Objective: To observe the effect of electroacupuncture (EA) on the pyrolysis of dopaminergic neurons in rats with Parkinson's disease (PD), so as to explore its underlying molecular mechanism.
Methods: Male SD rats were randomly divided into the sham operation, model, EA, MCC950 and EA+MCC950 groups, with 12 rats in each group. The PD rat model was established by two-point injection of 6-OHDA. Rats in the MCC950 group were injected with MCC950 at the dose of 10 mg/kg, once a day; for rats of EA group, EA was applied to "Taichong" (LR3) and "Fengfu"(GV16) for 30 min, once a day; rats in the EA+ MCC950 group were given MCC950 injection and EA once a day. All above interventions were performed for 2 weeks. After the intervention, the behavioral changes of rats were observed using rotating induction experiment, rotating rod experiment and open field experiment; the positive expressions of dopaminergic neuronal markers tyrosine hydroxylase (TH) and α-Synuclein (α-Syn) in substantia nigra striatum were detected by immunohistochemistry; the damage of dopaminergic neurons in substantia nigra striatum was observed after HE staining; immunopositive coexpression of brain nucleotide-binding oligomerization domain like receptor protein 3 (NLRP3), Caspase-1 and ionized calcium binding adapter1 (Iba-1) were detected by immunofluorescence double staining; the levels of interleukin (IL)-1β and IL-18 in brain tissues were detected by ELISA; the protein expression levels of NLRP3, Cleaved Caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) in the substantia nigra striatum were detected by Western blot.
Results: Compared with the sham operation group, the number of rotations of rotating induction experiment, the residence time in the central area of open field experiment, the positive expression of α-Syn, the positive co-expressions of NLRP3/Iba-1 and Caspase-1/Iba-1, the contents of IL-1β and IL-18 in brain tissues, and the protein expression levels of NLRP3, ASC and Cleaved Caspase-1 in substantia nigra striatum were significantly increased (P<0.05), while the drop latency of rotating rod experiment, the rearing times and the total distance of open field experiment, and the positive expression of TH in substantia nigra striatum were significantly decreased (P<0.05) in the model group. Compared with the model group, the above mentioned markers were reversed in EA, MCC950 and EA+MCC950 groups (P<0.05), and the improvement of the EA+MCC950 group was more obvious than those of the MCC950 and EA groups. In the model group, the neurons were disorderly arranged, the number of neurons was reduced, the cytoplasm was swollen, and some of them were vacuolar degeneration; while the degree of neuronal arrangement disorder, cytoplasmic swelling and the vacuolar degeneration were reduced in varying degrees in the MCC950, EA and EA+MCC950 groups.
Conclusion: The ameliorative effect of EA on dopaminergic neuron damage in PD rats may be related to its effects in inhibiting NLRP3/Caspase-1 mediated neuronal pyrosis.
目的：观察电针对帕金森病(PD)大鼠多巴胺能神经元焦亡的影响，探讨其可能的分子机制。方法：SD大鼠随机分为假手术组、模型组、电针组、MCC950组、电针+MCC950组，每组12只。采用6-羟基多巴胺两点注射法制备PD大鼠模型。MCC950组大鼠按10 mg/kg腹腔注射MCC950，1次/d；电针组予电针“太冲”及“风府”，每次30 min，1次/d；电针+MCC950组在电针组基础上按10 mg/kg给予腹腔注射MCC950，1次/d。以上干预均连续2周。干预后，采用诱导旋转实验、转棒实验和旷场实验观察大鼠行为学改变；免疫组织化学法检测中脑黑质多巴胺能神经元标志物酶酪氨酸羟化酶(TH)、α-突触核蛋白(α-Syn)的阳性表达；HE染色法观察大鼠中脑黑质纹状体多巴胺能神经元损伤程度；免疫荧光双染法检测脑组织核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、半胱天冬氨酸蛋白酶1(Caspase-1)和离子钙接头蛋白1(Iba-1)的免疫阳性共表达；ELISA法检测脑组织中白细胞介素(IL)-1β、IL-18的含量；Western blot法检测中脑黑质纹状体NLRP3、剪切的半胱天冬氨酸蛋白酶-1(Cleaved Caspase-1)、凋亡相关斑点样蛋白(ASC)的蛋白表达水平。结果：与假手术组比较，模型组大鼠的旋转圈数、中央区域停留时间、中脑黑质α-Syn阳性表达均增加(P<0.05)；掉落潜伏期、直立次数、运动总距离、中脑黑质TH阳性表达均减少(P<0.05)；脑组织NLRP3、Caspase-1阳性表达，IL-1β、IL-18含量，以及黑质纹状体中NLRP3、ASC、Cleaved Caspase-1蛋白表达水平均升高(P<0.05)。与模型组比较，电针组、MCC950组和电针+MCC950组大鼠的上述指标均发生逆转(P<0.05)。与MCC950组和电针组比较，电针+MCC950组的旋转圈数、中央区域停留时间减少(P<0.05)，掉落潜伏期、直立次数和运动总距离增加(P<0.05)；中脑黑质TH阳性表达增加(P<0.05)，α-Syn阳性表达减少(P<0.05)；脑组织NLRP3、Caspase-1阳性表达，IL-1β、IL-18含量，以及黑质纹状体中NLRP3、ASC、Cleaved Caspase-1蛋白表达水平均降低(P<0.05)。模型组大鼠神经元排列紊乱，数量减少，胞质肿胀，部分空泡样变性；MCC950组、电针组和电针+MCC950组大鼠神经元排列紊乱程度和胞质肿胀程度较模型组有不同程度的减轻，空泡样变性减少，且电针+MCC950组神经元损伤改善程度优于MCC950组和电针组。结论：电针对PD大鼠多巴胺能神经元损伤的改善作用可能与抑制NLRP3/Caspase-1介导的神经元焦亡有关。.
Keywords: Electroacupuncture; NLRP3/Caspase-1; Neuronal pyrosis; Parkinson’s disease.