9-Hydroxy-6,7-dimethoxydalbergiquinol suppresses hydrogen peroxide-induced senescence in human dermal fibroblasts through induction of sirtuin-1 expression

Objective:To investigate the potential anti-aging mechanism of 9-hydroxy-6,7-dimethoxydalbergiquinol (HDDQ) on hydrogen peroxide (H2O2)-induced oxidative stress in human dermal fibroblasts (HDFs). Methods:The effect of HDDQ on cell viability was assessed by MTT assay, and the effects of HDDQ on senescence-like phenotypes were determined by senescence-associated β-galactosidase (SA-β-gal) staining, Western blotting analysis, and a cell proliferation assay. The expression level and activity of sirtuin-1 (SIRT1) induced by HDDQ were also measured. Results:HDDQ reversed senescence-like phenotypes in the oxidant-challenged model, through reducing SA-β-gal activity and promoting cell growth. Meanwhile, decreases in ac-p53, p21Cip1/WAF1, and p16Ink4a and an increase in pRb were observed. HDDQ induced the expression of SIRT1 in a concentration- and time-dependent manner. Moreover, HDDQ inhibited H2O2-induced phosphorylation of Akt by SIRT1 up-regulation and reduced SA-β-gal staining. Conclusions:HDDQ inhibits H2O2-induced premature senescence and upregulation of SIRT1 expression plays a vital role in the inhibition of the senescence phenotype in HDFs.

Fenugreek (Trigonella foenum-graceum) increases postmenopausal fibroblast-associated COL1A1 and COL3A1 production dominantly through its binding to estrogen receptor beta

In postmenopausal women, oral or topical administration of estradiol increases skin thickness and collagen synthesis,such as collagen type 1 alpha 1 (COL1A1) and collagen type 3 alpha 1 (COL3A1). Due to undesirable side effectsof estradiol, such as risks of breast and endometrium pathology, topical phytoestrogens are alternative treatments foraging-related skin changes. Phytoestrogen is a nonsteroidal substance derived from plants, like fenugreek (Trigonellafoenum-graceum L.), which has an estrogen like composition that appears to mimic estradiol. The mechanism ofaction remains unknown, especially in fibroblast-associated COL1A1 and COL3A1 production. In vitro experimentswere conducted using postmenopausal women's fibroblasts with estrogen receptor (ER) antagonists. Cell isolationused explant and enzymatic techniques with ELISA kit (MyBioSource, California) for COL1A1 and COL3A1. Pairedstudent t-tests compared results between control (no treatment), fenugreek extract 2 µg/ml alone, fenugreek extract 2µg/ml with receptor antagonists for ERα, ERβ, and both receptors. Greater suppresion of COL1A1 and COL3A1 wereshown by both antagonists ERα / ERβ group and antagonist ERβ group compared to antagonist ERα group. Theseresults indicate that the fenugreek increases secretion of COL1A1 and COL3A1 through ERα, ERβ, and is mainlymediated by ERβ in post menopausal women’s fibroblasts.

Platelet-rich plasma ameliorates photo-aging phenotype of fibroblasts by adjusting the generation of reactive oxygen species / 中华医学美学美容杂志

Objective To explore the effect of platelet-rich plasma (PRP) on the generation of reactive oxygen species (ROS) and the phenotypes of photo-aging fibroblasts.Methods A photoaging cell model by repeating UVB irradiation was treated using appropriate concentration of PRP;Cell morphology and the rate of aging dying were observed under inverted microscope 24 hours later after establishment of the cell model;The expression of ROS between experimental and control group was detected using fluorescence microscope after single UVB irradiation.The relative intensity of fluorescence was analyzed using flow cytometry.Results PRP could ameliorate the large and sprawl appearance of photoaging fibroblasts obviously,reduce the generation of ROS as well as decrease the relative intensity of ROS.Conclusions PRP can decrease the level of intracellular oxidative stress caused by UVB irradiation,reduce the generation of ROS and ameliorate the senescence-like phenotypes of pho toaging fibroblasts.

Anti-aging effect of adipose-derived stem cell conditioned medium on senescent human dermal fibroblasts / 中华整形外科杂志

Objective@#To study the protective effect of adipose-derived stem cell conditioned medium against ultraviolet radiation B (UVB)-induced photoaging and replicative senescence in human dermal fibroblasts (HDFs).@*Methods@#HDFs were cultured and passaged in vitro, and treated with ADSC-CM after being irradiated once with UVB. The mRNA expression of CollagenⅠ(Col1), CollagenⅢ(Col3) and Elastin were detected by real-time PCR to define the anti-aging effects of ADSC-CM. The protein expressions of phosphorylated c-Jun N-terminal kinase (p-JNK) and p-p38 were detected by Western blot.@*Results@#①Both successive passage and UVB irradiation reduced the expression of Col1, Col3 and Elastin in HDFs.②ADSC-CM inhibited the reduction of Col1, Col3 and Elastin protein expressions induced by successive passage and UVB. ③ADSC-CM activated p38 and JNK pathway. Downregulation of p38 MAPK by SB203580 decreased the mRNA expression of Col1, Col3, and Elastin. Inhibiting JNK by SP600125 did not induce significant ECM changes.@*Conclusions@#Both intrinsic and extrinsic stimuli can decrease the expression of collagen and elastin, common markers of skin aging in HDFs, and ADSC-CM can attenuate the above decreasing and promoting the expression of ECM by p38 signaling pathway.

A standardized extract of Asparagus officinalis stem prevents reduction in heat shock protein 70 expression in ultraviolet-B-irradiated normal human dermal fibroblasts: an in vitro study

BACKGROUND@#Heat shock protein 70 (HSP70) exhibits protective effects against ultraviolet (UV)-induced premature skin aging. A standardized extract of Asparagus officinalis stem (EAS) is produced as a novel and unique functional food that induces HSP70 cellular expression. To elucidate the anti-photoaging potencies of EAS, we examined its effects on HSP70 expression levels in UV-B-irradiated normal human dermal fibroblasts (NHDFs).@*METHODS@#NHDFs were treated with 1 mg/mL of EAS or dextrin (vehicle control) prior to UV-B irradiation (20 mJ/cm). After culturing NHDFs for different time periods, HSP70 mRNA and protein levels were analyzed using real-time polymerase chain reaction and western blotting, respectively.@*RESULTS@#UV-B-irradiated NHDFs showed reduced HSP70 mRNA levels after 1-6 h of culture, which were recovered after 24 h of culture. Treatment with EAS alone for 24 h increased HSP70 mRNA levels in the NHDFs, but the increase was not reflected in its protein levels. On the other hand, pretreatment with EAS abolished the UV-B irradiation-induced reduction in HSP70 expression at both mRNA and protein levels. These results suggest that EAS is capable to preserve HSP70 quantity in UV-B-irradiated NHDFs.@*CONCLUSIONS@#EAS exhibits anti-photoaging potencies by preventing the reduction in HSP70 expression in UV-irradiated dermal fibroblasts.

Reprogramming human dermal fibroblast into induced pluripotent stem cells using non-integrative Sendai virus for transduction

@#Introduction: Induced pluripotent stem cells (iPSC) that exhibit embryonic stem cell-like properties with unlimited self-renewal and multilineage differentiation properties, are a potential cell source in regenerative medicine and cell-based therapy. Although retroviral and lentiviral transduction methods to generate iPSC are well established, the risk of mutagenesis limits the use of these products for therapeutic applications. Materials and Methods: In this study, reprogramming of human dermal fibroblasts (NHDF) into iPSC was carried out using non-integrative Sendai virus for transduction. The iPSC clones were characterised based on the morphological changes, gene expression of pluripotency markers, and spontaneous and directed differentiation abilities into cells of different germ layers. Results: On day 18-25 post-transduction, colonies with embryonic stem cell-like morphology were obtained. The iPSC generated were free of Sendai genome and transgene after passage 10, as confirmed by RT-PCR. NHDF-derived iPSC expressed multiple pluripotency markers in qRT-PCR and immunofluorescence staining. When cultured in suspension for 8 days, iPSC successfully formed embryoid body-like spheres. NHDF-derived iPSC also demonstrated the ability to undergo directed differentiation into ectoderm and endoderm. Conclusion: NHDF were successfully reprogrammed into iPSC using non-integrating Sendai virus for transduction.

A pilot study on indunction and differentiation of human fibroblasts to iPS cells and iPS-RPE cells mediated by retrovirus infection / 中华实验眼科杂志

Background Retinal pigment epithelium (RPE) cell transplantation is the primary means of human trial for the treatment of retinal degeneration.Induced pluripotent stem cells (iPSCs) will become an important source for cell transplantation.In addition,iPS-RPE cells may provide a personalized treatment platform for the patient's own cells treatment.Objective This study was to evaluate the feasibility of human fibroblasts differentiate toward iPSCs from retinitis pigmentosa (RP) patients and toward iPSC-RPE cells from non-RP individual by retroviral transfection of Oct4,Sox2,c-Myc and KLF4 genes.Methods Human thigh skin tissues were obtained from a RP patient with hotspot mutation of SNRNP200 p.S1087L and individual without SNRNP200 p.S1087L mutation,respectively,with the size 2 c m×3 cm.Human dermal fibroblasts were isolated and cultured by trypsin digestion and explant method.The fibroblasts were transfected by a series of retrovirus and cultured by human embyonic cellconditioned medium to generate and induce iPSCs,and then the iPSCs were identified by morphology,alkaline phosphatase (AP) staining and immunofluorescence assay of specific markers of pluripotent stem cells.iPSCs suspension were injected into SCID mouse to observe the tumorigenesis.The iPSCs from non-RP subject were induced to differentiate toward iPS-RPE cells by embryonic body (EB) inducing method,and iPS-RPE cells were identified by detecting the expression of RPE65,zonula occludens protein 1 (ZO-1) and lecithin retinol acyltransferase (LRAT).Results Cultured human dermal fibroblasts showed fusiform or polygon shape and intercellular close arrangement,and Vimentin was positively expressed in the cells.Small cell colonies were harvested 5-7 days after infected by retroviruses,and the morphology changed from spindle into round mass.The hESC-like iPSCs clonies appeared 20 days after cultivation,and the positive expressions of hESC-specific surface antigens including SSEA3,SSEA4,TRA-1-60,TRA-1-81 and Nanog were found in the cells 25-30 days after cultivation,and the positive staining of AP was obtained 12 weeks after cultivation.A teratoma was formed at the injection site of iPSCs suspension in SCID mouse.Immunofluorescence technique showed that RPE cell-specific proteins including RPE65,ZO-1 and LRAT proteins were positively expressed in iPS-RPE cells at 30 days after differentiation.Conclusions Mutation SNRNP200 p.S1087L of RP patient-specific iPSCs can be induced from human dermal fibroblast by retrovirus infection method.The function and morphology of the iPSCs were similar to hESCs.Human iPSCs cell line generated from the dermal fibroblasts of non-RP individuals can differentiate into iPS-RPE cells.

Effects of Er-Miao-San extracts on TNF-alpha-indueed MMP-1 expression in human dermal fibroblasts

BACKGROUND: Various health benefits have been attributed to Er-Miao-San (EMS), a traditional Chinese herbal formulation that contains equal amounts of cortex phellodendri (Phellodendron amurense Ruprecht) and rhizoma atractylodis (Atractylodes lancea D.C). However, its effect on the anti-inflammatory activity in human dermal fibroblasts (HDFs) and the mechanism underlying this effect are unknown. RESULTS: This study investigated the effects of EMS on TNF-α-induced MMP-1 expression in HDFs. Our data show that EMS inhibited TNF-α-induced MMP-1 expression in a concentration-dependent manner. Interestingly, EMS maintained IkB content without inhibiting the phosphorylation of MAPKs, which are well-established upstream kinases of NF-kB. Moreover, EMS reduced the level of nuclear p65 protein in HDFs. Luciferase assay revealed that EMS inhibits the transcriptional activity of NF-kBbystabilizing IkB. Our results show that EMS exerts its anti-inflammatory effect by inhibiting NF-kB-regulated genes such as IL-1ß and IL-8. Moreover, EMS effectively inhibited TNF-α-induced expression of MMP-1 via the NF-kBpathway. CONCLUSIONS: Taken together, our data suggest that EMS could potentially be used as an anti-inflammatory and anti-aging treatment.

Transcriptional Regulation of Proteoglycans and Glycosaminoglycan Chain-synthesizing Glycosyltransferases by UV Irradiation in Cultured Human Dermal Fibroblasts

Various kinds of glycosaminoglycans (GAGs) and proteoglycans (PGs) have been known to be involved in structural and space-filling functions, as well as many physiological regulations in skin. To investigate ultraviolet (UV) radiation-mediated regulation of GAGs and PGs in cultured human dermal fibroblasts, transcriptional changes of many types of PGs and GAG chain-synthesizing enzymes at 18 hr after 75 mJ/cm2 of UV irradiation were examined using quantitative real-time polymerase chain reaction methods. Hyaluronic acid synthase (HAS)-1, -2, and -3 and hyaluronidase-2 mRNA expressions were significantly increased by UV irradiation. Expressions of lumican, fibromodulin, osteoglycin, syndecan-2, perlecan, agrin, versican, decorin, and biglycan were significantly decreased by UV irradiation, while syndecan-1 was increased. Expressions of GAG chain-synthesizing glycosyltransferases, xylosyltransferase-1, beta1,3-glucuronyltransferase-1, beta1,4-galactosyltransferase-2, -4, exostosin-1, chondroitin polymerizing factor, and chondroitin sulfate synthase-3 were significantly reduced, whereas those of beta1,3-galactosyltransferase-6, beta1,4-galactosyltransferase-3, -7, beta-1,3-N-acetylglucosaminyltran sferase-2, and -7 were increased by UV irradiation. Heparanase-1 mRNA expression was increased, but that of heparanase-2 was reduced by UV irradiation. Time-course investigation of representative genes showed consistent results. In conclusion, UV irradiation may increase hyaluronic acid production through HAS induction, and decrease other GAG productions through downregulation of PG core proteins and GAG chain-synthesizing glycosyltransferases in cultured human dermal fibroblasts.

Resveratrol protects against oxidative damage of fibroblasts irradiated with UVB / 中华物理医学与康复杂志

Objective To explore the protective effect of resveratrol against oxidative damage to cultivated fibroblasts irradiated with UVB. Methods Fibroblasts from normal human skin cultured in vitro were divided into 5 groups (a normal control group, a group irradiated with UVB, a group treated with resveratrol before UVB irradiation, and a group treated after irradiation). A monolayer of fibroblasts was irradiated with UVB at 60 mJ/cm2. The vitality of the cells was measured using the methylthiazol tetrazolium (MTT) method. The activity of superoxide dis-mutase (SOD) and malondialdehyde (MDA) content were determined using enzyme biochemistry. Results Resveratrol over 100 μM inhibited the proliferation of fibroblasts. Resveratrol under 100 μM improved the proliferation of cells. The optimal concentration was 50 μM. UVB irradiation decreased the vitality of the cells and SOD activity, and it significantly enhanced MDA content. Conclusions Resveratrol treatment before or after UVB irradiation elevates the survival rate of fibroblasts, enhances the activity of SOD, and decreases MDA content. Resveratrol at low concentration could improve the proliferation of fibroblasts, and at high concentration could inhibit their proliferation. Res-veratol at 50 μM relieves the inhibited proliferation of fibroblasts damaged by UVB irradiation.

Effect of Oncostatin M on Proliferation and Matrix Synthesis of Dermal Fibroblasts

PURPOSE: Oncostatin M(OSM) is a multifunctional cytokine that belongs to the interleukin(IL)-6 family. Although there have been a number of studies that focused on the role and mechanism of OSM in various organs and tissues, there are few reports on its effect on wound healing. The final purpose of this project is to evaluate the effect of OSM on wound healing. This pilot study was designed to investigate the effect of OSM on proliferation and matrix synthesis of human dermal fibroblasts, which are the major components of the wound healing. METHODS: Excess skin that was obtained from patients who underwent skin grafts, was used for this study. From this material, fibroblasts were isolated and cultured. The cultured fibroblasts were treated with one of four concentrations of OSM. The OSM concentrations used were 0, 50, 100, and 200ng/ml, respectively. After the OSM treatment, cell proliferation was determined by the MTT assay, collagen synthesis by the C1CP method, GAG levels by the Blyscan Dye method. The parameter levels of each group were compared. RESULTS: OSM treatment increased all the components tested in the study. In particular, cell proliferation, GAG synthesis demonstrated statistically significant increases(p<0.05 in the Mann-Whitney U-test). The highest increase in all the components was obtained at a 100ng/ml concentration of OSM. CONCLUSION: The results of the present study indicate that OSM stimulates proliferation and matrix synthesis of human dermal fibroblast and the optimal concentration for wound healing is 100ng/mL.

Protective actions of Rubus coreanus ethanol extract on collagenous extracellular matrix in ultraviolet-B irradiation-induced human dermal fibroblasts

Solar ultraviolet (UV) irradiation leads to distinct changes in the skin connective tissues by degradation of collagen, which is a major structural component in the extracellular matrix. UV irradiation induces the production of matrix metalloproteinases (MMP) capable of attacking native fibrillar collagen and responsible for inhibiting the construction of collagenous extracellular matrix. In this study, we attempted to investigate the protective actions of Rubus coreanus ethanol extract (RCE) on the MMP production and the consequent procollagen/collagen degradation in UV-B-irradiated human dermal fibroblasts. The analytical data showed that Rubus coreanus ethanol extract was mostly comprised of cyanidin 3-rutinoside. Pre-treatment of fibroblasts with this extract inhibited UV-B-induced production of MMP-1, MMP-8 and MMP-13 in dose-dependent manners. In addition, Western blot analysis and immunocytochemical staining assay revealed that RCE markedly augmented the cellular levels of procollagen/collagen declined in UV-B-exposed dermal fibroblasts. These results demonstrate that RCE blocks UV-B-induced increase of the collagen degradation by inhibiting MMP production. Thus, RCE may act as an agent inhibiting excessive dermal collagen degradation leading to the skin photoaging.

Effects of Pharmacological Modulators of Ca2+ -activated K+ Channels on Proliferation of Human Dermal Fibroblast

Employing electrophysiological and cell proliferation assay techniques, we studied the effects of Ca2+ -activated K+ channel modulators on the proliferation of human dermal fibroblasts, which is important in wound healing. Macroscopic voltage-dependent outward K+ currents were found at about -40 mV stepped from a holding potential of -70 mV. The amplitude of K+ current was increased by NS1619, a specific large-conductance Ca2+ -activated K+ (BK) channel activator, but decreased by iberiotoxin (IBTX), a specific BK channel inhibitor. To investigate the presence of an intermediate-conductance Ca2+ -activated K+ (IK) channels, we pretreated the fibroblasts with low dose of TEA to block BK currents, and added 1-EBIO (an IK activator). 1-EBIO recovered the currents inhibited by TEA. When various Ca2+ -activated K+ channel modulators were added into culture media for 1~3 days, NS1619 or 1-EBIO inhibited the cell proliferation. On the other hand, IBTX, clotrimazole or apamin, a small conductance Ca2+ -activated K+ channel (SK) inhibitor, increased it. These results suggest that BK, IK, and SK channels might be involved in the proliferation of human dermal fibroblasts, which is inversely related to the channel activation.

The Effect of Hyaluronic Acid on Fibroblast Proliferation in Vitro and Skin Wound Healing in Vivo

It is not determined yet whether hyaluronic acid up- regulates or down-regulates wound healing. This study was designed to define the effect of hyaluronic acid on proliferation of human dermal fibroblasts in vitro and on skin wound closing in vivo. Fibroblasts were isolated from the dermis of adults and cultivated in the presence of either one of 6 concentrations of hyaluronic acid(0, 0.1, 0.2, 0.5, 1.0, 2.0 mg/ml). The fibroblasts were seeded at 2.0 x 10(4) cells/well in Dulbecco's Modified Eagle's Medium/Ham's F-12 nutrient including 10% fetal bovine serum with either one of 6 different concentrations of hyaluronic acid in 24-well plates. The cells were incubated for 6 days. All concentrations of hyaluronic acid stimulated the proliferation of fibroblasts. The best proliferation was seen at 0.2 mg/ml of hyaluronic acid concentration(p = 0.01). For in vivo study, 10 white rats were used. A 5 mm round punch was employed to excise skin and subcutaneous tissue at eight sites on the back. After creating 8 open wounds, 8 concentrations(0, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mg/ml) of hyaluronic acid were applied. The degrees of wound closing were compared the 6th day under light microscope. Low concentration of hyaluronic acid(0 - 2.0 mg/ml) stimulated the wound closing. However, high concentration of hyaluronic acid(5.0 -10.0 mg/ml) delayed the wound closing. The best wound closing was seen at 0.5 mg/ml of hyaluronic acid (p = 0.032). These results demonstrated that hyaluronic acid influenced human dermal fibroblast proliferation and the skin wound closing in rats, and its concentration was critically important factor.