Calcium and potassium inhibit barrier recovery after disruption, independent of the type of insult in hairless mice

Abstract Disruption of the cutaneous permeability barrier induces metabolic responses in the epidermis which result in barrier recovery. Barrier disruption by either solvent treatment or tape stripping results in the loss of the epidermal calcium gradient. Previous studies in acetone treated hairless mice have shown that maintaining this calcium gradient inhibits barrier repair, suggesting that alterations in the epidermal calcium concentration may be an important signal for barrier homeostasis. In the present study, we show that in hairless mice disruption of the barrier by treatment with the detergent. SDS, also results in the loss of the calcium gradient, as demonstrated both semi-quantitatively with ultrastructural cytochemical localization and quantitatively using proton induced X-ray emission (PIXE). Additionally, immersion in calcium containing solutions delays barrier repair after either detergent (SDS treatment) or mechanical (tape stripping) disruption of the barrier, as reported previously for acetone treated skin. These results indicate that barrier disruption, regardless of the insult, induces changes in the epidermal calcium gradient which may play an important role in signaling the metabolic changes required for barrier homeostasis.     

Acute cutaneous barrier disruption activates epidermal p44/42 and p38 mitogen-activated protein kinases in human and hairless guinea pig skin

Acute cutaneous barrier disruption of the skin elicits various homeostatic repair responses in the epidermis. Although several candidates for the signaling mechanisms that induce these responses have been reported, e.g. the calcium and ion concentration, peroxisome proliferator-activated receptor-alpha, and TNF-alpha signaling mediated by sphingomyelinases, the exact nature of the signals remains undertermined. Therefore, assuming that an important group of serine/threonine-signaling kinases, mitogen- and SAPK/JNK, might link the barrier disruption to the subsequent homeostatic responses, the activation of three MAPKs in hairless guinea pig or in human skin after barrier disruption was investigated. The epidermal barrier was insulated with tape stripping or organic solvents, and Western blotting, and immune complex kinase assay. In the skin of hairless guinea pigs, p44/42 MAPK and p38 MAPK, but nor SAPK/JNK, were continued to be activated for at least 180 min. The activation of p44/42 which positively correlated with the number of tape strippings, whereas K+ sucrose solution suppressed its activation. The activation of p44/42 MAPK was also induced by treatment of the skin with organic solvents. In similar fashion, p44/42 and p38 MAPKs were found to be activated in human skin after tape stripping. These results for strongly suggest that the activation of p44/42 and p38 MAPKs links the stimuli of barrier disruption to the subsequent homeostatic responses to repair the barrier defect.     

Generation and distribution of reactive oxygen species in the skin of hairless mice under UVA: studies on in vivo chemiluminescent detection and tape stripping methods

Although the formation of reactive oxygen species (ROS) in the skin induced by the ultraviolet (UV) light has been shown to lead to many cutaneous disorders, skin cancer and photoageing, the mechanism and distribution of ROS generation has not yet been definitively determined. In the present study, we examined the distribution of UVA-induced ROS in the skin of live hairless mice, using our proposed in vivo imaging chemiluminescent (CL) method to detect ROS combined with a CL probe (cypridina hilgendorfii luciferin analogue; CLA) and tape stripping (TS) technique. The CL intensities in the skin of live hairless mice were confirmed to significantly increase by UVA exposure. When TS was conducted five times in a maximum level after CL measurement following UVA exposure and subsequent CLA application, CL intensities due to UVA-induced ROS generation in the residual skin decreased to 10% of the original levels; and those in the stripped skin on each tape decreased in the stripped order such as 52%, 16%, 11%, 6% and 5%. Next, CLA was applied and then CL intensities were measured in the residual skin after advance 1, 3 and 5 tape strippings, and CL intensities due to ROS were detected primarily in the outer layer of the skin. On the basis of these results, we concluded that ROS induced by UVA exposure occurs and distributes in the outermost layer of the stratum corneum.     

The efficacy of different moisturizers on barrier recovery in hairless mice evaluated by non-invasive bioengineering methods

Moisturizers (emollients) are used frequently on normal and diseased skin. However, only few studies have examined their effects in dynamic situations and in more clinically relevant settings. We evaluated the effect of 4 commonly used products in a hairless mice model after acute skin barrier perturbation with acetone. The efficacy was evaluated by measurement of the transepidermal water loss (TEWL) and electrical conductance at various time intervals during barrier repair. The test products were compared with acetone-treated air-exposed controls allowed to recover otherwise normally and with a known irritant product, chlorhexidine cream 1%. Locobase® was the most effective product in correcting barrier function and significantly improved barrier function during early stages of barrier recovery (<6 h) with out interfering with late stages of barrier recovery (>6 h). The irritant control product, chlorhexidine cream 1%, delayed barrier recovery in the late stages. The model makes it possible to evaluate the combined effects of exogenous and endogenous components on barrier repair and to select the potentially most effective products before perforating more cumbersome and time-consuming field studies.     

Variation in barrier impairment and inflammation of human skin as determined by sodium lauryl sulphate penetration rate

BACKGROUND: Skin irritability after a brief exposure to the model skin irritant, sodium lauryl sulphate (SLS), is known to vary considerably between individuals. A difference in the skin barrier to SLS may contribute to this variation. To date, no human in vivo data have been available on SLS penetration into the skin. OBJECTIVES: We studied whether the SLS penetration rate into the stratum corneum (SC) is related to impairment of the water barrier function and inflammation of the skin. METHODS: The penetration of SLS into the SC was assessed using a noninvasive tape-stripping procedure in 20 volunteers after a 4-h exposure to 1% SLS. Additionally, the effect of a 24-h exposure to 1% SLS on the skin water barrier function was assessed by measuring the transepidermal water loss (TEWL). The accompanying inflammation was quantified by measuring erythema. RESULTS: The mean +/- SD diffusivity of SLS (D) and the SLS permeability coefficient (Kp) were 1.4 +/- 0.6 x 10(-8) cm2 h(-1) and 1.5 +/- 0.7 x 10(-3) cm h(-1), respectively. A multiple regression analysis showed that the baseline TEWL, SC thickness and SLS penetration parameters K (SC/water partition coefficient) and D clearly influenced the increase in TEWL after the 24-h irritation test (explained variance: r2 = 0.80). Change in erythema was mainly influenced by SC thickness. CONCLUSIONS: We found that variation in the barrier impairment and inflammation of human skin depends on the SLS penetration rate, which was mainly determined by SC thickness.     

Molecular events associated with apoptosis and proliferation induced by ultraviolet-B radiation in the skin of hairless mice

BACKGROUND: It is recognized that UV radiation produced apoptotic cells (sun burn cells) in the epidermis of mice. However, the relationship between apoptosis and cell proliferation after UV exposure in the skin of hairless mice are still unclear. OBJECTIVE: To investigate the effects of ultraviolet (UV) radiation on molecular events associated with apoptosis and proliferation in SKH1-hr mouse skin. METHODS: Mice were irradiated with daily UVB exposure of 0.1 or 0.25 J/cm(2) for 14 days. The skin tissues were analyzed at 2 and 24 h after the end irradiation for the presence of apoptotic cells and Bromodeoxyuridine (BrdU)-positive cells. We measured the expression of p53, p21, bcl-2, bax and E2F-1. RESULTS: The results indicated that UVB irradiation caused to increase apoptotic cells in the epidermis of mice. The expression of p53 and p21 was increased at 2 and 24 h after irradiation compared with the control. UV radiation induced high levels of bax at 2 and 24 h after irradiation with a concomitant decrease in bcl-2 expression. The expression of E2F-1 in the skin was also increased at 2 and 24 h after irradiation. Coinciding with these changes, BrdU positive cells increased at 2 and 24 h after UVB exposure at the epidermis of hairless mice, which observed the apoptotic expression. CONCLUSION: These results suggest that UVB irradiation of mouse skin induces apoptosis and is mediated by the p53/p21/E2F-1/bax pathway and that the dead cells are replaced by hyperproliferative cells, leading to epidermal hyperplasia.     

抗原致敏树突状细胞抗小鼠白念珠菌系统感染的实验研究

目的研究不同形态白念珠菌致敏的小鼠骨髓来源树突状细胞(DC)对免疫抑制小鼠白念珠菌系统感染的免疫保护作用及所对应的细胞因子改变。方法孢子相和菌丝相白念珠菌在体外分别致敏小鼠骨髓来源的DC(BM-DC),测定混合培养上清IL-12水平;尾静脉回输免疫抑制小鼠体内后,ELISA法测定各组小鼠脾IFN-γ及IL-4水平,并检测肾携菌量。结果DC孢子致敏组上清IL-12水平(380.2±104.13)pg/mL明显高于DC菌丝致敏组和对照组(P<0.05);而DC菌丝致敏组(74.79±23.47)pg/mL与单纯DC培养组上清IL-12水平(19.71±9.21)pg/mL差异无统计学意义(P>0.05)。孢子致敏DC、菌丝致敏DC分别过继免疫小鼠后,前者脾脏IFN-γ水平(269.43±17.34)pg/g明显高于其他组(P<0.05),IL-4水平(6.23±0.37)pg/g则明显低于其他对照组(P<0.05);荷菌一周后孢子致敏DC回输组小鼠肾携菌量(3.58±2.32)×102CFUs与健康小鼠荷菌组比较无统计学意义(P>0.05);其他各组间肾携菌量比较则有统计学意义(P<0.05)。结论尾静脉回输白念珠菌孢子体外致敏的小鼠骨髓来源DC可有效诱导免疫抑制小鼠抗白念珠菌保护性免疫。     

Immunohistochemical characterization of cellular infiltrates in epidermal tumors induced by two-stage and complete chemical carcinogenesis in mouse skin.

We investigated the population and pattern of the infiltrated cells in both benign and malignant epidermal tumors which were induced chemically with benzo(a)pyrene (B(a)P) in murine skin. In benign papillomas, which were evolved by a two stage carcinogenesis regimen, a slight to mild inflammatory infiltration around the tumors was observed, and cells infiltrating into the tumor nests were rarely seen. In carcinomas, which were produced by a complete carcinogenesis regimen, a dense inflammatory infiltration was observed around the tumor nests. The infiltrated cells were characterized as T-lymphocytes, macrophages, and neutrophils. Natural killer (NK) cells were found around and in the tumor nests, but their number was small. Both T-lymphocytes and macrophages were found to invade the tumor nests in squamous cell carcinoma whose duration was more than four weeks. This experimental carcinogenesis animal model allows the detailed quantitative and functional analysis of the infiltration of immunocompetent cells into epidermal tumors.     

Membrane expression of annexin I is enhanced by calcium and TPA in cultured human keratinocytes

Abstract  Annexin I plays an important role in the process of keratinization as a component of the cornified envelope. To elucidate the function of annexin I in keratinization, we investigated the effects of calcium, epinephrine, hydrocortisone, and 12-O-tetradecanoyl phorbol 13-acetate (TPA) on the expression and localization of annexin I in cultured human keratinocytes. Normal human keratinocytes were cultured in serum-free culture medium (0.15 mM calcium) until 70% confluence. After incubation with a higher concentration of calcium (1.8 mM), TPA (100 nM), epinephrine (50 ìM), or hydrocortisone (10 ìM) for 24 h, the expression of annexin I protein and mRNA was examined using immunofluorescence, Western blot, and Northern blot techniques. Immunofluorescence microscopy showed increased membrane staining of annexin I by calcium, which was inhibited by the addition of epinephrine or hydrocortisone. Western blotting confirmed elevated annexin I on the cell membrane. It was increased in the cell membrane fraction, but not in the cytosol fraction. Interestingly, the mRNA level of annexin I was slightly reduced after incubation with calcium, whereas TPA upregulated both membrane expression and the mRNA level. Secretion of annexin I was increased by TPA but inhibited by calcium. Because calcium and TPA are known to promote keratinization, our data suggest that annexin I expression on the cell membrane is involved in the process of keratinization. Received: 23 March 2000 / Revised: 14 June 2000 / Accepted: 21 July 2000     

扇贝多肽对UVB损伤无毛小鼠皮肤结构及其抗氧化剂含量的影响

目的 :　本文探讨扇贝多肽 (PCF)对中波紫外线 (UVB)照射下小鼠皮肤的氧化损伤有无保护作用。方法 :　建立UVB(辐照强度为 5 .15× 10 - 2 J cm2 × 30天 )对无毛小鼠皮肤氧化损伤模型。昆明种无毛小鼠 ,随机分为双蒸水未照射组和UVB模型组 (双蒸水对照组、5 %PCF组、2 0 %PCF组、10 %维生素C组 )。光镜下测表皮平均厚度及成纤维细胞数目 ;电镜观察皮肤组织超微结构。酶法测定皮肤匀浆上清液中抗氧化酶 (谷胱甘肽GSH -Px、超氧化物歧化酶SOD、)的活性和丙二醛 (MDA)的含量及总抗氧化能力(T -AOC)。结果 :　①UVB损伤的对照组小鼠皮肤光镜下可见表皮变薄、成纤维细胞数量减少 ;电镜下表皮细胞胞质内可见空泡形成 ,真皮的成纤维细胞内可见囊泡状扩张的滑面内质网、粗面内质网等细胞器减少。②PCF能增加表皮的平均厚度和皮肤成纤维细胞的数量 ;超微结构显示PCF组表皮细胞结构正常 ,成纤维细胞的粗面内质网明显增多 ,与维生素C的抗氧化作用一致。③PCF可提高UVB辐射损伤小鼠皮肤组织的总抗氧化能力、SOD活性 ,降低MDA含量 ;与维生素C的抗氧化作用一致。结论 :　扇贝多肽与VitC一样具有抗UVB氧化损伤的作用。其作用机制可能与扇贝多肽提高抗氧化酶含量、清除自由基有关     

Opposing effects on immune function and skin barrier regulation by the proteasome inhibitor bortezomib in an allergen‐induced eczema model

Proteasome inhibition (PI) has been reported to interfere with antibody‐driven autoimmune diseases. The impact of PI on the allergic immune response and on skin diseases like atopic dermatitis (AD) has not been thoroughly explored, however. Here, we examined whether the PI bortezomib interferes with the allergic immune response and the severity of AD by using an established mouse model of allergen‐driven dermatitis, to which bortezomib was applied after the establishment of systemic sensitization to ovalbumin. The treatment indeed resulted in a remarkable decrease in total and allergen‐specific plasma cells/antibody‐secreting cells, as evidenced by flow cytometry and ELISpot, respectively. This was accompanied by rapid reductions in serum antibody titres, including a prominent reduction of the IgE isotype. CD4+ and CD8+ cells were greatly diminished in lesional skin on immunohistological staining. The impressive effects at the level of immune modulation did not result in any improvement in the eczema, however. Following up on this unexpected result, we found that the skin itself was susceptible to bortezomib, by which it was instructed to lower the expression of critical skin barrier genes, especially transglutaminase‐1 and filaggrin. Together, bortezomib eliminates plasma cells and decreases immunoglobulin responses, including allergenic IgE. Although anti‐inflammatory effects are detectable in the skin, counter‐regulatory effects from PI on resident skin cells likely undermine improvement in the eczema. These results caution against the therapeutic use of bortezomib for inflammatory skin disorders, which are characterized by inherently impaired barrier function, especially AD.     

“菟丝子-枸杞子”药对修复生精功能障碍大鼠血睾屏障的机制研究

目的观察"菟丝子-枸杞子"药对对雷公藤多苷(GTW)诱导的生精障碍大鼠模型血睾屏障的影响,初步探讨其改善模型大鼠生殖功能的机理。方法将56只SD大鼠随机分为空白对照组、模型对照组、阳性对照组和药对组。以GTW 40mg/(kg·d)灌胃4周构建SD大鼠生精障碍模型,成模后阳性对照组每日给予左卡尼汀悬液[0.2g/(kg·d)]灌胃,药对组每日给予菟丝子-枸杞子悬液[6g/(kg·d)]灌胃,治疗4周后检测各组大鼠的精子质量、生殖激素和抑制素B、附睾肉毒碱,观察支持细胞形态,并采用Western blot检测睾丸组织血睾屏障相关蛋白的表达,分析比较不同组间的差异。结果药对组大鼠精子浓度[(39.17±13.75)×10~6/mL]高于模型对照组[(28.23±8.05)×10~6/mL],其差异具有统计学意义(P<0.05)。药对组[(29.92±5.25)%]和左卡尼汀组精子活动率[(29.31±6.33)%]均显著高于模型对照组大鼠[(20.13±6.63)%],其差异具有统计学意义(P<0.01);药对组和左卡尼汀组大鼠卵泡生成素(FSH)、黄体生成素(LH)和泌乳素(PRL)与模型对照组相比均显著降低,附睾肉毒碱含量显著升高,其差异具有统计学意义(P<0.05或P<0.01)。药对组与模型对照组比较,支持细胞结构和形态改善,生精小管结构、排列较为正常,生精细胞明显增多,形态改善;药对组Occludin、ZO-1和connexin 43的蛋白表达比模型对照组显著增多,其差异具有统计学意义(P<0.05);β-catenin和N-cadherin的蛋白表达也有不同程度增高,虽然未见明显差异,但增高趋势明显。结论 "菟丝子-枸杞子"药对能够有效减轻GTW造成的生精功能障碍,其机制可能与修复血睾屏障损伤有关。     

Physiological and functional changes in the stratum corneum restored by oestrogen in an ovariectomized mice model of climacterium

Significant decreases in hormonal levels at menopause induce physiological and functional discomfort in the skin. Representative changes at menopause are based on so‐called dry skin. However, there is no evidence to explain the mechanism, even though hydration of the stratum corneum (SC) in women at menopause is comparable with that at premenopause but is enhanced by hormone replacement therapy. This study objective was to evaluate structural and functional changes in the SC in ovariectomized mice model of menopause. Hydration of the SC, recovery of the permeability barrier function, integrity and cohesion of the SC, and irritant dermatitis were analysed in mice that underwent ovariectomy with or without replacement of 17ß‐estradiol. In ovariectomized mice, hydration of the SC was reduced, recovery of permeability barrier function after acute disruption was impaired, and integrity of the SC was weakened and was associated with increased cohesion and increased levels of irritant dermatitis. Oestrogen replacement treatment restored all changes. Immunohistochemistry revealed reduced levels of expression of desmoglein‐1 and differentiation markers of epidermis in ovariectomized mice compared with control mice and mice with oestrogen replacement treatment. These changes might be directly associated with weakened integrity and impaired permeability barrier function of the SC in ovariectomized mice. This study results reveal that so‐called dry skin at menopause is caused by not only lower hydration of the SC but also complicated structural and functional changes in the SC and skin.