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

Abstract

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 icariin) on neurotoxicity in PC12 cells induced by amyloid beta-protein (Abeta). Injury of PC12 cells was induced by incubating with Abeta(25-35) in vitro. Ovariectomized rats were intragastrically administered with GNC decoction twice daily for 5 days and then sera were obtained. PC12 cells were cultured with different concentrations of serum containing GNC decoction and its main monomers including paeoniflorin, berberine, timosaponin and icariin and the monomer mixtures to determine the best concentration of the drugs by methyl thiazolyl tetrazolium (MTT) method. The effective concentration of Abeta(25-35) was determined by culturing PC12 cells with different concentrations of Abeta(25-35). Then, the activity of PC12 cells with Abeta(25-35)-induced 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 Abeta(25-35)-induced early apoptosis of PC12 cells. After Abeta(25-35) induction, the PC12 cells were fewer in number, less viable with shrinked cel1 body and nuclei, and many fragments, and the cells were less adhered. The cell proliferation was inhibited by Abeta(25-35) concentration- and time-dependently. Abeta(25-35) at concentration of 20 micromol/ 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 micromol/L berberin and monomer mixture 3 including 1 mg/mL paeoniflorin, 1 micromol/L berberine, 1 micromol/L timosaponin and 1 ng/mL icariine could antagonize neurotoxicity induced by Abeta(25-35). Moreover, they could inhibit Abeta(25-35)-induced early apoptosis of PC12 cells, with the effect of 20% serum containing GNC decoction better than 0.1 micromol/L berberine and monomer mixture 3. Serum containing GNC decoction at 20% concentration has a potential neuroprotective effect on Abeta-induced cellular impairment. The serum containing GNC decoction was found to be stronger in action than the main monomers.