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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 neuroprotective effects of Danggui Shaoyao San on cerebrospinal fluid and its possible mechanism. Methods: The oxidative injury model of PC12 cells was established by FeSO 4 and H 2 O 2 induced free radicals. The rats were induced with 1.5 g · kg -1 of Danggui Shaoyao San (1.5 g · kg -1) PC12 cells were treated with cerebrospinal fluid (5%, 10% and 20%, respectively) on 14th day. Cell viability was assayed by MTT assay and assayed by thiobarbituric acid (TBA) The content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) by xanthine oxidase method and the level of intracellular Ca2 + by methylthiotoluene blue (MTB) colorimetric assay, and the activity of α7 nicotine by immunocytochemistry Acetylcholine receptor (nAchR) positive expression, BCA protein quantification and dot blot assay α7nAchR subunit levels. Results: Compared with injured group (0.986 ± 0.051), the activity of PC12 cells containing 20% Danggui Shaoyao San (PC12) in a dose of 1 d (1.241 ± 0.117) and 14 d (1.297 ± 0.213) (P <0.05, P <0.01), significantly increased the activity of SOD (19.48 ± 0.34), (19.52 ± 0.33) U · mL-1 vs 18.18 ± 0.12 U · mL- , Decreased the content of MDA and decreased the level of Ca2 + in the cells. Compared with the injury group, the level of α7nAChR was significantly increased (P <0.01). There was a significant difference between the 14th and the 1st days (P <0.05). Conclusion: Danggui Shaoyao Sanzhao can protect PC12 cells against oxidative damage induced by FeSO4 and H2O2, and its mechanism is related to the protection of α7nAchR.