[Electroacupuncture improves myocardial ischemia inflammatory injury by regulating the homeostasis of myocardial Th17/Treg cells through JAK1/STAT3 signaling in rats].

摘要

OBJECTIVES: To observe the effect of electroacupuncture (EA) on the homeostasis of Th17/Treg cells and the expressions of key proteins of the janus kinase 1/signal transducer and activator of transcription 3 signaling pathway in myocardial tissue of rats with myocardial ischemia(MI), so as to explore its mechanisms underlying amelioration of inflammatory injury following MI. METHODS: SD rats were randomly divided into control, model, and EA groups, with 6 rats in each group. The MI model was established by subcutaneous injection of isoproterenol hydrochloride(5 mg·kg-1·d-1), once daily for 7 consecutive days. After successful modeling, the rats of EA group received EA stimulation (2 Hz/10 Hz, 2 to 3 mA) of unilateral "Neiguan" (PC6) and "Zusanli" (ST36) for 20 min, once daily for 21 d. The standard limb lead II electrocardiogram (ECG) was recorded to analyze the height of ST and amplitude of T wave for assessing the degree of ischemic myocardial injury. The hematoxylin-eosin (H.E.) staining was used to observe histopathological changes in the myocardial tissue, and the Masson staining performed to examine the collagen deposition of the myocardial cells. The concentrations of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-17, and IL-10 in the serum and myocardial tissue were detected using enzyme-linked immunosorbent assays (ELISA), and the proportions of Th17 cells and Treg cells in the myocardial tissue analyzed using flow cytometry. The expression levels of signal transducers and activators of transcription 3 (STAT3), phosphorylated (p)- STAT3, forkhead box protein 3 (Foxp3), Janus kinase 1 (JAK1), retinoic acid-related orphan nuclear receptor-α(RORα), suppressor of cytokine signaling 3 (SOCS3) proteins in the myocardial tissue were detected using Western blot. RESULTS: Compared with the control group, the model group had a significant increase in the ST height and T-wave amplitude, myocardial collagen volume fraction (CVF), TNF-α and IL-17 contents in the serum and myocardial tissue, myocardial Th17/Treg ratio, and expression levels of myocardial JAK1, STAT3, p-STAT3, and RORα proteins (P<0.01, P<0.05), and a striking decrease in the proportion of Treg cells, contents of myocardial and serum IL-10, and expression levels of myocardial FOXP3 and SOCS3 (P<0.01, P<0.05). In contrast to the model group, both the increase and the decrease of the indexes mentioned above were reversed in the EA group (P<0.05, P<0.01). H.E. staining showed necrosis and dissolution of a large number of myocardial cells, with increased cell spacing and blurred boundaries, inflammatory infiltration and fibrous tissue hyperplasia in the model group, which was relatively and significantly milder in the degree of myocardial injury in the EA group. CONCLUSIONS: EA at PC6 and ST36 can improve myocardial inflammation injury in rats with MI, which may be related to its function in regulating the homeostasis of Th17/Treg cells in myocardial tissue through JAK1/STAT3 signaling, balancing the secretion of pro-inflammatory factors TNF-α, IL-17 and anti-inflammatory factor IL-10. 目的: 观察电针对心肌缺血（MI）大鼠心肌组织辅助性T细胞17（Th17）/调节性T细胞（Treg）细胞稳态及炎性相关Janus激酶1（JAK1）/信号转导及转录激活因子（STAT）3信号通路关键蛋白表达的影响，探讨电针改善MI后炎性损伤的机制。方法: SD大鼠随机分为对照组、模型组和电针组，每组6只。模型组、电针组连续7 d皮下多点位注射盐酸异丙肾上腺素（5 mg·kg-1·d-1）制备MI大鼠模型。电针组电针“内关”“足三里”，20 min/次，1次/d，共21 d。心电图记录大鼠ST段高度和T波振幅;HE染色观察心肌组织病理变化;Masson染色观察心肌细胞胶原沉积情况;ELISA法检测大鼠血清和心肌组织中肿瘤坏死因子-α（TNF-α）、白细胞介素-17（IL-17）、白细胞介素-10（IL-10）的含量;流式细胞术测定大鼠心肌组织中Th17细胞和Treg细胞比例;Western blot法检测心肌组织JAK1、STAT3、磷酸化（p）-STAT3、维甲酸相关孤儿受体α（RORα）、叉头样转录因子3（Foxp3）、细胞因子信号转导抑制因子3（SOCS3）蛋白表达水平。结果: 与对照组比较，模型组大鼠ST段高度、T波振幅升高（P<0.01）;心肌组织炎性损伤明显，大量心肌细胞溶解坏死，纤维组织增生;Masson染色可见心肌胶原纤维数量增加，胶原容积分数（CVF）升高（P<0.01）;心肌组织和血清中TNF-α、IL-17含量升高（P<0.01），IL-10含量降低（P<0.01）;心肌组织Treg细胞比例降低（P<0.01），Th17/Treg比值升高（P<0.01）;心肌组织中JAK1、STAT3、p-STAT3、RORα蛋白表达水平升高（P<0.01，P<0.05），Foxp3、SOCS3蛋白表达水平降低（P<0.05）。与模型组比较，电针组大鼠ST段高度、T波振幅降低（P<0.05）;心肌组织炎性损伤缓解，胶原纤维数量明显减少，CVF降低（P<0.01）;心肌组织和血清中TNF-α、IL-17含量降低（P<0.05，P<0.01），IL-10含量升高（P<0.01）;心肌组织Th17细胞比例显著降低（P<0.01），Treg细胞比例升高（P<0.01），Th17/Treg比值降低（P<0.01）;心肌组织JAK1、STAT3、p-STAT3、RORα蛋白表达水平下降（P<0.01，P<0.05），Foxp3、SOCS3蛋白表达水平升高（P<0.01）。结论: 电针“内关”“足三里”可改善MI大鼠心肌炎性损伤，其机制可能与电针通过JAK1/STAT3信号通路良性调控MI大鼠心肌组织Th17/Treg细胞稳态，以平衡促炎因子TNF-α、IL-17及抗炎因子IL-10的分泌，减轻炎性损伤有关。.