Expression of nitric oxide synthases in keratinocytes after UVB irradiation

The importance of nitric oxide (NO) in mediating vasodilation, neurotransmission, and immune and inflammatory responses has been demonstrated. Human keratinocyte express inducible nitric oxide synthase (iNOS) and the neuronal constitutive isoform of NOS (ncNOS). We established an in vitro model in keratinocytes to investigate changes in NO, iNOS and ncNOS expression after UVB exposure. We demonstrated a large induction of NO after UVB exposure and that the source of NO produced in UVB-exposed keratinocytes was increased expression of iNOS and ncNOS. The increased NO production with increased expression of iNOS and ncNOS may contribute to the pathological and physiological features of UVB-induced erythema and skin inflammation.     

长波紫外线照射对HaCaT细胞iNOS/NO表达的影响

目的 探讨不同剂量长波紫外线 （UVA） 照射HaCaT细胞后不同时间点诱导型一氧化氮合成酶（iNOS）的表达情况。方法 1 J/cm2、5 J/cm2和10 J/cm2 UVA照射HaCaT细胞后继续培养24 h、48 h和72 h,倒置相差显微镜下观察细胞形态的变化;分别用RT-PCR、Western印迹和Griess法检测HaCaT细胞iNOS mRNA、蛋白及NO的表达。结果 所有UVA剂量组HaCaT细胞iNOSmRNA在光照后24 h有表达,48 h达高峰,72 h后下降,各时间点间表达量差异有统计学意义（P < 0.05）;1 J/cm2 UVA照射后3个时间点均未见iNOS蛋白表达,而5 J/cm2和10 J/cm2 UVA照射后iNOS蛋白在24 h增加,48 h达高峰且显著高于24 h（P < 0.05）,照射后72 h无iNOS蛋白表达。所有UVA剂量组HaCaT细胞NO表达量在24 h升高,48 h显著升高,72 h平稳升高,3个时间点NO表达量均比正常对照组明显增加（P < 0.05）。对照组HaCaT细胞无iNOS mRNA和蛋白表达,NO表达量低。结论 HaCaT细胞iNOS和NO的表达变化与UVA照射存在时间和剂量关系。     

银屑病患者皮肤组织一氧化氮的检测分析

目的:研究皮肤组织一氧化氮(NO)水平与银屑病之间的关系。方法:选择寻常型银屑病患者54例,利用Griss化学比色法检测患者皮损组织及非皮损组织中NO水平。结果:银屑病患者皮损组织及非皮损组织中NO水平明显高于正常人且与疾病严重程度有相关性(P＜0.01)。结论:银屑病患者皮肤组织中存在诱导型一氧化氮合成酶(iNOS)的异常表达,与疾病的严重程度呈一定正相关。     

P物质对HaCaT细胞一氧化氮合成的影响

目的 观察P物质、NK1受体拮抗剂和一氧化氮合酶（NOS）抑制剂对体外培养的人永生化角质形成细胞株HaCaT细胞一氧化氮（NO）分泌和诱生型NOS（iNOS）表达的影响。方法 用不同浓度P物质（10-9 ～ 10-6 mol/L）或联合应用10-8 mol/L P物质和3 × 10-7 mol/L spantide、10-7 mol/L氨基胍、10-6 mol/L 7-硝基吲唑、10-5 mol/L L-NAME处理HaCaT细胞24 h,硝酸还原酶法测定培养上清液中NO含量。HaCaT细胞中加入10-8 mol/L P物质,1 h、24 h、48 h后用RT-PCR检测iNOS mRNA表达。结果 10-9 ～ 10-6 mol/L P物质可诱导HaCaT细胞分泌NO,其中以10-8 mol/L P物质效应最明显。Spantide在各个时间点上（30 min及1、3、6、12、24 h）均可明显抑制P物质诱导的HaCaT细胞NO合成（P < 0.01）,L-NAME组在3个时间点上（30 min、1 h、24 h）、7-硝基吲唑组在30 min、1 h后HaCaT细胞NO水平明显低于P物质组（P < 0.05）,但氨基胍组与P物质组在各个时间点上差异均无统计学意义（P > 0.05）。10-8 mol/L P物质处理HaCaT细胞后24 h、48 h,iNOS mRNA表达量分别为0.199 ± 0.018、0.516 ± 0.030,两个时间点之间差异有统计学意义（P < 0.01）。结论 P物质可通过激活细胞上NK1受体促进HaCaT细胞分泌NO,但iNOS可能不是P物质诱导HaCaT细胞分泌NO的主要来源。     

一氧化氮在P物质诱导BALB/c小鼠搔抓反应中的作用

目的 探讨一氧化氮（NO）在P物质诱导小鼠搔抓反应中的作用。方法 40只BALB/c小鼠随机分为5组,颈背部皮内注射不同剂量P物质（20 ~ 160 nmol/部位）建立BALB/c小鼠急性瘙痒模型。另取40只小鼠随机分为5组,分别在颈背部皮内注射生理氯化钠溶液、L-精氨酸、spantide、L-NAME、氨基胍,10 min后皮内注射P物质（80 nmol/部位）,观察这些试剂对P物质诱导小鼠搔抓反应的影响,并用免疫组化和硝酸还原酶法分别检测注射部位皮肤中一氧化氮合酶（iNOS）、NO表达。结果 皮内注射生理氯化钠溶液和20、40、80、160 nmol/部位P物质引起小鼠1 h内搔抓次数分别为4.38 ± 4.07、5.38 ± 3.78、12.75 ± 6.52、23.50 ± 7.84、42.38 ± 15.84;与生理氯化钠溶液组相比,40 ~ 160 nmol/部位P物质可使小鼠出现剂量依赖性搔抓反应（P < 0.01 ~ 0.05）。P物质组、L-精氨酸组、spantide组、L-NAME组和氨基胍组小鼠1 h内搔抓次数分别为67.13 ± 32.79、70.75 ± 34.80、10.75 ± 8.14、29.00 ± 21.19、35.38 ± 22.83;与P物质组相比,spantide、L-NAME、氨基胍可明显减少P物质诱导的小鼠搔抓次数（P < 0.01 ~ 0.05）,但L-精氨酸无明显影响（P > 0.05）。与P物质组比较,spantide、L-NAME、氨基胍可下调皮肤中iNOS表达和NO水平（P < 0.01 ~ 0.05）,而L-精氨酸无明显作用（P > 0.05）。结论 P物质可能通过激活皮肤中神经激肽1受体来促进NO合成,从而引起BALB/c小鼠搔抓反应。     

The role of nitric oxide synthases in pemphigus vulgaris in a mouse model

Background  Pemphigus vulgaris (PV) is a blistering autoimmune disease characterized by IgG autoantibodies against desmoglein 3. Nitric oxide synthases (NOS) may contribute to the increase of inflammation in tissues by the generation of nitrotyrosine residues (NTR).
Objectives  To investigate whether the production of NTR mediated by NOS may participate in the development of inflammation and acantholysis in PV.
Methods  Mice were pretreated or not with NOS, tyrosine-kinase (TK) or nuclear factor (NF)-κB inhibitors, and then injected with PV-IgG. PV manifestations were examined in all mice. The expression of NTR, constitutive NOS (cNOS) [endothelial NOS (eNOS) and neuronal NOS (nNOS)], inducible NOS (iNOS) and NF-κB factor were studied in epidermis of mice using immunohistochemical techniques.
Results  After PV-IgG injection, expressions of NTR, iNOS, eNOS and nNOS increased in acantholytic cells, as did nuclear translocation of NF-κB in the basal cells of the epidermis. Pretreatment of mice with inhibitors of TK, nNOS and nonselective NOS, completely prevented NTR expression and the clinical and histological findings of PV in mice. TK inhibitor genistein inhibited both nNOS and iNOS expression on the membrane of basal keratinocytes, and nuclear translocation of NF-κB.
Conclusions  Upregulation of cNOS and iNOS, NTR generation and nuclear translocation of NF-κB may contribute to increased inflammation and tissue damage in PV lesions. The absence of the clinical and histological findings of PV and NTR expression in mice injected with PV-IgG, through pretreatment with TK and nNOS inhibitors, provides compelling evidence that these signalling molecules should be considered as potential therapeutic targets in PV.     

皮肤恶性黑素瘤中诱导型一氧化氮合酶mRNA及其蛋白的表达

目的探讨皮肤恶性黑素瘤中诱导型一氧化氮合酶（iNOS）的表达及其临床意义。方法免疫组化方法检测30例皮肤恶性黑素瘤患者肿瘤组织中iNOS蛋白的表达，逆转录聚合酶链反应（RT-PCR）检测其中6例患者肿瘤组织和肿瘤邻近正常组织中iNOSmRNA的表达。结果iNOS在肿瘤组织中无论蛋白或mRNA阳性表达率皆明显高于邻近正常组织（P〈0．05）。其中iNOS蛋白在无转移的恶性黑素瘤中阳性表达率为69．2％，有转移的恶性黑素瘤中阳性表达率为100％。结论iNOS表达异常在恶性黑素瘤发生、发展与转移过程中可能起重要作用。     

SLE患者外周血单个核细胞一氧化氮合酶mRNA的表达

探讨原生型一氧化氮合酶(cNOS)和诱生型一氧化氮合酶(iNOS)在系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMC)中的表达水平及其意义,应用逆转录-聚合酶链反应(RT-PCR)检测了34例活动期SLE患者和30名正常人PBMC中cNOS和iNOSmRNA的表达水平。结果表明,活动期SLE患者iNOS和cNOS的阳性表达率与正常人对照组相比均无明显差异(P>0.05);活动期SLE患者PBMC中iNOSmRNA的平均表达水平(0.7710±0.1050)明显高于正常人对照组(0.5249±0.0770),差异非常显著(P<0.01);活动期SLE组cNOS的平均表达水平与正常人对照组处于同一表达水平,差异无统计学意义(P>0.05)。提示活动期SLE患者外周血单个核细胞中iNOSmRNA的表达增加,可能通过诱导一氧化氮(NO)及其代谢产物的产生参与SLE的发病过程。特异性iNOS抑制剂的开发与研制可能是治疗SLE新药发展的一个方向。     

Differential expression of nitric oxide synthases in human scalp epidermal and hair follicle pigmentary units: implications for regulation of melanogenesis

BACKGROUND: Nitric oxide (NO) is a ubiquitous gaseous lipophilic molecule generated from the conversion of L-arginine to L-citrulline by the NO synthases (NOSs). Ultraviolet radiation (UVR)-induced NO production appears to stimulate epidermal melanogenesis. However, given their relative protection from UVR, it is unclear whether NO plays a similar role in hair bulb melanocytes. OBJECTIVES: We aimed to identify the expression profiles of the NOS isoforms endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) and of phosphorylated eNOS and nitrotyrosine within the epidermal and follicular melanin units of normal human haired scalp during the hair growth cycle. METHODS: This study employed single and double immunohistochemical and immunofluorescence staining techniques using haired scalp from 10 healthy individuals (six women and four men). RESULTS: Melanocytes in the basal layer of the epidermis expressed eNOS, nNOS and nitrotyrosine. By contrast, melanogenically active melanocytes of the anagen hair bulb were wholly negative for these markers. However, other follicular melanocytes not actively involved in pigment production, including undifferentiated melanocytes located in the outer root sheath and melanocytes surviving the apoptosis-driven hair follicle (HF) regression during catagen/telogen, expressed eNOS, nNOS and nitrotyrosine. While iNOS was only weakly expressed in the basal layer of the human epidermis, it was highly expressed in keratinocytes of the inner root sheath (IRS), where it colocalized with trichohyalin, a differentiation-associated protein of the IRS that requires enzyme-catalysed conversion of arginine to citrulline. CONCLUSIONS: The NOS isoforms and nitrotyrosine are differentially expressed in different cutaneous melanocyte subpopulations. Results of this study suggest a possible role for eNOS, nNOS, iNOS and nitrotyrosine in melanocyte biology, particularly with respect to melanogenesis and melanocyte survival during HF regression. Another example of possible NO involvement in HF biology is the postsynthetic modification of trichohyalin in differentiating keratinocytes of the IRS. These results suggest that NO may influence several aspects of HF biology.     

寻常型银屑病患者皮肤中诱生型一氧化氮合酶、核因子-κBp65表达及其临床意义

目的 研究诱生型一氧化氮合酶(iNOS)mRNA、iNOS和核因子(NF)-κBp65蛋白在寻常型银屑病患者皮肤中的表达。方法 用原位杂交和免疫组化的方法检测了20例寻常型银屑病患者皮损区、非皮损区和12例正常人表皮中iNOSmRNA、iNOS和NF-κBp65蛋白的表达和分布;用银屑病面积和严重程度指数计分法评定患者的临床病情程度。结果 ①寻常型银屑病患者皮损区表皮中iNOSmRNA和iNOS蛋白表达均较非皮损区和正常人表皮中的表达明显增强(P<0.01),正常人表皮中仅少部分基底层有微弱表达,而寻常型银屑病患者皮损区的表达为基底层全层和棘层局灶性表达。②寻常型银屑病患者皮损中有一致性的iNOS和NF-κBp65高表达,两者表达水平呈显著正相关(P<0.01)。③寻常型银屑病患者皮损中iNOSmRNA的表达水平与相应患者的PASI计分呈显著正相关(P<0.01)。结论 ①寻常型银屑病患者皮损中有iNOS和NF-κBp65的高水平表达。②活化的NF-κB可能参与了寻常型银屑病的病理过程,高水平的iNOS表达可能导致了NF-κB的活化。③寻常型银屑病患者皮损中iNOS的表达水平与临床病情呈正相关。     

Lipopolysaccharide and cytokines induce nitric oxide synthase and produce nitric oxide in cultured normal human melanocytes

Abstract The role of nitric oxide in normal and pathological conditions of human skin is still poorly understood. In this study we have demonstrated by immunobloting the expression of an inducible nitric oxide synthase isoform (iNOS) in cultured normal human melanocytes treated with bacterial lipopolysaccharide, tumor necrosis factor-· and interferon-á. Nitric oxide was also detected in the culture medium and its formation was abolished upon treatment with N^G-monomethyl-l-arginine(l-NMMA), a competitive inhibitor of nitric oxide synthase. These results suggest that nitric oxide could led to autodestruction of melanocytes causing skin depigmentation. The therapeutic relevance of nitric oxide synthase inhibitors in treatment of vitiligo was suggested. Received: 15 September 2000 / Revised: 7 October 2000 / Accepted: 20 January 2001     

缺氧在银屑病发病机制中的作用

银屑病是一种以鳞屑性红斑、丘疹、斑块为主要临床表现的皮肤病,其表皮角质形成细胞的过度增殖使其具备类似肿瘤的生物学行为,与肿瘤组织一样,均存在局部缺氧现象.缺氧可使角质形成细胞低氧诱导因子1 α及其相关的多种基因,如血管内皮生长因子、一氧化氮合酶、基质金属蛋白酶2、环氧合酶2、胰岛素样生长因子2、神经生长因子、组蛋白去乙酰化酶1和LL37等因子的表达升高,从而促进炎症细胞浸润、表皮细胞增生和血管形成.缺氧还可使角质形成细胞糖酵解有关酶的表达增高,使得细胞糖酵解功能增强,从而满足细胞快速增殖的能量需求.     

脑梗死患者血浆-氧化氮水平的相关性研究

目的探讨脑梗死患者血浆-氧化氮（NO）水平的变化。方法37例脑梗死患者（病例组），男25例，女12例，年龄（62．3±11．4）岁，接受头颅CT和双侧颈部血管超声检查。根据颈动脉内膜中层厚度（IMT）分为内膜增厚组（1．0mm〈IMT≤1．2mm）14例，斑块形成组（IMT〉1．2mm）23例。于发病急性期（3d内）和恢复期（2周）清晨空腹采静脉血，用硝酸还原酶法测定NO2^-／NO3^-，间接反映-氧化氮的含量。选取同期来门诊接受健康查体的性别、年龄匹配者20例作为对照组。结果脑梗死急性期血NO水平为（42．89±7．75）μLmol/L，显著低于对照组[（70．85±7．56）μmol／L，P〈0．051；恢复期上升至（57．09±8．26）μmol／L，仍显著低于对照组（P〈0．05）。斑块形成组的血N0水平急性期为（39．31±6．74）μmol／L，显著低于内膜增厚组[（48．78±5．40）μmol／L，P〈0．05）；恢复期为（53．71±6．95）μmol／L，仍显著低于内膜增厚组[（62．63±7．36）μmol／L，P〈0．05]。结论脑梗死患者血浆NO水平下降。颈动脉硬化程度越重，NO水平越低。NO水平下降可能与脑梗死的发生发展相关。NO可作为动脉粥样硬化严重程度的参考指标。     

The expression of interleukin-8 receptor in untreated and treated psoriasis

The expression of IL-8 in psoriasis has been clearly shown with the use of immunocytochemical, RT-PCR and in situ hybridization methods. The presence of its ligand, the IL-8 receptor, has been demonstrated by the RT-PCR technique. We report here a study of the expression of both IL-8 type A and B receptors by immunohistochemical techniques, using one polyclonal and four monoclonal antibodies. By this technique, we found that the neutrophilic granulocytes express the IL-8 type A receptor, whereas the IL-8 type B receptor was present on the keratinocytes. The type B receptor on the keratinocytes was localized in the suprabasal layers of the epidermis. Following therapy, the expression of the IL-8 type B receptor on the keratinocytes was reduced. This could suggest that IL-8 in psoriasis is involved in the disturbed differentiation rather than in proliferation, probably via an autocrine loop. Received: 18 October 1996     

Skin wound healing in the SKH-1 female mouse following inducible nitric oxide synthase inhibition

BACKGROUND: The inducible isoform of the nitric oxide (NO) synthase (NOS) enzyme (iNOS) is upregulated by inflammatory mediators and/or other pathological stresses, generating high, sustained levels of NO. Cumulative data suggest a role for NO in the regulation of skin wound healing, although it is not clear to what extent NO generated by iNOS, and possibly endothelial NOS (eNOS), contribute to that healing process. Because of the current lack of understanding regarding the contribution of iNOS in wound healing, as well as the lack of wound healing data available for SC-842, an iNOS inhibitor, this in vivo study was conducted to investigate the possible role of SC-842 in interfering with wound healing. OBJECTIVES: This study evaluated whether inhibition of iNOS affects incisional skin wound healing. METHODS: Using a cutaneous full-thickness, sutured, incisional wound model in hairless SKH-1 mice, the role of iNOS in the wound healing process was evaluated by comparing in vivo effects of the iNOS inhibitor, SC-842, at various doses that result in selective inhibition of iNOS as well as nonselective NOS inhibition (as evidenced by elevated blood pressure resulting in inhibition of eNOS and/or neuronal NOS). Dexamethasone was used as a positive control. RESULTS: There were no differences in wound healing at day 28 postwounding, as evaluated by tensile strength and histology, between SC-842- and vehicle-treated animals. A decrease in tensile strength was noted at day 14 postwounding in wounds from the mid- and high-dose-treated animals as compared with vehicle-treated animals, but this difference was slight and was not associated with histological differences from vehicle-treated controls. CONCLUSIONS: These data indicate that iNOS inhibition does not adversely affect the healing of incisional wounds in SKH-1 mice as assessed over 28 days by wound tensile strength and histology.     

局部外用一氧化氮供体对屏障功能破坏的小鼠表皮增生的影响

目的:探讨一氧化氮（NO）对屏障功能破坏的小鼠表皮增生的影响。方法:将15只SKH1无毛小鼠按随机数字表法分为正常对照组（3只）、S-亚硝基-N-乙酰青霉胺（SNAP）处理组（4只）、屏障破坏组（4只）、屏障破坏+SNAP处理组（4只）;将15只C57BL/6J小鼠随机分为正常对照组、屏障破坏组、屏障破坏+硝普钠（SN...     

银屑病患者外周血一氧化氮合酶同功酶的检测

目的　探讨一氧化氮合酶 (NOS)同功酶在进行期斑块型银屑病患者 (PPP)外周血单个核细胞中 (PBMC)的表达及其意义。方法　用逆转录 聚合酶链反应 (RT PCR)检测了 3 0例PPP患者和 3 0例对照PBMC中组成性一氧化氮合酶 (cNOS)和诱导性一氧化氮合酶 (iNOS)mRNA的表达。结果　PPP患者PBMC中iNOSmRNA的平均水平高于对照组 (P <0 .0 5 ) ,且与PASI(银屑病面积严重度积分 )呈正相关 (P <0 .0 5 )。结论　PPP患者PBMC中iNOSmRNA的表达增高 ,可能通过诱导一氧化氮 (NO)的产生参与PPP患者的发病。