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 investigate the effect of extracellular signal-regulated kinase (ERK) signaling pathway on the endothelial differentiation of periodontal ligament stem cells (PDLSC).

METHODSHuman PDLSC was cultured in the medium with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) to induce endothelial differentiation. Endothelial inducing cells was incubated with U0126, a specific p-ERK1/2 inhibitor. PDLSC from one person were randomly divided into four groups: control group, endothelial induced group, endothelial induced+DMSO group and endothelial induced+U0126 group. The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0, 1, 3, 6 and 12 hours during endonthelial induction. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) after a 7-day induction. The proportion of CD31(+) to VE-cadherin(+) cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction. The measurement data were statistically analyzed.

RESULTSPhosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively, during the endothelial induction (P<0.01). The mRNA expressions of CD31 and VEGF in induced+U0126 group were decreased to 0.09±0.18 and 0.49±0.17, which were both significantly different with those in induced group (P<0.05). The proportion of CD31(+) to VE-cadherin(+) cells of induced+U0126 group were decreased to 5.22±0.85 and 3.56±0.87, which were both significantly different with those in induced group (P<0.05). In Matrigel assay, the branching points, tube number and tube length were decreased to 7.0±2.7, 33.5±6.4, and (15 951.0±758.1) pixels, which were all significantly different with those in induced group (P<0.05).

CONCLUSIONSThe endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSC.

Antigens, CD ; genetics ; metabolism ; Butadienes ; pharmacology ; Cadherins ; genetics ; metabolism ; Cell Differentiation ; Endothelial Cells ; cytology ; physiology ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; physiology ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; metabolism ; Phosphorylation ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Signal Transduction ; Stem Cells ; cytology ; physiology ; Time Factors ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; pharmacology

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase (MAPK) signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1β (IL-1β)-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1β induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1β restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-1β restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.
3T3 Cells ; Animals ; Cartilage, Articular ; cytology ; Cell Survival ; physiology ; Cells, Cultured ; Chondrocytes ; drug effects ; enzymology ; Coculture Techniques ; Culture Media, Conditioned ; Gelatinases ; drug effects ; Interleukin-1beta ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; MAP Kinase Signaling System ; physiology ; Matrix Metalloproteinase 2 ; drug effects ; Matrix Metalloproteinase 9 ; drug effects ; Mice ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; drug effects ; Monocytes ; cytology ; NF-kappa B ; antagonists & inhibitors ; Osteoclasts ; physiology ; Protease Inhibitors ; analysis ; Tissue Inhibitor of Metalloproteinase-1 ; drug effects ; Tissue Inhibitor of Metalloproteinase-2 ; drug effects ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

OBJECTIVETo investigate the effect of CAL-101, a selective inhibitor of PI3Kδ, in combination with bortezomib on the proliferation and apoptosis in human mantle cell lymphoma cell lines Z138, HBL-2 and Jeko-1 in vitro, to explore its mechanisms and provide the foundation for effective treatment strategies against mantle cell lymphoma.

METHODSMTT assay was applied to detect the inhibitory effects of CAL-101 and bortezomib either alone or combined on Z138, HBL-2 and Jeko-1 cells. Calcusyn software was used to analyze the synergistic cytotoxicity. Western blot was used to detect the expression of PI3K-p110σ and p-Akt, Akt, p-ERK and ERK proteins after the cells were exposed to different concentrations of CAL-101. Flow cytometry was employed to assess the apoptosis rate. NF-κB kit was used to determine the changes of location of NF-κB P65, and Western blot was applied to detect the level of caswpase-3 and the phosphorylation of Akt in different groups.

RESULTSCAL-101 and BTZ inhibited the proliferation of Z138, HBL-2 and Jeko-1 cells in a dose- and time-dependent manner. CAL-101/BTZ combination induced significantly synergistic cytotoxicity in the MCL cells. The results of Western blot assay showed that CAL-101 significantly blocked the phosphorylation of Akt and ERK in the MCL cell lines. In addition, CAL-101 combined with BTZ induced pronounced apoptosis (P < 0.01). For example, after the Z138 cells exposed to the drugs for 48 h, the apoptosis rates of the control, CAL-101, BTZ and CAL-101 + BTZ groups were: (2.6 ± 1.8)%, (40.0 ± 3.0)%, (34.0 ± 1.0)%, and (67.4 ± 1.0)%, respectively; and when drug treatment was given to HBL-2 cells over 96 h, the apoptosis rates of these four cell groups were (7.4 ± 0.6)%, (30.7 ± 5.7)%, (12.0 ± 1.0)%, and (85.0 ± 4.0)%, respectively. The combination therapy contributed to the enhanced inactivity of nuclear factor-κB (NF-κB) and Akt inactivation in the MCL cell lines (P < 0.05), however, the casepase-3 activity was up-regulated.

CONCLUSIONSThe combination of CAL-101 and bortezomib is muchmore effective in inhibiting proliferation and promoting apoptosis of mantle cell lymphoma cell lines (Z138, HBL-2 and Jeko-1), which may be mediated through inhibiting PI3K/Akt signaling pathway and the transcription of NF-κB.

Antineoplastic Agents ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Boronic Acids ; Bortezomib ; pharmacology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Class Ia Phosphatidylinositol 3-Kinase ; antagonists & inhibitors ; Dose-Response Relationship, Drug ; Drug Synergism ; Formazans ; Humans ; Lymphoma, Mantle-Cell ; drug therapy ; pathology ; MAP Kinase Signaling System ; drug effects ; NF-kappa B ; metabolism ; Neoplasm Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Purines ; administration & dosage ; pharmacology ; Pyrazines ; Quinazolinones ; administration & dosage ; pharmacology ; Signal Transduction ; Software ; Tetrazolium Salts

Angiogenesis, the formation of new blood vessels, is critical for tumor growth and metastasis. Notably, tumors themselves can lead to angiogenesis by inducing vascular endothelial growth factor (VEGF), which is one of the most potent angiogenic factors. Inhibition of angiogenesis is currently perceived as one of the most promising strategies for the blockage of tumor growth. In this study, we investigated the effects of Acer tegmentosum maxim water extract (ATME) on angiogenesis and its underlying signal mechanism. We studied the antiangiogenic activity of ATME by using human umbilical vein endothelial cells (HUVECs). ATME strongly inhibited VEGF-induced endothelial cell proliferation, migration, invasion, and tube formation, as well as vessel sprouting in a rat aortic ring sprouting assay. Moreover, we found that the p44/42 mitogen activated protein (MAP) kinase signaling pathway is involved in the inhibition of angiogenesis by ATME. Moreover, when we performed the in vivo matrigel plug assay, VEGF-induced angiogenesis was potently reduced when compared to that for the control group. Taken together, these results suggest that ATME exhibits potent antiangiogenic activity in vivo and in vitro and that these effects are regulated by the extracellular regulated kinase (ERK) pathway.
Acer/*metabolism ; Angiogenesis Inhibitors/*pharmacology ; Animals ; Cell Line, Tumor ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Cell Survival ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Hep G2 Cells ; Human Umbilical Vein Endothelial Cells/*drug effects ; Humans ; MAP Kinase Signaling System/drug effects ; Mice ; Mice, Inbred C57BL ; Mitogen-Activated Protein Kinase 1/metabolism ; Neoplasm Invasiveness/pathology ; Neovascularization, Pathologic/*drug therapy/prevention & control ; Nitric Oxide Synthase Type III/metabolism ; Phosphorylation/drug effects ; Plant Extracts/pharmacology ; Rats ; Rats, Sprague-Dawley ; Transcription Factors/metabolism ; Vascular Endothelial Growth Factor A/antagonists & inhibitors/metabolism

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

The aim of the present study was to investigate the effect of precursor brain-derived neurotrophic factor (proBDNF) on survival and neurite outgrowth of cultured rat spiral ganglion neurons (SGNs). Spiral ganglions (SG) were collected from postnatal day 5 Sprague Dawley (SD) rats, then enzymatically digested and suspended. Dissociated SGNs were plated on poly-D-lysine/laminin coated eight-well chamber plates and maintained at 37 °C for 4 h to promote the attachment of the neurons. Cultured SGNs were randomly divided into five groups: control group, BDNF group (BDNF 10 ng/mL), C10 group (proBDNF 10 ng/mL), C50 group (proBDNF 50 ng/mL), and C100 group (proBDNF 100 ng/mL). All groups were incubated in a serum-free medium. 48 h after incubation, SGNs were fixed and stained for βIII tubulin. Immunostaining of the cultured SGNs showed that, compared with the control group, the cellular survival of C50 group and C100 group were significantly reduced (P < 0.001). Furthermore, surviving numbers of the three proBDNF-treated groups were all lower than the BDNF group. In order to assess the effect of proBDNF on cell morphology, SGNs were divided into two categories: SGNs with or without neurites. The results demonstrated that proBDNF significantly increased the proportions of SGNs without neurites in C10, C50 and C100 groups compared with that in control group (P < 0.001). In addition, c-Jun N-terminal kinase (JNK) inhibitor, SP600125 (20 μmol/L) significantly increased the surviving number of SGNs in C50 group. These results suggest that proBDNF reduces the survival rate of cultured SGNs and inhibits the sprouting of neurites. Furthermore, the inhibition of JNK signaling attenuates the effect of proBDNF on SGNs survival.
Animals ; Axons ; physiology ; Brain-Derived Neurotrophic Factor ; pharmacology ; Cell Survival ; Cells, Cultured ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; MAP Kinase Signaling System ; Neurites ; physiology ; Neurons ; cytology ; Protein Precursors ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Spiral Ganglion ; cytology

OBJECTIVETo demonstrate the effects and mechanisms of Huangkui capsule (HKC) on renal fibrosis in rats with diabetic nephropathy (DN).

METHODRats were randomly divided into 5 groups, the sham-operated group (Sham group, n = 5), the vehicle-given group (Vehicle group, n = 7), the low dose of HKC-treated group (L-HKC group, n = 7), the high dose of HKC-treated group (H-HKC group, n = 7) and the lipoic acid (LA)-treated group (LA group, n = 7). DN models were induced by intraperitoneal injection of streptozotocin (STZ,35 mg x kg(-1)) twice and unilateral nephrectomy. After models were successfully established, the rats in HKC and LA groups were daily administrated with HKC suspensions (0.75, 2 g x kg(-1)) or LA suspensions (60 mg x kg(-1)) respectively, and at the same time, the rats in Vehicle group were daily administrated with distilled water (2 mL) for 8 weeks. All rats were sacrificed at the end of week 8 to collect blood and renal tissues. UAlb, renal function, renal fibrotic morphologic characteristics, as well as oxidative stress (OS)-related markers, the protein expressions of the key signaling molecules in p38 mitogen-activated protein kinase (p38MAPK) signaling pathway, fibrogenic cytokines and inflammatory factors were examined respectively.

RESULTHKC, similar to LA, improved the general state of health, body weight, UAlb, BUN, UA and Alb in DN model rats. Of note, renal fibrosis was ameliorated in HKC groups,especially in H-HKC group which was better than that in LA group. In addition, HKC not only improved the main indexes of OS in the kidney like LA, but also down-regulated the protein expressions of phosphorylated-p38MAPK (p-p38MAPK), transforming growth factor (TGF)-β1 and tumor necrosis factor(TNF)-α in the kidney, whereas, LA only decreased the protein expression of TNF-α in the kidney in DN model rats.

CONCLUSIONHKC, similar to LA, has the actions of anti-OS in vivo. Moreover, HKC could attenuate renal fibrosis by suppressing the activation of p38MAPK signaling pathway and the protein expressions of fibrogenic cytokines and inflammatory factors in the kidney in DN model rats, which is different from LA.

Abelmoschus ; chemistry ; Animals ; Capsules ; Diabetic Nephropathies ; drug therapy ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Fibrosis ; Kidney ; drug effects ; pathology ; MAP Kinase Signaling System ; drug effects ; Male ; Oxidative Stress ; drug effects ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

OBJECTIVETo demonstrate the effects and mechanisms of Qifu decoction( QFD) on renal interstitial fibrosis (RIF) in model rats with yang-deficiency syndrome.

METHODThe rats were randomly divided into 3 groups, the Sham group (Group A), the Model group (Group B), the Qifu decoction group (Group C) and the Enalapril group (Group D). The RIF model was established by adenine administrated and unilateral ureteral obstruction (UUO) of the left ureter. After the model was successfully established, the rats in Group C and D were administrated with QFD or the Enalapril suspension,while the rats in Group A and B were administrated with distilled water. All rats were administrated for 3 weeks. Before administration and at the end of week 1, 2 and 3, the rats were weighted, and 24 h urinary protein excretion (Upro), urinary β2-microglobulin (Uβ2-MG) and urinary N-acetyl-D-glucosaminidase (NAG) were examined, respectively. All rats were killed after administration for 3 weeks. Blood and renal tissues were collected, renal morphology and tubulointerstitial morphology were evaluated, respectively. Serum cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), blood urea nitrogen (BUN), serum creatinine (Scr) and uric acid (UA) were detected, respectively. The protein expressions of E-cadherin, α-smooth muscle actin(α-SMA), transforming growth factor-β1 (TGF-β1), onnective tissue growth factor (CTGF) extracellular signal-regulated protein kinase 1/2(ERK1/2) and phosphorylated-ERK1/2 (p-ERK1/2) in kidney were evaluated, respectively.

RESULTQFD ameliorated serum cAMP level and the rate of cAMP/cGMP, attenuated urinary β2-MG level, NAG level and renal tubulointerstitial fibrosis, increased E-cadherin protein expression, and reduced α-SMA, TGF-β1, CTGF and p-ERK1/2 protein expressions in the kidney. However, QFD had no influence on renal function in vivo. In addition, these effects were better than those of the model rats treated by Enalapril.

CONCLUSIONQFD could alleviate yang-deficiency parameters, as well as urinary β2-MG level and NAG level in model rats induced by adenine administration and UUO. Moreover, QFD could improve EMT and RIF by up-regulating E-cadherin protein expression, and down-regulating α-SMA, TGF-β1, CTGF and p-ERK1/2 protein expressions, the key molecular in ERK1/2 signaling pathway.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Fibrosis ; Kidney ; drug effects ; pathology ; Kidney Diseases ; drug therapy ; pathology ; MAP Kinase Signaling System ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Ureteral Obstruction ; complications ; Yang Deficiency ; complications