花青素调节JAK2/STAT3信号通路对自身免疫性心肌炎大鼠Th17/Treg平衡的影响

Influence of Anthocyanins on Th17/Treg Balance in Autoimmune Myocarditis Rats by Regulating JAK2/STAT3 Signaling Pathway

目的:探讨花青素对自身免疫性心肌炎(AE)大鼠T细胞17/调节性T细胞(Th17/Treg)平衡及Janus激酶2/信号转导和转录激活因子3(JAK2/STAT3)信号通路的影响。方法:将60只SD大鼠分为对照(NC)组、模型(Model)组、花青素组(50 mg/kg花青素)、STAT3激活剂Colivelin组(1 mg/kg Colivelin)、花青素+Colivelin组(50 mg/kg花青素+1 mg/kg Colivelin),每组12只。采用酶联免疫吸附实验(ELISA)检测血清炎性因子水平;苏木素-伊红(HE)染色检测心肌病理变化;末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)染色检测心肌细胞凋亡;实时荧光定量聚合酶链式反应(RT-qPCR)检测脾脏维甲酸相关孤儿受体γt(RORγt)、叉头蛋白p3(Foxp3)mRNA表达情况;蛋白质免疫印迹法(Western Blot)检测心肌JAK2/STAT3通路相关蛋白表达。结果:Model组心肌明显坏死和水肿,大量炎性细胞浸润;花青素组心肌无明显的坏死和细胞肿胀;Colivelin组心肌损伤加重;与花青素组相比,花青素+Colivelin组大鼠炎性细胞浸润现象增加。与NC组相比,Model组白细胞介素(IL)-17、IL-23水平、RORγt mRNA水平、Th17细胞水平、Th17/Treg比例、心肌细胞凋亡率及磷酸化STAT3(p-STAT3)/STAT3、磷酸化JAK2(p-JAK2)/JAK2比例明显升高(P<0.05),IL-10、IL-4水平、Treg细胞水平、Foxp3 mRNA水平明显下降(P<0.05);与Model组比较,花青素组IL-17、IL-23水平、RORγt mRNA水平、Th17细胞水平、Th17/Treg比例、心肌细胞凋亡率以及p-STAT3/STAT3、p-JAK2/JAK2比例明显下降(P<0.05),IL-10、IL-4水平、Treg细胞水平、Foxp3 mRNA水平明显升高(P<0.05);Colivelin组对应指标呈相反趋势(P<0.05);与花青素组相比,花青素+Colivelin组IL-17、IL-23水平、RORγt mRNA水平、Th17细胞水平、Th17/Treg比例、心肌细胞凋亡率及p-STAT3/STAT3、p-JAK2/JAK2比例明显升高(P<0.05),IL-10、IL-4水平、Treg细胞水平、脾脏Foxp3 mRNA水平明显下降(P<0.05)。结论:花青素可能通过抑制JAK2/STAT3通路,恢复Th17/Treg平衡,实现对AE大鼠心肌的保护作用。

Objective:To investigate the influences of anthocyanins on T helper-17 cells(Thl7)/regulatory T cell(Treg)balance and Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway in autoimmune myocarditis(AE)rats.Methods:Sixty SD rats were randomly grouped into control(NC)group,Model group,anthocyanin group(50 mg/kg anthocyanin),STAT3 activator Colivelin group(1 mg/kg Colivelin),anthocyanin+Colivelin group(50 mg/kg anthocyanin+1 mg/kg Colivelin),with 12 rats in each group.Enzyme linked immunosorbent assay(ELISA)was used to detect the level of serum inflammatory factor.Hematoxylin-eosin(HE)staining was applied to detect myocardial pathological changes.Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL)staining was applied to detect cardiomyocyte apoptosis.Real-time quantitative polymerasechain reaction(RT-qPCR)was applied to detect the expression of retinoid-related orphan receptorγt(RORγt)and fork-head box protein(Foxp3)mRNA in spleen.Western Blot was used to detect the expression of JAK2/STAT3 pathway related proteins in the myocardium.Results:In the Model group,myocardial necrosis and edema were obvious,and a large number of inflammatory cells were infiltrated;the myocardium in the anthocyanin group showed no evident necrosis or cell swelling;the myocardial injury was aggravated in the Colivelin group;inflammatory cell infiltration increased in the anthocyanin+Colivelin group.Compared with NC group,the levels of interleukin(IL)-17,IL-23,RORyt mRNA,Th17 cells,ratio of Th17/Treg,cardiomyocyte apoptosis rate,and ratios of p-STAT3/STAT3 and p-JAK2/JAK2 in Model group were greatly increased(P<0.05),the levels of IL-10,IL-4,Treg cells and FoxP3 mRNA were greatly decreased(P<0.05).Compared with the Model group,the levels of IL-17,IL-23,RORγt mRNA,Th17 cells,ratio of Th17/Treg,cardiomyocyte apoptosis rate,and ratios of p-STAT3/STAT3 and p-JAK2/JAK2 in anthocyanin group decreased(P<0.05),the levels of IL-10,IL-4,Treg cells and Foxp3 mRNA increased(P<0.05);the corresponding indicators in the Colivelin group showed the opposite trends(P<0.05).Compared with anthocyanin group,the levels of IL-17,IL-23,RORγt mRNA,Th17 cells,ratio of Th17/Treg,cardiomyocyte apoptosis rate,and ratios of p-STAT3/STAT3 and p-JAK2/JAK2 in anthocyanin+Colivelin group increased(P<0.05),the levels of IL-10,IL-4,Treg cells and Foxp3 mRNA decreased(P<0.05).Conclusion:Anthocyanins may restore the balance of Th17/Treg by inhibiting the JAK2/STAT3 pathway and achieve the protective effect on the myocardium of AE rats.