation can increase the expression of c-Jun, inhibit the expression of collagen I, and cause disturbance in collagen III expression in human skin fibroblasts, which may be one of the mechanism of IR radiation to initiate and promote skin photoaging.

Cell Proliferation ; Cell Survival ; Cells, Cultured ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Fibroblasts ; metabolism ; radiation effects ; Humans ; Infrared Rays ; Proto-Oncogene Proteins c-jun ; metabolism ; RNA, Messenger ; metabolism ; Skin ; cytology ; Skin Aging ; Ultraviolet Rays

OBJECTIVETo study the ability of aqueous extract of Hericium erinaceus mushroom in the treatment of nerve injury following peroneal nerve crush in Sprague-Dawley rats.

METHODSAqueous extract of Hericium erinaceus was given by daily oral administration following peroneal nerve crush injury in Sprague-Dawley rats. The expression of protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) signaling pathways; and c-Jun and c-Fos genes were studied in dorsal root ganglia (DRG) whereas the activity of protein synthesis was assessed in peroneal nerves by immunohistochemical method.

RESULTSPeripheral nerve injury leads to changes at the axonal site of injury and remotely located DRG containing cell bodies of sensory afferent neurons. Immunofluorescence studies showed that DRG neurons ipsilateral to the crush injury in rats of treated groups expressed higher immunoreactivities for Akt, MAPK, c-Jun and c-Fos as compared with negative control group (P <0.05). The intensity of nuclear ribonucleoprotein in the distal segments of crushed nerves of treated groups was significantly higher than in the negative control group (P <0.05).

CONCLUSIONH. erinaceus is capable of promoting peripheral nerve regeneration after injury. Potential signaling pathways include Akt, MAPK, c-Jun, and c-Fos, and protein synthesis have been shown to be involved in its action.

Agaricales ; chemistry ; Animals ; Axons ; pathology ; Female ; Ganglia, Spinal ; metabolism ; Glucans ; analysis ; MAP Kinase Signaling System ; Nerve Crush ; Nerve Regeneration ; physiology ; Peripheral Nerves ; enzymology ; physiology ; Peroneal Nerve ; physiology ; Protein Biosynthesis ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-jun ; genetics ; metabolism ; Rats, Sprague-Dawley

BACKGROUNDNajanalgesin, a toxin isolated from the venom of Naja naja atra, has been shown to exert significant analgesic effects in a neuropathic pain model in rats. However, the molecular mechanism underlying this protective effect of najanalgesin is poorly understood. The present study sought to evaluate the intracellular signaling pathways that are involved in the antinociceptive effect of najanalgesin on neuropathic pain.

METHODSThe antinociceptive properties of najanalgesin were tested in hind paw withdrawal thresholds in response to mechanical stimulation. We analyzed the participation of the mitogen-activated protein kinase p38, extracellular-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) by western blot analysis. This inhibition of JNK was confirmed by immunohistochemistry.

RESULTSThe phosphorylation levels of JNK (as well as its downstream molecule c-Jun), p38, and ERK were significantly increased after injury. Najanalgesin only inhibited JNK and c-Jun phosphorylation but had no effect on either ERK or p38. This inhibition of JNK was confirmed by immunohistochemistry, which suggested that the antinociceptive effect of najanalgesin on spinal nerve ligation-induced neuropathic pain in rats is associated with JNK activation in the spinal cord.

CONCLUSIONThe antinociceptive effect of najanalgesin functions by inhibiting the JNK in a neuropathic pain model.

Animals ; Elapid Venoms ; therapeutic use ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Immunohistochemistry ; JNK Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Male ; Neuralgia ; drug therapy ; enzymology ; Proto-Oncogene Proteins c-jun ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

In this study, the rescue effect of receptor activator for nuclear factor-kappaB ligand (RANKL) on zoledronate acid (ZOL) induced inhibition of osteoclastogenesis and gene expression of NF-kappaB p50 and c-Jun was investigated. Mice calvarial osteoblasts (OBs) were harvested and co-cultured with RAW264.7 cells and the cells were divided into 4 groups and received treatment with ZOL and RANKL, either single or combined. The formation of multi-nucleated osteoclast (OC) was examined and gene expression of NF-kappaB p50 and c-Jun was detected. Group B (ZOL) showed least multi-nucleated OC and resorption lacunae among the 4 groups (P < 0.05 or P < 0.01) and it was followed by group C (ZOL+RANKL). Group D (RANKL) showed highest OC and resorption lacunae while it was similar to Group A (control) (P > 0.05). Gene expression of NF-kappaB p50 and c-Jun was the lowest in group B (P < 0.05 or P < 0.01) among the four groups and was significantly increased in group C when compared with group B (P < 0.05). Group A and D showed highest gene expression and they were similar to each other (P > 0.05). This study suggest that RANKL might partly rescue ZOL induced inhibition of osteoclastogenesis, and the effect of RANKL and ZOL on osteoclastogenesis may be mediated by NF-kappaB p50 and c-Jun.
Animals ; Bone Resorption ; drug therapy ; Cell Line ; Diphosphonates ; pharmacology ; Gene Expression ; Imidazoles ; pharmacology ; Mice ; NF-kappa B p50 Subunit ; metabolism ; Osteoblasts ; drug effects ; Osteoclasts ; drug effects ; Proto-Oncogene Proteins c-jun ; metabolism ; RANK Ligand ; pharmacology

Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncology. The hypoxia-inducible factor (HIF)-1α plays a crucial role in radiosensitivity, but the detailed mechanisms remain elusive. A relationship has been suggested to exist between hypoxia and autophagy recently. In the current study, we studied the effect of hypoxia-induced autophagy on radioresistance in lung cancer cell lines. A549 and H1299 cells were cultured under normoxia or hypoxia, followed by irradiation at dosage ranging from 0 to 8 Gy. Clonogenic assay was performed to calculate surviving fraction. EGFP-LC3 plasmid was stably transfected into cells to monitor autophagic processes. Western blotting was used to evaluate the protein expression levels of HIF-1α, c-Jun, phosphorylated c-Jun, Beclin 1, LC3 and p62. The mRNA levels of Beclin 1 were detected by qRT-PCR. We found that under hypoxia, both A549 and H1299 cells were radio-resistant compared with normoxia. Hypoxia-induced elevated HIF-1α protein expression preferentially triggered autophagy, accompanied by LC3 induction, EGFP-LC3 puncta and p62 degradation. In the meantime, HIF-1α increased downstream c-Jun phosphorylation, which in turn upregulated Beclin 1 mRNA and protein expression. The upregulation of Beclin 1 expression, instead of HIF-1α, could be blocked by SP600125 (a specific inhibitor of c-Jun NH2-terminal kinase), followed by suppression of autophagy. Under hypoxia, combined treatment of irradiation and chloroquine (a potent autophagy inhibitor) significantly decreased the survival potential of lung cancer cells in vitro and in vivo. In conclusion, hypoxia-induced autophagy through evaluating Beclin1 expression may be considered as a target to reverse the radioresistance in cancer cells.
Animals ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Autophagy ; Beclin-1 ; Cell Hypoxia ; Cell Line, Tumor ; Cell Survival ; genetics ; radiation effects ; Gene Expression Regulation, Neoplastic ; radiation effects ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Immunoblotting ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Membrane Proteins ; genetics ; metabolism ; Mice, Nude ; Microscopy, Fluorescence ; Microtubule-Associated Proteins ; genetics ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-jun ; metabolism ; Radiation Tolerance ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transplantation, Heterologous ; Tumor Burden ; genetics