Analysis of protease-activated receptor 2 expression and function in cultured human keratinocytes / 中华皮肤科杂志

Objective To assess the expression pattern of protease-activated receptor 2 (PAR2) in human keratinocytes and to characterize its biological functions in the regulation of skin barrier.Methods Primary human keratinocytes and human N/TERT keratinocytes were used as the subject of this study.The expression and distribution of PAR2 in the keratinocytes were analyzed by using immunoflorescence staining and Western blot.Two different PAR2 agonists,trypsin and a PAR2-activating peptide (AP),as well as a PAR2-antagonistic peptide (H2N-FSLLRY-COOH) and a control peptide were used to induce the activation of PAR2 in the keratinocytes.Then,a fluorescence-based calcium mobilization assay was performed to evaluate the biological function of PAR2.Data were statistically analyzed by one-factor analysis of variance.Results Under normal culture conditions,PAR2 was weakly expressed in keratinocytes,and the expression was unaffected by culture medium composition or culture duration.Calcium mobilization was induced by trypsin of 50-250 nmol/L and the PAR2-activating peptide in a dose-and time-dependent pattern.The maximal activation of PAR2 was observed in keratinocytes treated with the PAR2 agonist HAN-SLIGKV-COOH of 75-250 μmol/L.The PAR2-antagonistic peptide (H2N-FSLLRY-COOH) obviously suppressed the increase in calcium mobilization induced by trypsin,while the control peptide PAR-RAP showed no inductive effect on the PAR2 activation based on the absence of calcium mobilization.The substrate-induced calcium release was complete within 250 seconds,and peaked at 50 seconds after the initial trypsin or PAR-AP stimulation.Moreover,the activation of PAR2 was accompanied by an increase in ERK phosphorylation and elicitation of MAPK signaling pathway in keratinocytes.Conclusions Human keratinocytes positively express PAR2,which can be activated by trypsin and PAR2-activating peptides,and the activation of PAR2 may influence the physiological function of keratinocytes by inducing intracellular calcium release.

Influences of extracellular matrices secreted by ultraviolet B-induced senescent fibroblasts on ERK signaling in HaCaT cells / 中华皮肤科杂志

Objective To explore the influences of extracellular matrices (ECM) secreted by ultraviolet B (UVB)-induced senescent fibroblasts on the proliferation of and extracellular signal-regulated kinase (ERK) signaling in HaCaT cells. Methods Fibroblasts were irradiated with UVB of 15 mJ/cm2 once daily for 5 days to induce premature senescence, which was identified by SA-β-gal staining 72 hours after the last irradiation.HaCaT cells were divided into 3 groups and inoculated into plates coated with extracellular matrices secreted by non-senescent (PRE-ECM) or senescent fibroblasts (SIPS-ECM) or into uncoated plates (NON-ECM), fol-lowed by additional culture. U0126, an inhibitor of ERK1/2, was used to treat the HaCaT cells 1 hour before inoculation. Then, MTT assay was carried out to detect the proliferation of HaCaT cells after a 3-day culture,Western blot to assess the phosphorylation of ERK at 0.5, 1, 2 and 4 hours after the inoculation, flow cytometry to analyse cell cycle and apoptosis after 24 hours of culture. Results The most rapid and intense phosphory-lation of ERK was observed in SIPS-ECM group. Inhibiting the activation of ERK pathway with U0126 could completely suppress the promoting effect of ECM from senescent fibroblasts on the proliferation of HaCaT cells.After the blocking of ERK activation, the proportion of HaCaT cells in S and G2/M phase decreased from 37.40%, 41.34% and 43.31% to 29.41%, 36.48% and 39.96%, respectively, in NON-ECM, PRE-ECM and SCIP-ECM group. Conclusion The ECM produced by UVB-induced senescent fibroblasts promote the prolifera-tion of HaCaT cells via inducing the phosphorylation of ERK.

Cognition and behavior of sun-protection and facial photoageing in a Nanjing population / 中华医学美学美容杂志

Objective To investigate the cognition and behaviors of sun-protection and the facial photoaging in a Nanjing population, and to analyze the relationships between them. Methods The objects being investigated in Nanjing (n=974) were divided into 10 groups according to age. The ordinary information, knowledge and behavior of sun-protection and Glogau photoageing type of face were studied by questionnaires. The results were analyzed by a logistic regression model to select the related factors to photoageing. Results The risk of skin photoageing increased with age. Most of the 46-65 years old crowds were type Ⅲ photoageing. Most of the objects being investigated had some knowledge and active awareness of sun-protection and could use some ways to protect themselves from sun. However, most people did not use the sunscreen correctly. Those who had higher level awareness and knowledge of sunprotection suffered less risk from skin photoageing. Sunbath without sunscreen for a long time outside activity was a high risk factor of development to skin photoageing. Shade, broad-brimmed hat and sun-protection in autumn were the protective factors. Conclusion Active awareness and correct methods could help prevent skin photoageing. In order to avoid the damages to skin from ultraviolet efficiently, the accurate ways of using sunscreens should be well understood.

Inhibitory Action of Antisense c-jun oligodeoxynucleotides on Ultraviolet B Induced Fibroblast Damage / 中华皮肤科杂志

Objective To explore the inhibitory action of antisense c-jun oligodeoxynucleotides(ODN) on matrix metalloproteinase(MMP) expression of fibroblasts induced by ultraviolet B (UVB). Methods The c-jun and c-fos protein expression induced by UVB were measured by Western blot. The mRNA expression of c-jun was determined by reverse transcriptase polymerase chain reaction (RT-PCR) after transfection with c-jun antisense ODN. The pro-MMP-1 and MMP-3 synthesis of fibroblasts was measured by ELISA after treatment with UVB and antisense c-jun ODN. Results The UVB-induced c-jun protein expression of fibroblasts increased to 1.8, 2.6, 3.3 times compared with that of non-irradiated controls,while there was no significant change in c-fos protein expression. The pro-MMP-1 and MMP-3 synthesis induced by UVB irradiation increased obviously. After transfection with different concentrations of c-jun antisense ODN, the UVB-induced c-jun mRNA expression could be significantly inhibited(P

Study on the Mechanism of UVB Irradiation and(-)-epigallocatechin-3-gallate Protection on Apoptosis of Cultured Human Keratinocytes / 中华皮肤科杂志

0.25)levels in the two groups.In the group treated with42mJ/cm 2 UVB irradiation followed by the addition of EGCG,the numbers of apoptotic and dead cells and Fas mRNA were decreased,but bcl-2protein was increased.Conclusions Low dosage of UVB irradiation could induce apoptosis of keratinocytes.High dosage of UVB irradiation might result in cell death.EGCG could reduce UVB-induced apoptosis of keratinocytes by increasing bcl-2protein and decreasing Fas mRNA.

Effects of Antisense NF-?B p65 Oligodeoxynucleotides on UV-induced IL-6 Secretion of Keratinocytes / 中华皮肤科杂志

Objective To explore the effects of antisense NF-?B p65 oligodeoxynucleotides on UV-induced IL-6 secretion of keratinocytes. Methods NF-?B p65 oligodeoxynucleotides were transfected to a keratinocyte cell line, HaCaT cells, mediated by lipofectamine. The NF-?B p65 protein in the cells was measured by Western blot assay, the mRNA level of NF-?B p65 was detected by RT-PCR, and UV-induced IL-6 level was determined by ELISA pre- and post-transfection and/or UVB irradiation. Results The NF-?B p65 protein expression was significantly increased in UVB (10, 20, 30 mJ/cm2 ) irradiation groups as compared with that of the control groups (P