OBJECTIVETo investigate the potential therapeutic effect of tamoxifen in treating abnormal skin scar contraction.

METHODSFibroblast-populated collagen lattices, which were made by embedding human dermal fibroblasts within type I collagen forming a three-dimensional culture system, were used as an invitro model. Then media either without or with addition of tamoxifen from 1 mumol/L to 50 mumol/L were added to the collagen lattices. Lattice areas were measured at intervals to assess the influence of tamoxifen on the lattice contraction. To visualize changes in the morphology and vitality of fibroblasts, MTT was added to the lattices.

RESULTSTamoxifen had an inhibitory effect on lattice contraction by a dose- and time-dependent pattern. 5 mumol/L or less of tamoxifen didn't show any influence on lattice contraction but 30 mumol/L or higher completely inhibited contraction. At intermediate concentrations from 10 mumol/L to 20 mumol/L the degree of lattice contraction was dose- and time-dependent, which was demonstrated by the reversibility of inhibition. Both the inhibition of contraction and the reversibility of inhibition appeared to correlate with changes in fibroblast morphology.

CONCLUSIONTamoxifen could inhibit the contraction of fibroblast-populated collagen lattices, indicating that tamoxifen may have potential effect on abnormal scar contraction in vivo.

Cicatrix ; drug therapy ; Collagen ; physiology ; Dose-Response Relationship, Drug ; Fibroblasts ; drug effects ; physiology ; Humans ; Skin ; cytology ; drug effects ; Tamoxifen ; pharmacology ; therapeutic use ; Time Factors

OBJECTIVETo investigate the influence of alpha smooth muscle actin fusion protein (alpha-SMA-FP) on fibroblast contraction, in order to find a new way to control scar contracture.

METHODSThree dimensional gel culture model of fibroblasts populated collagen lattices (FPCLs) was employed in the study. The fibroblasts were cultured in gel for 5 days. The cells in experimental group were processed by alpha-SMA-FP in dose of 5, 10, 50, 100 and 250 mg/L, respectively. The cells were therefore divided into E(1), E(2), E(3), E(4) and E(5) groups. Blank control was set to be C(1) group, and the cells processed by 250 mg/L of alpha-SMA-FP be C(2) group. The contraction rate was calculated by measuring the diameters of the gel before and after the procession. The change of contraction rate in E5 group was observed after the alpha-SMA-FP being rinsed out. Immunofluorescent staining of alpha-SMA-FP was carried out in fibroblasts.

RESULTSThe contraction rate in C1 and C2 groups showed no difference, being (58.6 +/- 3.1)% and (56.2 +/- 4.9)% respectively, while that in E1 to E5 groups was (45.56 +/- 4.1)%, (42.3 +/- 4.2)%, (41.8 +/- 3.6)%, (37.6 +/- 5.8)% and (26.4 +/- 4.7)%, respectively. However, the contraction rate in E5 was (53.3 +/- 5.6)% after the alpha-SMA-FP had been rinsed out. The difference of the rates among control group and experimental groups, especially in E5 after alpha-SMA-FP being rinsed out, was significant (P < 0.05 or 0.01). alpha-SMA-FP was located on the fibers of the fibroblasts as shown by staining, while the alpha-SMA was not stained. Nevertheless, the staining was obvious in control group.

CONCLUSIONalpha-SMA-FP could inhibit the contraction of fibroblasts specifically with dose dependent effect.

Actins ; administration & dosage ; pharmacology ; Animals ; Cells, Cultured ; Dose-Response Relationship, Drug ; Fibroblasts ; drug effects ; physiology ; Rats ; Rats, Wistar

OBJECTIVETo explore the mechanism of injurious effect of succinic acid on human fibroblast and it's role in bacteroides fragilis infection.

METHODSIn vitro cultured human fibroblasts were challenged by succinic acid in concentrations of 5, 10, 20 and 30 mmol/L (pH5.5), respectively. The cellular activity, apoptosis rate, the collagen synthesis in the supernatant of the cell culture, and the activity of caspase-3 were determined 24 hours after challenge. Isotonic saline challenged fibroblast were employed as control and the changes in the indices before and after succinic acid challenge were observed.

RESULTSAlong with the increase in the concentration of succinic acid, the fibroblast proliferation rate was decreased and so was the collagen synthesis. But the apoptosis rate and caspase-3 activity were increased. The activity of caspase-3 was markedly higher than that in normal control when the succinic acid concentration was 10-30 mmol/L. The cellular activity and collagen synthesis were significantly lower and the apoptosis rate was obviously higher than those in control group when the succinic acid concentration was 20 or 30 mmol/L (P < 0.05).

CONCLUSIONThe proliferation and collagen synthesis in fibroblast culture could be significantly inhibited and the cellular apoptosis could be promoted by succinic acid. The process of wound healing of the wounds infected by bacteroides fragilis would be delayed due to the production of succinic acid by the bacteria.

Apoptosis ; drug effects ; Caspase 3 ; Caspases ; metabolism ; Cells, Cultured ; Collagen ; biosynthesis ; Dose-Response Relationship, Drug ; Fibroblasts ; drug effects ; metabolism ; physiology ; Humans ; Succinic Acid ; pharmacology

BACKGROUNDThrombin is a multifunctional serine protease that plays a crucial role in hemostasis following tissue injury. In addition to its procoagulation effect, thrombin is also a potent mesenchymal cell mitogen, therefore it plays important roles in the local proliferation of mesenchymal cells in the tissue repair process. Reactive oxygen species (ROS) can induce some human cells to proliferate at lower rates while at higher concentrations they promote cells to undergo apoptosis or necrosis. Accumulative evidence suggests that thrombin can induce some cells to produce ROS. Based on these observations, we provide a hypothesis that thrombin can stimulate human lung fibroblasts to produce ROS, which play an important role in human lung fibroblast proliferation.

METHODSROS were detected in fibroblasts at 30 minutes and 60 minutes following thrombin (20 U/ml) exposure using flow cytometry. The ratio of reduced glutathione/oxidized glutathione (GSH/GSSG) was assayed in lung fibroblasts using a commercial kit following treatment with thrombin at different concentrations. NADPH oxidase and the extracellular regulated kinase1/2 (ERK1/2) signaling pathway were detected by Western blotting after thrombin stimulation to lung fibroblasts.

RESULTSThrombin, at 20 U/ml, stimulated human lung fibroblasts (HLF) to generate ROS in a time dependent manner. The ratio of GSH/GSSG in fibroblasts treated with thrombin showed a significant decrease. NADPH oxidase was activated and the ERK1/2 signal pathway was involved in the proliferation process of fibroblasts treated with thrombin.

CONCLUSIONThe activation of NADPH oxidase by thrombin leads to the production of ROS, which promotes fibroblasts proliferation via activation of the ERK1/2 signaling pathway.

Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; analysis ; physiology ; Fibroblasts ; drug effects ; physiology ; Flow Cytometry ; Glutathione ; metabolism ; Humans ; Lung ; cytology ; NADPH Oxidases ; analysis ; physiology ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology ; Thrombin ; pharmacology

OBJECTIVETo study the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in silica-induced DNA double-strand break repair in human embryo lung fibroblasts (HELF).

METHODSTwo stable transfectants, HELF transfected with DNA-PKcs siRNA (HELF-PKcs) and with negative control siRNA (HELF-NC), were established. HELF cells were treated with 0, 25, 50, 100, 200, 300 and 400 microg/ml silica for 12 h and with 200 microg/ml silica for different times (0, 1, 2, 6, 12 and 24 h). HELF-PKcs and HELF-NC were treated with 200 microg/ml silica for 0, 12 and 24 h. The expression levels of DNA-PKcs and phosphor-H2AX (H2AX) were determined by Western blot. DNA double strand breaks were measured by neutral comet assay.

RESULTSAfter treatment with different doses of silica for 12 h, the levels of H2AX and the percentages of tail DNA increased in concentration-dependent manner. After treatment with 200 microg/ml silica for different times, the levels of H2AX increased in a time-dependent manner. The percentages of tail DNA increased significantly at 6 h, and reaching maximum at 12 h and then decreasing at 24 h. The expression level of DNA-PKcs was suppressed in HELF-PKcs. After treatment with silica at 12 h, the level of H2AX was lower in HELF-PKcs than in HELF-NC, and the percentages of tail DNA increased obviously in both HELF-PKcs and HELF-NC compared with non-treated cells, but no significant difference was found in the percentages of tail DNA between them. The percentages of tail DNA decreased markedly in silica-treated HELF-NC and was significantly lower than in HELF-PKcs at 24 h (P < 0.05).

CONCLUSIONSilica can induce DNA double strand breaks in human embryo lung fibroblasts. DNA-PKcs might play a major role in silica-induced DNA double strand break repair. Silica-induced histone H2AX phosphorylation was dependent on DNA-PKcs.

Cell Line ; DNA Breaks, Double-Stranded ; drug effects ; DNA Repair ; DNA-Activated Protein Kinase ; genetics ; metabolism ; Fibroblasts ; drug effects ; physiology ; Histones ; metabolism ; Humans ; Phosphorylation ; Silicon Dioxide ; pharmacology ; Transfection

OBJECTIVETo explore the influence of NH2-terminal sequence Ac-EEED peptide of alpha-smooth muscle actin (alpha-SMA) which is the specific antibody of alpha-SMA on wound contraction.

METHODS(1) Full skin loss wounds were created on the backs of Wistar rats. The wound edge was fixed by a hard plastic frame. The wounds in experimental group (EG) were applied topically with alpha-SMA fusion peptide containing Ac-DEDE at N-terminal (alpha-SMA -FP, 1 mg/ml) during 8 to 10 days after the injury, while gel only (0.5 mg/ml) and alpha-SMA -FP (1 mg/ml) were topically applied to the wounds in control group 1 and 2, respectively. The wound areas were determined at 1, 6 and 24 hours after the removal at the fixing frame at 10 days after injury. The wound contraction rates were determined by comparing the wound area after and before the frame removal. (2) The fibroblasts in the granulation tissue were isolated 9 days after injury and were cultured in deformable silicone substrate dish. The changes in cell contraction were observed before and after the fibroblasts were treated with alpha-SMA -FP (1 mg/ml) and after alpha-SMA -FP was washed away.

RESULTS(1) The wound contraction rates exhibited no evident difference at 1, 6 and 24 hours after the removal of fixing frame in control group 1 and 2 (P < 0.05). (2) There exhibited numerous wrinkles within the fibroblasts under the microscope before alpha-SMA -FP processing. But the wrinkles decreased and became shallow remarkably at 5 mins after alpha-SMA -FP processing and disappeared completely 30 mins later. The wrinkles recovered gradually after alpha-SMA -FP was removed. But the cells treated by gel and alpha-SKA -FP exhibited no such phenomenon.

CONCLUSIONalpha-SMA-AcEEED might specifically inhibit the contraction of granulation tissue and inhibit the contraction of fibroblasts, which was reversible.

Actins ; pharmacology ; Animals ; Disease Models, Animal ; Fibroblasts ; drug effects ; physiology ; Granulation Tissue ; drug effects ; Male ; Muscle, Smooth ; chemistry ; physiology ; Peptide Fragments ; pharmacology ; Rats ; Rats, Wistar ; Wound Healing ; drug effects

OBJECTIVETo investigate the role of calcineurin (CaN) in the lung fibroblast proliferation and collagen synthesis induced by basic fibroblast growth factor (bFGF).

METHODSWe used Western blot and immunohistochemical methods for investigating the content and distribution of calcineurin in the lung tissue. Calcineurin activity in different tissues was measured using (32)P-labelled substrate. In the primary culture of lung fibroblasts, (3)H-thymidine ((3)H-TdR) and (3)H-proline incorporation methods were used to study the effect of cyclosporin A (CsA), an inhibitor of calcineurin, on the lung fibroblast DNA and collagen synthesis stimulated by bFGF.

RESULTSWe found that calcineurin was expressed in lung tissue and has phosphatase activity (7.1 +/- 2.0 pmol Pi/mg pr/min). CsA (10(-8) - 10(-6) mol/L) inhibited lung fibroblast (3)H-TdR incorporation induced by bFGF in a dose-dependent manner, with the inhibitory rates by 20%, 46% and 66% (P < 0.01). CsA (10(-7) - 10(-6) mol/L) inhibited (3)H-proline incorporation in lung fibroblasts stimulated by bFGF, with the inhibitory rates by 21% and 37% (P < 0.01). In a culture medium, CsA (10(-8) - 10(-6) mol/L) inhibited (3)H-proline secretion induced by bFGF in a dose-dependent manner, with the inhibitory rates by 19%, 29% (P < 0.05) and 56% (P < 0.01). CsA (10(-7) mol/L) could inhibit calcineurin activity by 44% in lung fibroblasts (P < 0.01).

CONCLUSIONSCalcineurin is expressed in lung tissue and has phosphatase activity. It is involved in the bFGF stimulated lung fibroblast DNA and collagen synthesis.

Animals ; Calcineurin ; analysis ; physiology ; Cell Division ; Cell Survival ; drug effects ; Collagen ; biosynthesis ; Fibroblast Growth Factor 2 ; pharmacology ; Fibroblasts ; drug effects ; physiology ; Lung ; cytology ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the role of AcSDKP on collagen synthesis and degradation in cultured rat cardiac fibroblasts.

METHODSNeonatal rat cardiac fibroblasts were isolated and stimulated by PDGF. The cell proliferation was observed by (3)H-TdR incorporation assay. The synthesis of collagen was measured by (3)H-proline incorporation assay. The expression of type I and type III collagen and MMP-1 protein were measured by Western blot. The MMP-2 and MMP-9 activity was evaluated with zymography assay.

RESULTSPDGF stimulated cardiac fibroblasts proliferation with increased collagen synthesis and type I and type III collagen protein expressions as well as MMP-2 and MMP-9 activities and MMP-1 expression. AcSDKP inhibited cardiac fibroblasts proliferation induced by PDGF and reduced collagen synthesis and type I and type III collagen protein expression. AcSDKP also further up-regulated MMP-2 and MMP-9 activities and MMP-1 expression in cardiac fibroblasts induced by PDGF.

CONCLUSIONAcSDKP inhibited proliferation and collagen synthesis and up-regulated matrix metalloproteinases activity or expression induced by PDGF, which was possibly related with the effect of AcSDKP anti-fibrosis.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen ; biosynthesis ; Fibroblasts ; drug effects ; metabolism ; Matrix Metalloproteinase 2 ; biosynthesis ; Matrix Metalloproteinase 9 ; biosynthesis ; Myocytes, Cardiac ; drug effects ; metabolism ; Oligopeptides ; physiology ; Platelet-Derived Growth Factor ; drug effects ; Rats ; Rats, Wistar

OBJECTIVETo observe the effects of Bushen Qianggu decoction proliferation and PCNA and Bcl-2 expression.

METHODSSerum containing BQD was made and synovial fibroblasts were separated and cultured and passaged in vitro. Four groups were divided as 20% blank control group, serum containing 20% Tripterygium wilfordii multi-glycosides drug (TWMD), 20% of serum containing high and low of BQD, respectively. Serum containing drugs of different concentration were added into the synovial fibroblasts of the third generation, and then the synovial fibroblasts were cultured continued. The effects of different drugs on synovial fibroblasts and PCNA and Bcl-2 expression were observed.

RESULTSCompared with the control serum, BQD-containing serum promoted the apoptosis of synovial fibroblasts (P < 0.000 1); especially, high dose could inhibit proliferation. The expression of PCNA and Bcl-2 was significantly lower in BQD-containing serum (P < 0.000 1 vs control group).

CONCLUSIONBQD can promote the apoptosis of synovial fibroblasts by improving of expression of PCNA and Bcl-2, which may be one of the mechanisms of BQD in preventing and treating osteoporosis of rheumatoid arthritis.

Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Fibroblasts ; drug effects ; physiology ; Proliferating Cell Nuclear Antigen ; analysis ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Rats ; Rats, Wistar ; Synovial Membrane ; chemistry ; cytology ; drug effects

An extracellular matrix protein plays an important role in skin wound healing. In the present study, we engineered a recombinant protein encompassing the 9th and 10th type III domains of fibronectin, and 4th FAS1 domain of beta ig-h3. This recombinant protein, in total, harbors four known-cell adhesion motifs for integrins: Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) in 9th and 10th type III domains of fibronectin, respectively, and Glu-Pro-Asp-Ile-Met (EPDIM) and Try-His (YH) in 4th FAS1 domain of big-h3, were designated to tetra-cell adhesion motifs (T-CAM). In vitro studies showed T-CAM supporting adhesion, migration and proliferation of different cell types including keratinocytes and fibroblasts. In an animal model of full-thickness skin wound, T-CAM exhibited excellent wound healing effects, superior to both 4th FAS1 domain of beta ig-h3 or 9th and 10th type III domains of fibronectin. Based on these results, T-CAM can be applied where enhancement of cell adhesion, migration and proliferation are desired, and it could be developed into novel wound healing drug.
Amino Acid Motifs ; Animals ; Cell Adhesion/*drug effects ; Cell Line ; Cell Movement/*drug effects ; Cell Proliferation/*drug effects ; Extracellular Matrix Proteins/chemistry/genetics/pharmacology ; Fibroblasts/cytology/drug effects/physiology ; Fibronectins/chemistry/genetics/*pharmacology ; Humans ; Keratinocytes/cytology/drug effects/physiology ; Mice ; NIH 3T3 Cells ; Rabbits ; Recombinant Fusion Proteins/chemistry/genetics/pharmacology ; Transforming Growth Factor beta/chemistry/genetics/pharmacology ; Wound Healing/*drug effects/physiology