系统性红斑狼疮患者外周血CD4^(+)T细胞中ALKBH3-AS1表达及其与Th17/Treg和疾病活动度的相关性研究

ALKBH3-AS1 Expression in Peripheral Blood CD4^(+)T Cells of Patients with Systemic Lupus Erythematosus and Its Correlation with Th17/Treg and Disease Activity

目的研究分析系统性红斑狼疮(SLE)患者外周血CD4^(+)T细胞中ALKBH3-AS1表达及其与辅助性T细胞17/调节性T细胞(Th17/Treg)和疾病活动度的相关性。方法回顾性收集乐山市中医医院2020年7月~2024年3月确诊的系统性红斑狼疮患者60例(SLE组),根据SLE疾病活动指数(SLEDAI)评分分为活动组(SLEDAI≥10分,n=33)和稳定组(SLEDAI<10分,n=27);同时选取同期健康体检者52例为对照组。收集三组一般资料,采集纳入者外周血离心得外周血单个核细胞(PBMC)。免疫磁珠法分离CD4^(+)T细胞,流式细胞仪检测Th17/Treg占比情况,荧光定量PCR检测CD4^(+)T细胞中ALKBH3-AS1,维甲酸相关孤核受体(retinoidrelated orphan receptorγt,RORγt)相对表达量;酶联免疫吸附法(ELISA)测定血清转化生长因子(transforming growth factor,TGF)-β,白细胞介素-17(IL-17)含量;速率散射免疫比浊法测定补体C3,C4水平变化。Pearson分析ALKBH3-AS1,Th17与SLE患者各临床指标的相关性;Logistic回归分析影响SLE患者活动程度的因素。结果对照组Hb,ALB,ALKBH3-AS1 mRNA,CD4^(+)T,补体C3,补体C4显著高于SLE组(t/Z=3.245,-11.169,-12.675,-17.829,-15.240,-19.212),RDW,TGF-β,RORγt,Th17/Treg,IL-17,CRP显著低于SLE组(t/Z=4.206,10.054,19.869,37.942,50.463,3.115),差异具有统计学意义(均P<0.05)。活动组ALB,ALKBH3-AS1 mRNA,CD4^(+)T显著低于稳定组(t/Z=-8.918,-2.483,-11.694),CRP,TGF-β,RORγt,Th17/Treg,IL-17显著高于稳定组(t/Z=3.121,5.671,1.787,14.720,12.044),差异具有统计学意义(均P<0.05)。Pearson分析结果显示,ALKBH3-AS1与CD4^(+)T呈正相关(r=0.663),与Th17/Treg,IL-17,TGF-β,RORγt,SLEDAI指数呈负相关(r=-0.687,-0.715,-0.705,-0.678,-0.671);Th17/Treg与CD4^(+)T呈负相关(r=-0.817),与IL-17,TGF-β,RORγt,SLEDAI指数呈正相关(r=0.687,0.767,0.598,0.704)。Logistics回归分析结果显示,CD4^(+)T[OR(95%CI):0.715(0.304~0.904)]占比增加、ALKBH3-AS1[OR(95%CI):0.654(0.320~0.987)]表达上调为影响SLE患者疾病活动度的保护因素,TGF-β[OR(95%CI):1.487(1.120~1.814)]和IL-17[OR(95%CI):1.294(1.217~1.887)]含量上调、Th17/Treg[OR(95%)CI:1.674(1.361~1.679)]占比上调、RORγt[OR(95%)CI:1.547(1.252~1.941)]相对表达量增加为影响SLE患者疾病活动度的危险因素。结论SLE患者CD4^(+)T细胞中ALKBH3-AS1表达下调,TGF-β,RORγt,IL-17表达上调均与患者疾病活动度相关,可作为SLE诊断及评估疾病活动及疗效的潜在生物标志物。

Objective To investigate the expression of ALKBH3-AS1 in peripheral blood CD4^(+)T cells of patients with systemic lupus erythematosus(SLE)and its correlation with T helper cell 17/regulatory T cells(Th17/Treg)and disease activity.Methods A total of 60 patients diagnosed with SLE in Leshan Hospital of Traditional Chinese Medicine from July 2020 to March 2024 were retrospectively collected.According to SLEDAI score,they were divided into active group(n=33,SLEDAI≥10 score)and stable group(n=27,SLEDAI<10 score).At the same time,52 healthy subjects were selected as control group.The general data of three groups were collected and peripheral blood mononuclear cell(PBMC)was obtained by centrifugation in peripheral blood.CD4^(+)T cells were isolated by immunomagnetic beads,and Th17/Treg ratio was detected by flow cytometry.The relative expression levels of ALKBH3-AS1 and retinoid-related orphan receptorγt(RORγt)in CD4^(+)T cells were detected by fluorescence quantitative PCR.The contents of transforming growth factor(TGF)-βand interleukin(IL)-17 in serum were determined by enzyme-linked immunosorbent assay(ELISA).The levels of C3 and C4 were determined by rate scattering immunoturbidimetry.Pearson analyzed the correlation between ALKBH3-AS1,Th17 and various clinical indicators in SLE patients.Logistic regression analysis of the influencing factors in patients with severe SLE showed that the difference was statistically significant.Results Hb,ALB,ALKBH3-AS1 mRNA,CD4^(+)T,complement C3 and C4 in the control group were significantly higher than those in SLE group(t/χ^(2)/Z=3.245,-11.169,-12.675,-17.829,-15.240,-19.212),RDW,TGF-β,RORγt,Th17/Treg,IL-17,CRP was significantly lower than that in SLE group(t/χ^(2)/Z=4.206,10.054,19.869,37.942,50.463,3.115),and the differences were statistically significant(all P<0.05).ALB,ALKBH3-AS1 mRNA,CD4^(+)T in the active group were significantly lower than those in the stable group(t/χ^(2)/Z=-8.918,-2.483,-11.694),CRP,TGF-β,RORγt,Th17/Treg,and IL-17 were significantly higher than those in the stable group(t/χ^(2)/Z=3.121,5.671,1.787,14.720,12.044),and the differences were statistically significant(all P<0.05).Pearson analysis showed that ALKBH3-AS1 was positively correlated with CD4^(+)T(r=0.663),and negatively correlated with Th17/Treg,IL-17,TGF-β,RORγt,SLEDAIindex(r=-0.687,-0.715,-0.705,-0.678,-0.671),Th17/Treg was negatively correlated with CD4^(+)T(r=-0.817),and positively correlated with IL-17,TGF-β,RORγt,SLEDAI index with statistical significance(r=0.687,0.767,0.598,0.704).Logistics regression analysis,the results show:Increased CD4^(+)T[OR(95%CI):0.715(0.304~0.904)]proportion and up-regulated ALKBH3-AS1[OR(95%CI):0.654(0.320~0.987)]expression were protective factors affecting disease activity in SLE patients.The contents of TGF-β[OR(95%CI):1.487(1.120~1.814)]and IL-17[OR(95%CI):1.294(1.217~1.887)]were up-regulated,the proportion of Th17/Treg[OR(95%)CI:1.674(1.361~1.679)]was up-regulated,and the relative expression of RORγt[OR(95%)CI:1.547(1.252~1.941)]was increased as risk factors affecting disease activity in SLE patients.Conclusion The down-regulated expression of ALKBH3-AS1 and upregulated expression of TGF-β,RORγt and IL-17 in CD4^(+)T cells of SLE patients are all correlated with disease activity,and can be potential biomarkers for diagnosis,disease activity and efficacy evaluation in SLE patients.