He Shuang, Han Li-Na, Wang Yu-Tang, Liu Jian-Wei, Ding Guo-Lei
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2014 Mar;30(2):119-23.
To investigate the therapeutic effect of IL-6 mAb on experimental autoimmune myocarditis (EAM) in rats, and search the mechanism of the role of IL-6, helper T cells 17 (Th17) and regulative T cells (Treg) in EAM pathogenesis.
Thirty-four Lewis rats were divided into three groups randomly, i.e. control group (n = 6), EAM group (n = 12), and IL-6 mAb intervention group (n = 16). Rats in EAM group and IL-6 mAb intervention group were injected intracutaneously with myosin to establish EAM model. Rats in IL-6 mAb intervention group were injected intraperitoneally with 1 mg IL-6 mAb on 1st, 7th to 20th day after cardiac myosin immune injection. Myocardial inflammation was examined by HE stain, Masson stain, and TdT assay (TUNEL reaction) on 21st and 84th day after IL-6 mAb therapy in order to assess the therapeutic role. Spleen cells were analyzed by flow cytometry to illustrate Th17 and Treg cells? number and function. The serum concentration of IL-6, IL-10, IL-17, and TGF-beta in each group was measured by ELISA, concentration of STAT3, RORgammat, and Foxp3 mRNA in each group was determined with RT-PCR. Spleen cells derived from EAM were stimulated by IL-6 mAb in vitro, and the concentration of IL-10, IL-17 and TGF-beta was measured by ELISA.
Inflammation score, fibrosis score, and apoptosis index in IL-6 mAb intervention group were significantly decreased as compared with those in EAM group (P < 0.01). The number of Th17 and Treg cells in EAM group on the 21st day (experimental acute peak stage) were increased, and those in intervention group on the 21st day were significantly inhibited (P < 0.01). The concentration of serum IL-6, IL-10, IL-17 and TGF-beta in intervention group on the 21st day was decreased dramatically in comparison with that in EAM group on the same day (P < 0.01). The levels of peripheral blood STAT3, RORgammat, Foxp3 mRNA in intervention group on the 21st day was decreased significantly as compared with that in EAM group (P < 0.01). The expression of IL-10, IL-17 and TGF-beta was increased significantly (P < 0.01) by stimulation of IL-6 mAb on spleen cells derived from EAM in vitro.
IL-6 mAb could neutralize IL-6, and ameliorate myocarditis and reduce heart autoimmune responses. IL-6 mAb has significantly protective effects on EAM by suppressing Th17 and Treg cells.
探讨白细胞介素-6单克隆抗体（IL-6 mAb）对大鼠实验性自身免疫性心肌炎（EAM）的治疗作用，探寻IL-6、辅助性T细胞17（Th17）及调节性T细胞（Treg）在EAM发病机制中的作用机制。
将34只Lewis大鼠随机分为三组，即对照组（n = 6）、EAM组（n = 12）和IL-6 mAb干预组（n = 16）。EAM组和IL-6 mAb干预组大鼠经皮内注射肌球蛋白以建立EAM模型。IL-6 mAb干预组大鼠在心肌肌球蛋白免疫注射后的第1、7至20天腹腔注射1 mg IL-6 mAb。在IL-6 mAb治疗后的第21天和第84天，通过苏木精-伊红（HE）染色、Masson染色及末端脱氧核苷酸转移酶介导的缺口末端标记法（TdT法，TUNEL反应）检测心肌炎症，以评估治疗作用。采用流式细胞术分析脾细胞，以阐明Th17和Treg细胞的数量及功能。通过酶联免疫吸附测定（ELISA）法检测每组血清中IL-6、IL-10、IL-17和转化生长因子-β（TGF-β）的浓度，采用逆转录聚合酶链反应（RT-PCR）法测定每组中信号转导子和转录激活子3（STAT3）、维甲酸相关孤儿受体γt（RORγt）及叉头框蛋白3（Foxp3）mRNA的浓度。体外使用IL-6 mAb刺激EAM来源的脾细胞，并通过ELISA法检测IL-10、IL-17和TGF-β的浓度。
与EAM组相比，IL-6 mAb干预组的炎症评分、纤维化评分及凋亡指数均显著降低（P < 0.01）。在第21天（实验性急性高峰期），EAM组的Th17和Treg细胞数量增加，而干预组在第21天的Th17和Treg细胞数量受到显著抑制（P < 0.01）。与同一天的EAM组相比，干预组在第21天的血清IL-6、IL-10、IL-17和TGF-β浓度显著降低（P < 0.01）。与EAM组相比，干预组在第21天外周血STAT3、RORγt、Foxp3 mRNA水平显著降低（P < 0.01）。体外使用IL-6 mAb刺激EAM来源的脾细胞后，IL-10、IL-17和TGF-β的表达显著增加（P < 0.01）。
IL-6 mAb可中和IL-6，改善心肌炎并减轻心脏自身免疫反应。IL-6 mAb通过抑制Th17和Treg细胞对EAM具有显著的保护作用。
