[논문]신경세포에서 신경성장인자(nerve growth factor)의 조절에 미치는 천문동(Asparagus cochinchinensis) 열수추출물의 영향

이현아; 김지은; 송성화; 성지은; 정민기; 김동섭; 손홍주; 이충열; 이희섭; 황대연

doi:10.5352/jls.2016.26.5.509

신경세포에서 신경성장인자(nerve growth factor)의 조절에 미치는 천문동(Asparagus cochinchinensis) 열수추출물의 영향
Effects of an Aqueous Extract of Asparagus cochinchinensis on the Regulation of Nerve Growth Factor in Neuronal Cells 원문보기

생명과학회지 = Journal of life science, v.26 no.5 = no.193, 2016년, pp.509 - 518

초록
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천문동(Asparagus cochinchinensis)은 북아시아 지역에서 열병, 감기, 신장질환, 유방암, 염증질환, 뇌질환 등의 치료에 오랫동안 사용되어온 약용식물(medicinal plant)이다. 비록, 천문동의 항염증(ani-inflammatory) 효능에 대한 일부 연구들이 수행되었지만, 신경세포에서 항염증작용과 신경성장인자(nerve growth factor, NGF)의 연관성에 대한 연구는 수행된바 없다. 따라서, 본 연구에서는 신경세포에서 신경성장인자의 분비와 작용기전에 대한 천문동 열수추출물(aqueous extract from A. cochinchinensis, AEAC)의 영향을 연구하였다. AEAC로 처리된 B35세포의 배양액에 NGF단백질의 농도는 대조물질(vehicle) 처리군에 비하여 유의적으로 증가하였으며, 특별한 독성은 관찰되지 않았다. 또한, NGF mRNA의 발현도 단백질의 농도변화와 유사한 양상을 나타내었다. 더불어, B35세포로부터 분비된 NGF의 생리활성을 확인하기 위해, AEAC-조정배지(conditioned medium)를 미분화된 PC12세포에 처리한 후 이들 세포의 신경염성 성장(neuritic outgrowth)을 관찰하였다. PC12세포의 수상돌기 길이(dendritic length)는 vehicle처리군에 비하여 AEAC-조정배지처리군에서 유의적으로 증가하였다. 또한, High affinity NGF 수용체의 하위신호전달에 포함된 p-TrkA와 p-ERK의 발현은 AEAC-조정배지처리군에서 높았지만, low affinity NGF 수용체의 하위신호전달에서는 낮은 수준으로 관찰되었다. 이러한 결과는 AEAC가 신경세포에서 NGF발현과 분비의 조절에 기여하기 때문에 신경퇴행성질환(neurodegenerative disease) 치료제로서 우수한 후보물질임을 제시하고 있다.

Abstract ▼ AI-Helper

Asparagus cochinchinensis is a medical plant that has long been used to treat fever, cough, kidney disease, breast cancer, inflammatory disease and brain disease in northeast Asian countries. Although several studies have been conducted on the anti-neuroinflammatory effects of A. cochinchinensis, the correlation between these effects and nerve growth factor (NGF) has not yet been examined. In this study, we investigated the effects of an aqueous extract of A. cochinchinensis (AEAC) on the secretion and action mechanism of NGF in neuronal cells. The concentration of the NGF protein in the supernatant collected from cultured cells increased significantly in B35 cells treated with AEAC in comparison with the vehicle-treated group without any specific cytotoxicity. Furthermore, the mRNA expression of NGF showed a very similar pattern to its protein concentration. To examine the bioactivity of NGF secreted from B35 cells, undifferentiated PC12 cells were cultured in an AEAC-conditioned medium and neuritic outgrowth was observed. The dendrite length of PC12 cells in the AEAC-treated group was significantly higher than that in the vehicle-treated group. Moreover, the level of the downstream effectors p-TrkA and p-ERK of the high-affinity NGF receptor was significantly higher in the AEAC-treated group, while the expression of the downstream effectors of the low-affinity NGF receptor was significantly lower in the same group. These results suggest that AEAC may contribute to the regulation of NGF expression and secretion in neuronal cells; it is therefore an excellent candidate for further investigation as a therapeutic drug for neurodegenerative diseases.

주제어

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가설 설정

platyphylla in- duced the secretion and synthesis of NGF in B35 cells [9, 10, 24]. In this study, we investigated the effects of A. cochin- chinensis on NGF secretion and synthesis ability of neuro- blastoma cells. The results presented herein provide addi- tional evidence that AEAC purified from A.

제안 방법

In this study, we investigated the effects of AEAC on the NGF secretion ability and NGF receptor signaling pathway in B35 neuroblastoma and PC₁₂ cells to provide scientific evidence of the therapeutic effects of A. cochinchinensis on neurodegenerative disorders.
Among these, toxicity has long been considered one of the most im- portant problems during preclinical investigations of natural products [5]. In this study, we investigated the toxicity of AEAC in B35 cells to determine the optimal concentration of AEAC. As shown in Fig.
The primer sequences for target gene expression identification were as follows: NGF, sense primer: 5'-CAT GTT GTT CTA CAC TCT GAT CAC-3', an- ti-sense primer: 5'-CTC CTT GCC CTT GAT GTC TGT GG-3’; β-actin, sense primer: 5'-GTG GGG CGC CCC AGG CAC CAG GGC-3', anti-sense primer: 5'-CTC CTT AAT GCT ACG CAC GAT TTC-3'. The experiment was repeated three times, and all samples were analyzed in triplicate. The final PCR products were separated on 1% agarose gel and then visualized by ethidium bromide staining.
To verify the activity of secreted NGF, AEAC-conditioned media were inoculated with undifferentiated PC₁₂ cells and their dendritic outgrowth was measured based on micro- scopic analysis. Dendrites were longer in the AEAC-con- ditioned medium treated goups than the vehicle-conditioned medium treated group (Fig.

대상 데이터

They were grown with Dulbecco Modified Eagle's Medium (DMEM, Welgene, Gyeongsan-si, Korea) containing 10% fetal bovine serum (FBS, Welgene, Gyeongsan-si, Korea), L-glutamine, pen- icillin, and streptomycin (Thermo Scientific, Waltham, MA, USA) in a humidified incubator at 37℃ under 5% CO₂ and 95% air. The PC₁₂ cell line, which is a pheochromocytoma that originated from the adrenal medulla of rats, was also used in this study and provided by the Korean Cell Line Bank (Seoul, Korea). PC₁₂ cells were grown in Roswell Park Memorial Institute medium (RPMI, Welgene, Gyeongsan-si, Korea) containing 10% FBS (Welgene), L-glutamine, pen- icillin, and streptomycin (Thermo Scientific, Waltham, MA, USA) in a humidified incubator at 37℃ under 5% CO₂ and 95% air.
, Dongducheon, Korea) at 60℃. Voucher specimens of A. co- chinchinensis (WPC-14-003) were deposited in the functional materials bank of the PNU-Wellbeing RIS Center at Pusan National University. Dry roots of A.

데이터처리

One-way ANOVA was used to identify significant differ- ences between vehicle and AEAC treated groups (SPSS for Windows, Release 10.10, Standard Version, Chicago, IL, USA). All values are reported as the means ± standard devi- ation (SD) and a p value of <0.

이론/모형

Total phenolics in AEAC were measured by the Folin- Ciocalteu method, with slight modification [32]. Briefly, 1 ml of AEAC solution was mixed with 5 ml of Folin- Ciocalteu reagent (Sigma-Aldrich Co.

성능/효과

Therefore, we investigated whether the in- crease in NGF concentration induced by AEAC treatment could affect the two NGF receptor signaling pathways in PC₁₂ cells. Analysis of the high affinity receptor revealed that the level of p-TrkA was slightly higher in the AEAC- conditioned medium treated group than the vehicle-con- ditioned medium treated group. A similar enhancement was observed in the level of p-ERK and p-Akt among down- stream members of the high affinity receptor.
cochinchinensis in B35 cells and PC₁₂ cells. Our results showed that AEAC could significantly stimulate the secretion ability and synthesis of NGF in B35 cells, and that secreted NGF induced the differentiation of PC₁₂ cells through regulation of the high affinity and low affinity re- ceptor signaling pathway.
6). Taken together, these results showed that NGF secreted by AEAC treatment may stim- ulate proliferation and inhibit apoptosis of PC₁₂ cells through regulation of the NGF receptor TrkA and p75^NTR signaling pathways.

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저자의 다른 논문 :

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