Objective: To observe the impact of electro-scalp acupuncture (ESA) on the neural function and inflammatory response of ischemic cortex in the model rats with ischemic stroke and explore the anti-inflammation mechanism of ESA in treatment of ischemic stroke from the perspective of modulating the interleukin 12 (IL-12) mediated JAK (Janus kinase)/STAT (signal transduction and transcription activator) signal pathway.
Methods: Ninety male SD rats were randomized into a normal group (n =16) and a model preparation group (n=74). In the model preparation group, the model of middle cerebral artery occlusion (MCAO) was duplicated with suture-occlusion method. After modeled successfully, 48 rats with neurological deficit score of 1-3 were divided into a model group, an inhibitor group and an ESA group, 16 rats in each one. In the inhibitor group, IL-12 inhibitor (apilimod, 5 mg/kg) was used via intragastric administration. In the ESA group, the anterior oblique line of vertex-temporal (MS6) was stimulated bilaterally with electric acupuncture, with disperse-dense wave, 2 Hz/100 Hz in frequency, 1 mA in current intensity. The needles were retained for 30 min. The treatment was given once daily and for 7 days in above two intervention groups. Before and after intervention, the neurological deficit score (NDS) and neurobehavioral score (NBS) were assessed in each group. HE staining method was adopted to observe the morphological manifestations of ischemic cortical lesion; the concentrations of IL-12 and IL-12R of the brain tissue in the ischemic cortical lesion were measured by enzyme-linked immunosorbent assay (ELISA); the mRNA expression levels of STAT4 and Tbx21 were detected by real-time PCR technique; and the protein expression of IL-2, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-4 were detected using immunohistochemistry.
Results: NDS and NBS in the model group, the inhibitor group and the ESA group were all higher than those in the normal group before intervention (P<0.01). After intervention, NDS and NBS in the model group were higher than the normal group (P<0.01); the two scores were all reduced when compared with those before intervention in the inhibitor group and the ESA group (P<0.01), and lower than those of the model group (P<0.01). NDS in the ESA group was lower than the inhibitor group (P<0.05). In the model group, the cells were shrunk and vacuolated in the ischemic cortical lesion. Many normal cells were visible in the ESA group and the inhibitor group. Compared with the normal group, the concentrations of IL-12 and IL-12R , the mRNA expression levels of STAT4 and Tbx21 and the protein expression levels of IL-2, TNF-α and IFN-γ in brain tissue of ischemic cortical lesion were all increased in the model group (P<0.01), while the protein expression level of IL-4 decreased (P<0.01). The concentrations of IL-12 and IL-12R, the mRNA expression levels of STAT4 and Tbx21 and the protein expression levels of IL-2, TNF-α and IFN-γ were all reduced (P<0.01), while the protein expression level of IL-4 increased (P<0.01) in the ESA group and the inhibitor group when compared with the model group. The concentration of IL-12, the mRNA expression levels of STAT4 and Tbx21 and the protein expression levels of IL-2, TNF-α and IFN-γ in the ESA group were all higher than those of the inhibitor group (P<0.05); while the concentration of IL-12R and the protein expression level of IL-4 were lower than the inhibitor group (P<0.05).
Conclusion: Electro-scalp acupuncture may improve the neurological function of the rats with ischemic stroke. The modulation to IL-12 mediated JAK/STAT signaling pathway is the potential molecular mechanism of this therapy for the inflammatory response in ischemic cortical lesion.
目的：观察电针头穴对缺血性脑卒中模型大鼠神经功能及缺血皮质区炎性反应的影响，从调控白细胞介素（IL）-12介导的Janus激酶（JAK）/信号转导和转录激活因子（STAT）信号通路角度探讨电头针治疗缺血性脑卒中的抗炎机制。方法：将90只雄性SD大鼠随机分为正常组（16只）和造模组（74只）。造模组大鼠以线栓法复制大脑中动脉闭塞（MCAO）模型，将造模成功且神经功能缺损评分1~3分的48只大鼠分为模型组、抑制剂组和电头针组，各16只。抑制剂组予IL-12抑制剂（apilimod，5 mg/kg）灌胃；电头针组电针双侧顶颞前斜线，予疏密波，频率2 Hz/100 Hz，电流强度1 mA，留针30 min，两组均每日1次，共干预7 d。分别于干预前后评定各组大鼠神经功能缺损评分和神经行为学评分。HE染色法观察各组大鼠缺血皮质区形态学表现，ELISA法检测各组大鼠缺血皮质区脑组织IL-12、IL-12R含量，real-time PCR法检测缺血皮质区脑组织STAT4、Tbx21 mRNA表达，免疫组化法检测缺血皮质区脑组织IL-2、肿瘤坏死因子（TNF）-α、干扰素（IFN）-γ、IL-4蛋白表达。结果：干预前，模型组、抑制剂组和电头针组大鼠神经功能缺损评分及神经行为学评分均高于正常组（P<0.01）。干预后，模型组大鼠神经功能缺损评分及神经行为学评分均高于正常组（P<0.01）；抑制剂组与电头针组大鼠两项评分均较干预前降低（P<0.01），且均低于模型组（P<0.01）；电头针组大鼠神经功能缺损评分低于抑制剂组（P<0.05）。模型组缺血皮质区细胞皱缩，可见空泡形成；电头针组与抑制剂组可见较多正常细胞。与正常组比较，模型组大鼠缺血皮质区脑组织IL-12、IL-12R含量，STAT4、Tbx21 mRNA表达及IL-2、TNF-α、IFN-γ蛋白表达升高（P<0.01）；IL-4蛋白表达降低（P<0.01）。与模型组比较，电头针组与抑制剂组大鼠IL-12、IL-12R含量，STAT4、Tbx21 mRNA表达及IL-2、TNF-α、IFN-γ蛋白表达降低（P<0.01）；IL-4蛋白表达升高（P<0.01）。电头针组IL-12含量，STAT4、Tbx21 mRNA表达及IL-2、TNF-α、INF-γ蛋白表达高于抑制剂组（P<0.05）；IL-12R含量及IL-4蛋白表达低于抑制剂组（P<0.05）。结论：电头针可改善缺血性脑卒中大鼠神经功能，调控IL-12介导的JAK/STAT信号通路是其改善缺血皮质区炎性反应的可能分子机制。.
Keywords: JAK/STAT signaling pathway; anti-inflammatory mechanism; electro-scalp acupuncture; inflammatory response; interleukin 12 (IL-12); ischemic stroke.