摘要
目的:探讨含当归芍药散简方脑脊液的神经保护作用及其可能的机制。方法:采用FeSO4和H2O2作用产生自由基的方法诱导建立PC12细胞氧化损伤模型,分别用当归芍药散简方(0.135 g·mL-1)ig家兔(1.5 g·kg-1),于1,14 d时取脑脊液添加于培养液中(每组分5%,10%,20%3个体积分数)处理PC12细胞,MTT法测定细胞活性,硫代巴比妥酸(TBA)比色法测定丙二醛(MDA)含量,黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活力,甲基百里香酚蓝(MTB)比色法测定细胞内Ca2+水平,免疫细胞化学法测定α7烟碱型乙酰胆碱受体(nAchR)阳性表达,BCA蛋白定量及斑点印迹法测定α7nAchR亚单位水平。结果:含20%当归芍药散简方脑脊液对PC12细胞活性(以吸光度表示,A)1 d组(1.241±0.117)和14 d组(1.297±0.213)与损伤组(0.986±0.051)比较能明显增加PC12细胞活性(P<0.05,P<0.01)、显著提高SOD活力(19.48±0.34),(19.52±0.33)U·mL-1对(18.18±0.12)U·mL-1(P<0.01),有效降低MDA含量及细胞内Ca2+水平均降低,与损伤组比较P<0.01;明显上调α7nAChR的表达,14 d组与1 d组比较有显著性差异(P<0.05)。结论:当归芍药散简方对FeSO4和H2O2诱导建立PC12细胞氧化损伤模型具有保护作用,其机制与保护α7nAchR有关。
Objective： To explore the protective effect of cerebrospinal fluid（CSF） with the optimized Danggui Shaoyao San（FBD） and its underlying mechanisms.Method： PC12 cells were treated with H2O2 / Fe2＋ to reproduce the cell injury model.The rabbits were randomly divided into 1 d group and 14 d group and they were given FBD by intragastric administrate.PC12 cells were treated with different concentrations of CSF containing FBD（5%,10%,20%）.Cell survival was assessed by Thiazzolyl blue（MTT） assay.The content of maleic diadehyde（MDA） and superoxide dismutase（SOD） activity were determined respectively by thiobarbituric acid and xanthine oxidation enzyme.The Ca2＋content within cell was measured by MTB colorimetry.The protein expression of α7 nicotinic acetylcholine receptor（nAchR） was examined by using immunocytochemical detecting kit,bicinchoninic acid（BCA） protein kit and Dot blot assay.Result： The 20% group for 1 day（1.241±0.117） and 14 days（1.297±0.213） of FBD improved the survival of the PC12.The content of SOD increased from（18.18±0.12） U·mL-1 in the 20% group for 1 d to（19.48±0.34）U·mL-1 and 14 d（19.52±0.33） U·mL-1（P0.01）.In addition,α7nAChR in PC12 cells was significantly up-regulated.The expression of α7nAChR in 20% group for 14 d（0.161±0.024） was significantly higher than that in the group for 1 d（0.132±0.021）（P0.05）.These groups reduced MDA and Ca2＋within cell from（3.26±0.13）mmol·L-1,（74.89±3.66）nmol/105 cell and（2.77±0.05）mmol·L-1,（74.25±2.64）nmol/105 cell to（4.41±0.23）mmol·L-1,（82.42±4.25） nmol/105 cells）.Conclusion： The CSF containing FBD can prevent from the neurotoxicity induced by H2O2 / Fe2＋.It may be related to α7nAchR up-regulation.
出处
《中国实验方剂学杂志》
CAS
北大核心
2012年第9期165-168,共4页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81060231,81160338)
国家中医药管理局项目(04-05zp62)
