中性粒细胞明胶酶相关载脂蛋白在银屑病患儿血清中的表达及致病机制研究

Expression of serum neutrophil gelatinase-associated lipocalin in children with psoriasis and its pathogenic mechanisms

目的观察中性粒细胞明胶酶相关载脂蛋白(NGAL)与儿童银屑病患者皮损严重程度和外周血中性粒细胞数目的关系,及其体外对永生化人角质形成细胞株HaCaT表达分泌肿瘤坏死因子α(TNF-α)和白细胞介素22(IL-22)的影响。方法2017年1月1日至2018年12月31日在全国6家医院皮肤科收集98例新发银屑病患儿,其中男51例,女47例,年龄3~14(7.00±2.99)岁,病程3~28(7.4±5.85)d。检测治疗前和治疗2周后所有患儿血清NGAL表达水平,分析NGAL与银屑病皮损面积及严重程度指数(PASI)积分和外周血中性粒细胞的关系。Western印迹法和反转录PCR检测0(对照组)、0.125、0.25、0.5、1 mg/L NGAL作用12 h后HaCaT细胞IL-22、TNF-α蛋白和mRNA的表达水平。统计分析采用t检验及单因素方差分析。结果治疗2周后,银屑病患儿PASI积分、中性粒细胞数和NGAL水平分别为1.80±1.19、(6.16±0.76)×10^9/L、(90.86±0.75)μg/L,与治疗前10.38±3.42、(11.01±2.85)×10^9/L、(113.48±21.26)μg/L相比均显著下降(t=31.42、18.34、16.37,均P<0.001);治疗前患儿血清中NGAL水平与PASI积分、外周血中性细胞数呈正相关(r=0.918、0.799,P<0.05)。不同浓度NGAL组HaCaT细胞IL-22 mRNA及蛋白水平差异均有统计学意义(F=176.31、296.96,均P<0.001),TNF-αmRNA及蛋白水平差异亦有统计学意义(F=193.28、318.80,均P<0.001),0.125、0.25、0.5、1 mg/L NGAL组细胞IL-22、TNF-α蛋白和mRNA表达的水平均高于对照组(均P<0.05);NGAL与TNF-α、IL-22蛋白及mRNA的表达均呈正相关(均P<0.05)。结论银屑病患儿血清中NGAL高表达,与皮损的严重程度和外周血中性粒细胞数呈正相关;NGAL体外能够上调HaCaT细胞内TNF-α和IL-22的表达。

Objective To explore the relationships of neutrophil gelatinase-associated lipocalin(NGAL)with severity of skin lesions in children with psoriasis and peripheral neutrophil count,and to evaluate in vitro effect of NGAL on expression of tumor necrosis factor-α(TNF-α)and interleukin-22(IL-22)by a human immortalized keratinocyte cell line HaCaT.Methods From January 1st 2017 to December 31st 2018,98 children who newly developed psoriasis were enrolled from Department of Dermatology of 6 hospitals in China,including 51 males and 47 females.Their age was 7.00±2.99 years(range:3-14 years),and their course of disease was 7.4±5.85 days(range:3-28 days).The serum level of NGAL was detected in all the patients before and two weeks after treatment,and the relationships of NGAL with psoriasis area and severity index(PASI)scores and peripheral neutrophil count were evaluated.Western blot analysis and reverse-transcription(RT)-PCR were performed to determine the protein and mRNA expression of TNF-αand IL-22 in HaCaT cells,respectively,after 12-hour treatment with NGAL at concentrations of 0(control group),0.125,0.25,0.5,1 mg/L.Statistical analysis was carried out with SPSS 16 software.by using t test and one-way analysis of variance.Results After 2-week treatment,the PASI score,neutrophil count and NGAL level in children with psoriasis significantly decreased(1.80±1.19,[6.16±0.76]×10^9/L,90.86±0.75μg/L,respectively)compared with those before the treatment(10.38±3.42,[11.01±2.85]×10^9/L,113.48±21.26μg/L,respectively;t=31.42,18.34,16.37 respectively,all P<0.001).Before the treatment,the serum level of NGAL in the patients was positively correlated with the PASI score and peripheral neutrophil count(r=0.918,0.799 respectively,both P<0.05).The mRNA and protein expression of IL-22 in HaCaT cells significantly differed among these groups treated with different concentrations of NGAL(F=176.31,296.96 respectively,both P<0.001),so did the mRNA and protein expression of TNF-α(F=193.28,318.80 respectively,both P<0.001).Additionally,the protein and mRNA expression of IL-22 and TNF-αin HaCaT cells was significantly higher in the 0.125-,0.25-,0.5-and 1-mg/L NGAL group than in the control group(all P<0.05).The NGAL level was positively correlated with the protein and mRNA expression of TNF-αand IL-22 in HaCaT cells(all P<0.05).Conclusions The serum level of NGAL was high in children with psoriasis,and positively correlated with severity of skin lesions and peripheral neutrophil count.NGAL can upregulate the expression of TNF-αand IL-22 in HaCaT cells in vitro.