Objective To investigate the role of infiltrating inflammatory cells in photoaging process by comparing the type and number of these cells in sun-exposed and-unexposed skin.Methods The expression of CD3,CD45RO and CD68 were detected by immunohistochemieal staining in 46 paraffin-embeded skin samples from the extensor forearms(sun-exposed)and upper-inner arms(sun-unexposed) of 23 healthy female volunteers.The number of positive cells in sun-exposed and -unexposed sites was counted and statistically tested by paired samples t test,and Pearson correlation analysis was performed to assess the relationship between the number of positive cells and age of these volunteers.Results The number of cells positive for CD3,CD45RO and CD68 per square millimetre in sun-exposed skin was significantly higher than that in sun-unexposed skin(48.91±13.173 vs.40.61±11.571,46.83±12.915 vs.38.00±10.109,85.43±22.346 vs.73.48±16.208,respectively,P<0.01 or 0.05).The number of cells positive for CD3 and CD45RO increased significantly with age (r=0.557,0.555,respectively,both P<0.01) in the sun-exposed skin but not in sun-unexposed skin,and the number of CD68-positive cells was uncorrelated with age in either sunexposed or -unexposed skin.Conclusion T lymphocytes and macrophages may play a role in the process of photoaging.

Objective To observe the expression changes of CD34 and M30 in skin homogenate from a mouse model of scleroderma after irradiation with different doses of UVA1, and to investigate the effect of UVA1 phototherapy on vascular endothelial cell function in scleroderma. Methods The experimental mouse models of scleroderma were established by the injection with bleomycin and randomly divided into model control group (n = 10), UVA1 irradiation group (n = 30) and unirradiated group (n = 10). The UVA1 irradiation group was further equally divided into 3 groups, HD-UVA1 group irradiated with UVA1 at 100 J/cm2, MD-UVA1group with UVA1 at 60 J/cm2, and LD-UVA1 group with UVA1 at 20 J/cm2; phototherapy was performed thrice weekly for 10 weeks followed by the sacrifice of mice. The mice in model control group were killed immediately after the establishment of models, and the mice in unirradiated group received no irradiation after the establishment of models and were maintained till the killing of mice in UVA1 irradiation groups. Skin specimens were obtained from the bleomycin-induced scleroderma lesions of mice and separated into two parts, one was subjected to histopathological examination, and the other one was used to prepare skin homogenate for the detection of CD34 and M30 content with ELISA assay. Results After 30 sessions of treatment with UVA1,the softening and thinning of sclerotic skin were seen by the naked eye, with the most obvious changes in HDUVA1 group; pathological examination revealed a reduction in dermal thickness and the presence of hair follicular structures in subcutaneous fat tissue with no obvious proliferation of collagen in these mice. Compared with the mice in model control group and unirradiated group, there was an increase in CD34 and decrease in M30 content in skin homogenate from UVA 1-irradiated mice, with the most marked changes in mice irradiated with UVA1 at 100 J/cm2. The concentration of CD34 and M30 in skin homogenate from unirradiated group and model control group was significantly different from that in HD-UVA1 group (22.25 ± 8.91 μg/L and 31.97 ±17.97 μg/L vs. 72.39 ± 13.04 μg/L, 162.41 ± 58.00 U/L and 195.71 ± 71.09 U/L vs. 38.06 ± 19.89 U/L, all P ＜ 0.01 ). Additionally, significant differences were observed between the three UVA1 groups in the concentration of CD34 and M30 (F = 21.23, 15.32, respectively, both P ＜ 0.01 ). Conclusions UVA1 phototherapy could up-regulate the expression of CD34 but down-regulate that of M30 in skin homogenate from the mouse model of scleroderma, and the effect is correlated with the intensity and cumulative dose of irradiation.

Objective To study the expression of matrix metalloproteinase(MMP)-1,-3 and-9 in sun-exposed and-unexposed skin as weil as its significance in the mechanism of skin photoaging.Methods Skin samples were resected from the exmnsor side of forearm(sun-exposed area)and flexor side of upper arm(sun-unexposed area)of 23 healthy female volunteers.The expression of MMP-1,-3 and-9 was detected by immunohistochemical staining in 46 skin samples.Immunoreactive intensity distribution index (IRIDI)was calculated to assess the expression of MMP-1,-3 and-9.Wilcoxon signed ranks test,Mann-Whitney U-test and Spearman rank correlation analysis were performed.Results MMP-1,-3 and-9 were expressed in both sun-exposed and -unexposed skin.The average IRIDI value for MMP-1,-3 and-9 was 7.70(range,3 to 12).9.22(range,6 to 12),8.30(range,6 to 12)in sun-exposed skin,and 4.26 (range,2 to 6),5.39(range,2 to 9),4.04(range,1 to 6)in sun-unexposed skin,respectively;significant difierence existed between sun.exposed and-unexposed skin in the three parameters(all P＜0.01).A significant inerease was observed in the average IRIDI value for MMP-1,-3 and-9 in sun-exposed skin vs.sun-unexposed skin in women above 50 years of age (9.17 vs 4.75,10.58 vs 6.42,8.92 vs 4.33,respectively,all P＜0.05).In women younger than 50 years,the average IRIDI value for MMP-1,-3 and-9 was 6.09(range,3 to 8),7.73(range,6 to 9),7.64(range,6 to 12)in sun-exposed skin,significantly higher than that in sun-unexposed skin[3.73(range,2 to 6),4.27(range,2 to 8),3.73(range,1 to 6),all P＜0.05].Increased IRIDI scores of MMP-1,-3 and -9 were noticed in sun-exposed skin in women above 50 years of age vs.those younger than 50 years.but there was no statistical difrerence in MMP-I or MMP-9 between the two aged groups in sun-unexposed skin(all P＞0.05).The IRIDI scores of MMP-1,MMP-3 and MMP-9 were positively correlated with age(r=0.66,0.69,0.74,all P＜0.01)in sun-exposed skin,but the IRIDI scores of MMP-1 and MMP-9 uncorrelated with age in sun-unexposed skin.Conclusions There isan elevated expression of MMP-1,-3 and.9 in sun-exposed skin VS.SUn.unexposed skin.hinting that these three MMPs play a role in the occurrence and development of photoaging,but their biological mechanism may be different.

Objective To evaluate the sunscreen application and the level of photoprotective knowledge in both dermatologists and photosensitive patients. Methods The style, sites and amount of the sunscreen applied were examined by 0. 05 % dipyridamole cream in 39 dermatologists and 41 photosensitive patients with Wood's light. The participants were asked to fill in a questionnaire about the photoprotective knowledge. Results Frequent mistakes made by participants in this study were as follow: (1) using an inadequate amount of sunscreen; (2) putting sunscreen in the palm of the hand and rubbing the hands together before application; (3) lacking a systematic approach to sunscreen application. The median quantity of individual sites ranged from 0. 5 mg/cm2 to 1 mg/cm2 except for the forehead of the female dermatologist that had a median thickness of 1. 5 mg/cm2. The questionnaire survey showed that dermatologists also had less knowledge on sun protection even though better than photosensitive patients. Conclusions Dermatologists and photosensitive patients always fail to apply sunscreen in some prominently exposed sites and to paint the average thickness of sunscreen used far less than that of experimentally measured dose (2 mg/cm2). Continuing education and training about pho-toprotection for dermatologists should be carried out to provide better education for the patients on sun protection.

Objective To study the relationship of sunlight exposure and photoprotection with clinical activity in systemic lupus erythematosus. Methods A structured questionaire was administered to 107 SLE patients, to assess their attitudes and behavior regarding sunlight exposure and photoprotection. The clinical manifestations, laboratory findings and treatment were evaluated. Results All patients were classified into two groups based on the duration of exposure to sunlight per day. Fourty-eight (44.86%) patients were exposed to direct sunlight for an average of less than one hour per day in one group and 59 (55.14%) for one hour or more in the other group. Twenty-four (22.43%) patients reported use of photoprotective measures in spring and summer. The patients in the former group had significantly lower incidences of photosensitivity, arthritis, alopecia, exacerbations, presence of anti-dsDNA antibody, decrease of complement C3, C4 and CH50 than those in the latter group(P

Objective To study the precipitating factors, clinical features, histopathological changes and treatment in patients with localized and generalized granuloma annulare (GA). Methods Clinical data of 24 cases of localized GA and 19 cases of generalized GA were analyzed retrospectively. Results Some cases of GA were found to be related to the exposure of sunlight, especially in the generilized patients. In the patients with localized GA, lesions usually distributed on the dorsal surface of the hands, nape of the neck, dorsum of feet. Annular lesions with 1-2 centimeters in diameter were formed by small papules. The largest lesion was 7 centimeter in diameter. Generalized GA presented as a diffuse papular eruption, 0.5 ~ 1 cm in diameter, and the lesions favoured the trunk and four limbs. The histopathological study showed that palisading granuloma pattern accounted for 61.9% in all patients, and scattered histiocytic infiltration accounted for 38.1%. Ultraviolet light avoidance, topical steroids, cryotherapy, surgical excision, systemic vitamin E or nicotinamide were effective for localized lesions. Systemic administration of chloroquine in low dosage was an alternative way for the stubborn localized GA. Systemic chloroquine, dapsone, corticosteroids, isotretinoin were effective in most generalized GA cases, but some cases relapsed when treatment was stopped. Conclusions Ultraviolet may be associated with the development of generalized GA. The histopathological changes were variable, the palisading granuloma pattern is the most common pattern. Topical therapy is effective in localized GA, and systemic therapy is mainly used for generalized GA.

Objective To study the effects of different stimulators on the production of matrix metalloproteinases (MMPs) by peripheral blood mononuclear cells (PBMCs),and to evaluate the effects of the culture supernatant of activated PBMCs,named conditioned media (CM),on the proliferation of and production of MMPs by cultured human fibroblasts.Methods PBMCs were isolated from the venous blood samples of healthy volunteers and divided into three groups to be stimulated by phytohemagglutinin (PHA group),the combination of antibodies against CD3 and CD28 (double-antibody group),or the RPMI 1640 medium containing 10％ fetal calf serum (control group).After 72-hour stimulation,CM was collected from all the three groups,diluted to several different degrees.Cultured human fibroblasts were classified into several groups to be treated with different dilutions of CM from the three groups for 48 or 24 hours,with the fibroblasts untreated with CM serving as the control group.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cellular proliferative activity,semi-quantitative reverse transcription (RT)-PCR to detect the expressions of MMP-1,MMP-3 and MMP-9 mRNAs in cells,and enzyme-linked immunosorbent assay (ELISA) to measure the levels of interleukin (IL)-6,MMP-1,MMP-3 and MMP-9 proteins in the culture supernatant of cells.Statistical analysis was carried out mainly by using one-way analysis of variance (ANOVA),Tukey HSD test,and GamesHowell test.Results Compared with the control group,the PHA group showed increased cellular proliferative activity,IL-6 and MMP-3 protein levels in the culture supernatant of activated PBMCs (all P ＜ 0.05).Significant differences were observed among the PHA group,double-antibody group and control group in the relative mRNA expression level (expressed as the ratio of target mRNA to β-actin mRNA) of MMP-1 in activated PBMCs (0.083 ± 0.016 vs.0.188 ± 0.030 vs.0.714 ± 0.104,F =85.905,P ＜ 0.05),but neither MMP-3 nor MMP-9 mRNA was expressed by activated PBMCs.MMP-3 protein was detectable in the culture supernatant of fibroblasts after the treatment with CM,and the level of MMP-3 protein was highest in that of fibroblasts treated with undiluted CM,and lowest with 1/10 diluted CM;at the same dilutions,the level of MMP-3 protein was highest in the culture supernatant of fibroblasts treated with CM from the PHA group,but lowest with that from the control group.Neither MMP-1 nor MMP-9 protein was detected in the culture supernatant of activated PBMCs or treated fibroblasts.There were no significant differences in cellular proliferative activity of and mRNA expressions of MMP-1 or MMP-3 in fibroblasts among these groups (all P ＞ 0.05),and MMP-9 mRNA expression was undetected in the treated fibroblasts.Conclusions PBMCs can be induced to express MMP-1 mRNA and secret MMP-3 protein after activation.However,the culture supernatant of activated PBMCs has no capacity to stimulate the expressions of MMP-1,MMP-3 and MMP-9 mRNAs or proteins by fibroblasts,suggesting that inflammatory cells may function through self-production of MMPs.

Objective To investigate the effects of sirolimus and 3-methyladenine (3-MA) on the autophagy in,as well as matrix matalloproteinase (MMP)-1 and MMP-3 secretion by,human skin fibroblasts (HSFs).Methods HSFs were isolated from the circumcised foreskin of a healthy male,and subjected to primary culture.After 3 to 10 passages,HSFs were incubated with sirolimus of 20,50,100,250 nmol/L and 3-MA of 0.5,2.0,5.0,10.0 mmol/L respectively for 4 hours followed by the observation of autophagy and detection of MMP-1 and MMP-3 levels in the supernatant by enzyme linked immunosorbent assay (ELISA).Those HSFs remaining untreated or treated with dimethyl sulfoxide served as the control.Results The percentage of autophagy-positive cells was 59.075％ ± 6.884％,76.350％ ± 5.226％,85.063％ ± 6.002％,86.288％ ± 5.558％ and 96.825％ ± 1.500％ respectively in HSFs treated with sirolimus of 0,20,50,100 and 250 nmol/L; significant differences were observed between the 5 groups (P ＜ 0.01 ) but not between the cells treated with sirolimus of 50 and 100 nmol/L (P ＞ 0.05).After being treated with 3-MA of 0,0.5,2.0,5.0 and 10.0 mmol/L,the percentage of autophagy-positive cells in HSFs was 63.037％ ± 5.876％,34.425％ ± 5.183％,19.700％ ± 3.028％,12.900％ ± 3.334％ and 7.775％ ± 2.293％ respectively with a significant difference between these groups (all P ＜ 0.01 ).Elevated levels of MMP-1 and MMP-3 were observed in the supernatant of HSFs treated with sirolimus of 250 nmol/L and 3-MA of 10.0 mmol/L (all P ＜ 0.05).Conclusions The autophagy in HSFs can be upregulated by sirolimus,but downregulated by 3-MA.For the secretion of MMPs by HSFs,3-MA and high concentrations of sirolimus exert a promotive effect,and the effect of 3-MA is in a concentration-related manner,but the influences of sirolimus at lower concentrations remain unclear.

The purpose of this study is to investigate the transdermal delivery characteristics of Gentiana macrophylla complex components system through different parts of the skin under micro-needles conditions. Two-chamber diffusion cells were used, different parts of isolated skin and micro-needle pretreated isolated mouse skin were applied separately, high performance liquid chromatography (HPLC) similarity evaluation methods were used to evaluate transdermal delivery characteristics of Gentiana macrophylla complex components system on receiving pool and the permeation rate and penetration amount of Gentiopicroside at different parts of mouse skin. In the 24 h, the similarity between receiving fluid which was on passive transdermal delivery and micro-needle transdermal delivery conditions and original fluid were ranged from 83.0% to 98.9%; By the micro-needle pretreatment with different parts of the mouse skin, the time that Gentiana macrophylla complex components system though abdominal skin to the receiving fluid which reached 90% similarity compared with that of original fluid was 4 h, which was 18 h at back skin and 12 h at neck skin separately. Micro-needles can be used as the ideal ingredients for traditional Chinese medicine complex transdermal delivery; transdermal absorption time delay could be greatly reduced and its bioavailability was improved. The permeation rate and similarity to original liquid of Chinese medicine complex components increased significantly in the abdominal skin relative to the neck and back skin under micro-needle conditions.

Objective To observe the changes of autophagy in human skin fibroblast (HSF) model for photoaging. Methods HSF model for photoaging was established through repeated exposure to ultraviolet B (UVB). Those HSFs receiving no irradiation served as the control. The degree of aging was evaluated by p-galactosidase assay, and autophagy level was detected. Results After repeated exposure to UVB, most pho-toaged HSFs were deformed and distorted, and some of them even died. The percentage of P-galactosidase-positive cells was 50.60% ± 5.04% and 14.58% ± 2.69%, respectively in photoaged and control HSFs (P< 0.01). Significant difference was also observed in the proportion of cytophagosome-positive cells between photoaged and control HSFs (14.91% ± 4.59% vs 68.45% ± 8.25%, P < 0.01). Conclusion The HSF model for photoaging shows obviously abnormal appearance and stagnant growth with increased degree of senescence and decreased autophagy compared with normal control HSFs.