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Neuroactivity and Its Mechanism of Cuscuta Chinensis Lam in Rat Pheochromocytoma PC12 Cells

Author: LiuJianHui
Tutor: AnLiJia
School: Dalian University of Technology
Course: Biochemical Engineering
Keywords: Cuscuta chinensis Lam Growth associated protein Acetylcholinesterase Mitogen-activated protein kinase Differentiation
CLC: Q25
Type: PhD thesis
Year: 2003
Downloads: 223
Quote: 0
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Abstract


Drug screening, based on sound experimental evidence in cell lines or animal models, is a rapid and effective way. For the purpose of exploiting the affluent resource of traditional Chinese medicines, rat pheochromocytoma PC 12 cells were used as a model to screen the neuroactive components from natural products. Most of all, the effect of it on inducing the differentiation of PC 12 cells and/or execute the neuroprotective effect challenged by serum-deprivation and oxidative pressure, and its mechanism were studied in this paper.Powered seeds of Cuscuta Chinensis Lam. were soaked with 75% ethanol at room temperatures and the extract was evaporated under reduced pressure to afford brown syrup. According to the polarity, the residue was isolated with petroleum ether, chloroform, ethyl acetate, and n-BuOH, respectively. The n-BuOH fraction, confirmed as neuroactive component, was subjected to Sephadex LH-20 column chromatography to provide an extract fraction, as a buff powder, which could induce neurite outgrowth in rat pheochromocytoma PC12 cells in a dose-dependent manner up to 50 mg/L.Serum-deprivation can trigger the program of cell apoptosis, induces cells apoptosis, even necrosis in neuronal differentiated PC 12 cells. C. chinensis extract prevents the neuronal differentiated PC 12 cells apoptosis, DNA fragmentation, and enhances the cells viability at concentration up to 20 mg/L in serum-deprivation condition. Oxidative stress plays an important role in cell death associated with many diseases. In the present study, after treated with xanthine/xanthine oxidase system, cells viability, cells cycle, the expression of apoptosis proteins p53, Bax and Bcl-2, and the level of superoxidase, glutathione, malondialdehyde of neuronal differentiated PC 12 cells in the absence and /or presence of C. chinensis extract were also compared. The results show that C. chinensis extract can prevent PC 12 cells apoptosis induced by reactive oxygen species (ROS), intensify cells viability, inhibit proteins expression related to apoptosis, regulate cell cycle, increase the level of SOD and GSH, and decrease the product of MDA. So, it is evident that C. chinensis extract may protect neuronal differentiated PC 12 cells death from serum-deprivation and ROS challenge.After added into PC 12 cells medium, C. chinensis extract is confirmed asneuroactive components of inducing differentiation of PC 12 cells up to 50 mg/L. With the results of analysis of cells morphology and cell cycle determination, C. chinensis extract, inhibiting PC 12 cells proliferation, inducing neurite outgrowth, was testified. At the same time, C. chinensis extract can also amplify expression of neuroregenerating maker molecule, growth associated protein-43, increase the activity of acetylcholinesterase in rat pheochromocytoma PC 12 cells.Mixing experiments with NGF and C. chinensis extract revealed an additive effect on neuritogenesis when low dose of both agents were used. Whereas saturating dose NGF were used, there was no distinct additive effect. These findings suggest that C. chinensis extract may interact with cell surface membranes in a different way from NGF, and indirectly activate NGF receptor. Consequently, C. chinensis extract shares the common pathway induced by NGF, leading to activation of specific gene required for the differentiation process.Because of the importance of mitogen-acivated protein kinase (MAPK) in cell growth, proliferation, differentiation and death, PD98059, a specific inhibitor of MAPKK, is utilized to explore the linkage between the differentiation of rat pheochromocytoma PC 12 cells induced by C. chinensis extract and MAPK signaling cascade. Although the intracellular mechanism of C. chinensis extract inducing differentiation of rat pheochromocytoma PC 12 cells remains to be clarified, the current results show that C. chinensis extract, enhancing the phosphorylation of MAPK and inducing differentiation of PC 12 cells, might be involved in MAPK-mediated signaling pathway.

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