BACKGROUND： Neuronal loss, synapse mutilation, and increasing malnourished axons are pathologically related to Alzheimer＇s disease. Microtubule-associated protein 2 （MAP2） is of importance for neuronal, axonal, an...BACKGROUND： Neuronal loss, synapse mutilation, and increasing malnourished axons are pathologically related to Alzheimer＇s disease. Microtubule-associated protein 2 （MAP2） is of importance for neuronal, axonal, and dendritic generation, extension, and stabilization, as well as for the regulation of synaptic plasticity. OBJECTIVE： To investigate the antagonistic effects of natural-cerebrolysin-containing serum on beta amyloid protein 1-40 （Aβ1-40）-induced neurotoxicity from the standpoints of cell proliferation, synaptogenesis, and cytoskeleton formation （MAP2 expression）. DESIGN, TIME AND SETTING： A paralleled, controlled, neural cell, and molecular biology experiment was performed at the Institute of Integrated Chinese and Western Medicine, Shenzhen Hospital, Southern Medical University between February 2006 and April 2008. MATERIALS： PC12 cells, derived from the rat central nervous system, were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China. A β1-40 was provided by Sigma, USA. Natural-cerebrolysin was provided by Shenzhen Institute of Integrated Chinese and Western Medicine, China. The natural-cerebrolysin was predominantly composed of Renshen （Radix Ginseng）, Tianma （Rhizoma Gastrodiae）, and Yixingye （Ginkgo Leaf） in a proportion of 1：2：2. Following conventional water extraction technology, an extract （1：20） was prepared. Each gram of extract equaled 20 grams of crude drug. In a total of 12 adult male New Zealand rabbits, six underwent intragastric administration of natural-cerebrolysin extract for 1 month to prepare natural-cerebrolysin-containing serum, and the remaining six rabbits received intragastric administration of physiological saline to prepare normal blank serum. METHODS： An AIzheimer＇s disease in vitro model was induced in PC12 cells using Aβ1-40. The cells were incubated with varying doses of natural-cerebrolysin-containing serum （2.5%, 5%, and 10%）. Normal blank serum-treated PC12 cells served as a blank control group. MAIN OUTCOME MEASURES： Through the use of inverted phase contrast microscope, cell morphology and neurite growth were observed, neurite length was measured, and the percentage of neurite-positive cells was calculated. Cell proliferation rate was determined by MTT assay, and MAP 2 expression was detected by fluorescent immunocytochemistry. RESULTS： Following Aβ1-40 treatments, some PC12 cells were apoptotic/dying, and only a few short neurites were observed. Following interventions with natural-cerebrolysin-containing serum, the PC12 cells proliferated, there was an increased number of neurites, and neurite length was enhanced. After middle- and high-dose natural-cerebrolysin treatments, the percentage of neurite-positive cells, as well as the average length of neurites, was significantly greater than the normal blank serum-treated PC12 cells （P 〈 0.05 or P 〈 0.01）. Compared with the blank control group, MAP2 expression in the Aβ1-40-treated PC12 cells was significantly inhibited, and the cell proliferation rate was significantly decreased （P 〈 0.01）. Following incubations with natural-cerebrolysin-containing serum, MAP2 expression and cell proliferation rate in the PC12 cells were significantly increased in a dose-dependent manner, compared with treatments with blank control serum （P 〈 0.05 or P 〈 0.01 ）. CONCLUSION： Natural-cerebrolysin exhibited antagonistic effects on neurotoxicity in Aβ1-40 induced Alzheimer＇s disease in vitro models. These effects were likely related to cell proliferation and the upregulation of intracellular MAP2 expression.展开更多
基金Supported by:Scientific and Technological Foundation of the National Administration of Traditional Chinese Medicine of China,No.02-03LP41the Scientific and Technological Key Project of Guangdong Province,No. 2006B35630007
文摘BACKGROUND： Neuronal loss, synapse mutilation, and increasing malnourished axons are pathologically related to Alzheimer＇s disease. Microtubule-associated protein 2 （MAP2） is of importance for neuronal, axonal, and dendritic generation, extension, and stabilization, as well as for the regulation of synaptic plasticity. OBJECTIVE： To investigate the antagonistic effects of natural-cerebrolysin-containing serum on beta amyloid protein 1-40 （Aβ1-40）-induced neurotoxicity from the standpoints of cell proliferation, synaptogenesis, and cytoskeleton formation （MAP2 expression）. DESIGN, TIME AND SETTING： A paralleled, controlled, neural cell, and molecular biology experiment was performed at the Institute of Integrated Chinese and Western Medicine, Shenzhen Hospital, Southern Medical University between February 2006 and April 2008. MATERIALS： PC12 cells, derived from the rat central nervous system, were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China. A β1-40 was provided by Sigma, USA. Natural-cerebrolysin was provided by Shenzhen Institute of Integrated Chinese and Western Medicine, China. The natural-cerebrolysin was predominantly composed of Renshen （Radix Ginseng）, Tianma （Rhizoma Gastrodiae）, and Yixingye （Ginkgo Leaf） in a proportion of 1：2：2. Following conventional water extraction technology, an extract （1：20） was prepared. Each gram of extract equaled 20 grams of crude drug. In a total of 12 adult male New Zealand rabbits, six underwent intragastric administration of natural-cerebrolysin extract for 1 month to prepare natural-cerebrolysin-containing serum, and the remaining six rabbits received intragastric administration of physiological saline to prepare normal blank serum. METHODS： An AIzheimer＇s disease in vitro model was induced in PC12 cells using Aβ1-40. The cells were incubated with varying doses of natural-cerebrolysin-containing serum （2.5%, 5%, and 10%）. Normal blank serum-treated PC12 cells served as a blank control group. MAIN OUTCOME MEASURES： Through the use of inverted phase contrast microscope, cell morphology and neurite growth were observed, neurite length was measured, and the percentage of neurite-positive cells was calculated. Cell proliferation rate was determined by MTT assay, and MAP 2 expression was detected by fluorescent immunocytochemistry. RESULTS： Following Aβ1-40 treatments, some PC12 cells were apoptotic/dying, and only a few short neurites were observed. Following interventions with natural-cerebrolysin-containing serum, the PC12 cells proliferated, there was an increased number of neurites, and neurite length was enhanced. After middle- and high-dose natural-cerebrolysin treatments, the percentage of neurite-positive cells, as well as the average length of neurites, was significantly greater than the normal blank serum-treated PC12 cells （P 〈 0.05 or P 〈 0.01）. Compared with the blank control group, MAP2 expression in the Aβ1-40-treated PC12 cells was significantly inhibited, and the cell proliferation rate was significantly decreased （P 〈 0.01）. Following incubations with natural-cerebrolysin-containing serum, MAP2 expression and cell proliferation rate in the PC12 cells were significantly increased in a dose-dependent manner, compared with treatments with blank control serum （P 〈 0.05 or P 〈 0.01 ）. CONCLUSION： Natural-cerebrolysin exhibited antagonistic effects on neurotoxicity in Aβ1-40 induced Alzheimer＇s disease in vitro models. These effects were likely related to cell proliferation and the upregulation of intracellular MAP2 expression.
