OBJECTIVE: To assess the role of p38 in fibroblast orientation and to explore the cell signal transduction mechanism of cyclic strain induced cell orientation. METHODS: Fibroblasts were seeded onto collagen coated flexible membranes. Membranes were then deformed at 10 cycles per minute under 135 mmHg subatmospheric pressure. Orientation angles of cells treated with or without SB203580 were measured with inverted microscope. P38 phosporylation was analyzed with Western blot. RESULTS: Eighty percent cyclic strain induced cells rotated from 60 degree to 90 degree perpendicular to stretch direction after 4 h strain exposure. P38 phosphorylation reached the peak at 5 min. Fibroblast orientation was inhibited after SB203580 treatment. CONCLUSION Fibroblast orientation in response to cyclic strain is mediated by p38 phosporylation.
Cell Movement ; Cells, Cultured ; Enzyme Inhibitors ; pharmacology ; Fibroblasts ; cytology ; enzymology ; Humans ; Imidazoles ; pharmacology ; Male ; Mechanotransduction, Cellular ; physiology ; Phosphorylation ; Pyridines ; pharmacology ; Skin ; cytology ; enzymology ; Stress, Mechanical ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

OBJECTIVETo investigate the influence of Bushenguchiwan on expression of matrix metalloproteinase-13 (MMP-13) in periodontium of rats with experimental periodontitis.

METHODSThe model of experimental periodontitis of rats was established and treated by Bushenguchiwan with different doses. The periodontal tissues from groups of different doses were immunohistochemically stained by antibody of MMP-13. The expression of MMP-13 was examined and semi-quantitative analysis of signals performed by integrated absorbance.

RESULTSMMP-13 was intensely positive in gingival epithelial cells and periodontal fibroblasts in periodontitis models and negative in normal rat periodontal tissues. After 30 days of Bushenguchiwan treatment with high dose, middle dose and low dose, the expression of MMP-13 (2.9103 ± 0.5534, 3.6588 ± 0.4330, 4.4550 ± 0.4255) was down-regulated respectively compared with model rats (5.3233 ± 0.7993), P < 0.05. After 60 days of treatment the expression of MMP-13 (2.1855 ± 0.5381, 2.8558 ± 0.4759, 3.8980 ± 0.5885) was down-regulated more significantly. with model rats (6.2693 ± 0.4538), P < 0.05.

CONCLUSIONSBushenguchiwan could down-regulate the expression of MMP-13 in rats' periodontium and the high dose group had better effect.

Animals ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; therapeutic use ; Epithelial Cells ; enzymology ; Female ; Fibroblasts ; enzymology ; Gingiva ; cytology ; enzymology ; Male ; Matrix Metalloproteinase 13 ; metabolism ; Periodontitis ; drug therapy ; enzymology ; microbiology ; Periodontium ; cytology ; enzymology ; Porphyromonas gingivalis ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the roles of the cyclin D1/CDK4 and E2F-1/4 pathways and compare their work patterns in cell cycle changes induced by different doses of B[a]P.

METHODSHuman embryo lung fibroblasts (HELFs) were treated with 2 micromol/L or 100 micromol/L B[a]P which were provided with some characteristics of transformed cells (T-HELFs). Cyclin D1, CDK4 and E2F-1/4 expressions were determined by Western blotting. Flow cytometry was used to detect the distribution of cell cycle.

RESULTSAfter B[a]P treatment, the proportion of the first gap (G1) phase cells decreased. CDK4 and E2F-4 expression did not change significantly. In 2 micromol/L treated cells, a marked overexpression of cyclin D1 and E2F-1 was observed. However, in T-HELFs overexpression was limited to cyclin D1 only, and no overexpression of E2F-1 was observed. The decreases of G1 phase in response to B[a]P treatment were blocked in antisense cyclin D1 and antisense CDK4 transfected HELFs (A-D1 and A-K4) and T-HELFs (T-A-D1 and T-A-K4). After 2 micromol/L B[a]P treatment, overexpression of E2F-1 was attenuated in A-D1, and E2F-4 expression was decreased significantly in A-K4. In T-A-D1 and T-A-K4, E2F-4 expression was increased significantly, compared with T-HELFs. The E2F-1 expression remained unchanged in T-A-D1 and T-A-K4.

CONCLUSIONSCyclin D1/CDK4-E2F-1/4 pathways work in different patterns in response to low dose and high dose B[a]P treatment. In HELFs treated with 2 micromol/L B[a]P, cyclin D1 positively regulates the E2F-1 expression while CDK4 negatively regulates the E2F-4 expression; however, in HELFs treated with 100 micromol/L B[a]P, both cyclin D1 and CDK4 negatively regulate the E2F-4 expression.

Benzo(a)pyrene ; pharmacology ; Cell Cycle ; drug effects ; Cell Line ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Dose-Response Relationship, Drug ; E2F4 Transcription Factor ; metabolism ; Fibroblasts ; drug effects ; enzymology ; metabolism ; Humans ; Lung ; cytology ; drug effects ; embryology ; enzymology ; metabolism

OBJECTIVETo study the inhibitory effect of Jinye Baidu Preparation (JBP), a Chinese medicinal preparation, on human cytomegalovirus protein kinase pu197 and to explore its molecular mechanism in treating human cytomegalovirus (HCMV) infection.

METHODSExpression of the HCMV pu197mRNA in infected cells was measured by semi-quantitative RT-PCR before and after intervention of JBP or Ganciclovir (GCV), and effect of the two medicines on the proliferation activity of the infected cells was observed by MTT.

RESULTSBoth JBP and GCV showed obvious inhibitory action on HCMV pu197mRNA. They could significantly enhance the proliferation activity of the cells 72 hours after HCMV infection.

CONCLUSIONJBP could inhibit the gene expression and duplication of HCMV by inhibiting the gene expression of HCMV protein kinase pu197 to enhance the proliferation activity of the infected cells so as to achieve its anti-virus action.

Antiviral Agents ; pharmacology ; Cytomegalovirus ; drug effects ; enzymology ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Embryo, Mammalian ; Fibroblasts ; cytology ; virology ; Humans ; Lung ; cytology ; Protein Kinases ; biosynthesis ; drug effects ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Viral Proteins ; biosynthesis ; drug effects ; genetics ; Virus Replication ; drug effects

OBJECTIVETo observe the effects of artemisiae annuae CQ-189 (AACQ-189) on proliferation of hNSC and HELF in vitro, and the main organ toxicity and the median lethal dose (LD50) of kunming mouse in vivo. The purpose is to approach that the toxicity and side effects of AACQ-189.

METHODUsing techniques of the colorimetric 5-diphenyl tetrazolium bromide (MTT) to detect the effects of AACQ-189 on proliferation of hNSC, and to detect the number of HELF survival by using techniques of trypan blue exclusion. To detect LD50 by tail vein injection in kunming mouse and using histomorphology method to observe the mouse main organ damage by AACQ-189.

RESULTAACQ-189 has low poisonous function on hNSC and HELF that our experimental concentration (3.125-12.5 mg x L(-1)) has already achieve an effective dose to inhibit the proliferation of Leukemia cells obviously. LD50 concentration of kunming mouse is 550 mg x kg(-1). Moreover, AACQ-189 has little effect to main organs at higher concentration.

CONCLUSIONAACQ-189 has low poisonous function, which is a natural anti-tumor drug and has a promising prospect for potential application. However we should do more research on its mechanism.

Animals ; Artemisia ; chemistry ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drug-Related Side Effects and Adverse Reactions ; Female ; Fibroblasts ; cytology ; enzymology ; Humans ; Lung ; cytology ; Male ; Mice ; Plant Extracts ; adverse effects ; pharmacology ; Stem Cells ; drug effects

Apoptosis has an important role in maintaining tissue homeostasis in cellular stress responses such as inflammation, endoplasmic reticulum stress, and oxidative stress. T-cell death-associated gene 51 (TDAG51) is a member of the pleckstrin homology-like domain family and was first identified as a pro-apoptotic gene in T-cell receptor-mediated cell death. However, its pro-apoptotic function remains controversial. In this study, we investigated the role of TDAG51 in oxidative stress-induced apoptotic cell death in mouse embryonic fibroblasts (MEFs). TDAG51 expression was highly increased by oxidative stress responses. In response to oxidative stress, the production of intracellular reactive oxygen species was significantly enhanced in TDAG51-deficient MEFs, resulting in the activation of caspase-3. Thus, TDAG51 deficiency promotes apoptotic cell death in MEFs, and these results indicate that TDAG51 has a protective role in oxidative stress-induced cell death in MEFs.
Animals ; *Apoptosis ; Embryo, Mammalian/*cytology ; Fibroblasts/enzymology/*metabolism/pathology ; Gene Expression Regulation ; Intracellular Space/metabolism ; Mice ; Mitogen-Activated Protein Kinases/metabolism ; NF-kappa B/metabolism ; *Oxidative Stress/genetics ; Reactive Oxygen Species/*metabolism ; Signal Transduction ; Transcription Factors/*deficiency/genetics/metabolism

OBJECTIVETo investigate the role of NADPH oxidase (Nox) in the oxidative stress injury of human dermal fibroblasts (HFbs).

METHODSAn oxidative stress injury model was established in HFbs by exposure to H(2)O(2). Normal HFbs and HFbs exposed to H(2)O(2) with and without pretreatment with NADPH oxidase inhibitor were tested for cell viability using MTT assay, and the intracellular reactive oxygen species (ROS) were determined with a DCFH-DA fluorescent probe. Western blotting was used to measure the protein expressions of membrane-bound subunit gp91phox of NADPH oxidase in the cells.

RESULTH(2)O(2) time- and concentration-dependently induced oxidative stress injury in the fibroblasts, causing a reduction of the cell viability to 40% after a 24-h exposure at 700 µmol/L (P<0.05) and an increase of ROS by 2 folds after a 2-h exposure at 700 µmol/L (P<0.05). Compared with the cells with oxidative stress injury, the cells with NADPH oxidase inhibitor pretreatment showed a 20% higher cell viability (P<0.05) and normal ROS level (P<0.05) following H(2)O(2) exposure. Western blotting demonstrated increased expression of gp91phox in the cells exposed to increasing H(2)O(2) concentrations, but gp91phox expression remained normal in cells pretreated with NADPH oxidase inhibitor.

CONCLUSIONH(2)O(2) can induce oxidative stress injury in the fibroblasts by affecting NADPH oxidase, especially its membrane-bound subunit gp91phox.

Cell Survival ; Cells, Cultured ; Fibroblasts ; cytology ; enzymology ; Humans ; Hydrogen Peroxide ; Membrane Glycoproteins ; metabolism ; NADPH Oxidase 2 ; NADPH Oxidases ; antagonists & inhibitors ; metabolism ; Oxidation-Reduction ; Oxidative Stress ; Reactive Oxygen Species ; metabolism

OBJECTIVETo study the role of Phosphatidylinositol 3 kinase (PI3K) in silica-induced DNA double strand break repair in human embryo lung fibroblasts (HELF).

METHODSControl HELF cells and DN-Deltap85 (HELF transfected with Dominant negative mutant of PI3K) were treated with 200 microg/ml silica for different times. The expression levels of phosphor-H2AX (H2AX), Ku70, Ku80 and DNA-PKcs were determined by Western blot. Furthermore, DNA double strand breaks were measured by neutral comet assay after cells were treated with 200 microg/ml silica for 0, 12 and 24 h.

RESULTSAfter treatment with 200 microg/ml silica for different times, the levels of H2AX were increased in a time-dependent manner and the expression levels of H2AX were obviously suppressed in DN-Deltap85 compared with control cells. The levels of Ku70 and Ku80 were also significantly suppressed in DN-Deltap85 (0.37 +/- 0.14, 0.55 +/- 0.17) compared with control cells (0.58 +/- 0.09, 0.95 +/- 0.21) after treatment with 200 microg/ml silica for 12 h (P < 0.05). Both the percentage of tail DNA in HELF and DN-Deltap85 increased significantly at 12 h (9.78 +/- 1.15, 11.79 +/- 4.90) compared with groups without treatment with silica (2.40 +/- 0.69, 3.31 +/- 1.35) and then decreased at 24 h (4.19 +/- 0.47, 7.58 +/- 4.32), but only the decrease of HELF at 24 h was significant compared with HELF at 12 h (P < 0.05). DNA repair competence of HELF was 75.74% and that of DN-Deltap85 declined to 49.64%.

CONCLUSIONSilica dust can induce DNA double strand breaks in human embryo lung fibroblasts. PI3K might play a role in silica-induced DNA double strand break repair by regulating the expression levels of Ku70 and Ku80.

Antigens, Nuclear ; metabolism ; Calcium-Binding Proteins ; metabolism ; Cells, Cultured ; Comet Assay ; DNA Breaks, Double-Stranded ; DNA Damage ; DNA Repair ; DNA-Binding Proteins ; metabolism ; Fibroblasts ; enzymology ; Histones ; metabolism ; Humans ; Ku Autoantigen ; Lung ; cytology ; Phosphatidylinositol 3-Kinase ; metabolism ; Silicon Dioxide ; toxicity

OBJECTIVETo investigate the effects of Panax notoginseng saponins (PNS) on the proliferation and differentiation in NIH3T3 cells.

METHODSNIH3T3 cells were treated by various concentrations of PNS 0, 0.05, 0.10, 0.20, and 0.40 g/L. The vitality and proliferation potential of cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, the alkaline phosphatase (ALP) activity was measured by p-nitrophenyl phosphate (pNPP) assay, and the mineralization formation ability was tested for the cellular differentiation toward osteoblast, as well as the expression level of phosphorylated extracellular signal-regulated kinase1/2(P-ERK1/2), extracellular signal-regulated kinase1/2 (ERK1/2) protein kinase was analyzed by Western blot with total cell lysate of NIH3T3 cells treated by PNS.

RESULTSBoth MTT and pNPP assay showed that optical density (OD) values were increased in response to PNS treatment at a dose-dependent pattern. The mineralization formation ability was enhanced in PNS-treated NIH3T3 cells compared with untreated cells. Meanwhile, the expression level of P-ERK1/2 protein kinase was up-regulated in PNS-treated NIH3T3 cells, while, the expression level of ERK1/2 protein kinase revealed no obvious difference with or without PNS treated cells.

CONCLUSIONPNS could pay a role to promote the proliferation and differentiation in NIH3T3 cells by means of up-regulation of P-ERK1/2 protein kinase.

Alkaline Phosphatase ; metabolism ; Animals ; Calcium ; metabolism ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Fibroblasts ; cytology ; drug effects ; enzymology ; Mice ; NIH 3T3 Cells ; Osteocalcin ; metabolism ; Panax notoginseng ; chemistry ; Saponins ; pharmacology

The role of nitric oxide (NO) in the ocular surface remains unknown. We investigated the conditions leading to an increase of NO generation in tear and the main sources of NO in ocular surface tissue. We evaluated the dual action (cell survival or cell death) of NO depending on its amount. We measured the concentration of nitrite plus nitrate in the tears of ocular surface diseases and examined the main source of nitric oxide synthase (NOS). When cultured human corneal fibroblast were treated with NO producing donor with or without serum, the viabilities of cells was studied. We found that the main sources of NO in ocular surface tissue were corneal epithelium, fibroblast, endothelium, and inflammatory cells. Three forms of NOS (eNOS, bNOS, and iNOS) were expressed in experimentally induced inflammation. In the fibroblast culture system, the NO donor (SNAP, S-nitroso-N-acetyl-D, L-penicillamine) prevented the death of corneal fibroblast cells caused by serum deprivation in a dose dependent manner up to 500 micrometer SNAP, but a higher dose decreased cell viability. This study suggested that NO might act as a doubleedged sword in ocular surface diseases depending on the degree of inflammation related with NO concentration.
Animals ; Apoptosis/drug effects/physiology ; Aqueous Humor/metabolism ; Blood Proteins/pharmacology ; Cell Survival/drug effects/physiology ; Cells, Cultured ; Epithelium, Corneal/*cytology/*enzymology ; Fibroblasts/cytology/enzymology ; Humans ; Nitric Oxide/biosynthesis/*physiology ; Nitric Oxide Donors/pharmacology ; Nitric Oxide Synthase/metabolism ; Nitric Oxide Synthase Type I ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; Penicillamine/*analogs & derivatives/pharmacology ; Peroxynitrous Acid/biosynthesis ; Rabbits ; Tears/metabolism ; Uveitis/metabolism