MiR-155在砷致肝损伤大鼠Th17/Treg稳态失衡中的作用及机制研究

The role and mechanism of miR-155 in Th17/Treg homeostasis imbalance in arsenic-induced liver injury rats

目的探讨微小RNA(miRNA,miR)-155在砷致肝损伤大鼠辅助性T细胞17(Th17)/调节性T细胞(Treg)稳态失衡中的作用。方法选取32只健康清洁级Wistar大鼠(体质量为80~100 g),采用随机数字表法分为对照和低、中、高砷剂量组,每组8只,雌雄各半。对照组给予去离子水灌胃,低、中、高砷剂量组按体质量分别给予2.5、5.0、10.0 mg/kg亚砷酸钠溶液灌胃,每周灌胃6 d,持续饲养4个月。实验终期采集大鼠外周血及肝组织样本,采用流式细胞术检测大鼠外周血淋巴细胞中Th17及Treg亚群比例,酶联免疫吸附测定法(ELISA)检测血浆中Th17、Treg主要效应因子白细胞介素(IL)-17、IL-10含量;实时荧光定量PCR检测血浆miR-155水平及肝组织细胞因子信号转导抑制蛋白1(SOCS1)、Janus激酶3(JAK3)、信号转导和转录激活因子5(STAT5)mRNA表达水平;蛋白质印迹法检测肝组织SOCS1、JAK3、STAT5、磷酸化(p)-JAK3、p-STAT5蛋白表达水平;并采用Pearson相关性分析进行各指标间的关联性分析。同时,利用miRNA靶基因在线预测数据库(TargetScan、miRanda及miRBase)分析miR-155与SOCS1 mRNA靶向结合情况,并进一步利用STRING数据库进行通路蛋白互作网络分析。结果(1)对照,低、中、高砷剂量组大鼠外周血Th17/Treg比值(中位数:0.12、0.15、0.18、0.24),IL-17/IL-10比值(中位数:0.20、0.28、0.35、0.37)及miR-155水平(中位数:1.03、1.60、2.07、3.34)比较,差异均有统计学意义(H=9.65、17.15、17.30,P=0.022、0.001、0.001);且中、高砷剂量组各指标水平均高于对照组,高砷剂量组各指标水平均高于低砷剂量组(均P<0.05)。(2)与对照组比较,中、高砷剂量组大鼠肝组织SOCS1 mRNA及蛋白表达水平均较低,而中、高砷剂量组JAK3、STAT5 mRNA表达水平及高砷剂量组JAK3、p-JAK3、STAT5、p-STAT5蛋白表达水平均较高(均P<0.05)。(3)关联性分析发现,大鼠外周血miR-155水平与外周血Th17/Treg、IL-17/IL-10比值均呈正相关(r=0.42、0.56,P=0.016、0.001);与肝组织SOCS1 mRNA、蛋白表达水平均呈负相关(r=-0.38、-0.41,P=0.032、0.018),而与肝组织JAK3、p-JAK3、STAT5、p-STAT5蛋白表达水平均呈正相关(r=0.39、0.37、0.50、0.47,P=0.028、0.039、0.004、0.007)。(4)miRNA靶基因在线预测数据库分析结果显示,miR-155与SOCS1 mRNA的3′非编码区存在碱基互补配对序列。STRING数据库分析结果显示,SOCS1可通过JAK3-STAT5通路作用于Treg特异转录因子FOXP3,其主要作用部位为Src同源2结构域。结论异常表达的miR-155通过靶向SOCS1调控JAK3-STAT5通路,进而参与砷暴露致肝损伤大鼠Th17/Treg稳态失衡。

Objective To study the role of microRNA(miRNA,miR)-155 in the imbalance of T helper cell 17(Th17)/regulatory T cell(Treg)homeostasis in arsenic-induced liver injury rats.Methods Thirty-two healthy and clean grade Wistar rats(body weight:80-100 g)were selected and divided into control and low,medium and high arsenic dose groups using a random number table method,with 8 rats in each group,half male and half female.The control group was given deionized water by gavage,while the low,medium and high arsenic dose groups were given 2.5,5.0 and 10.0 mg/kg sodium arsenite solution by gavage according to body weight,respectively,for 6 days a week,and continued feeding for 4 months.At the end of the experiment,peripheral blood and liver tissue samples of rats were collected.Flow cytometry was used to detect the proportion of Th17 and Treg subsets in peripheral blood lymphocytes,and enzyme-linked immunosorbent assay(ELISA)was used to detect the content of Th17 and Treg effector interleukin(IL)-17 and IL-10 in plasma.The miR-155 level in plasma and the mRNA expression levels of cytokine signal transduction inhibitor 1(SOCS1),Janus kinase 3(JAK3)and signal transducers and activators of transduction 5(STAT5)in liver tissue were detected by real-time fluorescence quantitative PCR,and the protein expression levels of SOCS1,JAK3,STAT5,phosphorylated(p)-JAK3 and p-STAT5 in liver tissue were detected by Western blotting.Pearson correlation analysis was used to analyze the correlation among each index.Meanwhile,the targeted binding of miR-155 and SOCS1 mRNA was analyzed using miRNA target gene online prediction databases(TargetScan,miRanda and miRBase),and the pathway protein interaction network was further analyzed using STRING database.Results(1)There were significant differences in Th17/Treg ratio(median:0.12,0.15,0.18,0.24),IL-17/IL-10 ratio(median:0.20,0.28,0.35,0.37)and miR-155 level(median:1.03,1.60,2.07,3.34)in peripheral blood of rats in control and low,medium and high arsenic dose groups(H=9.65,17.15,17.30,P=0.022,0.001,0.001).All the indexes in the medium and high arsenic dose groups were higher than those in the control group,and the indexes in the high arsenic dose group were higher than those in the low arsenic dose group(P<0.05).(2)Compared with the control group,the mRNA and protein expression levels of SOCS1 in liver tissue of rats in medium and high arsenic dose groups were lower,while the mRNA expression levels of JAK3 and STAT5 in medium and high arsenic dose groups and the protein expression levels of JAK3,p-JAK3,STAT5 and p-STAT5 in high arsenic dose group were higher(P<0.05).(3)Correlation analysis showed that miR-155 level in peripheral blood of rats was positively correlated with Th17/Treg and IL-17/IL-10 ratios in peripheral blood(r=0.42,0.56,P=0.016,0.001).The miR-155 level in peripheral blood was negatively correlated with SOCS1 mRNA and protein expression levels in liver tissue(r=-0.38,-0.41,P=0.032,0.018),while positively correlated with the protein expression levels of JAK3,p-JAK3,STAT5 and p-STAT5 in liver tissue(r=0.39,0.37,0.50,0.47,P=0.028,0.039,0.004,0.007).(4)The results of miRNA target gene online prediction databases analysis showed that there was a complementary pairing sequence between miR-155 and SOCS1 mRNA in the 3'non-coding region.The STRING database analysis showed that SOCS1 acted on Treg-specific transcription factor FOXP3 through JAK3-STAT5 pathway,and its main action site was the Src homologous 2 domain.Conclusion The abnormally expressed miR-155 regulates JAK3-STAT5 pathway by targeting SOCS1,thereby participating in the Th17/Treg homeostasis imbalance in arsenic-induced liver injury rats.