Screening of natural medicines that efficiently activate neurite outgrowth in PC12 cells in C2C12-cultured medium

Abstract

We have studied the effects of natural medicines on neurite outgrowth in PC12D cells in a cultured medium of C2C12 cells. Derived from mouse myoblasts, the C2C12 cells secrete neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). The secretion of these neurotrophins from C2C12 cells stimulate neurite outgrowth in PC12D cells. We have screened a total of 120 samples and found five natural medicines: Trichosanthes Root, Asiasarum Root, Lycium Bark, Sinomenium Stem, and Dictamni radicis Cortex, that enhance the activity of C2C12-cultured medium to stimulate neurite outgrowth in PC12D cells. These natural medicines promoted not only neurite outgrowth but also stabilized the neurite formation in PC12D cells for several days. RT-PCR analysis showed that NGF was significantly increased with Trichosanthes and Lycium Bark. However, BDNF was slightly decreased with Lycium Bark, Sinomenium Stem, and Dictamni radicis Cortex. NT-3 was increased slightly by all of these natural medicines except Sinomenium Stem. All these five natural medicines significantly increased the number and length of neurites in PC12D cells in co-culture with C2C12 cells.Neurite genesis and retraction are associated with the development and pathogenesis of the nervous system. The neurotrophins-nerve growth factor (NGF), brain-derived neurotrophin factor (BDNF) and neurotrophin-3 (NT-3)-are the factors mainly responsible for neurite outgrowth. However, because of their high molecular weights, these neurotrophins cannot cross the blood-brain barrier. Thus, the emphasis has been given to the availability of the neurotrophic factors as therapeutic agents. There are hundreds of natural medicines that have a long history of curative properties against various diseases. Recently, several synthetic compounds and natural medicines are known to enhance the action of NGF or show a similar mode of action as NGF in PC12 cells by generating neurite outgrowth (10,13,20). In this study, we have used C2C12 cells to monitor the effect of natural medicines on neurite outgrowth in PC12D cells. NGF, BDNF and NT-3 are expressed and secreted by myoblast C2C12 cells (15,17). These neurotrophins bind to the low-affinity or the high-affinity receptors in a highly specific manner on PC12 cells (3,14). The neurotrophin secretion from the C2C12 cells induces neurites in PC12 cells and neural differentiation in co-cultured systems (1, 2). For example, C2C12 cells co-cultured with neuronal ganglia promoted neurite outgrowth (17). Thus, the C2C12/PC12 system serves as a useful tool for studying neurite-promoting activators in in vitro systems (23). We investigated the effects of natural medicines on promoting and inducing neurotrophin secretion in C2C12 cells. Here we present the evidence that five natural medicines out of 120 samples promote neurite outgrowth and stabilize neurite genesis in PC12D cells co-cultured with C2C12 cells.

In vitroandin vivostudy of effects of fermented soybean product (chungkookjang) on NGF secretion ability and NGF receptor signaling pathway

“…Especially, Uezato et al [27] screened a total of 120 samples to identify the effects of natural medicines on neuritic outgrowth in PC12 cells. Five natural medicines, including Trichosanthes root, Asiasarum root, Lycium bark, Sinomenium stem, and Dictamni radices, significantly promoted not only neuritic outgrowth but also stabilized neuritic formation in PC12 cells.…”

Three-dimensional co-culture of C2C12/PC12 cells improves skeletal muscle tissue formation and function

“…To ease translation into the clinic, a co‐culture model without the use of animal sera has been established and primary muscle cells have been co‐cultured with human stem cells differentiated into motor neurons (Das et al , ; Guo et al , , ). Another neuromuscular model, which is often used in particular for studying molecular events or neurite outgrowth, is the co‐culture system composed of C2C12 myoblasts and rat phaeochromocytoma PC12 cells (Uezato et al , ). In such a model, C2C12 cells secreted nerve growth factor (NGF) (Uezato et al , ), allowing the PC12 cells to differentiate into neurons (Greenberg et al , ; Greene and Tischler, ).…”

“…Another neuromuscular model, which is often used in particular for studying molecular events or neurite outgrowth, is the co‐culture system composed of C2C12 myoblasts and rat phaeochromocytoma PC12 cells (Uezato et al , ). In such a model, C2C12 cells secreted nerve growth factor (NGF) (Uezato et al , ), allowing the PC12 cells to differentiate into neurons (Greenberg et al , ; Greene and Tischler, ).…”

Real-Time Electrochemical Recording of Dopamine Release under Optogenetic Stimulation

“…Studies on dopamine regulation have shown that PC12 cells, a rat pheochromocytoma cell line, synthesize, release, and reuptake dopamine in a manner similar to that of dopaminergic neurons [5], [6]. Compared to brain neuron cultures or tissue slices, PC12 cell culture consists of a homogeneous dopamine-containing population, which had been widely used in dopaminergic cell investigations, including those on cell differentiation, neural protection, drug screening, and cell implantation therapies [7], [8], [9], [10], [11], [12], [13], [14]. Conventional approaches for regulating dopamine release in PC12 cells are pharmaceutical or electrical stimulation (ES) techniques, which have critical limitation on controlling dopamine release.…”