Objectives: To observe the effect of electroacupuncture (EA) of scalp acupoint (Dingnieqian-xiexian, MS6) on expression of retinoid-related orphan receptor γT (ROR γ t), interleukin (IL)-17A, IL-10, transfor-ming growth factor-β1 (TGF-β1), IL-6, IL-21, and IL-17A+ Thelper cells(Th) 17 and forkhead transcription factor P3 (FOXP3)+ regulatory T cells (Treg) differentiation of ischemic cortex in ischemic stroke rats, so as to explore its molecular mechanisms underlying relief of inflammatory injury of ischemic stroke.
Methods: A total of 120 male SD rats were randomly assigned to sham operation, model, EA, inhibitor, agonist and EA+agonist groups, with 15 rats in each group. The ischemic stroke model was established by occlusion of the left middle cerebral artery according to Longa's methods. For rats of the EA group and EA+agonist group, EA (2 Hz/100 Hz, 1 mA) was applied to bilateral MS6 for 30 min, once daily for 7 days. Rats of the inhibitor group received intraperitoneal injection of solution of SR1001 (RORγt inhibitor) (2.5 mg/mL, 10 mg/kg), once daily for 7 days. Rats of the agonist and EA+agonist groups received intraperitoneal injection of solution of SR1078 (RORγt agonist) (5 mg/mL, 5 mg/kg) before EA, once daily for 7 days. Rats of the sham operation and model groups were grabbed and fixed in the same way with the other groups. The Zea-longa's score, modified neurological severity score (mNSS) and the neurobehavioral score were assessed before and after the intervention. At the end of experiments, the ischemic cortex tissue was collected. The 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining was used to detect the volume of cerebral infarction. The expression of RORγt mRNA was detected by real-time quantitative PCR；the protein expression levels of RORγt, IL-17A, IL-10 and TGF-β1 were detected by Western blot；the immunoactivity of IL-6 and IL-21 were detected by immunohistochemistry；the fluorescence areas of IL-17A+Th17 and FOXP3+Treg cells were measured by immunofluorescence and their ratio was calculated in the tissue of ischemic cortex.
Results: Relevant to the sham operation group, the model group had a significant increase in the Zea-Longa's score, mNSS score, neurobehavioral score, cerebral infarct volume, expression levels of RORγt mRNA and protein, IL-17A protein, IL-6 and IL-21 immunoactivity, IL-17A+Th17 immunofluorescence intensity, and the ratio of IL-17A+Th17/FOXP3+Treg (P<0.01), and an obvious decrease in the expression levels of TGF-β1 and IL-10 proteins and FOXP3+Treg immunofluorescence intensity (P<0.01). In contrast to the model group, both EA and inhibitor groups had a significant decrease in the Zea-Longa's score, mNSS score, neurobehavioral score, cerebral infarct volume, expression levels of RORγt mRNA and protein, IL-17A protein, IL-6 and IL-21 immunoactivity, IL-17A+Th17 immunofluorescence intensity, and the ratio of IL-17A+Th17/FOXP3+Treg (P<0.01, P<0.05), and a marked increase in the expression levels of TGF-β1 and IL-10 proteins and FOXP3+Treg immunofluorescence intensity (P<0.05, P<0.01), while the above indicators of the agonist group were all reversed (P<0.01, P<0.05). Comparison between the agonist and EA+agonist groups showed that the Zea-Longa's score, mNSS score, neurobehavioral score, cerebral infarct volume, expression levels of RORγt mRNA and protein, IL-17A protein, IL-6 and IL-21 immunoactivity, IL-17A+Th17 immunofluorescence intensity, and the ratio of IL-17A+Th17/FOXP3+Treg were significantly lower (P<0.01, P<0.05), and the expression of TGF-β1 and IL-10 proteins and FOXP3+Treg immunofluorescence intensity were obviously higher (P<0.01, P<0.05) in the EA+agonist group than in the agonist group, suggesting that EA intervention can effectively weaken the effects of RORγt agonist.
Conclusions: EA of scalp acupoint MS6 can effectively improve the neurological function, behavior reaction and reduce cerebral infarct volume in ischemic stroke rats, which may be associated with its functions in down-regulating the expression of RORγt and promoting the balance of IL-17A+Th17/FOXP3+Treg to alleviate inflammatory injury after ischemic stroke.
目的: 观察电头针干预对局灶性脑缺血大鼠缺血皮层区维甲酸相关孤儿受体γt（RORγt）、辅助性T细胞（Th）17和调节性T细胞（Treg）的影响，从调控免疫细胞平衡的角度探讨电头针缓解缺血性脑卒中炎性损伤的分子机制。方法: SD大鼠随机分为假手术组、模型组、电头针组、抑制剂组、激动剂组、电头针+激动剂组，每组15只。采用线栓法复制大脑中动脉栓塞大鼠模型。电头针组取双侧顶颞前斜线电针干预，每次30 min；抑制剂组给予RORγt抑制剂SR1001溶液腹腔注射；激动剂组给予RORγt激动剂SR1078溶液腹腔注射；电头针+激动剂组在腹腔注射SR1078溶液基础上再行电头针干预；所有干预均1次/d，连续7 d。干预前、后对各组大鼠行Zea-Longa评分，神经功能缺损量表（mNSS）评分和神经行为学评分。TTC染色法检测脑梗死体积；实时荧光定量PCR法检测脑缺血区皮层中RORγt mRNA的表达水平；Western blot法检测脑缺血区皮层中RORγt、白细胞介素（IL）-17A、IL-10和转化生长因子β1（TGF-β1）的蛋白表达水平；免疫组织化学法检测脑组织中IL-6、IL-21的阳性表达；免疫荧光染色法检测脑缺血区皮层中IL-17A+Th17和叉头状转录因子P3（FOXP3）+Treg细胞的阳性表达面积并计算其比值。结果: 与假手术组比较，模型组Zea-Longa评分、mNSS评分与神经行为学评分升高（P<0.01），脑梗死体积增大（P<0.01），缺血区皮层RORγt mRNA及蛋白、IL-17A蛋白表达水平升高（P<0.01），IL-10、TGF-β1蛋白表达水平降低（P<0.01），IL-6、IL-21阳性表达升高（P<0.01），IL-17A+Th17/FOXP3+Treg阳性表达面积比值升高（P<0.01）。与模型组比较，电头针组和抑制剂组Zea-Longa评分、mNSS评分与神经行为学评分降低（P<0.01），脑梗死体积显著缩小（P<0.01），RORγt mRNA及蛋白、IL-17A蛋白表达水平降低（P<0.01，P<0.05），IL-10、TGF-β1蛋白表达水平升高（P<0.05，P<0.01），IL-6、IL-21阳性表达降低（P<0.01），IL-17A+Th17/FOXP3+Treg阳性表达面积比值降低（P<0.01）；而激动剂组以上指标的结果均呈相反趋势（P<0.01，P<0.05）。与激动剂组比较，电头针+激动剂组Zea-Longa评分、mNSS评分与神经行为学评分降低（P<0.01），脑梗死体积显著缩小（P<0.05），RORγt mRNA及蛋白、IL-17A蛋白表达水平降低（P<0.01，P<0.05），IL-10、TGF-β1蛋白表达水平升高（P<0.01，P<0.05），IL-6、IL-21阳性表达降低（P<0.05，P<0.01），IL-17A+Th17/FOXP3+Treg阳性表达面积比值降低（P<0.01）。结论: 下调RORγt的表达，促进IL-17A+Th17/FOXP3+ Treg比例恢复平衡可能是电头针缓解缺血性脑卒中炎性神经损伤的机制之一。.
Keywords: Acute ischemic stroke; Electroacupuncture of scalp acupoint; Regulatory T cells; Retinoid-related orphan receptor γt; T helper cells.