Green tea polyphenol (–)-epigallocatechin-3-gallate (EGCG) is a potent antioxidant with protective effects against neurotoxicity. However, it is currently unclear whether EGCG protects neuronal cells against radiation-induced damage. Therefore, the objective of this study was to investigate the effects of EGCG on ultraviolet (UV)-induced oxidative stress and apoptosis in PC12 cells. The effects of UV irradiation included apoptotic cell death, which was associated with DNA fragmentation, reactive oxygen species (ROS) production, enhanced caspase-3 and caspase-9 activity, and poly (ADP-ribose) polymerase cleavage. UV irradiation also increased the Bax/Bcl-2 ratio and mitochondrial pathway-associated cytochrome c expression. However, pretreatment with EGCG before UV exposure markedly decreased UV-induced DNA fragmentation and ROS production. Furthermore, the UV irradiationinduced increase in Bax/Bcl-2 ratio, cytochrome c upregulation, and caspase-3 and caspase-9 activation were each ameliorated by EGCG pretreatment. Additionally, EGCG suppressed UV-induced phosphorylation of p38 and rescued UV-downregulated phosphorylation of ERK. Taken together, these results suggest that EGCG prevents UV irradiationinduced apoptosis in PC12 cells by scavenging ROS and inhibiting the mitochondrial pathways known to play a crucial role in apoptosis. In addition, EGCG inhibits UV-induced apoptosis via JNK inactivation and ERK activation in PC12 cells. Thus, EGCG represents a potential neuroprotective agent that could be applied to prevent neuronal cell death induced by UV irradiation.

Cognitive impairment is considered to be key research topics in the field of neurodegenerative diseases and in understanding of learning and memory. In the present study, we investigated neuroprotective effects of Schisandra chinensis (SC) and Ribes fasciculatum (RF) extracts in hydrogen peroxide-induced neuronal cell death in vitro and scopolamine-induced cognitive impairment in Sprague Dawley® (SD) rat in vivo. Apoptotic cell death in neuroblastic PC12 cell line was induced by hydrogen peroxide for 1 hour at 100 μM. However, mixture of SC and RF treatment prevented peroxide induced PC12 cell death with no neurotoxic effects. For in vivo experiment, the effect of SC and RF extracts on scopolamine-induced cognitive impairment in SD rat was evaluated by spontaneous alternation behavior in Y-Maze test. After 30 min scopolamine injection, the scopolamine-induced rats presented significantly decreased % spontaneous alteration and acetylcholine level, compared to non-induced group. However, treatment of SC+RF extracts rescued the reduced % spontaneous alteration with acetylcholine concentration from hippocampus in scopolamineinduced rats. These results suggested that mixture of SC and RF extract may be a potential natural therapeutic agent for the prevention of cognitive impairment.

The purpose of this study was to investigate the protective effect on oxidative stress induced PC12 cells, and volatile flavor composition of essential oils derived from medicinal plant seeds- Gossypium hirsutum L. (G. hirsutum), Coix lachryma-jobi (C. lachryma-jobi) and Oenothera biennis (O. biennis). The essential oils were obtained by the solvent (hexane) extraction method from the seeds. The essential oils of the seeds were analyzed by the solid-phase micro-extraction gas chromatography mass spectrometry (SPME-GC/MS). The major compounds of G. hirsutum, C. lachryma-jobi and O. biennis were cyclonexanol (16.65%), β-asarone (14.29%) and ylangene (50.01%). The DPPH radical scavenging activity (IC50) was the highest value of 8.52 mg/mL in the O. biennis. Additionally, IC50 values of G. hirsutum and C. lachryma-jobi were 26.76 mg/mL and 36.81 mg/mL. For the oxidative stress on PC12 cells, we treated with hydrogen peroxide (H2O2). The pretreatment of oxidative stress induced PC12 cells with all the essential oils preserved or increased their cell viability and G. hirsutum and O. biennis attenuated the ROS generation (by 68.75% and 56.25% vs. H2O2 control). The results of this study suggest that the essential oils derived from medicinal plant seeds could be used as valuable back data as a natural essential oil material to prevent neurodegenerative diseases by protecting neuro-cells.

본 연구는 동충하초 추출물을 PC12 및 BV2 세포에서 인지능력 개선에 대한 효능을 평가하고자 하였다. 동충하초 추출물을 증류수로 추출하여 PC12 및 BV2 세포로 MTT 분석을 통해 세포 생존율을 확인하고 L-glutamate로 유도한 PC12 세포를 통해 세포 보호 효능과 아세틸콜린 함량 및 아세틸콜린에스테아제 활성을 평가하였다. 또한, LPS로 유도한 BV2 세포를 통해 nitric oxide (NO) 및 prostaglandin E2 (PGE2) 생성량 등의 항염증 효능을 측정하고 western blot 분석을 통해 NK-ĸB, p38, JNK, caspase-3 등의 단백질 발현량을 확인하였다. 동충하초 추출물은 200 ㎍/㎖ 농도를 제외하고 1, 10, 100 (㎍/㎖) 농도에서 세포 독성이 나타나지 않았다. L-glutamate로 유도한 PC12 세포에서 유의성있게 세포 보호 효능과 아세틸콜린 함량의 증가, 아세틸콜린에스테아제 활성 감소가 나타났다. 또한, 동충하초 추출물은 NO 및 PGE2 생성량과 NK-ĸB, p38, JNK, caspase-3 등의 단백질 발현을 억제하였다. 이와 같은 결과는 동충하초 추출물이 인지능력에 대한 예방 및 개선 효능이 있음을 나타낸다. 따라서 동충하초 추출물은 인지능력 개선을 위한 새로운 천연 소재로 활용될 수 있다.

This study was designed to evaluate the antioxidant activities and protective effects on PC12 cells of the extract of Epimedium koreanum and its main constituents icariin and icariside I. After screening the seven identified flavonoid glycosides from E. koreanum through DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay, E. koreanum, Icariin and Icariside I exhibited significant effect on radical scavenging activity. E. koreanum, icariin and icariside I were examined using DPPH, ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) and FRAP (ferric reducing ability power) assay. In all antioxidant assays, E. koreanum, icariin and icariside I showed high radical scavenging activities in a dose-dependent manner. Protective effects against H2O2-induced PC12 cells were assessed with MTT assay. The results indicated that cell viability and protection on PC12 cells of icariside I and icariin increased dose dependently. These study results suggest that E. koreanum, icariin and icariside showed high antioxidant capacities and cell protective effects. Icariside I, one of the metabolites of icariin, may be a new and effective flavonoid compound as a functional component.

Inhibition of Rho-associated coiled coil-containing kinase (ROCK) has been reported to promote differentiation of neuronal cells. Here, we examined the effect of Y-27632, a ROCK inhibitor, on the outgrowth of neurites in PC12 cells. Y-27632 caused a rapid induction of neurite outgrowth in PC12 cells in a time-dependent manner. The neurite outgrowth, triggered by Y-27632, was accompanied by Rac1 activation, and was attenuated by Rac1 inhibitor NSC23766, in a concentration-dependent manner. Y-27632 also induced an increase in the production of reactive oxygen species (ROS). Pretreatment with N-acetylcysteine, an ROS scavenger, inhibited the ROS generation and neurite outgrowth in response to Y-27632. These results indicate that the activation of Rac1 and the generation of ROS contribute to the neurite outgrowth triggered by Y-27632 in PC12 cells.

Neuronal differentiation is a complex biological process accompanying cytoskeletal reorganization, including neurite outgrowth and growth cone formation. Therefore, neuronal differentiation is critically regulated by actin-related signaling proteins, such as small Rho GTPases, guanine nucleotide exchange factors (GEFs), and myosins. This study will demonstrate the change in activity of three small Rho GTPases, Rac, Cdc42, and Rho A, by treatment with blebbistatin (BBS), a specific inhibitor for myosin, during bFGF-induced neurite outgrowth in PC12 cells. Treatment with BBS induced morphological changes in growth cones and neurites during differentiation. A marked increase in protrusion and filopodia structures in growth cones, the shaft of neuritis, and cell membranes was observed in the cells treated with BBS. Activity of Rho GTPases showed the alterations in response to BBS. Activities of both Rac and Rho A were inhibited by BBS in a time-dependent manner. By contrast, Cdc42 activity was not changed by BBS. These results suggest that inactivation of myosin II by BBS induced morphological changes in neurites and growth cones and distinct regulation of three Rho GTPases during differentiation of PC12 cells.

Cyclosporin A (CsA) has been used clinically as an immunosuppressive drug to prevent organ transplant rejection and in basic research as a mitochondrial permeability blocker. It has been reported that CsA has a protective role in severed neurons and a neurotrophic effect in neuronal cells. However, the molecular mechanisms underlying the stimulation of neuronal cell proliferation by CsA have not yet been elucidated. In our current study, we investigated CsA responsive proteins in PC12 cells using a systematic proteomic approach. The viability of these cells following CsA treatment increased in a dose- and time-dependent manner. Proteins in the CsA-treated PC12 cells were profiled by two-dimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) and electrospray ionization quadupole time-of-flight mass spectrometries (EIQ-TOFMS). This differential expression analysis showed significant changes for 10 proteins (6 up-regulated and 4 down-regulated) upon CsA treatment that were related to cell proliferation, metabolism and the stress response. These proteomics data further our understanding of the proliferation mechanisms of PC12 cells exposed to CsA and demonstrate that our methodology has potential to further elucidate the mechanisms and pathways involved.

Cyclosporin A (CsA) plays an important role in clinical medicine and basic biology as an immunosuppressant and a mitochondrial permeability blocker, respectively. It was reported that CsA has a protective role by preventing apoptosis and promoting the proliferation in severed neurons. However, the molecular mechanisms for CsA-induced neuronal cell proliferation are unclear. In this study, we examined the mechanisms underlying the CsA-induced proliferation of PC12 cells. CsA increased the viability of PC12 cells in a dose(over 0.1~10 μM)-and time-dependent manner. The level of ROS generation was decreased in the CsA-treated PC12 cells. Expression of Bcl-2, an antiapoptotic molecule that inhibits the release of cytochrome c from the mitochondria into the cytosol, was upregulated, whereas Bax, a proapototic molecule, was not changed in the CsA-treated PC12 cells. CsA downregulated the mRNA expression of VDAC 1 and VDAC 3, but VDAC 2 was not changed in the CsA-treated PC12 cells. The level of cytosolic cytochrome c released from the mitochondria and the caspase-3 activity were attenuated in the CsA-treated PC12 cells. These results suggest that the mitochondria-mediated apoptotic signal and Bcl-2 family may play an important role in CsA-induced proliferation in PC12 cells.

산화적 스트레스로부터 참다래 과실 추출물의 신경세포 보호효과에 미치는 영향을 알아보기 위하여 신경세포주인 PC12 세포를 이용하여 참다래 과실추출물의 전처리가 산화적 손상으로부터 유발되는 신경세포사멸을 억제할 수 있는지 조사하였다. t-BHP에 의해 유도된 신경세포손상으로부터 세포사멸을 억제하여 세포생존도를 증가시켰으며 세포사멸로부터 형성되는 핵의 농축현상과 단편화가 현저히 감소함을 확인 할 수 있었다. 그리고 Bcl-2 단백의 발현 증가, Bax 단백의 발현 감소, caspase-3의 활성, PARP 분해 단백(85KDa)감소, ERK, p38 활성을 감소시켰다. 따라서 참다래 과실의 추출물은 신경세포증식효과를 통해 신경세포손상으로부터 유발되는 다양한 퇴행성 뇌질환의 예방에 도움이 될 것으로 나타났다.