Objective: To observe the effect of moxibustion on learning-memory ability and expression of hippocampal inflammatory factors and microtubule-associated protein doublecortin (DCX, a marker of neuronal regeneration) in vascular dementia (VD) rats, so as to explore its mechanisms underlying improvement of VD.
Methods: SD rats were randomly divided into normal control, sham operation, VD model, moxibustion and medication groups (n=15 rats in each group). The VD model was established by repeated occlusion of the bilateral common carotid arteries and reperfusion. Moxibustion was applied to "Guanyuan" (CV4), "Mingmen" (GV4) and "Dazhui"(GV14) for 15 min, once a day, 6 days a week for 4 weeks. Rats of the medication group were treated by gavage of Nimodipine (2mg·kg-1·d-1) 3 times daily for 4 weeks. Morris water maze test was used to detect the average escape latency of location navigation tasks for assessing the rats' learning-memory ability. H.E. staining was used to detect histopathological changes of the hippocampus tissue. The number of DCX-positive neurons (DCX/NeuN co-expression) in the dentate gyrus (DG) region of hippocampus was counted under microscope after immunofluorescence double staining, the immunoactivity of hippocampal DCX detected by using immunohistochemistry stain and the expression of DCX, TNF-α, IL-1β, MPO, NF-κB p65 and IL-6 proteins in the hippocampus tissue detected using Western blot.
Results: Following modeling, the average escape latency was significantly longer in the model group than in the normal control and sham operation groups (P<0.01), and notably shorter in both the moxibustion and medication groups than in the model group after the treatment (P<0.01, P<0.05). The number of DCX-positive neurons, and the expression levels of DCX, TNF-α, IL-1β, MPO, NF-κB p65 and IL-6 proteins in the hippocampus were significantly increased in the model group in comparison with the normal control and sham operation groups (P<0.01, P<0.05). After the interventions and in comparison with the model group, the number of DCX-positive neurons and the expression level of DCX were further up-regulated in both moxibustion and medication groups (P<0.01), while the expression levels of hippocampal TNF-α, IL-1β, MPO, NF-κB p65 and IL-6 proteins were considerably down-regulated in the moxibustion and medication groups (P<0.01). The effect of moxibustion was weaker than that of medication in down-regulating the expression of TNF-α,MPO, NF-κB p65, IL-6 and IL-1β, and in up-regulating DCX-positive neuron number and DCX expression (P<0.05, P<0.01). H.E. staining showed loose arrangement of neurons (with vague neuronal membrane in some cells), uneven organelle chromatin, disappearance of partial nucleolus, necrocytosis, and infiltration of small number of lymphocytes after modeling, which was relatively milder in both moxibustion and medication groups.
Conclusion: Moxibustion can improve learning-memory ability in VD rats, which may be related to its effect in down-regulating the expression of inflammatory factors and up-regulating the expression of DCX to promote neuronal repair and regeneration.
目的：观察艾灸对血管性痴呆(VD)大鼠炎性因子及神经元再生标志物微管相关蛋白(DCX)表达的影响，探讨艾灸抑制炎性反应，促进神经元修复，改善VD的作用机制。方法：将SD大鼠随机分为正常组、假手术组、模型组、艾灸组、西药组，每组15只。用双侧颈总动脉缺血再灌注法建立VD大鼠模型。艾灸组给予悬灸“关元”“命门”“大椎”治疗，每穴15 min，每日1次，每周休息1 d，连续治疗4周；西药组给予尼莫地平灌胃治疗，每日3次，连续治疗4周。应用Morris水迷宫实验检测大鼠逃避潜伏期，HE染色法检测大鼠海马组织病理形态，免疫荧光双标记法检测大鼠海马DG区DCX与NeuN共表达强度，用免疫组织化学法检测DG区DCX阳性表达，Western blot法检测大鼠海马组织DCX、肿瘤坏死因子-α(TNF-α)、髓过氧化物酶(MPO)、核转录因子κB p65(NF-κB p65)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)表达。结果：造模后模型组大鼠逃避潜伏期较正常组与假手术组延长(P<0.01)，治疗后艾灸组与西药组大鼠逃避潜伏期较模型组明显缩短(P<0.01，P<0.05)。模型组海马组织神经元较正常组、假手术组排列紊乱，可见大量坏死神经元及少量淋巴细胞浸润；艾灸组与西药组细胞形态接近正常。免疫荧光双标记检测显示，与正常组、假手术组比较，模型组大鼠DCX/NeuN共表达细胞数显著增加(P<0.01)；与模型组比较，艾灸组与西药组DCX/NeuN共表达细胞数均显著增加(P<0.05，P<0.01)，艾灸组DCX/NeuN共表达细胞少于西药组(P<0.05)。免疫组织化学染色结果显示，模型组DCX阳性表达高于正常组与假手术组(P<0.01，P<0.05)，艾灸组、西药组高于模型组(P<0.01)，艾灸组略低于西药组(P<0.05)。模型组海马组织促炎因子TNF-α、MPO、NF-κB p65、IL-6、IL-1β及DCX蛋白相对表达量明显高于正常组和假手术组(P<0.01)；与模型组比较，艾灸组和西药组大鼠各炎性因子表达明显下降(P<0.01)，DCX相对表达量显著增加(P<0.01)；与西药组比较，艾灸组大鼠海马组织中促炎因子表达下降(P<0.05，P<0.01)，DCX相对表达量下降(P<0.01)。结论：艾灸可通过抑制炎性损伤，促进神经细胞修复再生，改善VD大鼠记忆与学习能力。.
Keywords: Doublecortin; Inflammatory factors; Learning-memory ability; Moxibustion; Vascular dementia.