蕨麻正丁醇提取物抑制大鼠海马神经元缺氧所致一氧化氮生成的研究

An experimental study on inhibitory effects and mechanisms of N-butanol extract of Potentilla anserine L. against hypoxia-induced nitric oxide on hippocampus neurons of rats

目的研究蕨麻正丁醇提取物（NP）对大鼠海马神经元急性缺氧所致一氧化氮（NO）生成的抑制作用。方法建立体外原代培养海马神经元缺氧损伤实验模型，随机分为空白对照组、缺氧模型组、尼莫地平组（2μmol/L）及NP高（250.0 mg/L）、中（62.5 mg/L）、低（15.6 mg/L）剂量组。用四甲基偶氮唑盐（MTT）比色法检测海马神经元细胞活性，同时检测其一氧化氮（NO）含量；用半定量逆转录-聚合酶链反应（RT-PCR）、蛋白质免疫印迹试验（Western blotting）分别检测各组神经型一氧化氮合酶（nNOS）mRNA及蛋白的表达，用免疫细胞化学染色法检测nNOS阳性率。结果与空白对照组比较，缺氧模型组神经元细胞活性〔吸光度（A）值〕明显降低（0.0826±0.0095比0.3315±0.0105），NO含量（μmol/g：0.0509±0.0027比0.0291±0.0032）、nNOS mRNA表达（A值：0.1463±0.0081比0.0801±0.0058）、nNOS阳性率〔（74.4238±3.9423）%比（28.3714±4.1361）%〕及nNOS蛋白表达（A值：1.9130±0.0471比0.5068±0.0368）均明显升高（均P＜0.01）。与缺氧模型组比较，各药物组缺氧神经元细胞活性均增加，NO含量、nNOS mRNA及蛋白表达均降低，以高剂量组变化更显著〔神经元细胞活性：0.1681±0.0118，NO含量：0.0319±0.0044，nNOS mRNA表达：0.0648±0.0032， nNOS阳性率：（40.1240±6.4900）%，nNOS蛋白表达：1.3924±0.0621，均P＜0.01〕；而NP低剂量组与模型组比较差异均无统计学意义（均P＞0.05）。结论 NP能减轻缺氧对体外培养大鼠海马神经元的损伤，其机制可能为有效抑制nNOS合成NO的过度升高。

Objective To study in vitro the inhibitory effects and mechanisms of N-butanol extract of Potentilla anserine L.（NP）against hypoxia-induced nitric oxide（NO）in hippocampus neuron of rats. Methods The models of hippocampus neurons hypoxia injury of Sprague-Dawley（SD）neonatal rats were cultured in vitro. The cultured hippocampus neurons were divided randomly into blank control group, hypoxia injury model group, nimodipine group（2 μmol/L）and NP high（250.0 mg/L）,middle（62.5 mg/L）,low（15.6 mg/L）dose groups. The activities of hippocampus neurons were examined by methyl thiazolyl tetrazolium（MTT）assay,and meanwhile their contents of nitrogen monoxidum（NO）were detected. Half quantity reverse transcription-polymerase chain reaction（RT-PCR）and Western blotting were used to detect neuronal nitric oxide synthetase（nNOS）mRNA and protein expression levels respectively in each group,immunocytochemistry stain was used to detect protein positive rate. Results Compared with blank control group,the activity of neuron〔absorbance（A）value〕was significantly decreased（0.0826±0.0095 vs. 0.3315±0.0105）,content of NO（μmol/g：0.0509±0.0027 vs. 0.0291±0.0032）, the expression levels of nNOS mRNA （0.1463±0.0081 vs. 0.0801±0.0058）, the positive rate of nNOS〔（74.4238±3.9423）%vs.（28.3714±4.1361）%〕,the expression levels of nNOS protein（A value：1.9130±0.0471 vs. 0.5068±0.0368）were all significantly increased in the hypoxia injury model group（all P0.05）. Conclusions NP can ameliorate the injury of rat hippocampus neurons induced by hypoxia in vitro. The possible mechanisms might be related to the effective inhibition of the synthesis of nNOS and NO excessive generation.