白细胞介素-2/Janus激酶3/信号转导和转录激活因子5在强直性脊柱炎患者外周血中的表达及临床意义

Expression an d clinical significance of interleukin-2/Janus kinase 3/signal transduction and transcriptional activator 5 in peripheral blood of patients with ankylosing spondylitis

目的检测IL-2/Janus激酶3(JAK3)/信号转导和转录激活因子(STAT)5信号通路在AS患者外周血中的表达并探讨其在AS发生发展中的作用机制。方法收集30例AS活动期患者(ASA)、30例AS稳定期患者(ASS)和50名健康体检者(HC)的临床资料、外周血标本及实验室检查指标。采用实时荧光定量PCR(RT-qPCR)检测JAK3、STAT5a及STAT5b mRNA表达水平;采用蛋白质印迹法检测JAK3、STAT5a和STAT5b蛋白及磷酸化蛋白表达水平;采用ELISA检测血浆中IL-2浓度。计量资料符合正态分布采用两独立样本t检验或单因素方差分析, 3组间两两比较采用LSD-t检验, 非正态分布用Mann-WhitneyU检验或Kruskal-WallisH检验, 分类变量关联性分析采用χ^(2)检验, 变量间相关分析采用Spearman相关分析, 采用受试者工作特征(ROC)曲线评估JAK3、STAT5a和STAT5b mRNA表达水平监测AS活动性的价值。结果①JAK3、STAT5a和STAT5b的mRNA表达水平在3组间的差异均有统计学意义(F=65.98, P<0.001;F=21.15, P<0.001;F=13.67, P<0.001), ASA组JAK3 mRNA表达水平(2.5±0.9)明显高于ASS组(1.1±0.4)和健康体检者组(1.0±0.5), 差异有统计学意义(P均<0.001), ASA组STAT5a mRNA表达水平(1.4±0.3)明显高于ASS组(0.9±0.3)和健康体检者组(1.0±0.3), 差异均有统计学意义(P<0.001), ASA组STAT5b mRNA表达水平(1.5±0.6)明显高于ASS组(1.0±0.4)和健康体检者组(1.0±0.4), 差异均有统计学意义(P<0.001), AS患者中HLA-B27阳性组JAK3 mRNA表达水平(1.92±1.01)高于HLA-B27阴性组(1.44±0.60), 差异有统计学意义(t=-2.22, P=0.032)。JAK3、STAT5a和STAT5b蛋白磷酸化水平在3组间的差异均有统计学意义(F=91.56, P<0.001;F=25.15, P<0.001;F=178.59, P<0.001), ASA组JAK3蛋白磷酸化水平(1.035±0.076)明显高于ASS组(0.568±0.019)和健康体检者组(0.536±0.064), 差异均有统计学意义(P<0.001), ASA组STAT5a蛋白磷酸化水平(1.166±0.096)明显高于ASS组(0.923±0.018)和健康体检者组(0.911±0.017), 差异均有统计学意义(P<0.001), ASA组STAT5b蛋白磷酸化水平(0.81±0.05)明显高于ASS组(0.21±0.03)和健康体检者组(0.24±0.07), 差异均有统计学意义(P<0.001)。血浆中IL-2浓度在3组间的差异有统计学意义(F=3.32, P=0.040), ASA组患者IL-2浓度[(110±40)pg/ml]明显高于ASS组[(89±40)pg/ml]和健康体检者组[(88±39)pg/ml], 差异有统计学意义(P=0.044, P=0.016)。②Spearman相关分析显示:在AS患者中STAT5a mRNA表达水平与血小板呈正相关(r=0.353, P=0.006);ASA组JAK3 mRNA表达水平与IL-2浓度呈正相关(r=0.766, P<0.001), 与肾小球滤过率估计值呈负相关(r=-0.485, P=0.007), STAT5a mRNA表达水平与ESR呈正相关(r=0.680, P<0.001), STAT5b mRNA表达水平与hs-CRP呈正相关(r=0.823, P<0.001)。③ROC曲线显示:JAK3 mRNA表达水平预测ASA的ROC曲线下面积(AUC)95%CI为0.920(0.853, 0.987), 灵敏度和特异度分别是86.7%和90.0%;STAT5a mRNA表达水平预测ASA的AUC 95%CI为0.874(0.787, 0.961), 灵敏度和特异度分别是96.7%和66.7%;STAT5b mRNA表达水平预测ASA的AUC 95%CI为0.749(0.617, 0.881), 灵敏度和特异度分别是73.3%和80.0%。结论 IL-2/JAK3/STAT5可能参与了AS的发病, 并且JAK3 mRNA可作为监测AS疾病活动性的生物学指标。

Objective To detect the expression of interleukin 2(IL-2)/Janus kinase 3/signal transduction and transcriptional activator 5(JAK3/STAT5)signaling pathway in peripheral blood of patients with ankylosing spondylitis(AS)and explore its mechanism in the development and progression of AS.Methods Clinical data,peripheral blood and laboratory tests of 30 patients with active AS(ASA),30 patients with stable AS(ASS)and 50 healthy subjects(HC)were collected.The mRNA expression levels of JAK3,signal transduction and transcription activator 5a(STAT5a)and signal transduction and transcription activator 5b(STAT5b)were detected by quantitative real-time-polymerase chain reaction(RT-qPCR).The expression levels of JAK3,STAT5a and STAT5b proteins and phosphorylated proteins were detected by Western-blot.Plasma IL-2 concentration was determined by enzyme-linked immunosorbent assay(ELISA).Two independent samples t-test or one-way analysis of variance were used for measurement data consistent with normal distribution,LSD-t test was used for pairwise comparison between the three groups,Mann-Whitney U test or Kruskal-Wallis H test was used for non-normal distribution,χ^(2) test was used for correlation analysis of categorical variables.Spearman correlation analysis was used for correlation analysis between variables,and receiver operating characteristic(ROC)curve was used to evaluate the value of JAK3,STAT5a and STAT5b mRNA expression levels in monitoring AS activity.Results①The mRNA expression levels of JAK3,STAT5a and STAT5b were significantly different among the three groups(F=65.98,P<0.001;F=21.15,P<0.001;F=13.67,P<0.001).JAK3 mRNA expression in ASA group(2.5±0.9)was significantly higher than that in ASS group(1.1±0.4)and healthy subjects(1.0±0.5),the difference was statistically significant(both P<0.001).The mRNA expression level of STAT5a in ASA group(1.4±0.3)was significantly higher than that in ASS group(0.9±0.3)and healthy subjects group(1.0±0.3),the difference was statistically significant(both P<0.001).STAT5b mRNA expression level in ASA group(1.5±0.6)was significantly higher than that in ASS group(1.0±0.4)and healthy subjects(1.0±0.4),the difference was statistically significant(both P<0.001).The expression level of JAK3 mRNA in HLA-B27 positive group(1.9±1.0)was higher than that in HLA-B27 negative group(1.4±0.6),and the difference was statistically significant(t=-2.22,P=0.032).The phosphorylation levels of JAK3,STAT5a and STAT5b showed statistically significant differences among the three groups(F=91.56,P<0.001;F=25.15,P<0.001;F=178.59,P<0.001).The phosphorylation level of JAK3 protein in ASA group(1.04±0.08)was significantly higher than that in ASS group(0.568±0.019)and healthy subjects(0.536±0.064),the difference was statistically significant(both P<0.001).The phosphorylation level of STAT5a protein in ASA group(1.166±0.096)was significantly higher than that in ASS group(0.923±0.018)and healthy subjects(0.911±0.017),the difference was statistically significant(both P<0.001).The phosphorylation level of STAT5b protein in ASA group(0.81±0.05)was significantly higher than that in ASS group(0.21±0.03)and healthy subjects(0.24±0.07),the difference was statistically significant(both P<0.001).The difference of plasma IL-2 concentration among the three groups was statistically significant(F=3.32,P=0.040).The IL-2 concentration in the ASA group[(110±40)pg/ml]was significantly higher than that in the ASS group[(89±40)pg/ml]and the healthy group[(88±39)pg/ml],the difference was statistically significant(P=0.044,P=0.016).②Spearman correlation analysis showed that STAT5a mRNA expression level was positively correlated with platelets in AS patients(r=0.353,P=0.006).JAK3 mRNA expression level in ASA group was positively correlated with IL-2 concentration(r=0.766,P<0.001),and negatively correlated with estimated glomerular filtration rate(r=-0.485,P=0.007).STAT5a mRNA expression level was positively correlated with erythrocyte sedimentation rate(r=0.680,P<0.001),and STAT5b mRNA expression level was positively correlated with hypersensitive C-reactive protein(CRP)(r=0.823,P<0.001).③The ROC curve showed that JAK3 mRNA expression level predicted the area under ROC curve(AUC)of ASA with a 95%CI of 0.920(0.853,0.987),sensitivity and specificity of 86.7%and 90.0%,respectively.STAT5a mRNA expression level predicted the AUC 95%CI of ASA was 0.874(0.787,0.961),and the sensitivity and specificity were 96.7%and 66.7%,respectively.STAT5b mRNA expression level predicted the AUC 95%CI of ASA was 0.749(0.617,0.881),and the sensitivity and specificity were 73.3%and 80.0%,respectively.Conclusion This study suggests that IL-2/JAK3/STAT5 may be involved in the pathogenesis of AS,and JAK3 mRNA can be used as a biological indicator to monitor the activity of AS disease.