Caesalpinia sappan L., belonging to the family Leguminosae, is a medicinal plant that is distributed in Southeast Asia. The dried heartwood of this plant is used as a traditional ingredient of food, red dyes, and folk medicines in the treatment of diarrhea, dysentery, tuberculosis, skin infections, and inflammation. Brazilin is the major active compound, which has exhibited various pharmacological effects, including anti-platelet activity, anti-hepatotoxicity, induction of immunological tolerance, and anti-inflammatory and antioxidant activities. The present study aimed to evaluate the antioxidant activity and expression of antioxidant enzymes of C. sappan L. extract and its major compound, brazilin, in human epidermal keratinocytes exposed to UVA irradiation. Our results indicated that C. sappan L. extract reduced UVA-induced HO production via GPX7 activation. Moreover, brazilin exhibited antioxidant effects that were similar to those of C. sappan L. via glutathione peroxidase 7 (GPX7), suggesting that C. sappan L. extract and its natural compound represent potential treatments for oxidative stress-induced photoaging of skin.
Antioxidants ; pharmacology ; Benzopyrans ; pharmacology ; Caesalpinia ; chemistry ; Humans ; Hydrogen Peroxide ; toxicity ; Keratinocytes ; cytology ; drug effects ; enzymology ; radiation effects ; Oxidative Stress ; drug effects ; radiation effects ; Peroxidases ; genetics ; metabolism ; Plant Extracts ; pharmacology ; Protective Agents ; pharmacology ; Ultraviolet Rays

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 observe the effects of estrogen on epidermis growth of mice and proliferation of keratinocytes (human epidermal cell line HaCaT), and to explore its mechanism.

METHODS(1) Five adult C57BL/6 mice in estrus cycle were identified by vaginal exfoliative cytology diagnosis and set as estrus group, while another 5 adult C57BL/6 mice with ovary resected before sexual development were set as ovariectomized group. The full-thickness skin from the tail root of mice in two groups were collected. The thickness of epidermis was observed and measured after HE staining. The distribution of proliferating cell nuclear antigen (PCNA)-positive cells in epidermis was observed by immunohistochemical staining, the number of which was counted. (2) HaCaT cells in logarithmic growth phase were cultured with RPMI 1640 nutrient solution containing 10% fetal bovine serum, and they were divided into negative control group (NC), pure estradiol group (PE), protein kinase B (Akt) inhibitor group (AI), and extracellular signal-regulated kinase (ERK) inhibitor group (EI) according to the random number table, with 20 wells in each group. To nutrient solution of each group, 1 μL dimethyl sulfoxide, 1 μL 17β-estradiol (100 nmol/L), 1 μL LY294002 (10 μmol/L), and 1 μL PD98059 (30 μmol/L) were added in group NC, group PE, group AI, and group EI respectively, and the last two groups were added with 1 μL 17β-estradiol (100 nmol/L) in addition. At post culture hour (PCH) 0 (immediately after culture), 24, 48, 72, 5 wells of cells from each group were collected to detect the proliferation activity of cells by cell counting kit 8 and microplate reader. (3) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 wells in each group. At PCH 72, cell cycle distribution was detected by flow cytometer to calculate proliferation index (PI) of cells. (4) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 dishes in each group. At PCH 72, the protein levels of phosphorylated Akt (p-Akt), phosphorylated ERK (p-ERK), and PCNA were determined with Western blotting. The cell experiments were repeated for 3 times. Data were processed with t test, one-way analysis of variance, analysis of variance of factorial design, and LSD test.

RESULTS(1) The epidermis thickness of mice in ovariectomized group was (33.5±3.0) μm, which was obviously thinner than that in estrus group [(51.4±3.1) μm, t=20.7, P<0.01]. The PCNA-positive cells mainly aggregated in the basal layer of epidermis of mice in two groups. The number of PCNA-positive cells in epidermis of mice in ovariectomized group was 37±12 per 200 fold visual field, obviously fewer than that in estrus group (96±15 per 200 fold visual field, t=15.3, P<0.01). (2) During PCH 0 to 48, there were no significant differences in the proliferation activity of cells between group PE and group NC (with P values above 0.05). At PCH 72, compared with that in group NC, the proliferation activity of cells in group PE was obviously increased (P<0.01). The proliferation activity of cells in groups AI and EI was obviously lower than that in the previous two groups (with P values below 0.01). (3) Compared with that in group NC [(51.6±1.1)%], the PI of cells in group PE was obviously increased [(58.5±0.8)%, P<0.05]. The PI values of cells in groups AI and EI were (34.9±0.8)% and (48.2±0.4)% respectively, both obviously lower than those in the previous two groups (with P values below 0.01). (4) Compared with that of group NC (0.566±0.034), the protein level of p-Akt in cells of group PE was significantly increased (1.048±0.077, P<0.01). Compared with that of group PE, the protein level of p-Akt was obviously decreased in cells of groups AI and EI (respectively 0.682±0.095 and 0.672±0.019, with P values below 0.01). Compared with that of group NC (0.469±0.013), the protein level of p-ERK obviously increased in cells of groups PE, AI, and EI (respectively 1.064±0.089, 1.010±0.038, 0.778±0.065, with P values below 0.01). The protein level of p-ERK in cells of group EI was obviously lower than that in group PE (P<0.01). Compared with that of group NC (0.386±0.053), the protein level of PCNA was obviously increased in cells of group PE (0.743±0.043, P<0.01). The protein levels of PCNA in cells of groups AI and EI were 0.264±0.019 and 0.223±0.065 respectively, both obviously lower than those in the previous two groups (with P values below 0.01).

CONCLUSIONSLack of estrogen damages the growth ability of epidermis of mice. Estrogen (17β-estradiol) can promote the proliferation of HaCaT cells by increasing the expression of PCNA via activating ERK/Akt signaling pathway.

Animals ; Cell Cycle ; Cell Line ; Cell Proliferation ; drug effects ; Epidermis ; cytology ; drug effects ; growth & development ; Estradiol ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Female ; Humans ; Keratinocytes ; cytology ; drug effects ; Mice ; Mice, Inbred C57BL ; Phosphorylation ; Proliferating Cell Nuclear Antigen ; metabolism ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; Signal Transduction

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 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

The purpose of this study was to identify the differences in angiogenesis gene expression between normal and diabetic keratocytes stimulated with interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha). Primarily cultured normal and diabetic keratocytes were treated with 20 ng/mL of IL-1a and TNF-alpha for 6 hr. cDNA was hybridized to an oligonucleotide microarray. Microarray analysis was used to identify differentially expressed genes that were further evaluated by real-time polymerase chain reaction (RT-PCR). Diabetes keratocytes overexpressed vital components of angiogenesis including Agtr1, and under-expressed components related to the blood vessel maturation, including Dcn. Cytokine-treated diabetic keratocytes differentially expressed components of angiogenesis. OLETF keratocytes after treatment with IL-1alpha and TNF-alpha showed the newly expressed 15 and 14 genes, respectively. Newly and commonly under-expressed five genes followed by treatment with both IL-1alpha and TNF-alpha were also evident. RT-PCR showed results similar to the microarray results. Agtr1 and Itga1 showed an increased expression in diabetic keratocytes compared with normal corneal keratocytes, especially after TNF-alpha treatment. Il6 appeared strong expression after interleukin-1alpha treatment, but showed down expression after TNF-alpha treatment. Further studies to analyze and confirm the significance of the identified angiogenetic genes of diabetes are needed.
Animals ; Cells, Cultured ; Gene Expression Regulation/drug effects ; Interleukin-1alpha/pharmacology ; Keratinocytes/cytology/drug effects/*metabolism ; Neovascularization, Physiologic/*genetics ; *Oligonucleotide Array Sequence Analysis ; Rats ; Real-Time Polymerase Chain Reaction ; Receptor, Angiotensin, Type 1/genetics/metabolism ; Tumor Necrosis Factor-alpha/pharmacology

Meisoindigo is an indigo natural derivative commonly used in anti-cancer therapy. In the clinical application, it was also found to have good therapeutic effect on psoriasis. In order to further understand its mechanism of action, human normal keratinocyte cell line HaCaT and RAW 264.7 were used to identify if meisoindigo could affect the inflammatory factors such as NO and other important cytokines which were highly involved in psoriasis. Our results indicated that meisoindigo decreased the production of NO in LPS-stimulated RAW 264.7 cells and reduced the expression of cytokines in LPS-stimulated HaCaT cells. And TLR4-TAK-NF-kappaB was a possible pathway mainly involved in the attenuation of iNOS and pro-inflammatory cytokine production by meisoindigo, which may take part in the therapeutic effect of meisoindigo on psoriasis.
Animals ; Cell Line ; Cell Survival ; drug effects ; Cyclooxygenase 2 ; metabolism ; Down-Regulation ; Humans ; Indoles ; pharmacology ; Keratinocytes ; cytology ; MAP Kinase Kinase Kinases ; metabolism ; Macrophages ; cytology ; Mice ; NF-kappa B ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Psoriasis ; drug therapy ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; metabolism

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

In this paper, the preliminary study on antioxidant, enhancement of antioxidant enzymes activity, reducing the content of oxygen free radicals, delaying skin aging of the recombination cytoglobin (rCygb) purified by our lab were investigated through human keratinocyte cell line (HaCAT) H2O2 oxidative stress model, mouse skin aging model caused by continuous subcutaneous injection D-gal, rat acute liver injury model induced by CCl4 and rat skin wound healing model. The results showed that rCygb improved the activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), reduced the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) as well as decreased the content of malondialdehyde (MDA). Skin biopsy showed that rCygb promoted angiogenesis, increased expression of collagen and improved the anti-inflammatory ability. All results displayed that rCygb improved the oxygen free radical scavenging ability, delayed skin aging and promoted wound healing.
Adenoviridae ; genetics ; Aging ; drug effects ; Alanine Transaminase ; metabolism ; Animals ; Antioxidants ; pharmacology ; Carbon Tetrachloride ; Catalase ; metabolism ; Cells, Cultured ; Chemical and Drug Induced Liver Injury ; metabolism ; Collagen ; Female ; Genetic Vectors ; Globins ; pharmacology ; Glutathione Peroxidase ; metabolism ; Humans ; Keratinocytes ; cytology ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Male ; Malondialdehyde ; metabolism ; Mice ; Oxidative Stress ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; pharmacology ; Superoxide Dismutase ; metabolism ; Wound Healing ; drug effects

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