通窍活血汤含药血清对谷氨酸损伤的PC12细胞的保护作用

Neuroprotective effects of serum with Tongqiao Huoxue decoction (TQHXD) against glutamate-induced neurotoxicity in PC12 cells

目的:从细胞水平研究通窍活血汤含药血清(TQHXD)对谷氨酸(Glu)损伤的PC12细胞的保护作用,为该方剂的临床应用提供依据。方法:SD大鼠灌胃给予通窍活血汤提取液,每日2次,每次4 g.kg-1,连续5 d,制备通窍活血汤含药血清。采用PC12细胞和终浓度为12.5 mmol.L-1的谷氨酸共培养,造成神经细胞损伤模型。光镜观察5%,10%,20%的TQHXD对损伤PC12细胞形态改变的影响;台盼兰染色法、AO/EB染色法及LDH法观察TQHXD对损伤的细胞膜通透性的变化影响;MTT法检测TQHXD对PC12细胞增殖及活性的影响及对培养上清液中NO浓度的影响。结果:倒置显微镜下可见,正常组细胞胞体较损伤组略大,呈强折光性,细胞数量多,突起长。模型组细胞数量显著减少,折光性明显减弱,细胞突起减少、缩短。TQHXD和PC12细胞共培养24 h后,活细胞数明显增加,细胞折光性增加,损伤细胞形态接近于正常细胞;台盼兰染色后,TQHXD组活细胞比率明显上升,AO/EB染色后被EB染色的数量较少。MTT法检测结果显示,与模型组比较,各浓度TQHXD组的吸光度A均升高且有显著性差异。各浓度的TQHXD组细胞培养上清中LDH,NO含量相对模型组明显减少,两者有显著性差异。结论:TQHXD对Glu损伤的PC12细胞有明显的保护作用。

Objective： To observe the protective effect of serum with Tongqiao Huoxue decoction（TQHXD） on PC12 cells damaged by glutamate（Glu）and provide clinical proof of the formulae.Method： Sprague Dawley rats underwent intragastric administration of Tongqiao Huoxue decoction twice a day for five days,the administration dose for rats was 4 g·kg-1.Thus the serum containing TQHXD was prepared.The model of neurocytes damaged by Glu had been established by adding 12.5 mmol·L-1 Glu to culture medium of PC12 cells.Photic microscope had been used to observe the morphologic changes of cells,the proliferation and activity of PC12 cells had been detected by MTT.Cell membrane permeability had been investigated by the methods of the coloration of trypan blue and AO/EB,the activities of LDH and the contents of NO in the supernatant of PC12 cells had been detected after 5%,10%,20% TQHXD being added to the culture medium of PC12 cells damaged by Glu.Result： The morphologic changes in cells were observed under the inverted microscope.Normal PC12 cells in control group were full and bright,but the cells exposed to Glu were shrank;moreover,some cells were disrupted,configuration of the cells in the TQHXD group was closed to that of normal group.Compared with model group,the ratio of living cells in the group being treated by TQHXD after trypan blue staining had a significant increase.After AO/EB staining,cells in TQHXD groups were less being stained by EB.The absorbance（A） values of TQHXD group increased largely.LDH and NO in cell culture supernatant had decreased,it had shown the marked difference.Conclusion： TQHXD showed an apparent protective effect on neurocytes damaged by Glu on proliferation activity and membrane permeability.