Acute rosmarinic acid treatment enhances long-term potentiation, BDNF and GluR-2 protein expression, and cell survival against scopolamine treatment in rat organotypic hippocampal slice cultures

다국어 초록 (Multilingual Abstract)

Introduction : Various medicinal plants have been widely used in public that are known to have an effect on brain function. Rosmarinic acid (RA) isan ester of caffeic acid and subfamily Nepetoideae of the Lamiaceae are rich in it. RA is a natural polyphenol with bioactivity : antioxidant, anti-apoptotic, and anti-inflammatory. The current study was conducted to evaluate and anti-depressant effect of RA using behavior test and proteomics analysis. RA were demonstrated decreasing immobility time in tail suspension test (TST) and regulation of several neurotransmitters (dopamine, norepinephrine, serotonin and acetylcholine) and gene expression in mice brain like TH, PC, BNDF and MAPK phosphatase (MKP-1).
Objective : Our study provides the evidence that RA increase Long-term plasticity (LTP) and has neuroprotective activity in scopolamine-induced memory impairment model.
Materials and methods : LTP experiment group were divided into control, RA treatment (doses of 1, 10 and 100 ug/ml respectively), blocker (AP5, CNQX and NFA at doses of 10 uM, 10uM and 100uM respectively), blocker + RA (10ug/ml), scopolamine (300 uM) and scopolamine + RA (10 ug/ml). Western blotting and propidiumiodide (PI) staining experiment group were divided into contol, scopolamine (300 uM) and scopolamine + RA (10 ug/ml). Two weeks afters organotypic hippocampal slice cultures (OHSCs), starting the experiments. The intensity of test pulse stimulation was set at 100 mA. Baseline responses were evoked for at least 30 min, of which the last 40 minutes were recorded, before the high-frequency stimulation (HFS) was applied to induce LTP. After the stimulation, field excitatory postsynaptic potentials (fEPSPs) were recorded every 60 sec for another 50 min. For western blotting and PI staining experiments, OHSCs were exposed to the culture medium containing PI, scopolamine and RA for 24h or 48h.
On western blotting experiment, hippocampal tissues dissected from 6-well insert and then analyzed protein level of brain-derived neurotrophic factor (BDNF) and Glutamate receptor-2 (GluR-2) using anti-BDNF and anti-GluR-2specific antibody.
The cell death was assessed by fluorescent image analysis of PI staining. Tissues were washed with culture medium and detected the cell death in the pre-phase which has no PI uptake in the hippocampal layers. Treatment for 24, 48h were observed by PI.
Results and discussion : Our results indicated that RA treatment enhanced LTP in CA1 synapses in a dose-dependently. (1 ug/ml and 10 ug/ml but not 100 ug/ml) and blocker (CNQX, NFA) and blocker + RA treatment group were blocked LTP.
But AP5 + RA group’s fEPSP higher than AP5 only treatment group. RA also increased expression BDNF and GluR-2 protein level and inhibited cell deaths on scopolamine-induced memory impairment model by western blotting and PI staining analysis.

국문 초록 (Abstract)

로즈마리산은 카페인산과 살바인산A가 에스터 결합을 하고 있는 폴리페놀 물질로, 꿀풀과에 속한 허브 식물에서 많이 발견된다. 이전 연구들에서 in vivo 및 in vitro 실험을 통해 로즈마리산이 항산화, 항우울, 신경보호 효과 등을 가진 것으로 보고되고 있다. 이 실험에서는 해마조직에 로즈마리산을 처리하여 해마의 장기증강 (LTP), 신경가소성과 연관된 단백질의 발현, 스코폴라민에 의해 손상된 조직에 미치는 영향에 대해 알아보고자 했다.
첫 번째로 해마조직을 multi-electrode arry (MEA) 에 올려 1, 10, 100µg/ml의 농도의 로즈마리산을 aCSF와 함께 처리하며 장기증강을 측정하였다. 그 결과 로즈마리산 1, 10µg/ml 농도에서 LTP가 증가하였으나 100µg/ml에서는 오히려 정상군보다 감소하였다. 로즈마리산이 LTP 메커니즘에서 어떤 수용체와 연관되어 있는지 알아보기 위해 일련의 길항제 실험을 진행한 결과 CNQX 와 niflumic acid에서는 그렇지 못했지만 AP-5의 LTP 감소 효과가 로즈마리산에 의해 저해되었다. 또한 웨스턴 블롯 실험을 통해 해마조직에서 로즈마리산의 상황에서 LTP와 연관된 단백질인 BDNF와 GluR-2의 발현을 증가시킴을 확인하였다. 마지막으로 PI 염색실험을 진행한 결과, 스코폴라민에 의한 세포사멸이 로즈마리산에 의해 억제됨을 확인하였고, LTP 실험을 통해서도 스코폴라민에 의한 LTP 감소가 로즈마리산에 의해 저해되는 것을 알 수 있었다.
이 결과들은 로즈마리산이 해마조직의 glutamatergic signal pathway에영향을 주어 LTP를 증가시키는 것으로 사려된다. 또한 스코폴라민에 의한 병증모델에서 신경보호적인 효과가 있어 신경병성질환 치료제 개발을 위한 좋은 후보 물질이 될 것이라 사려된다.

목차 (Table of Contents)

• Ⅰ. Introduction……………………………………………………………………….1
• Ⅱ. Materials and Methods…………………………………………………………5
• 1. Materials……………………………………………………………………...5
• 2. Organotypic hippocampal slice culture…………………………………..5
• 3. Preparation of organotypic hippocampal slice tissue on the MEA
• probe............................................................................... 6
• 4. Induction of LTP for hippocampal slice………………………………7
• 5. Electrophysiology data processing …………………………………..8
• 6. Western blot analysis …………………………………………………..12
• 7. Measurement of propidium iodide (PI) uptake …………………………13
• 8. Statistical analysis………………………………………………………14
• Ⅲ. Results………………………………………………………………………….15
• 1. The effect of rosmarinic acid on CA1 region in hippocampus...….15
• 2. Rosmarinic acid block the effect of AP-5 in hippocampus but not CNQX and niflumic acid …………………………………………….…….18
• 3. Rosmarinic acid attenuated scopolamine-induced memory impairment
• model in hippocampus ………………………………………………….25
• 4. Rosmarinic acid increases BDNF and GluR-2 levels in hippocampusRosmarinic acid increase of BDNF and Glu2 receptor level in hippocampus …………..…………………………...…………………..28
• 5. Rosmarinic acid inhibition of scopolamine-induced cell death in hippocampal tissue …………...…………………….…………………..31
• IV. Discussion……………………………………………………………………….34
• V. Reference……………………………………………………………………37
• Abstract…………………………………………………………………………….41
• Abstract in Korean………………………………………………………………….43