安宫牛黄丸及朱砂和雄黄对脂多糖诱导的神经损伤的作用(英文)

目的探讨朱砂和雄黄在安宫牛黄丸(AGNH)配方中对脂多糖(LPS)介导的神经损伤的保护作用及机制。方法制备大鼠中脑原代神经元-神经胶质细胞联合培养模型。实验分为正常对照组、LPS 10μg.L-1模型组、LPS +朱砂(4和40 mg.L-1)组、LPS +雄黄(4和40 mg.L-1)组和LPS +AGNH(40和400mg.L-1)组。药物与细胞作用30 min后加入LPS;7 d后进行实验指标检测。采用免疫组织化学染色法检测酪氨酸羟化酶阳性的细胞数量和形态学变化以及小神经胶质细胞的激活情况,实时RT-PCR检测细胞中肿瘤坏死因子α(TNF-α)mRNA和诱导型一氧化氮合酶(iNOS)mRNA表达水平,酶联免疫吸附试验(ELISA)与Griess试剂分别检测细胞培养上清液中TNF-α和一氧化氮(NO)的含量。结果与正常对照组比较,LPS10μg.L-1作用于细胞,多巴胺能神经元的数量明显下降了40%(P<0.05);LPS显著诱导小神经胶质细胞的激活,TNF-αmRNA和iNOS mRNA的表达分别增加了9倍和2倍(P<0.05),同时神经元-胶质细胞联合培养上清液中TNF-α和NO的含量分别增加了20倍和30倍(P<0.05)。与LPS模型组比较,AGNH400mg.L-1和雄黄40 mg.L-1能明显拮抗LPS对多巴胺能神经元的毒性作用,多巴胺神经元数量分别上升了40%和30%(P<0.05);AGNH400 mg.L-1和雄黄40 mg.L-1能抑制小神经胶质细胞的激活,小胶质神经细胞中TNF-αmRNA分别下降了61%和52%(P<0.05)和iNOS mRNA的表达分别降低了58%和51%(P<0.05),而且神经元-神经胶质细胞联合培养上清液中TNF-α的含量分别降低了55%和43%(P<0.05)以及NO的含量分别下降了53%和34%(P<0.05)。而朱砂对LPS的作用无影响。结论 AGNH能改善LPS介导的神经元损伤,雄黄是其抗炎作用的有效成分之一,朱砂未见明显的神经保护作用。

Effect of Angong Niuhuang Wan, cinnabar and realgar against lipopolysaccharide-mediated neurotoxicity

OBJECTIVE To investigate the role of cinnabar and realgar in Angong Niuhuang Wan (AGNH) -produced neuroprotection against lipopolysaccharide ( LPS) -mediated neuronal damage and further explore the corresponding mechanisms. METHODS Primary rat midbrain neuron-glia cultures were used as an in vitro model to investigate effects of AGNH on LPS-mediated degeneration of dopamine (DA) neurons. The experiment was divided into normal control group, LPS model group, LPS + cinnabar (4 and 40 mg·L-1) groups, LPS + realgar (4 and 40 mg·L-1 ) groups and LPS + AGNH (40 and 400 mg·L-1 ) group. Drugs were added 30 min before LPS treatment. After 7 d, dopaminergic neurotoxicity was assessed through the quantification of tyrosine hydroxylase (TH)-positive neurons and morphological analysis of TH-positive neurons; the activation of microglia was evaluated using OX-42 antibody; the gene expression of tumor necrosis factor-α (TNF-α) and induced nitric oxide synthase (iNOS) mRNA in microglia was performed by real-time RT-PCR analysis, and the release of TNF-α and nitric oxide (NO) in the supernatant of neuron-glia cultures was determined respectively by the ELISA and Griess reagent. RESULTS Compared with normal control group, DA neurons in LPS model group decreased by 40% (P <0.05) , microglial activation was induced, the expression of TNF-α mRNA and iNOS mRNA in microglia increased 9 and 2 times, respectively ( P < 0. 05 ) , and subsequent production of TNF-α and NO in the supernatant of neuron-glia cultures increased 20 and 30 times, respectively (P<0.05). Compared with LPS model group, AGNH 400 mg·L-1 and realgar 40 mg·L-1 significantly attenuated LPS-mediated DA neuronal loss by 40% and 30% , respectively (P<0.05) and inhibited activation of microglia and expression of TNF-α mRNA by 61% and 52% (P <0.05). iNOS mRNA was reduced by 58% and 51% (P <0.05 ) in microglia. The subsequent release of TNF-α was reduced by 55% and 43% (P<0.05) and NO reduced by 53% and 34% (P<0.05) in the supernatant of neuron-glia cultures. Cinnabar had no inhibitory effect on LPS-induced changes. CONCLUSION AGNH protects LPS-induced neurotoxicity through its anti-inflammatory properties and realgar might be the key contributor to the neuroprotective action of AGNH, while cinnabar fails to show any neuroprotection.