OBJECTIVETo investigate the origin of myofibroblasts in oral submucous fibrosis.

METHODSThe oral keratinocytes and fibroblasts were isolated and cultured. The expression of the alpha-smooth muscle actin in the fibroblasts was examined by immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR).

RESULTSNo difference was found in expression of alpha-smooth muscle actin between the fibroblasts that were directly stimulated by arecoline and the control. The expression of alpha-smooth muscle actin in the keratinocyte and fibroblast-cocultured group was higher than in the control group, and higher in fibroblasts cocultured with keratinocytes preprocessed by arecoline than in fibroblasts cocultured with keratinocytes without preprocessed by arecoline.

CONCLUSIONSThe differentiation of myofibroblasts from fibroblasts in oral submucous fibrosis might be induced by the interaction of arecoline and keratinocyte.

Actins ; metabolism ; Arecoline ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Fibroblasts ; cytology ; metabolism ; Humans ; Keratinocytes ; cytology ; Mouth Mucosa ; cytology ; Oral Submucous Fibrosis ; metabolism ; pathology

OBJECTIVETo investigate the effects of bradykinin (BK) on the proliferation, apoptosis and differentiation of human keratinocyte (HKC) and the underlying mechanisms.

METHODSHKCs were cultured together with 1 x 10(-4) - 1 x 10(-9) mol/L of BK. With methyl thiotetrazole (MTT) and trypan blue staining it was shown that the BK in dose of 1 x 10(-4) mol/L possessed most powerful inhibitory effect, and the survival rate of HKC was 69.3%. Therefore, BK was employed in the dose of 1 x 10(-4) mol/L in the following studies. When the growth of HKCs reached the logarithmic phase, BK in the concentration of 1 x 10(-4) mol/L was added, and it was categorized as the test group (E). HKCs without BK served as the control group (C). The cell cycle and apoptosis were detected by flow cytometry after being cultured for 24 and 48 hours. The change in intracellular calcium [Ca(2+)](i) was determined by means of laser scanning confocal microscopy with calcium fluorescence probe Fluo-3/AM technique. The expression of HKC differentiation labeling protein keratin10 (K10) and involucrin were detected with Strept Avidin-Biotin Complex (SABC) immunocytochemical assay.

RESULTSThe cell ratio in G0/G1 phase in E group increased by 34.57% while in S phase decreased by 58.91% in reference to that in C group. The G1/S phase switching of HKCs was obviously inhibited by BK, and apoptosis was stimulated (apoptotic rate of 15.34% in E group vs 5.60% in C group, P < 0.05). The [Ca(2+)](i) increased transiently in HKCs by 163.0% in E group after 3 minutes of BK activation and decreased thereafter in reference to that in C group. The K10 expression in HKC was down-regulated in E group with positive cell rate of 2.20%, which was lower than that of C group (6.89%, P < 0.05).

CONCLUSIONThe cell cycle process of HKC could be inhibited by high concentration of BK with increased apoptosis and an increase in [Ca(2+)](i), which might be the mechanism of inhibition of growth of HKC in vitro. Furthermore, the epithelial regeneration and HKC differentiation can also be inhibited by BK.

Apoptosis ; drug effects ; Bradykinin ; pharmacology ; Cell Cycle ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Humans ; Keratinocytes ; cytology ; metabolism ; Keratins ; metabolism

Silver-containing preparations are widely used in the management of skin wounds, but the effects of silver ions on skin wound healing remain poorly understood. This study investigated the effects of silver ions (Ag) on the proliferation of human skin keratinocytes (HaCaT) and the production of intracellular reactive oxygen species (ROS). After treating HaCaT cells with Ag and/or the active oxygen scavenger N-acetyl cysteine (NAC), cell proliferation and intracellular ROS generation were assessed using CCK-8 reagent and DCFH-DA fluorescent probe, respectively. In addition, 5-bromo-2-deoxyUridine (BrdU) incorporation assays, cell cycle flow cytometry, and proliferating cell nuclear antigen (PCNA) immunocytochemistry were conducted to further evaluate the effects of sub-cytotoxic Ag concentrations on HaCaT cells. The proliferation of HaCaT cells was promoted in the presence of 10 and 10 mol/L Ag at 24, 48, and 72 h. Intracellular ROS generation also significantly increased for 5-60 min after exposure to Ag. The number of BrdU-positive cells and the presence of PCNA in HaCaT cells increased 48 h after the addition of 10 and 10 mol/L Ag, with 10 mol/L Ag markedly increasing the cell proliferation index. These effects of sub-cytotoxic Ag concentrations were repressed by 5 mmol/L NAC. Our results suggest that sub-cytotoxic Ag concentrations promote the proliferation of human keratinocytes and might be associated with a moderate increase in intracellular ROS levels. This study provides important experimental evidence for developing novel silver-based wound agents or dressings with few or no cytotoxicity.
Apoptosis ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Fluorescent Antibody Technique ; Humans ; Keratinocytes ; cytology ; drug effects ; Proliferating Cell Nuclear Antigen ; metabolism ; Reactive Oxygen Species ; metabolism ; Silver ; pharmacology

OBJECTIVETo study the cellular and molecular mechanism of gasoline-induced adverse effects on skin, particularly on keratinocyte and fibroblast in vitro.

METHODSThe primary cell culture of keratinocyte and fibroblast were treated with 0, 0.001%, 0.01%, 0.1% and 1.0% gasoline, respectively. (3)H-thymidine ((3)H-TdR), (3)H-leucine ((3)H-Leu), (3)H-proline ((3)H-Pro) and (14)C-linoleic acid incorporation tests were applied to elucidate their capacity of synthesizing DNA, protein and sebum.

RESULTSThe incorporation of (3)H-TdR in keratinocyte and (3)H-TdR and (3)H-Pro in fibroblast inhibited significantly after exposure to 0.01% gasoline (P < 0.05), with inhibition rates 68.5%, 45.1% and 40.6% for (3)H-TdR in keratinocyte, and (3)H-TdR and (3)H-Pro in fibroblast, respectively. Significant depression in incorporation of (3)H-Leu and (14)C-linoleic acid in keratinocyte were found even in the group treated with 0.001% gasoline (P < 0.05), with inhibition rates of 20.2% and 41.2%, respectively.

CONCLUSIONSSolvent gasoline has certain toxic effect on keratinocyte and fibroblast, intervening their normal metabolic and physiological process and affecting their ability of synthesizing DNA, protein and sebum, and their physiological functions, which could be one of the mechanisms causing skin damage by gasoline. The results also indicated that keratinocyte was more susceptible to gasoline than fibroblast.

Animals ; Cells, Cultured ; DNA ; biosynthesis ; genetics ; Dose-Response Relationship, Drug ; Fibroblasts ; cytology ; drug effects ; metabolism ; Gasoline ; toxicity ; Keratinocytes ; cytology ; drug effects ; metabolism ; Proteins ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sebum ; drug effects ; metabolism

OBJECTIVETo observe the effect of shikonin on the proliferation of human keratinocytes induced by IL-17 and secretion of chemokines, in order to discuss the mechanism of Shikonin in the treatment of psoriasis.

METHODIn vitro cultured HaCaT cells were stimulated by IL-17A (200 μg x L(-1)) and mixed with different concentrations (2, 1 mg x L(-1)) of shikonin for 24 hours. The cell proliferation was detected by CCK-8 assay. Cell secretion inflammatory factor interleukin-23 (IL-23) was detected by ELISA. The expressions of intracellular chemokines CXCL1, CXCL2, CCL20 and 6-defensin 4 (DEFB4) were detected by Real-time PCR.

RESULTShikonin (2,1 mg x L(-1)) could distinctly inhibit HaCaT cell proliferation induced by IL-17A, with statistical difference (P < 0.01). Each shikonin group showed decreases in the secretion of IL-23 and inhibition in expressions of intracellular CXCL1, CXCL2, CCL20 and DEFB4.

CONCLUSIONShikonin could inhibit HaCaT cells proliferation induced by IL-17 and secretion of relevant cytokines and recruit leukocytes by inhibiting chemokines, so as to show the effect in treating psoriasis.

Cell Line ; Cell Proliferation ; drug effects ; Chemokines ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Interleukin-17 ; genetics ; metabolism ; Keratinocytes ; cytology ; drug effects ; metabolism ; Naphthoquinones ; pharmacology

This study is to investigate the effect and mechanism of puerarin on DNA damage of HaCaT cells induced by UVB. Puerarin pre-treated cells were irradiated with UVB at 30 mJ x cm(-2). Twenty four hours after irradiation, DNA damage was detected by comet assay, ceramide was measured by thin layer chromatography and gas chromatography, intracellular free calcium ion was analyzed by flow cytometry, the phosphorylation level of p38 protein was examined by Western blotting method. Levels of DNA damage, ceramide, free calcium ion and p-p38 protein were elevated in UVB model cells. Contrary to the model group, all indicators above were reduced in all groups pre-treated by puerarin. Puerarin restrains the ceramide accumulation to block downstream p38 MAPK pathway and calcium ion rising, therefore reduces DNA damage in HaCaT cells induced by UVB.
Calcium ; metabolism ; Cell Line ; Ceramides ; metabolism ; DNA Damage ; drug effects ; radiation effects ; Down-Regulation ; Humans ; Isoflavones ; pharmacology ; Keratinocytes ; cytology ; metabolism ; Phosphorylation ; Signal Transduction ; drug effects ; Ultraviolet Rays ; adverse effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

Articular cartilage, which is mainly composed of collagen II, enables smooth skeletal movement. Degeneration of collagen II can be caused by various events, such as injury, but degeneration especially increases over the course of normal aging. Unfortunately, the body does not fully repair itself from this type of degeneration, resulting in impaired movement. Microfracture, an articular cartilage repair surgical technique, has been commonly used in the clinic to induce the repair of tissue at damage sites. Mesenchymal stem cells (MSC) have also been used as cell therapy to repair degenerated cartilage. However, the therapeutic outcomes of all these techniques vary in different patients depending on their age, health, lesion size and the extent of damage to the cartilage. The repairing tissues either form fibrocartilage or go into a hypertrophic stage, both of which do not reproduce the equivalent functionality of endogenous hyaline cartilage. One of the reasons for this is inefficient chondrogenesis by endogenous and exogenous MSC. Drugs that promote chondrogenesis could be used to induce self-repair of damaged cartilage as a non-invasive approach alone, or combined with other techniques to greatly assist the therapeutic outcomes. The recent development of human induced pluripotent stem cell (iPSCs), which are able to self-renew and differentiate into multiple cell types, provides a potentially valuable cell resource for drug screening in a "more relevant" cell type. Here we report a screening platform using human iPSCs in a multi-well plate format to identify compounds that could promote chondrogenesis.
Cell Differentiation ; drug effects ; Chondrocytes ; cytology ; drug effects ; metabolism ; Chondrogenesis ; drug effects ; Drug Evaluation, Preclinical ; methods ; Genes, Reporter ; genetics ; Humans ; Induced Pluripotent Stem Cells ; cytology ; drug effects ; metabolism ; Keratinocytes ; cytology ; drug effects ; metabolism ; Luciferases ; genetics ; Peptides ; chemical synthesis ; metabolism ; Reproducibility of Results ; Small Molecule Libraries ; pharmacology

BACKGROUNDShikonin is a major active chemical component extracted from Lithospermi Radix, an effective traditional herb in various types of wound healing. Shikonin can accelerate granulomatous tissue formation by the rat cotton pellet method and induce neovascularization in granulomatous tissue. The purpose of the study was to investigate its mechanism of action in human skin cells.

METHODSMTS assay was used to measure cell growth. The collagen type I (COL1 ) mRNA expression and procollagen type I C-peptide (PIP) production were detected by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Immunofluorescence and western blot analyses were carried out to investigate nuclear factor-κB (NF-κB) signaling pathway. Cell-based proteasome activity assay was used to determine proteasome activity.

RESULTSIn this study, we found that 10 μmol/L shikonin stimulated the growth of normal human keratinocytes and 1 μmol/L shikonin promoted growth of human dermal fibroblasts. However, shikonin did not directly induce COL1 mRNA expression and PIP production in dermal fibroblasts in vitro. In addition, 1 μmol/L shikonin inhibited translocation of NF-κB p65 from cytoplasm to nucleus induced by tumor necrosis factor-α stimulation in dermal fibroblasts. Furthermore, shikonin inhibited chymotrypsin-like activity of proteasome and was associated with accumulation of phosphorylated inhibitor κB-α in dermal fibroblasts.

CONCLUSIONSThese results suggested that shikonin may promote wound healing via its cell growth promoting activity and suppress skin inflammation via inhibitory activity on proteasome. Thus, shikonin may be a potential therapeutic reagent both in wound healing and inflammatory skin diseases.

Cell Proliferation ; drug effects ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts ; drug effects ; Humans ; Keratinocytes ; drug effects ; NF-kappa B ; metabolism ; Naphthoquinones ; pharmacology ; Polymerase Chain Reaction ; Proteasome Endopeptidase Complex ; drug effects ; Skin ; cytology

OBJECTIVETo explore the effect of epidermal growth factor (EGF) on apoptosis induced by TNF-alpha and the expression of PPARbeta in HaCaT keratinocytes.

METHODSHaCaT keratinocytes were cultured and randomly divided into A (normal control), B (with treatment of 10 ng/ml TNF-alpha for 24 hours), C (with treatment of 20 ng/ml TNF-alpha for 24 hours), D (with treatment of 10 ng/ml TNF-alpha after 20 ng/ml EGF treatment for 4 hours), E (with treatment of 20 ng/ml TNF-alpha after 20 ng/ml EGF treatment for 4 hours) groups. The apoptosis of HaCaT keratinocytes was observed by flow cytometry. The proliferative activity of HaCaT keratinocytes was evaluated by MTT method. The activity of caspase-3 was analyzed with caspase colorimetric assay Kit. The changes in the mRNA and protein expression of PPARbeta in HaCaT keratinocytes were observed by RT-PCR and western-blotting after treatment with different concentrations (5, 10, 20, 40 ng/ml) of EGF for 4 or 24 hrs.

RESULTSCompared with A and B groups [(32 +/- 6)%, (57 +/- 6)%], the apoptosis of HaCaT keratinocytes in D and E groups were significantly increased [(20 +/- 3)%, (28 +/- 4)%, respectively, P < 0.01], while the survival rate of HaCaT keratinocytes in D and E groups increased, and the caspase-3 activity were decreased (P < 0.01). The expression of PPARbeta mRNA and protein in HaCaT keratinocytes reached the peak with the treatment of 20 ng/ml EGF.

CONCLUSIONEGF can inhibit the apoptosis of HaCaT keratinocytes induced by TNF-alpha, and it can also increase the expression of PPARbeta.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line ; Epidermal Growth Factor ; pharmacology ; Humans ; Keratinocytes ; cytology ; drug effects ; metabolism ; PPAR-beta ; metabolism ; RNA, Messenger ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo observe the effect of the supernatant of heat injured keratinocytes (KC) on biological behavior of the dermal fibroblasts (Fb).

METHODSHuman dermal Fb were isolated and cultured. A model of heat injured KC (HaCaT) was reproduced in vitro. Supernatant of normal KC and the supernatant of KC culture 12 hours after heat injury were collected and diluted with non-serum DMEM in 1:1 volume ratio to make normal KC conditioned medium (NKCM) and heat injury KC conditioned medium (HKCM) respectively. Fb was respectively treated with non-serum DMEM and 2 kinds of conditioned medium. (1) The proliferation of Fb was detected with MTT method at post culture hour (PCH) 12, 24, 36, 48. (2) The apoptosis of Fb was determined by flow cytometry at PCH 12 (Fb were heat injured in advance; Fb without heat treatment was used as control). (3) At PCH 24, expression of a-SMA in Fb cytoplasm was determined with immunofluorescence method; expression of a-SMA mRNA in Fb was determined with real-time quantified PCR. Data were processed with one-way analysis of variance, and pairwise comparison among groups with LSD-t test.

RESULTS(1) The proliferation of Fb: the absorbance value of Fb cultured with HKCM at PCH 12, 24, 36, 48 was respectively higher than that of Fb cultured with non-serum DMEM (with t value respectively 1.89, 2.35, 2.02, 1.94, and P values all below 0.01). There were significant statistical differences between the absorbance values of Fb cultured with HKCM and those of Fb cultured with NKCM at PCH 12, 24, and 48 (at PCH 12, t = 1.83, P < 0.01; at PCH 24, t = 2.91, P < 0.05; at PCH 48, t = 1.83, P < 0.05). (2) Apoptosis of Fb cultured with HKCM was diminished as compared with that of Fb cultured with NKCM and of Fb without treatment (t = 3.31, P < 0.05; t = 1.47, P < 0.01). (3) The expression of alpha-SMA (red fluorescence) in Fb cultured with non-serum DMEM or NKCM was less as seen under fluorescence scope, and it was obviously increased in Fb cultured with HKCM. (4) The relative expression amount of alpha-SMA mRNA in Fb cultured with HKCM was 1.32 +/- 0.06, which was higher than that both in Fb cultured with NKCM (1.14 +/- 0.07, t = 2.51, P < 0.05) and in Fb cultured with non-serum DMEM (1.00 +/- 0.09, t = 1.77, P < 0.05).

CONCLUSIONSThe supernatant of KC 12 hours after heat injury can obviously promote the proliferation of Fb, inhibit its apoptosis and accelerate transdifferentiation of Fb to myofibroblasts.

Actins ; metabolism ; Apoptosis ; Cell Differentiation ; Cells, Cultured ; Culture Media, Conditioned ; pharmacology ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flow Cytometry ; Heat Stress Disorders ; Hot Temperature ; adverse effects ; Humans ; Keratinocytes ; cytology ; RNA, Messenger ; genetics