Puerarin protective effect on neural stem cells
|School||Shaanxi Normal University|
|Keywords||Neural stem cells β - amyloid Puerarin Apoptosis|
Neural stem cells (neural stem cells, NSCs) have the capacity of self-renewal, proliferation and differentiation into neurons, astrocytes and oligodendrocytes cells. The discovery of neural stem cells were isolated and cultured stem cell research and neurobiological fields of scientific research is an important milestone. They not only exist in the nervous system of the mammalian developmental stages, but also exist in the adult mammalian nervous system. This poses a challenge before mature nervous system regeneration theory impossible, also brought hope to the nervous system, damage repair and functional reconstruction. Alzheimer's disease (Alzheimer's disease, AD) is a progressive neurodegenerative disease, is caused by the death of brain cells and neurological diseases. The main pathological features of cortical and hippocampal beta-amyloid protein (amyloidβ-protein, Aβ) aggregate to form senile plaques (senile plaques, SP), Tau protein abnormalities aggregate formation of neurofibrillary tangles (neurofibrillary tangle (NFT)) as well as a decrease in the nerve cells of the cerebral cortex and hippocampus. High concentrations of Aβ on neurons neurotoxic effects of Aβ deposition is the main pathogenic factors result in AD brain cholinergic neuronal loss, that the deposition of Aβ is the central link of the pathogenesis of AD. Aβ can cause oxidative stress Ca 2 sup> influx, and thus damage the mitochondria, resulting in dysfunction of the nerve cells, activation of apoptosis-related proteins and factors, and finally start the process of apoptosis. In addition, Aβ also can cause brain inflammation reaction and neurofibrillary tangles indirectly lead to nerve cell apoptosis. Puerarin (puerarin, Pue) is extracted and isolated from the roots of the legume kudzu an isoflavone-type compound. The puerarin main pharmacological effects dilate blood vessels, improve microcirculation, antioxidant, repair the damage of endothelial cells and the inhibition of apoptosis of brain cells. Studies have shown that puerarin has a protective effect on the nerve cells of the damage, can suppress Aβ-induced apoptosis. Puerarin to treat vascular dementia, but no treatment of AD clinical study reports. Puerarin on the role of neural stem cells has not been reported. Puerarin different concentrations of Aβ 25-35 induced apoptosis of neural stem cells in the intervention study, the protective effect of puerarin neural stem cells, and its mechanism of action research, in order to The clinical will puerarin for neurodegenerative diseases such as AD prevention and treatment to provide experimental evidence and reference. In this study, 15-16 days fetal rat brain tissue as experimental material, neural stem cells in serum-free culture isolated and cultured in vitro. Immunocytochemical methods the neural stem cells were identified with NSE of GFAP antibody identification cell differentiation. The primary cultured neural stem cells after 3 passages, these cells cultured holes divided into three groups: (1) without control group of Aβ 25-35 and puerarin; (2) an increase of only of Aβ 25-35 treatment group, added 20μmol / L concentration in wells the condensed matter Aβ 25-35 ; the ③ puerarin protection group, that is, at the same time adding 20μmol / The L-Condensed Matter of Aβ 25-35 > and different concentrations of puerarin (10 -5 sup>, 10 -4 sup> 10 - 3 sup> mol / L concentration added). After 48 h incubation, using an inverted phase contrast microscope, Giemsa staining and DNA agarose gel electrophoresis was used to detect cell apoptosis, and by RT-PCR technology to detect cell apoptosis related gene bcl-2 and bax mRNA expression levels change. The main results and conclusions are as follows: 1. Neural stem cells were isolated, cultured and identified: cells were isolated from 15-16 days of gestation fetal rat brain tissue round, refraction, neurospheres cultured for 6-7 days after the border than clear, strong sense of three-dimensional passages. The second-generation and third-generation neurospheres immunocytochemical staining method more Nestin-positive cells within neurospheres. Addition of culture medium containing 10% fetal bovine serum induction of differentiation, the majority of neurospheres adherent growth and around protrusion grow, 5-6 days after the induction of differentiation, formation longer protrusion intertwined, single cells; after Immunocytochemistry NSE, GFAP positive expression. Isolated and cultured cells passaged expressed Nestin, induced differentiated cells expressed NSE, GFAP. That the experiments were isolated and cultured cells have self-renewal and differentiation capacity, has the basic characteristics of neural stem cells. Show that we get isolated and cultured in vitro cells are neural stem cells. Apoptotic morphology observed: neurospheres passaged 3 times, join 20μmol / L Aβ 25-35 continue to 48h, compared with the control group, the cell body rounded, protruding decrease in the number of only partially adherent cells, showing that a large number of cell suspension; gradually increased the number of prompts with time 20μmol / L Aβ 25-35 -induced cell apoptosis. 10 -4 sup> mol / L puerarin protection group, the increase in the number of cell processes, most adherent cells, and only see a small amount of suspended cells, suggesting that 10 -4 sup> mol / L puerarin factors can inhibit Aβ 25-35 -induced apoptosis, thereby better protecting cells. Giemsa staining showed that of Aβ 25-35 treatment group, the cell body is small, dark blue, the synaptic connections between the cells rupture; 10 -4 sup> mol / L puerarin protection group, the cell body and round stained lighter obvious synaptic connections between the cells. Aβ 25-35 cell damaging puerarin can protect cells from damage. Indicating that puerarin play a protective effect against cell by inhibiting Aβ 25-35 -induced apoptosis. Apoptosis-related gene bcl-2 and bax mRNA levels of expression change: RT-PCR results showed that control cells, bcl-2, bax no expression; Aβ 25-35 group of cells strong expression of apoptotic genes bax, to inhibit apoptosis gene bcl-2 expression is weak; puerarin protect the cells bcl-2, bax expression, and found that 10 -4 sup> mol / L puerarin protection group of bcl-2 expression levels, while the expression intensity of bax and bcl-2 quite. Showed that Aβ 25-35 in vitro can inhibit the proliferation of neural stem cells, promotion of apoptosis, the apoptotic genes bax mRNA upregulation. Puerarin has the antagonistic of Aβ 25-35 of neural stem cells induce apoptosis, and this antagonism is achieved by upregulating the suppression of apoptosis gene bcl-2 mRNA expression.