中药复方更年春含药血清及其主要单体对β淀粉样蛋白所致细胞损伤的保护作用

An in vitro study on neuroprotective effects of serum containing Gengnianchun decoction and its main monomers against amyloid beta protein-induced cellular toxicity

目的:观察中药复方更年春含药血清及其主要单体成分对β淀粉样蛋白(amyloidβ-protein,Aβ)所致肾上腺嗜铬细胞瘤细胞系PC12细胞损伤的保护作用。方法:采用Aβ25-35作用PC12细胞构建阿尔茨海默病(Alzheimers disease,AD)细胞模型,相差显微镜观察细胞形态改变。运用中药血清药理学方法制备大鼠更年春含药血清,以四甲基偶氮唑盐法观察更年春含药血清及其主要单体芍药苷、黄连素、知母皂苷A-Ⅲ、淫羊藿苷对体外培养的PC12细胞活力的影响及其拮抗Aβ损伤的作用,采用流式细胞仪观察更年春复方含药血清及复方主要单体对Aβ诱导的PC12细胞凋亡的影响。结果:PC12细胞在Aβ25-35作用后,有活力细胞数目减少,细胞间连接较松,胞浆较暗淡,细胞碎片较多,贴壁细胞欠透明,部分发生皱缩,胞浆中有较多颗粒。Aβ25-35剂量、时间依赖性地抑制PC12细胞的活力。更年春含药血清可以增强PC12细胞活力,不同浓度更年春含药血清培养细胞24、48、72h后,其增强PC12细胞活力均在20%浓度作用最强。更年春复方含药血清及复方主要单体之一的黄连素及主要单体混合液(包括芍药苷、黄连素、知母皂苷A-Ⅲ、淫羊藿苷)均有拮抗Aβ对PC12细胞损伤的作用,且均能抑制Aβ诱导的PC12细胞早期凋亡,其中更年春含药血清作用最强,中药单体混合液作用其次,而单体黄连素作用最弱。结论:更年春对AD细胞模型有保护作用,其含药血清作用强于主要中药单体及主要单体的混合液。

Obiective. To observe the effects of serum containing Gengnianchun （GNC） decoction, a compound traditional Chinese herbal medicine, and its monomers （paeoniflorin, berberine, timosaponin A-Ⅲ and ioariin） on neurotoxicity in PC12 cells induced by amyloid beta-protein （Aβ）. Metheds. Injury of PC12 cells was induced by incubating with Aβ2-35 in vitro. Ovariectomized rats were intragastrically administered with GNC decoction twice daily for 5 days and then sera were obtained. PC12cells were cultured with different concentrations of serum containing GNC decoction and its main monomers including paeoniflorin, berberine, timosaponin and icariine and the monomer mixtures to determine the best concentration of the drugs by methyl thiazolyl tetrazolium （MTT） method. The effective concentration of Aβ25-35 was determined by culturing PC12 cells with different concentrations of Aβ25-35, Then, the activity of PC12 cells with Aβ25-35induced injury was observed with MTT method. Cellular morphological change was observed by phase contrast microscopy. Flow cytometry and fluorescence microscopy were employed to observe the Aβ25-35induced early apoptosis of PC12 cells.Results： After Aβ25-35 induction, the PC12 cells were fewer in number, less viable with shrinked cell body and nuclei, and many fragments, and the cells were less adhered. The cell proliferation was inhibited by Aβ25-35 concentration- and time-dependently. Aβ25-35 at concentration of 20 μmol/ L was selected to construct the Alzheimer＇s disease model in vitro. The sera containing GNC decoction could reinforce PC12 cell activity, and concentration at 20% was better than other concentrations after 24-, 48- and 72-hour culture. The 20% serum containing GNC decoction, 0.1 μmol/L berberin and monomer mixture 3 including 1 mg/mL paeoniflorin, 1 μmol/L berberine, 1 μmol/L timosaponin and 1ng/mL icariine could antagonize neurotoxicity induced by Aβ25-35 Moreover, they could inhibit Aβ25-35-induced early apoptosis of PC12 cells, with the effect of 20% serum containing GNC decoction better than 0.1μmol/L berberine and monomer mixture3 Conclusion： Serum containing GNC decoction at 20% concentration has a potential neuroprotective effect on Aβ-induced cellular impairment. The serum containing GNC decoction was found to be stronger in action than the main monomers.