降钙素基因相关肽对HaCaT细胞表达血管内皮生长因子的调控

目的 探讨降钙素基因相关肽(CGRP)对角质形成细胞表达和分泌血管内皮生长因子(VEGF)的调控.方法 用实时定量PCR(RT-PCR)方法检测CGRP、CGRP1型受体拮抗剂CGRP8-37、细胞外信号调节激酶ERK1/2特异性的抑制剂PD98059、p38MAPK特异性拮抗剂SB203580对HaCaT角质形成细胞表达VEGFmRNA水平的影响;用酶联免疫吸附方法检测HaCaT细胞分泌至培养液中VEGF蛋白的水平.结果 CGRP时间依赖性地促进HaCaT细胞表达VEGFmRNA和分泌VEGF蛋白;CGRP8-37和PD98059均可明显抑制CGRP刺激的HaCaT细胞表达和分泌VEGF,SB203580不能减弱CGRP刺激的VEGF表达和分泌.结论 CGRP可以上调HaCaT细胞表达和分泌VEGF,CGRP1型受体及其相关的ERK1/2信号通路参与其调控.     

白芍总苷对角质形成细胞增殖及血管内皮生长因子和白介素-23表达的影响

目的 研究白芍总苷（TGP）对角质形成细胞增殖和分泌血管内皮生长因子（VEGF）和白介素（IL）-23的影响,探讨可能涉及的信号转导通路。方法 不同浓度TGP作用于体外培养的HaCaT细胞株,噻唑蓝（MTT）法观察TGP对HaCaT细胞增殖活性的影响。将HaCaT细胞分为三组,即对照组不加任何刺激因素,TGP组分别加入6种不同浓度的TGP,SB203580组在加入10 mol/L SB203580预处理2 h后加入125 mg/L TGP。实时定量PCR（RT-PCR）方法和ELISA方法检测TGP对HaCaT细胞VEGF和IL-23表达的影响;免疫印迹技术观察TGP作用于HaCaT细胞后p38的磷酸化及SB203580对p38磷酸化的影响。结果 TGP在低浓度（0.5、2.5 mg/L）时对HaCaT细胞增殖有促进作用,浓度≥12.5 mg/L时反而对细胞的增殖有抑制作用,至125 mg/L时抑制作用最强。TGP在低浓度（0.5、2.5 mg/L）时对HaCaT细胞VEGF和IL-23 mRNA和蛋白的表达有促进作用,12.5 ～ 125 mg/L时内可抑制HaCaT细胞VEGF mRNA和蛋白的表达,62.5 ～ 125 mg/L时可抑制IL-23 mRNA和蛋白的表达。TGP可时间依赖性地诱导HaCaT细胞p38的磷酸化,磷酸化p38 蛋白于125 mg/L TGP作用5 min后达到高峰,表达水平为0.3314 ± 0.0245,10 min后减弱至0.2173 ± 0.0189,但均高于对照组水平;30 min后表达水平降为0.1664 ± 0.0201;SB203580可减弱其作用,SB203580预处理组磷酸化p-p38 表达水平为0.1529 ± 0.0147。结论 TGP可抑制HaCaT细胞的增殖及VEGF和IL-23 mRNA和蛋白的表达,p38MAPK信号途径可能介导其抑制作用。     

Sphingosylphosphorylcholine-induced interleukin-6 production is mediated by protein kinase C and p42/44 extracellular signal-regulated kinase in human dermal fibroblasts

BACKGROUND: Sphingosylphosphorylcholine (SPC) has been reported as a novel lipid mediator that exerts various actions on wound healing process. OBJECTIVE: The aim of this study is to evaluate the involvement of interleukin-6 (IL-6) in SPC-induced wound healing acceleration. METHODS: We performed immunohistochemical analysis to demonstrate the IL-6 induction by SPC. To analyze the signaling events, skin fibroblasts were treated with SPC, and then RT-PCR, ELISA and Western blot analyses were carried out. RESULTS: SPC markedly induced interleukin-6 (IL-6) expression in rabbit ear wound. SPC also induced IL-6 expression at both the mRNA and protein levels in human dermal fibroblasts cultured in vitro. SPC rapidly phosphorylated p42/44 extracellular signal-regulated kinase (ERK). Pretreatment with PD 98059, a specific MAPK kinase 1/2 inhibitor, markedly suppressed SPC-induced IL-6 expression in a dose-dependent manner. Protein kinase C (PKC) activation by phorbol myristate acetate (PMA) potentiated IL-6 mRNA expression, whereas PKC inhibition by bisindolylmaleimide blocked SPC-induced p42/44 ERK phosphorylation and IL-6 expression. Over-expression of PKCalpha markedly induced the IL-6 expression and p42/44 ERK activation. CONCLUSION: These results suggest that SPC-induced IL-6 production is mediated by PKC-dependent p42/44 ERK activation in human dermal fibroblasts cultured in vitro.     

Deranged epidermal differentiation in kl/kl mouse and the effects of βKlotho siRNA on the differentiation of HaCaT cells

Mice deficient in the klotho gene (kl/kl mice) display the phenotypes of human ageing. We found that the expression of epidermal differentiation‐associated factors (keratin 1, keratin 10, filaggrin and loricrin) was lower in the skin of kl/kl mice than that of wild‐type mice. In vitro experiments showed that the expression of βKlotho, a family of klotho gene‐encoded protein, was induced concomitantly with the differentiation of an immortalized human epidermal keratinocyte cell line (HaCaT cells) when they were cultured in an air–liquid interface. βKlotho knockdown by small interfering ribonucleic acid suppressed the expression of the above differentiation‐associated factors in HaCaT cells. βKlotho small interfering ribonucleic acid increased the expression of keratin 14, which is expressed in mitotically active basal layer cells, and activated p44/p42 mitogen‐activated protein kinase in the HaCaT cells grown in the air–liquid interface. These findings suggest that the epidermal differentiation is deranged in kl/kl mice, and βKlotho is required for the differentiation of human epidermal keratinocytes.     

Particulate matter induces pro‐inflammatory cytokines via phosphorylation of p38 MAPK possibly leading to dermal inflammaging

Particulate matter (PM) is known to have harmful effects on human health. Epidemiological studies have suggested that PM exposure is related to skin diseases and extrinsic skin ageing. However, the mechanisms by which PM affects skin are unclear. The aim of this study was to investigate the mechanism of action of PMs on epidermal inflammation and skin ageing using a co‐culture of human keratinocytes (HaCaT) and fibroblasts (HDF). SRM 1648a (pmA) and 1649b (pmB), which mainly comprise heavy metals and polycyclic aromatic hydrocarbons, respectively, were used as reference PMs. Cytotoxic effects, activation of AhR, phosphorylation of p38 kinase and ROS generation were examined in PM‐treated HaCaT cells. The phosphorylation of p38 MAPK induced by PMs was shown to be critically important for the increases in IL‐1α and IL‐1β expression. Moreover, the mRNA and protein expression levels of MMP1 and COX2 were markedly increased in HDF cells co‐cultured with PM‐treated HaCaT cells. In conclusion, PMs induce the expression of pro‐inflammatory cytokines in keratinocytes via the p38 MAPK pathway, and these interleukins increase the expression of MMP1 and COX2 in HDF cells. These results suggest that PMs trigger skin ageing via p38 MAPK activation and interleukin secretion in epidermal keratinocytes.     

Insulin-like growth factor-II regulates the expression of vascular endothelial growth factor by the human keratinocyte cell line HaCaT

Psoriasis is a chronic, relapsing skin disease characterized by enhanced angiogenesis. The pathogenetic process resulting in hypervascularity remains to be further investigated. It has been reported that a potent angiogenic factor, vascular endothelial growth factor (VEGF) is overexpressed in psoriatic epidermis and that the level of insulin-like growth factor II (IGF-II) is significantly elevated in the tissue fluid and serum of the psoriatic lesion. We considered the possibility that IGF-II might function as a paracrine inducer of VEGF. Here, we demonstrated that exposure of HaCaT keratinocytes to IGF-II induced both mRNA and protein expression of VEGF through the MAP kinase (extracellular signal-regulated kinase (ERK2) pathway. Particularly, we determined that phosphorylation of ERK2 but not p38 and JNK1/2 was activated by IGF-II in a time-dependent manner. Additionally, we found that IGF-II treatment induced the expression of MDM2 through the MAP kinase pathway. Moreover, the increase of MDM2 resulted in decreased levels of p53 followed by increased expression of HIF-1alpha and VEGF. Taken together, these results suggest that IGF-II enhances the expression of VEGF in HaCaT cells by increasing HIF-1alpha levels.     

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.     

Transduced PEP-1-FK506BP ameliorates atopic dermatitis in NC/Nga mice

Immunophilin, FK506-binding protein 12 (FK506BP), is a receptor protein for the immunosuppressive drug FK506 by the FK506BP/FK506 complex. However, the precise function of FK506BP in inflammatory diseases remains unclear. Therefore, we examined the protective effects of FK506BP on atopic dermatitis (AD) in tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ)-induced HaCaT cells and 2,4-dinitrofluorobenzene-induced AD-like dermatitis in Nishiki-nezumi Cinnamon/Nagoya (NC/Nga) mice using a cell-permeable PEP-1-FK506BP. Transduced PEP-1-FK506BP significantly inhibited the expression of cytokines, as well as the activation of NF-κB and mitogen-activated protein kinase (MAPK) in TNF-α/IFN-γ-induced HaCaT cells. Furthermore, topical application of PEP-1-FK506BP to NC/Nga mice markedly inhibited AD-like dermatitis as determined by a histological examination and assessment of serum IgE levels, as well as cytokines and chemokines. These results indicate that PEP-1-FK506BP inhibits NF-κB and MAPK activation in cells and AD-like skin lesions by reducing the expression levels of cytokines and chemokines, thus suggesting that PEP-1-FK506BP may be a potential therapeutic agent for AD.     

miR-205-5p inhibits psoriasis-associated proliferation and angiogenesis: Wnt/β-catenin and mitogen-activated protein kinase signaling pathway are involved

Psoriasis is a chronic inflammatory skin disease, and the mechanism remains unknown. The present study found that the level of miR-205-5p was downregulated in psoriatic skin tissues. miR-205-5p inhibited proliferation in HaCaT cells. miR-205-5p impaired proliferation, migration and tube formation in human umbilical vein endothelial cells. Angiopoietin (Ang)-2, vascular endothelial growth factor (VEGFA) and bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) were confirmed as the targets of miR-205-5p. Moreover, miR-205-5p suppressed the phosphorylation of p38 and extracellular regulated protein kinase, and inhibited expression level of β-catenin. In vivo, miR-205-5p significantly alleviated imiquimod (IMQ)-induced psoriasis in mice, and deactivated mitogen-activated protein kinase (MAPK) and Wnt/β-catenin pathways. In summary, we demonstrated that miR-205-5p alleviated IMQ-induced psoriasis in mice by restraining epidermal hyperproliferation and excessive neovascularization. miR-205-5p may play its roles by targeting Ang-2, VEGFA and BAMBI, and deactivating the Wnt/β-catenin and MAPK signaling pathways. These findings may provide a potential therapeutic target for clinical treatment of psoriasis.     

All-trans retinoic acid antagonizes UV-induced VEGF production and angiogenesis via the inhibition of ERK activation in human skin keratinocytes

Incident UV radiation leads to the upregulation of vascular endothelial growth factor (VEGF), a potent angiogenic factor, in human skin. However, the molecular basis of UV-induced angiogenesis in skin remains to be elucidated. In this study, we investigated the roles of UV exposure on cutaneous angiogenesis, its associated signaling mechanisms, and the effect of all-trans retinoic acid (tRA) on UV-induced vascularization, and VEGF expression. Using a human epidermal cell line, HaCaT, we found that UV induces VEGF mRNA and protein expression via the MAPK/ERK kinase-ERK1/2 (extracellular signal-regulated kinase 1/2) pathway but not via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and that tRA pretreatment significantly inhibits UV-induced VEGF overexpression and ERK1/2 activation. In human skin in vivo, we confirmed that skin vascularization significantly increased after a single exposure to UV, as was evidenced by a prominent increase in vessel size, vascular density, and in the cutaneous area occupied by vessels, and we found that these events are associated with VEGF upregulation. Topical pretreatment with tRA under occlusion inhibited not only UV-induced VEGF upregulation and angiogenesis with a significant reduction of vessel density but also UV-induced ERK1/2 activation in human skin. Collectively, our data demonstrate that tRA inhibits the UV-induced angiogenic switch via downmodulation of ERK1/2 activation and consecutive VEGF overexpression. These findings may help us understand the molecular mechanisms that regulate skin angiogenesis due to UV exposure, and provide evidence of the potential of tRA in terms of preventing angiogenesis-associated skin damage following exposure to UV irradiation.     

Blocking MAPK signaling downregulates CCL21 in lymphatic endothelial cells and impairs contact hypersensitivity responses

CCL21 expression by lymphatic endothelial cells (LECs) is essential for migration of CCR7+ immune cells from skin to regional lymph nodes (LNs). We investigated the importance of mitogen-activated protein kinase (MAPK) signaling in CCL21 expression by ECs in vitro and in vivo. Normal human dermal lymphatic microvascular ECs (HMVEC-dLy) stimulated in vitro with oncostatin M (OSM) expressed high amounts of CCL21 mRNA. CCL21 protein expression by HMVEC-dLy was also markedly increased by OSM compared with unstimulated cultures. Marked phosphorylation of MAPK 44/42 was detected in HMVEC-dLy stimulated by OSM. CCL21 expression by HMVEC-dLy was blocked by a JAK inhibitor 1, JAK3 inhibitor, and U0126 (a MAPK kinase inhibitor) in vitro, all of which blocked phosphorylation of MAPK 44/42. In addition, injection of U0126 into murine skin significantly decreased CCL21 mRNA and protein expression. Moreover, injection of U0126 before sensitization decreased migration of dendritic cells to draining LNs and decreased contact hypersensitivity responses. In summary, these results suggest that the MAPK pathway is important for CCL21 expression by LECs in vitro and in vivo. Blocking MAPK signaling within skin may offer a novel approach to treatment of inflammatory skin diseases.     

UVB-induced interleukin-18 production is downregulated by tannic acids in human HaCaT keratinocytes

Tannic acids (TAs) are believed to be the key active components in plants, and are believed to be responsible for their anti-inflammatory, anti-viral effects and chemoprevention of cancer. However, the molecular mechanisms for the action of TA are unclear. This study examined the effects of TA on cutaneous inflammation with a human keratinocyte cell line (HaCaT). Interleukin-18 (IL-18) has multiple effects upon various cells involved in inflammatory response. In this study, the IL-18 mRNA expression and protein levels were reduced by a TA pretreatment. UV radiation can trigger the induction of the p38 mitogen-activated protein kinase (MAPK)-dependent signalling cascade. Immunoprecipitation and Western blot analysis was performed to determine if TA regulate the MAPK signalling pathway. TA significantly inhibited the activation of p38 MAPK and extracellular signal-regulated protein kinases. Moreover, TA-inhibited UVB enhanced the expression of the inflammatory mediators, IL-1, IL-6, tumor necrotic factor-alpha, cyclooxygenase-2 and prostaglandin E(2) in UVB-irradiated HaCaT cells. The topical application of TA on mouse skin treated with UVB irradiation has shown that TA inhibited the formation of erythema. These findings suggest that TA has significant anti-inflammatory effects on the UVB-induced response on the skin and may be a candidate natural compound for the regulation of cutaneous inflammation.     

葫芦素Ⅰ对HaCaT细胞体外增殖及对角蛋白17、STAT3、VEGF表达的影响

目的 探讨葫芦素Ⅰ对HaCaT细胞体外增殖及角蛋白17(K17)、信号转导及转录激活子3(STAT3)、血管内皮生长因子(VEGF)表达的影响.方法 采用不同浓度葫芦素Ⅰ(0.0125、0.025、0.05、0.1μmol/L)、含与0.1 μmol/L葫芦素Ⅰ等体积DMSO的DMEM培养液(溶媒组)、DMEM培养液(阴性对照组)、10 nmol/L卡泊三醇(阳性对照组)分别作用于HaCaT细胞.采用CCK8法检测葫芦素Ⅰ作用12、24、36 h后对HaCaT细胞体外增殖的影响,RT-PCR法检测葫芦素Ⅰ作用24 h对HaCaT细胞K17和VEGF mRNA表达的影响,Western印迹法检测葫芦素Ⅰ作用24h对HaCaT细胞K17、STAT3、磷酸化STAT3(P-STAT3)和VEGF蛋白表达的影响.结果 0.0125 μmol/L葫芦素Ⅰ作用12h即开始表现出对HaCaT细胞体外增殖的抑制作用,当葫芦素Ⅰ浓度增加到0.1 μmol/L时,24h和36h的抑制率分别为43.00％±2.11％和48.98％±2.27％.与阴性对照组相比,各浓度葫芦素Ⅰ组对HaCaT细胞体外增殖均有抑制作用(均P＜ 0.05),且该抑制作用随葫芦素Ⅰ浓度的增加及作用时间的延长逐渐增强.不同浓度葫芦素Ⅰ作用于HaCaT细胞24 h后,K17 mRNA及其蛋白、P-STAT3蛋白、VEGF mRNA及其蛋白表达量均随葫芦素Ⅰ浓度的增加而降低,差异有统计学意义(均P＜ 0.05).结论 葫芦素Ⅰ可抑制HaCaT细胞的体外增殖,并可在mRNA水平下调K17、VEGF的表达,在蛋白水平下调K17、P-STAT3、VEGF的表达.     

Effect of ultraviolet radiation on the expression of pp38MAPK and furin in human keratinocyte-derived cell lines

Background: Ultraviolet radiation (UVR) is known to induce the activation of stress‐inflammation signal transduction pathways, and to induce the activity of many proteases in skin cells. It is unknown whether the activation of proteases such as furin is related to changes in the phosphorylation status of p38MAPK. Methods: The effect of UVR on immortalized keratinocyte (HaCaT) and squamous cell carcinoma (Colo16) cells was investigated with respect to cell survival, phosphorylation of p38MAPK, and the proprotein convertase, furin. The cells were exposed to either a low or a high dose of UVA and/or UVB and the viability was monitored over 48 h, along with changes in the intracellular expression of p38MAPK and furin. Results: Low‐dose UVA (2 kJ/m²) and/or UVB (0.2 kJ/m²) radiation had no effect on cell viability, except in UVA‐irradiated Colo16 cells. High UVA (20 kJ/m²) caused a loss of cell viability in HaCaT cells, but not in Colo16 cells. The opposite effect was seen in cells exposed to a high UVB dose (2 kJ/m²). The viability of both cell cultures decreased when exposed to high‐dose UVA+B radiation. UV irradiation downregulated the expression of phosphorylated p38 (pp38) in HaCaT cells irrespective of the UV dose and type. In Colo16 cells, UV radiation induced pp38 expression in the cells following exposure, with the highest increase in cells exposed to high‐dose UVA. The expression of furin in UV‐irradiated HaCaT cells was similar to that seen for pp38 expression. In Colo16 cells, UV radiation induced furin expression, with the highest increase seen in cells 24 h after exposure to both high‐dose UVB and UVA+B radiation. Conclusion: The results show that there are differences between the effect of UV types and doses on cell function in the keratinocyte‐derived cell lines examined in this study. The level of furin expression in Colo16 cells correlated to changes in pp38 levels in the cells following exposure to UV radiation, but not in HaCaT cells. From an improved understanding of the signalling pathways and their downstream events and how these may differ as a result of tumorigenesis, it may enable the development of inhibitors, which may have therapeutic applications.     

Involvement of RAGE, MAPK and NF-κB pathways in AGEs-induced MMP-9 activation in HaCaT keratinocytes

Advanced glycation end products (AGEs) exert divergent effects on the pathogenesis of diabetes complications. Excessive expression of matrix metalloproteinases-9 (MMP-9) is deleterious to the cutaneous wound-healing process in the context of diabetes. However, the effect of AGEs on MMP-9 induction in skin cells and the exact molecular mechanisms involved are still poorly understood. In this study, we investigated the effect of AGEs on the production of MMP-9 in HaCaT keratinocytes and characterized the signal transduction pathways activated by AGEs that are involved in MMP-9 regulation. We showed that AGE-BSA increased MMP-9 expression in HaCaT cells at both the protein and mRNA levels. The stimulatory effect of AGE-BSA on MMP-9 was attenuated by inhibitors of extracellular-signal-regulated kinase (ERK1/2, U0126), p38 mitogen-activated protein kinase (MAPK, SB203580) and NF-κB, but not c-Jun N-terminal kinase. Furthermore, receptor for advanced glycation end products (RAGE) was expressed in keratinocytes, and incubation with AGE-BSA resulted in a significant upregulation of RAGE expression in a dose-dependent manner. Silencing of the RAGE gene prevented AGE-BSA-induced MMP-9 activation and the phosphorylation of ERK1/2 and p38 MAPK. We also observed the involvement of NF-κB in AGE-BSA-induced MMP-9 activation, which was not blocked by U0126 and SB203580. These results suggest that AGEs may play an important role in the impairment of diabetic wound healing by upregulating MMP-9 expression in keratinocytes via the RAGE, ERK1/2 and p38 MAPK pathways; activation of NF-κB is also involved in this process. These pathways may represent potential targets for drug interventions to improve diabetic wound healing, a process in which MMP-9 plays a critical role.     

Ethanol Extract of Peanut Sprout Exhibits a Potent Anti-Inflammatory Activity in Both an Oxazolone-Induced Contact Dermatitis Mouse Model and Compound 48/80-Treated HaCaT Cells

BackgroundWe developed an ethanol extract of peanut sprouts (EPS), a peanut sprout-derived natural product, which contains a high level of trans-resveratrol (176.75 µg/ml) and was shown to have potent antioxidant activity.ObjectiveWe evaluated the potential anti-inflammatory activity of EPS by measuring its antioxidant potential in skin.MethodsThe anti-inflammatory activity of EPS was tested using two models of skin inflammation: oxazolone (OX)-induced contact dermatitis in mice and compound 48/80-treated HaCaT cells. As biomarkers of skin inflammation, cyclooxygenase-2 (COX-2) and nerve growth factor (NGF) levels were measured.ResultsOX-induced contact dermatitis was suppressed markedly in mice that were treated with an ointment containing 5% EPS as evidenced by a decrease in the extent of scaling and thickening (p<0.05) and supported by a histological study. COX-2 (messenger RNA [mRNA] and protein) and NGF (mRNA) levels, which were upregulated in the skin of OX-treated mice, were suppressed markedly in the skin of OX+EPS-treated mice. Consistent with this, compound 48/80-induced expression of COX-2 (mRNA and protein) and NGF (mRNA) in HaCaT cells were suppressed by EPS treatment in a dose-dependent manner. As an inhibitor of NF-κB, IκB protein levels were dose-dependently upregulated by EPS. Fluorescence-activated cell sorting (FACS) analysis revealed that EPS scavenged compound 48/80-induced reactive oxygen species (ROS) in HaCaT cells.ConclusionEPS exerts a potent anti-inflammatory activity via its anti-oxidant activity in both mouse skin and compound 48/80-treated HaCaT cells in vitro. Compound 48/80-treated HaCaT cells are a useful new in vitro model of skin inflammation.     

节律基因隐花色素2在银屑病小鼠模型及HaCaT细胞中的表达变化及机制研究

【摘要】 目的 探讨节律基因隐花色素2（CRY2）在银屑病小鼠模型及HaCaT细胞中的表达变化及其机制。方法 咪喹莫特诱导小鼠模型实验：12只C57BL/6雌鼠随机均分为咪喹莫特组（连续外用咪喹莫特乳膏5 d诱导构建银屑病小鼠模型,6只）和对照组（不予任何处理,6只）,第6天处死小鼠,取其背部皮肤组织,免疫荧光染色检测表皮中CRY2的表达。HaCaT细胞转染实验：使用小干扰RNA（siRNA）技术在HaCaT细胞中敲减CRY2的表达（siRNA-CRY2组）,以siRNA-NC组作为对照,5-乙炔基-2′脱氧尿嘧啶核苷（EdU）染色检测HaCaT细胞增殖活力,实时荧光定量PCR（qPCR）检测HaCaT细胞中趋化因子mRNA表达水平,Western印迹检测细胞外信号调节激酶1/2（ERK1/2）蛋白的磷酸化水平。肿瘤坏死因子α（TNF-α）刺激动物和细胞实验：12只C57BL/6雌鼠随机均分为TNF-α组（小鼠耳部皮下连续注射TNF-α溶液6 d,6只）和磷酸盐缓冲液（PBS）组（注射等量的PBS,6只）,第7天处死小鼠,取其耳部皮肤组织,免疫荧光染色检测表皮中CRY2的表达;用50 ng/ml TNF-α刺激CRY2基因敲减的HaCaT细胞12 h（siRNA-CRY2 + TNF-α组）,以siRNA-NC + TNF-α组作为对照,qPCR检测各组细胞中趋化因子mRNA的表达。统计分析采用两独立样本t检验。结果 免疫荧光染色显示,咪喹莫特组小鼠背部表皮层中CRY2蛋白的表达（0.94 ± 0.23）显著低于对照组（2.30 ± 0.25,t = 3.99,P = 0.016）。HaCaT细胞转染实验：siRNA-CRY2组EdU阳性细胞比例（48.13% ± 10.97%）显著高于siRNA-NC组（38.23% ± 0.81%,t = 5.00,P = 0.007）,且siRNA-CRY2组趋化因子CXCL1、CXCL8 mRNA相对表达量及p-ERK1/2蛋白相对表达量均显著高于siRNA-NC组（均P < 0.05）,但两组间CCL20 mRNA表达量及ERK1/2蛋白表达量差异无统计学意义（均P > 0.05）。TNF-α刺激实验：免疫荧光染色显示,TNF-α组鼠耳表皮组织中CRY2蛋白表达水平（0.37 ± 0.34）显著低于PBS组（2.04 ± 0.17,t = 4.38,P = 0.012）;siRNA-CRY2 + TNF-α组HaCaT细胞趋化因子CXCL1、CXCL8、CCL20 mRNA相对表达量均显著高于siRNA-NC + TNF-α组（均P < 0.05）。结论 CRY2在银屑病小鼠模型中表达降低,促进了角质形成细胞的增殖以及趋化因子CXCL1、CXCL8和CCL20的表达,且TNF-α可能是下调CRY2表达的上游细胞因子。     

缺氧诱导因子-1α小干扰RNA对HaCaT细胞VEGF表达的影响

目的 探讨缺氧诱导因子-1α( HIF-1α )RNA干扰对低氧条件下HaCaT细胞HIF -1α和血管内皮细胞生长因子(VEGF)表达的影响。方法 将HaCaT细胞分为4组：常氧对照组(无干预因素)、低氧组(缺氧培养24h)、脂质体对照组(转染空载脂质体后缺氧培养24h)、RNA干扰组(转染脂质体介导的RNA干扰序列后缺氧培养24 h)。荧光实时定量PCR法检测各组HaCaT细胞HIF-1α和VEGF mRNA表达水平,Western印迹检测各组HaCaT细胞HIF-1α和VEGF蛋白表达水平。结果 低氧组和常氧对照组HIF-1α mRNA的表达水平分别为0.907±0.032和0.878±0.034,两者比较差异无统计学意义(F=1.108,P＞0.05),而低氧组VEGF mRNA和HIF-1α及VEGF蛋白的表达水平分别为0.935±0.032和0.813±0.047,0.791±0.030,均较常氧对照组(分别为0.652±0.053、0.236±0.014和0.316±0.013)明显增高(P值均＜ 0.05);RNA干扰组HIF-1α mRNA及蛋白的表达水平为0.230±0.044和0.213±0.026,VEGF为0.497±0.033和0.249±0.028,均较脂质体对照组(分别为0.978±0.030、0.817±0.049和0.806±0.040、0.833±0.052)显著降低(P值均＜0.05)。结论 低氧可以使HaCaT细胞HIF-1α和VEGF的表达增加,而抑制HIF-1α的表达可以使低氧条件下HaCaT细胞VEGF的表达减少。     

P38 and JNK signal pathways are involved in the regulation of phlorizin against UVB‐induced skin damage

Phlorizin is well known to inhibit sodium/glucose cotransporters in the kidney and intestine for the treatment of diabetes, obesity and stress hyperglycaemia. However, the effects of phlorizin against ultraviolet B (UVB) irradiation and its molecular mechanism are still unknown. We examined the effects of phlorizin on skin keratinocyte apoptosis, reactive oxygen species (ROS) production, pro‐inflammatory responses after UVB irradiation and the changes of some signal molecules by in vitro and in vivo assay. We observed that phlorizin pretreatments inhibited HaCaT cell apoptosis and overproduction of ROS induced by UVB. Phlorizin also decreased the expression of UVB‐induced pro‐inflammatory cytokines, such as interleukin‐1 beta (IL‐1β), interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8) at the mRNA level. Topical application of phlorizin on UVB‐exposed skin of nude mice prevented the formation of scaly skin and erythema, inhibited the increase of epidermal thickness and reduced acute inflammation infiltration in skin. Additionally, PCR, Western blot and immunohistochemical data showed that phlorizin reversed the overexpression of cyclooxygenase‐2 (Cox‐2) induced by UVB irradiation both in vitro and in vivo. The activation of p38 and JNK mitogen‐activated protein kinases (MAPK) after UVB irradiation was also inhibited by phlorizin. These findings suggest that phlorizin is effective in protecting skin against UVB‐induced skin damage by decreasing ROS overproduction, Cox‐2 expression and the subsequent excessive inflammation reactions. It seemed that p38 and JNK MAPK signal pathways are involved in the regulation of the protective function of phlorizin.